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Sample records for albumin fusion proteins

  1. Recombinant fusion protein of albumin-retinol binding protein inactivates stellate cells

    SciTech Connect

    Choi, Soyoung; Park, Sangeun; Kim, Suhyun; Lim, Chaeseung; Kim, Jungho; Cha, Dae Ryong; Oh, Junseo

    2012-02-03

    Highlights: Black-Right-Pointing-Pointer We designed novel recombinant albumin-RBP fusion proteins. Black-Right-Pointing-Pointer Expression of fusion proteins inactivates pancreatic stellate cells (PSCs). Black-Right-Pointing-Pointer Fusion proteins are successfully internalized into and inactivate PSCs. Black-Right-Pointing-Pointer RBP moiety mediates cell specific uptake of fusion protein. -- Abstract: Quiescent pancreatic- (PSCs) and hepatic- (HSCs) stellate cells store vitamin A (retinol) in lipid droplets via retinol binding protein (RBP) receptor and, when activated by profibrogenic stimuli, they transform into myofibroblast-like cells which play a key role in the fibrogenesis. Despite extensive investigations, there is, however, currently no appropriate therapy available for tissue fibrosis. We previously showed that the expression of albumin, composed of three homologous domains (I-III), inhibits stellate cell activation, which requires its high-affinity fatty acid-binding sites asymmetrically distributed in domain I and III. To attain stellate cell-specific uptake, albumin (domain I/III) was coupled to RBP; RBP-albumin{sup domain} {sup III} (R-III) and albumin{sup domain} {sup I}-RBP-albumin{sup III} (I-R-III). To assess the biological activity of fusion proteins, cultured PSCs were used. Like wild type albumin, expression of R-III or I-R-III in PSCs after passage 2 (activated PSCs) induced phenotypic reversal from activated to fat-storing cells. On the other hand, R-III and I-R-III, but not albumin, secreted from transfected 293 cells were successfully internalized into and inactivated PSCs. FPLC-purified R-III was found to be internalized into PSCs via caveolae-mediated endocytosis, and its efficient cellular uptake was also observed in HSCs and podocytes among several cell lines tested. Moreover, tissue distribution of intravenously injected R-III was closely similar to that of RBP. Therefore, our data suggest that albumin-RBP fusion protein comprises

  2. Retinol Binding Protein-Albumin Domain III Fusion Protein Deactivates Hepatic Stellate Cells

    PubMed Central

    Park, Sangeun; Choi, Soyoung; Lee, Min-Goo; Lim, Chaeseung; Oh, Junseo

    2012-01-01

    Liver fibrosis is characterized by accumulation of extracellular matrix, and activated hepatic stellate cells (HSCs) are the primary source of the fibrotic neomatrix and considered as therapeutic target cells. We previously showed that albumin in pancreatic stellate cells (PSCs), the key cell type for pancreatic fibrogenesis, is directly involved in the formation of vitamin A-containing lipid droplets, inhibiting PSC activation. In this study, we evaluated the anti-fibrotic activity of both albumin and retinol binding protein-albumin domain III fusion protein (R-III), designed for stellate cell-targeted delivery of albumin III, in rat primary HSCs and investigated the underlying mechanism. Forced expression of albumin or R-III in HSCs after passage 2 (activated HSCs) induced lipid droplet formation and deactivated HSCs, whereas point mutations in high-affinity fatty acid binding sites of albumin domain III abolished their activities. Exogenous R-III, but not albumin, was successfully internalized into and deactivated HSC-P2. When HSCs at day 3 after plating (pre-activated HSCs) were cultured in the presence of purified R-III, spontaneous activation of HSCs was inhibited even after passage 2, suggestive of a potential for preventive effect. Furthermore, treatment of HSCs-P2 with R-III led to a significant reduction in both cytoplasmic levels of all-trans retinoic acid and the subsequent retinoic acid signaling. Therefore, our data suggest that albumin deactivates HSCs with reduced retinoic acid levels and that R-III may have therapeutic and preventive potentials on liver fibrosis. PMID:23161170

  3. Fusion protein of retinol-binding protein and albumin domain III reduces liver fibrosis

    PubMed Central

    Lee, Hongsik; Jeong, Hyeyeun; Park, Sangeun; Yoo, Wonbaek; Choi, Soyoung; Choi, Kyungmin; Lee, Min-Goo; Lee, Mihwa; Cha, DaeRyong; Kim, Young-Sik; Han, Jeeyoung; Kim, Wonkon; Park, Sun-Hwa; Oh, Junseo

    2015-01-01

    Activated hepatic stellate cells (HSCs) play a key role in liver fibrosis, and inactivating HSCs has been considered a promising therapeutic approach. We previously showed that albumin and its derivative designed for stellate cell-targeting, retinol-binding protein–albumin domain III fusion protein (referred to as R-III), inactivate cultured HSCs. Here, we investigated the mechanism of action of albumin/R-III in HSCs and examined the anti-fibrotic potential of R-III in vivo. R-III treatment and albumin expression downregulated retinoic acid (RA) signaling which was involved in HSC activation. RA receptor agonist and retinaldehyde dehydrogenase overexpression abolished the anti-fibrotic effect of R-III and albumin, respectively. R-III uptake into cultured HSCs was significantly decreased by siRNA-STRA6, and injected R-III was localized predominantly in HSCs in liver. Importantly, R-III administration reduced CCl4- and bile duct ligation-induced liver fibrosis. R-III also exhibited a preventive effect against CCl4-inducd liver fibrosis. These findings suggest that the anti-fibrotic effect of albumin/R-III is, at least in part, mediated by downregulation of RA signaling and that R-III is a good candidate as a novel anti-fibrotic drug. PMID:25864124

  4. Expression and bioactivity of recombinant human serum albumin and dTMP fusion proteins in CHO cells.

    PubMed

    Ru, Yi; Zhi, Dejuan; Guo, Dingding; Wang, Yong; Li, Yang; Wang, Meizhu; Wei, Suzhen; Wang, Haiqing; Wang, Na; Che, Jingmin; Li, Hongyu

    2016-09-01

    The 14-amino acid (IEGPTLRQWLAARA) thrombopoietin mimetic peptide (TMP) shares no sequence homology with native thrombopoietin (TPO). When dimerized, it displays a high-binding affinity for the TPO receptor and has equipotent bioactivity with recombinant human TPO (rhTPO) in stimulating proliferation and maturation of megakaryocytes in vitro. However, TMP is limited for clinical usage because of its short half-life in vivo. In this study, fusion proteins that composed of tandem dimer of TMP (dTMP) genetically fused at the C- or N-terminus of human serum albumin (HSA) were separately expressed in Chinese hamster ovary (CHO) cells. In vitro bioactivity assays showed that purified fusion proteins promoted the proliferation of megakaryocytes in a dose-dependent manner and activated signal transducer and activator of transcription (STAT) pathway in TPO receptor-dependent manner. Following subcutaneous administration, both HSA-dTMP and dTMP-HSA significantly elevated peripheral platelet counts in normal mice in a dose-dependent manner. In addition, fusion with HSA successfully prolonged dTMP half-life in mice. However, when HSA was fused at the C-terminus of dTMP, the bioactivity of dTMP-HSA was about half of that of HSA-dTMP. In conclusion, these results suggested that HSA/dTMP fusion proteins might be potential drugs for thrombocytopenia and, when HSA was fused at the N-terminus of dTMP, the fusion protein had a higher activity. PMID:27115755

  5. Protein Crystal Serum Albumin

    NASA Technical Reports Server (NTRS)

    1998-01-01

    As the most abundant protein in the circulatory system albumin contributes 80% to colloid osmotic blood pressure. Albumin is also chiefly responsible for the maintenance of blood pH. It is located in every tissue and bodily secretion, with extracellular protein comprising 60% of total albumin. Perhaps the most outstanding property of albumin is its ability to bind reversibly to an incredible variety of ligands. It is widely accepted in the pharmaceutical industry that the overall distribution, metabolism, and efficiency of many drugs are rendered ineffective because of their unusually high affinity for this abundant protein. An understanding of the chemistry of the various classes of pharmaceutical interactions with albumin can suggest new approaches to drug therapy and design. Principal Investigator: Dan Carter/New Century Pharmaceuticals

  6. Development and Characterization of a Novel Fusion Protein of a Mutated Granulocyte Colony-Stimulating Factor and Human Serum Albumin in Pichia pastoris

    PubMed Central

    Huang, Yan-Shan; Wen, Xiao-Fang; Yang, Zhi-Yu; Wu, Yi-Liang; Lu, You; Zhou, Lin-Fu

    2014-01-01

    The purpose of the present work was to develop a novel, long-acting and potent human serum albumin/granulocyte colony stimulating factor (HSA/G-CSF) therapeutic fusion protein. The novel fusion protein, called HMG, was constructed by genetically fusing mutated human derived G-CSF (mG-CSF) to the C-terminal of HSA and then prepared in Pichia pastoris. The molecular mass of HMG was about 85 kDa and the isoelectric point was 5.3. Circular dichroism spectroscopy suggested that mG-CSF retained nearly all of its native secondary structure, regardless of fusion. The binding capabilities of mG-CSF moiety to G-CSF receptor and HSA moiety to warfarin showed very little change after fusing. The bioactivity of HMG (11.0×106 IU/mg) was more than twice that of rHSA/G-CSF (4.6×106 IU/mg). A mutation was made at the 718th amino acid of HMG, substituting Ala for Thr, to investigate the glycosylation of HMG expressed in P. pastoris. Data indicated that HMG was modified at Thr718, speculatively with the addition of a mannose chain. In conclusion, a novel HSA/G-CSF fusion protein was successfully constructed based on a mutated G-CSF. This protein showed more potent bioactivity than rHSA/G-CSF and thus may be a suitable long-acting G-CSF. PMID:25535738

  7. Transforming the treatment for hemophilia B patients: update on the clinical development of recombinant fusion protein linking recombinant coagulation factor IX with recombinant albumin (rIX-FP).

    PubMed

    Santagostino, Elena

    2016-05-01

    Recombinant fusion protein linking recombinant coagulation factor IX with recombinant albumin (rIX-FP; Idelvion®(†)) is an innovative new treatment designed to extend the half-life of factor IX (FIX) and ease the burden of care for hemophilia B patients. The rIX-FP clinical development program - PROLONG-9FP - is in its advanced phases, with pivotal studies in previously treated adults, adolescents, and pediatrics now completed. Across all age groups studied, rIX-FP has demonstrated a markedly improved pharmacokinetic profile compared with plasma-derived and recombinant FIX treatments, with a 30-40% higher incremental recovery, an approximately 5-fold longer half-life, a lower clearance, and a greater area under the curve. rIX-FP has been very well tolerated with an excellent safety profile. In the pivotal studies, there have been no reports of FIX inhibitors or antidrug antibodies, and few treatment-related adverse events have been observed. Prophylactic regimens of rIX-FP administered once weekly to once every 14 days have been highly effective. When used for surgical prophylaxis, a single infusion of rIX-FP has been sufficient to maintain hemostasis, even during major orthopedic surgery. An ongoing study is now enrolling previously untreated patients and evaluating the possibility of extending the dosing interval to every 21 days. There is little doubt that rIX-FP will transform the treatment of hemophilia B. PMID:27288064

  8. A novel exendin-4 human serum albumin fusion protein, E2HSA, with an extended half-life and good glucoregulatory effect in healthy rhesus monkeys

    SciTech Connect

    Zhang, Ling; Wang, Lin; Meng, Zhiyun; Gan, Hui; Gu, Ruolan; Wu, Zhuona; Gao, Lei; Zhu, Xiaoxia; Sun, Wenzhong; Li, Jian; Zheng, Ying; Dou, Guifang

    2014-03-07

    Highlights: • E2HSA has an extended half-life and good plasma stability. • E2HSA could improve glucose-dependent insulin secretion. • E2HSA has excellent glucoregulatory effects in vivo. • E2HSA could potentially be used as a new long-acting GLP-1 receptor agonist for type 2 diabetes management. - Abstract: Glucagon-like peptide-1 (GLP-1) has attracted considerable research interest in terms of the treatment of type 2 diabetes due to their multiple glucoregulatory functions. However, the short half-life, rapid inactivation by dipeptidyl peptidase-IV (DPP-IV) and excretion, limits the therapeutic potential of the native incretin hormone. Therefore, efforts are being made to develop the long-acting incretin mimetics via modifying its structure. Here we report a novel recombinant exendin-4 human serum albumin fusion protein E2HSA with HSA molecule extends their circulatory half-life in vivo while still retaining exendin-4 biological activity and therapeutic properties. In vitro comparisons of E2HSA and exendin-4 showed similar insulinotropic activity on rat pancreatic islets and GLP-1R-dependent biological activity on RIN-m5F cells, although E2HSA was less potent than exendin-4. E2HSA had a terminal elimation half-life of approximate 54 h in healthy rhesus monkeys. Furthermore, E2HSA could reduce postprandial glucose excursion and control fasting glucose level, dose-dependent suppress food intake. Improvement in glucose-dependent insulin secretion and control serum glucose excursions were observed during hyperglycemic clamp test (18 h) and oral glucose tolerance test (42 h) respectively. Thus the improved physiological characterization of E2HSA make it a new potent anti-diabetic drug for type 2 diabetes therapy.

  9. Results of a phase I/II open-label, safety and efficacy trial of coagulation factor IX (recombinant), albumin fusion protein in haemophilia B patients

    PubMed Central

    Martinowitz, U; Lissitchkov, T; Lubetsky, A; Jotov, G; Barazani-Brutman, T; Voigt, C; Jacobs, I; Wuerfel, T; Santagostino, E

    2015-01-01

    Introduction rIX-FP is a coagulation factor IX (recombinant), albumin fusion protein with more than fivefold half-life prolongation over other standard factor IX (FIX) products available on the market. Aim This prospective phase II, open-label study evaluated the safety and efficacy of rIX-FP for the prevention of bleeding episodes during weekly prophylaxis and assessed the haemostatic efficacy for on-demand treatment of bleeding episodes in previously treated patients with haemophilia B. Methods The study consisted of a 10–14 day evaluation of rIX-FP pharmacokinetics (PK), and an 11 month safety and efficacy evaluation period with subjects receiving weekly prophylaxis treatment. Safety was evaluated by the occurrence of related adverse events, and immunogenic events, including development of inhibitors. Efficacy was evaluated by annualized spontaneous bleeding rate (AsBR), and the number of injections to achieve haemostasis. Results Seventeen subjects participated in the study, 13 received weekly prophylaxis and 4 received episodic treatment only. No inhibitors were detected in any subject. The mean and median AsBR were 1.25, and 1.13 respectively in the weekly prophylaxis arm. All bleeding episodes were treated with 1 or 2 injections of rIX-FP. Three prophylaxis subjects who were treated on demand prior to study entry had >85% reduction in AsBR compared to the bleeding rate prior to study entry. Conclusion This study demonstrated the efficacy for weekly routine prophylaxis of rIX-FP to prevent spontaneous bleeding episodes and for the treatment of bleeding episodes. In addition no safety issues were detected during the study and an improved PK profile was demonstrated. PMID:25990590

  10. Long-acting recombinant coagulation factor IX albumin fusion protein (rIX-FP) in hemophilia B: results of a phase 3 trial

    PubMed Central

    Martinowitz, Uri; Lissitchkov, Toshko; Pan-Petesch, Brigitte; Hanabusa, Hideji; Oldenburg, Johannes; Boggio, Lisa; Negrier, Claude; Pabinger, Ingrid; von Depka Prondzinski, Mario; Altisent, Carmen; Castaman, Giancarlo; Yamamoto, Koji; Álvarez-Roman, Maria-Teresa; Voigt, Christine; Blackman, Nicole; Jacobs, Iris

    2016-01-01

    A global phase 3 study evaluated the pharmacokinetics, efficacy, and safety of recombinant fusion protein linking coagulation factor IX with albumin (rIX-FP) in 63 previously treated male patients (12-61 years) with severe hemophilia B (factor IX [FIX] activity ≤2%). The study included 2 groups: group 1 patients received routine prophylaxis once every 7 days for 26 weeks, followed by either 7-, 10-, or 14-day prophylaxis regimen for a mean of 50, 38, or 51 weeks, respectively; group 2 patients received on-demand treatment of bleeding episodes for 26 weeks and then switched to a 7-day prophylaxis regimen for a mean of 45 weeks. The mean terminal half-life of rIX-FP was 102 hours, 4.3-fold longer than previous FIX treatment. Patients maintained a mean trough of 20 and 12 IU/dL FIX activity on prophylaxis with rIX-FP 40 IU/kg weekly and 75 IU/kg every 2 weeks, respectively. There was 100% reduction in median annualized spontaneous bleeding rate (AsBR) and 100% resolution of target joints when subjects switched from on-demand to prophylaxis treatment with rIX-FP (P < .0001). The median AsBR was 0.00 for all prophylaxis regimens. Overall, 98.6% of bleeding episodes were treated successfully, including 93.6% that were treated with a single injection. No patient developed an inhibitor, and no safety concerns were identified. These results indicate rIX-FP is safe and effective for preventing and treating bleeding episodes in patients with hemophilia B at dosing regimens of 40 IU/kg weekly and 75 IU/kg every 2 weeks. This trial was registered at www.clinicaltrials.gov as #NCT0101496274. PMID:26755710

  11. Long-acting recombinant coagulation factor IX albumin fusion protein (rIX-FP) in hemophilia B: results of a phase 3 trial.

    PubMed

    Santagostino, Elena; Martinowitz, Uri; Lissitchkov, Toshko; Pan-Petesch, Brigitte; Hanabusa, Hideji; Oldenburg, Johannes; Boggio, Lisa; Negrier, Claude; Pabinger, Ingrid; von Depka Prondzinski, Mario; Altisent, Carmen; Castaman, Giancarlo; Yamamoto, Koji; Álvarez-Roman, Maria-Teresa; Voigt, Christine; Blackman, Nicole; Jacobs, Iris

    2016-04-01

    A global phase 3 study evaluated the pharmacokinetics, efficacy, and safety of recombinant fusion protein linking coagulation factor IX with albumin (rIX-FP) in 63 previously treated male patients (12-61 years) with severe hemophilia B (factor IX [FIX] activity ≤2%). The study included 2 groups: group 1 patients received routine prophylaxis once every 7 days for 26 weeks, followed by either 7-, 10-, or 14-day prophylaxis regimen for a mean of 50, 38, or 51 weeks, respectively; group 2 patients received on-demand treatment of bleeding episodes for 26 weeks and then switched to a 7-day prophylaxis regimen for a mean of 45 weeks. The mean terminal half-life of rIX-FP was 102 hours, 4.3-fold longer than previous FIX treatment. Patients maintained a mean trough of 20 and 12 IU/dL FIX activity on prophylaxis with rIX-FP 40 IU/kg weekly and 75 IU/kg every 2 weeks, respectively. There was 100% reduction in median annualized spontaneous bleeding rate (AsBR) and 100% resolution of target joints when subjects switched from on-demand to prophylaxis treatment with rIX-FP (P< .0001). The median AsBR was 0.00 for all prophylaxis regimens. Overall, 98.6% of bleeding episodes were treated successfully, including 93.6% that were treated with a single injection. No patient developed an inhibitor, and no safety concerns were identified. These results indicate rIX-FP is safe and effective for preventing and treating bleeding episodes in patients with hemophilia B at dosing regimens of 40 IU/kg weekly and 75 IU/kg every 2 weeks. This trial was registered atwww.clinicaltrials.govas #NCT0101496274. PMID:26755710

  12. Total Protein and Albumin/Globulin Ratio Test

    MedlinePlus

    ... limited. Home Visit Global Sites Search Help? Total Protein and Albumin/Globulin (A/G) Ratio Share this ... Globulin Ratio; A/G Ratio Formal name: Total Protein; Albumin to Globulin Ratio Related tests: Albumin ; Liver ...

  13. Elimination of the free sulfhydryl group in the human serum albumin (HSA) moiety of human interferon-alpha2b and HSA fusion protein increases its stability against mechanical and thermal stresses.

    PubMed

    Zhao, Hong Liang; Xue, Chong; Wang, Yang; Sun, Bo; Yao, Xue Qin; Liu, Zhi Min

    2009-06-01

    Interferon-alpha2b (IFN-alpha2b) and human serum albumin (HSA) fusion protein (IFN-alpha2b-HSA) is a promising long acting formulation of IFN-alpha2b for the treatment of hepatitis C. However, accelerated mechanical and thermal stress tests revealed that IFN-alpha2b-HSA was prone to disulfide-linked aggregation. The formation of aggregates was associated with an increase in immunogenicity in mice. The addition of non-ionic surfactant Tween 80 increased the stability of IFN-alpha2b-HSA against agitation, but its thermal stability was not improved. Moreover, Tween 80 prompted the aggregation of IFN-alpha2b-HSA during quiescent storage. To increase the stability of IFN-alpha2b-HSA, the unpaired cysteine residue in this fusion protein was substituted with serine by site-directed mutagenesis. The resultant fusion protein was designated as IFN-alpha2b-HSA(C34S). IFN-alpha2b-HSA(C34S) had significant higher stability over IFN-alpha2b-HSA, which was evidenced by the facts that after agitation for 72 h or incubation at 60 degrees C for 2 h, more than 90% of IFN-alpha2b-HSA(C34S) remained monomeric. Consistent with its improved stability, the immunogenicity of IFN-alpha2b-HSA(C34S) increased less significantly after agitation. Pharmacokinetics studies in rats revealed that both fusion proteins had similar pharmacokinetic behavior, both with a half-life of about 50 h. PMID:19462475

  14. (PCG) Protein Crystal Growth Human Serum Albumin

    NASA Technical Reports Server (NTRS)

    1989-01-01

    (PCG) Protein Crystal Growth Human Serum Albumin. Contributes to many transport and regulatory processes and has multifunctional binding properties which range from various metals, to fatty acids, hormones, and a wide spectrum of therapeutic drugs. The most abundant protein of the circulatory system. It binds and transports an incredible variety of biological and pharmaceutical ligands throughout the blood stream. Principal Investigator on STS-26 was Larry DeLucas.

  15. (PCG) Protein Crystal Growth Horse Serum Albumin

    NASA Technical Reports Server (NTRS)

    1995-01-01

    Horse Serum Albumin crystals grown during the USML-1 (STS-50) mission's Protein Crystal Growth Glovebox Experiment. These crystals were grown using a vapor diffusion technique at 22 degrees C. The crystals were allowed to grow for nine days while in orbit. Crystals of 1.0 mm in length were produced. The most abundant blood serum protein, regulates blood pressure and transports ions, metabolites, and therapeutic drugs. Principal Investigator was Edward Meehan.

  16. PEGylated Albumin-Based Polyion Complex Micelles for Protein Delivery.

    PubMed

    Jiang, Yanyan; Lu, Hongxu; Chen, Fan; Callari, Manuela; Pourgholami, Mohammad; Morris, David L; Stenzel, Martina H

    2016-03-14

    An increasing amount of therapeutic agents are based on proteins. However, proteins as drug have intrinsic problems such as their low hydrolytic stability. Delivery of proteins using nanoparticles has increasingly been the focus of interest with polyion complex micelles, prepared from charged block copolymer and the oppositely charged protein, as an example of an attractive carrier for proteins. Inspired by this approach, a more biocompatible pathway has been developed here, which replaces the charged synthetic polymer with an abundant protein, such as albumin. Although bovine serum albumin (BSA) was observed to form complexes with positively charged proteins directly, the resulting protein nanoparticle were not stable and aggregated to large precipitates over the course of a day. Therefore, maleimide functionalized poly(oligo (ethylene glycol) methyl ether methacrylate) (MI-POEGMEMA) (Mn = 26000 g/mol) was synthesized to generate a polymer-albumin conjugate, which was able to condense positively charged proteins, here lysozyme (Lyz) as a model. The PEGylated albumin polyion complex micelle with lysozyme led to nanoparticles between 15 and 25 nm in size depending on the BSA to Lyz ratio. The activity of the encapsulated protein was tested using Sprouty 1 (C-12; Spry1) proteins, which can act as an endogenous angiogenesis inhibitor. Condensation of Spry1 with the PEGylated albumin could improve the anticancer efficacy of Spry1 against the breast cancer cells lowering the IC50 value of the protein. Furthermore, the high anticancer efficacy of the POEGMEMA-BSA/Spry1 complex micelle was verified by effectively inhibiting the growth of three-dimensional MCF-7 multicellular tumor spheroids. The PEGylated albumin complex micelle has great potential as a drug delivery vehicle for a new generation of cancer pharmaceuticals. PMID:26809948

  17. Protein extracts from cultured cells contain nonspecific serum albumin.

    PubMed

    Miyara, Masatsugu; Umeda, Kanae; Ishida, Keishi; Sanoh, Seigo; Kotake, Yaichiro; Ohta, Shigeru

    2016-06-01

    Serum is an important component of cell culture media. The present study demonstrates contamination of intracellular protein extract by bovine serum albumin from the culture media and illustrates how this contamination can cause the misinterpretation of western blot results. Preliminary experiments can prevent the misinterpretation of some experimental results, and optimization of the washing process may enable specific protein detection. PMID:26967711

  18. Cellulose binding domain fusion proteins

    DOEpatents

    Shoseyov, O.; Yosef, K.; Shpiegl, I.; Goldstein, M.A.; Doi, R.H.

    1998-02-17

    A cellulose binding domain (CBD) having a high affinity for crystalline cellulose and chitin is disclosed, along with methods for the molecular cloning and recombinant production. Fusion products comprising the CBD and a second protein are likewise described. A wide range of applications are contemplated for both the CBD and the fusion products, including drug delivery, affinity separations, and diagnostic techniques. 16 figs.

  19. Cellulose binding domain fusion proteins

    DOEpatents

    Shoseyov, Oded; Shpiegl, Itai; Goldstein, Marc A.; Doi, Roy H.

    1998-01-01

    A cellulose binding domain (CBD) having a high affinity for crystalline cellulose and chitin is disclosed, along with methods for the molecular cloning and recombinant production thereof. Fusion products comprising the CBD and a second protein are likewise described. A wide range of applications are contemplated for both the CBD and the fusion products, including drug delivery, affinity separations, and diagnostic techniques.

  20. Biologically active protein fragments containing specific binding regions of serum albumin or related proteins

    NASA Technical Reports Server (NTRS)

    Carter, Daniel C. (Inventor)

    1998-01-01

    In accordance with the present invention, biologically active protein fragments can be constructed which contain only those specific portions of the serum albumin family of proteins such as regions known as subdomains IIA and IIIA which are primarily responsible for the binding properties of the serum albumins. The artificial serums that can be prepared from these biologically active protein fragments are advantageous in that they can be produced much more easily than serums containing the whole albumin, yet still retain all or most of the original binding potential of the full albumin proteins. In addition, since the protein fragment serums of the present invention can be made from non-natural sources using conventional recombinant DNA techniques, they are far safer than serums containing natural albumin because they do not carry the potentially harmful viruses and other contaminants that will be found in the natural substances.

  1. 21 CFR 862.1645 - Urinary protein or albumin (nonquantitative) test system.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Urinary protein or albumin (nonquantitative) test... Chemistry Test Systems § 862.1645 Urinary protein or albumin (nonquantitative) test system. (a) Identification. A urinary protein or albumin (nonquantitative) test system is a device intended to...

  2. 21 CFR 862.1645 - Urinary protein or albumin (nonquantitative) test system.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Urinary protein or albumin (nonquantitative) test... Chemistry Test Systems § 862.1645 Urinary protein or albumin (nonquantitative) test system. (a) Identification. A urinary protein or albumin (nonquantitative) test system is a device intended to...

  3. 21 CFR 862.1645 - Urinary protein or albumin (nonquantitative) test system.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Urinary protein or albumin (nonquantitative) test... Chemistry Test Systems § 862.1645 Urinary protein or albumin (nonquantitative) test system. (a) Identification. A urinary protein or albumin (nonquantitative) test system is a device intended to...

  4. 21 CFR 862.1645 - Urinary protein or albumin (nonquantitative) test system.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Urinary protein or albumin (nonquantitative) test... Chemistry Test Systems § 862.1645 Urinary protein or albumin (nonquantitative) test system. (a) Identification. A urinary protein or albumin (nonquantitative) test system is a device intended to...

  5. 21 CFR 862.1645 - Urinary protein or albumin (nonquantitative) test system.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Urinary protein or albumin (nonquantitative) test... Chemistry Test Systems § 862.1645 Urinary protein or albumin (nonquantitative) test system. (a) Identification. A urinary protein or albumin (nonquantitative) test system is a device intended to...

  6. Fusion protein of single-chain variable domain fragments for treatment of myasthenia gravis

    PubMed Central

    Li, Fangfang; Meng, Fanping; Jin, Quanxin; Sun, Changyuan; Li, Yingxin; Li, Honghua; Jin, Songzhu

    2014-01-01

    Single-chain variable domain fragment (scFv) 637 is an antigen-specific scFv of myasthenia gravis. In this study, scFv and human serum albumin genes were conjugated and the fusion protein was expressed in Pichia pastoris. The affinity of scFv-human serum albumin fusion protein to bind to acetylcholine receptor at the neuromuscular junction of human intercostal muscles was detected by immunofluorescence staining. The ability of the fusion protein to block myasthenia gravis patient sera binding to acetylcholine receptors and its stability in healthy serum were measured by competitive ELISA. The results showed that the inhibition rate was 2.0-77.4%, and the stability of fusion protein in static healthy sera was about 3 days. This approach suggests the scFv-human serum albumin is a potential candidate for specific immunosuppressive therapy of myasthenia gravis. PMID:25206900

  7. Chromatographic and traditional albumin isotherms on cellulose: a model for wound protein adsorption on modified cotton

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Albumin is the most abundant protein found in healing wounds. Traditional and chromatogrpahic protein isotherms of albumin binding on modified cotton fibers are useful in understanding albumin binding to cellulose wound dressings. An important consideration in the design of cellulosic wound dressin...

  8. Class II virus membrane fusion proteins

    SciTech Connect

    Kielian, Margaret . E-mail: kielian@aecom.yu.edu

    2006-01-05

    Enveloped animal viruses fuse their membrane with a host cell membrane, thus delivering the virus genetic material into the cytoplasm and initiating infection. This critical membrane fusion reaction is mediated by a virus transmembrane protein known as the fusion protein, which inserts its hydrophobic fusion peptide into the cell membrane and refolds to drive the fusion reaction. This review describes recent advances in our understanding of the structure and function of the class II fusion proteins of the alphaviruses and flaviviruses. Inhibition of the fusion protein refolding reaction confirms its importance in fusion and suggests new antiviral strategies for these medically important viruses.

  9. Photo selective protein immobilization using bovine serum albumin

    NASA Astrophysics Data System (ADS)

    Kim, Wan-Joong; Kim, Ansoon; Huh, Chul; Park, Chan Woo; Ah, Chil Seong; Kim, Bong Kyu; Yang, Jong-Heon; Chung, Kwang Hyo; Choi, Yo Han; Hong, Jongcheol; Sung, Gun Yong

    2012-11-01

    A simple and selective technique which immobilizes protein onto a solid substrate by using UV illumination has been developed. In protein immobilization, a Bovine serum albumin (BSA) performed bifunctional role as a cross-linker between substrate and proteins and as a blocker inhibiting a nonspecific protein adsorption. A new photo-induced protein immobilization process has been investigated at each step by fluorescence microscopy, ellipsometry, and Fourier transform infrared (FT-IR) spectroscopy. A UV photomask has been used to induce selective protein immobilization on target regions of the surface of the SiO2 substrates under UV illumination with negligible nonspecific binding. The UV illumination also showed improved photostability than the conventional methods which employed bifunctional photo-crosslinker molecules of photo-reactive diazirine. This new UV illumination-based photo-addressable protein immobilization provides a new approach for developing novel protein microarrays for multiplexed sensing as well as other types of bio-immobilization in biomedical devices and biotechnologies.

  10. Intracellular Delivery of Proteins via Fusion Peptides in Intact Plants

    PubMed Central

    Ng, Kiaw Kiaw; Motoda, Yoko; Watanabe, Satoru; Sofiman Othman, Ahmad; Kigawa, Takanori; Kodama, Yutaka; Numata, Keiji

    2016-01-01

    In current plant biotechnology, the introduction of exogenous DNA encoding desired traits is the most common approach used to modify plants. However, general plant transformation methods can cause random integration of exogenous DNA into the plant genome. To avoid these events, alternative methods, such as a direct protein delivery system, are needed to modify the plant. Although there have been reports of the delivery of proteins into cultured plant cells, there are currently no methods for the direct delivery of proteins into intact plants, owing to their hierarchical structures. Here, we demonstrate the efficient fusion-peptide-based delivery of proteins into intact Arabidopsis thaliana. Bovine serum albumin (BSA, 66 kDa) was selected as a model protein to optimize conditions for delivery into the cytosol. The general applicability of our method to large protein cargo was also demonstrated by the delivery of alcohol dehydrogenase (ADH, 150 kDa) into the cytosol. The compatibility of the fusion peptide system with the delivery of proteins to specific cellular organelles was also demonstrated using the fluorescent protein Citrine (27 kDa) conjugated to either a nuclear localization signal (NLS) or a peroxisomal targeting signal (PTS). In conclusion, our designed fusion peptide system can deliver proteins with a wide range of molecular weights (27 to 150 kDa) into the cells of intact A. thaliana without interfering with the organelle-targeting peptide conjugated to the protein. We expect that this efficient protein delivery system will be a powerful tool in plant biotechnology. PMID:27100681

  11. Fusion of an albumin-binding domain extends the half-life of immunotoxins.

    PubMed

    Guo, Rui; Guo, Wenjun; Cao, Li; Liu, Hui; Liu, Jieyu; Xu, Hua; Huang, Weiqiang; Wang, Fengwei; Hong, Zhangyong

    2016-09-10

    Immunotoxins have documented potential as a cancer treatment due to their extreme potency; a single toxin molecule delivered to the cytosol may be sufficient to kill a cell. However, their short half-life in the circulatory system may be one of the key problems associated with the clinical use of immunotoxins and may continue to limit their therapeutic activity. Herein, we genetically fused an albumin-binding domain (ABD) to the human epidermal growth factor receptor 2 (HER2)-specific immunotoxin ZHER2-PE38 to extend the circulation time and thus improve the therapeutic outcome of this immunotoxin. Furthermore, the fusion of an ABD to the immunotoxin was found to promote non-covalent interactions between the immunotoxin and serum albumin, which rescue the immunotoxin from lysosomal degradation through a serum albumin-mediated interaction with the neonatal Fc receptor (FcRn). This manuscript reports the construction, purification, and characterization of the ABD-fused HER2-specific immunotoxin, ABD-ZHER2-PE38, both in vitro and in vivo. Compared with non-fused ZHER2-PE38, this new construct exhibits a clearly increased half-life in plasma (330.8 versus 13.5min, approximately 24.4-fold extension) and remarkably improved antitumor effects in an NCI-N87 subcutaneous xenograft model. Therefore, the new construct represents a potentially attractive therapeutic modality, and the proposed strategy may also have useful applications for current immunotoxin designs. PMID:27457423

  12. Insulin Is Required to Maintain Albumin Expression by Inhibiting Forkhead Box O1 Protein.

    PubMed

    Chen, Qing; Lu, Mingjian; Monks, Bobby R; Birnbaum, Morris J

    2016-01-29

    Diabetes is accompanied by dysregulation of glucose, lipid, and protein metabolism. In recent years, much effort has been spent on understanding how insulin regulates glucose and lipid metabolism, whereas the effect of insulin on protein metabolism has received less attention. In diabetes, hepatic production of serum albumin decreases, and it has been long established that insulin positively controls albumin gene expression. In this study, we used a genetic approach in mice to identify the mechanism by which insulin regulates albumin gene transcription. Albumin expression was decreased significantly in livers with insulin signaling disrupted by ablation of the insulin receptor or Akt. Concomitant deletion of Forkhead Box O1 (Foxo1) in these livers rescued the decreased albumin secretion. Furthermore, activation of Foxo1 in the liver is sufficient to suppress albumin expression. These results suggest that Foxo1 acts as a repressor of albumin expression. PMID:26668316

  13. A Monoclonal IgM Protein with Antibody-like Activity for Human Albumin

    PubMed Central

    Hauptman, Stephen; Tomasi, Thomas B.

    1974-01-01

    The serum of a patient (L'ec) with an IgM lambda monoclonal protein was noted to bind albumin on immunoelectrophoresis. Analytical ultracentrifugation of the L'ec serum demonstrated 23S and 12S peaks, but no 4S (albumin) boundary. Immunologically identical 20S and 9S IgM proteins were isolated from the serum and the addition in vitro of either the patient's albumin or albumin isolated from normal serum was shown to reconstitute the 23S and 12S boundaries. The binding of high molecular weight IgM to albumin was demonstated by Sephadex G200 chromatography with 125I-labeled albumin and isolated IgM. Immunoelectrophoresis of the L'ec IgM developed with aggregated albumin (reverse immunoelectrophoresis) also demonstrated the binding of albumin to IgM. That all of the patient's IgM complexed with albumin was shown by affinity chromatography employing an aggregated albumin-immunoadsorbent column. Binding was shown to be of the noncovalent type by polyacrylamide gel electrophoresis in 8 M urea. With hot trypsin proteolysis, Fabμ and Fcμ5 fragments were isolated, and monomer albumin was shown to complex only with the Fabμ fragment by both analytical ultracentrifugation and molecular sieve chromatogaphy employing 125I-labeled Fab fragments. 1 mol of Fabμ fragment bound 1 mol of monomer albumin. Polymers of human albumin, produced by heat aggregation, precipitated with the isolated L'ec protein on gel diffusion analysis and, when coated on sheep red blood cells, gave a hemagglutination titer greater than 1 million with the whole L'ec serum. 50 additional monoclonal IgM, 33 IgA, and 80 IgG sera failed to show precipitation or hemagglutination with aggregated albumin. Native monomer albumin inhibited precipitation only at high concentrations (> 50 mg/ml); dimer albumin or fragments of albumin produced by trypsin digestion inhibited at low concentrations (0.4 mg/ml). No reactivity occurred with the albumin of five other mammalian species, including bovine. The L'ec protein

  14. Extending the Serum Half-Life of G-CSF via Fusion with the Domain III of Human Serum Albumin

    PubMed Central

    Zhao, Shuqiang; Zhang, Yu; Tian, Hong; Chen, Xiaofei; Cai, Di; Yao, Wenbing; Gao, Xiangdong

    2013-01-01

    Protein fusion technology is one of the most commonly used methods to extend the half-life of therapeutic proteins. In this study, in order to prolong the half-life of Granulocyte colony stimulating factor (G-CSF), the domain III of human serum albumin (3DHSA) was genetically fused to the N-terminal of G-CSF. The 3DHSA-G-CSF fusion gene was cloned into pPICZαA along with the open reading frame of the α-factor signal under the control of the AOX1 promoter. The recombinant expression vector was transformed into Pichia pastoris GS115, and the recombinant strains were screened by SDS-PAGE. As expected, the 3DHSA-G-CSF showed high binding affinity with HSA antibody and G-CSF antibody, and the natural N-terminal of 3DHSA was detected by N-terminal sequencing. The bioactivity and pharmacokinetic studies of 3DHSA-G-CSF were respectively determined using neutropenia model mice and human G-CSF ELISA kit. The results demonstrated that 3DHSA-G-CSF has the ability to increase the peripheral white blood cell (WBC) counts of neutropenia model mice, and the half-life of 3DHSA-G-CSF is longer than that of native G-CSF. In conclusion, 3DHSA can be used to extend the half-life of G-CSF. PMID:24151579

  15. Depletion of the highly abundant protein albumin from human plasma using the Gradiflow.

    PubMed

    Rothemund, Deborah L; Locke, Vicki L; Liew, Audrey; Thomas, Theresa M; Wasinger, Valerie; Rylatt, Dennis B

    2003-03-01

    Analysis of complex protein samples by two-dimensional electrophoresis (2-DE) is often more difficult in the presence of a few predominant proteins. In plasma, proteins such as albumin mask proteins of lower abundance, as well as significantly limiting the amount of protein that can be loaded onto the immobilized pH gradient strip. In this paper the Gradiflow, a preparative electrophoresis system, has been used to deplete human plasma of the highly abundant protein albumin under native and denatured conditions. A three step protocol incorporating a charge separation to collect proteins with an isoelectric point greater than albumin and two size separations to isolate proteins larger and smaller than albumin, was used. When the albumin depleted fractions were analysed on pH 3-10 2-DE gels, proteins that were masked by albumin were revealed and proteins not seen in the unfractionated plasma sample were visualised. Matrix-assisted laser desorption/ionisation-time of flight mass spectrometry analysis confirmed the identification of the protein that lies beneath albumin to be C4B-binding protein alpha chain. The liquid fractions from the Gradiflow separations were also analysed by liquid chromatography-tandem mass spectrometry to confirm the proteins were separated according to their size and charge mobility in an electric field. PMID:12627381

  16. Exo-endo cellulase fusion protein

    DOEpatents

    Bower, Benjamin S.; Larenas, Edmund A.; Mitchinson, Colin

    2012-01-17

    The present invention relates to a heterologous exo-endo cellulase fusion construct, which encodes a fusion protein having cellulolytic activity comprising a catalytic domain derived from a fungal exo-cellobiohydrolase and a catalytic domain derived from an endoglucanase. The invention also relates to vectors and fungal host cells comprising the heterologous exo-endo cellulase fusion construct as well as methods for producing a cellulase fusion protein and enzymatic cellulase compositions.

  17. Fusion to an albumin-binding domain with a high affinity for albumin extends the circulatory half-life and enhances the in vivo antitumor effects of human TRAIL.

    PubMed

    Li, Rui; Yang, Hao; Jia, Dianlong; Nie, Qianxue; Cai, Huawei; Fan, Qing; Wan, Lin; Li, Lin; Lu, Xiaofeng

    2016-04-28

    Clinical applications of recombinant human tumor necrosis factor-related apoptosis-inducing ligand (hTRAIL) have been limited by their poor pharmacokinetics. Using endogenous albumin as a carrier is an attractive approach for circulatory half-life extension. Here, we produced ABD-hTRAIL and hTRAIL-ABD by fusing the albumin-binding domain (ABD) from protein G to the N- or C-terminus of hTRAIL. We found that ABD-hTRAIL bound human serum albumin (HSA) with a high affinity (0.4±0.18nM) and formed nanoparticles with an average diameter (~12nm) above the threshold (~7nm) of renal filtration. ABD-hTRAIL also bound mouse serum albumin (MSA); thus, its half-life was 40-50-fold greater than that of hTRAIL (14.1±0.87h vs 0.32±0.14h). Tumor uptake of ABD-hTRAIL 8-48h post-injection was 6-16-fold that of hTRAIL. Consequently, the tumor suppression of ABD-hTRAIL in mice bearing subcutaneous xenografts was 3-4 times greater than that of hTRAIL. Additionally, the time period during which ABD-hTRAIL could kill circulating tumor cells was approximately 8 times longer than that of hTRAIL. These results demonstrate that ABD fused to the N-terminus endows hTRAIL with albumin binding ability; once it enters the vasculature, ABD mediates binding with endogenous albumin, thus prolonging the half-life and enhancing the antitumor effect of hTRAIL. However, hTRAIL-ABD did not show a high affinity for albumin and therefore did not display the prolonged circulatory half-life and enhanced antitumor effects. These results demonstrate that N-terminal, but not C-terminal, ABD-fusion is an efficient technique for enhancing the antitumor effects of hTRAIL by using endogenous albumin as a carrier. PMID:26951928

  18. Lipid transfer proteins and 2S albumins as allergens.

    PubMed

    Pastorello, E A; Pompei, C; Pravettoni, V; Brenna, O; Farioli, L; Trambaioli, C; Conti, A

    2001-01-01

    Plant lipid transfer proteins, a widespread family of proteins, have been recently identified as important food allergens. Their common structural features, such as eight conserved cysteines forming disulfide bridges, basic isoelectric point and high similarity in amino acid sequence, are the basis of allergic clinical cross-reactivity. This has been demonstrated for the LTP allergens of the Prunoideae subfamily, whose similarity is about 95% as demonstrated for the purified allergens of peach, apricot, plum and apple. A relevant aspect is the existence of sequence homology of LTPs of botanically unrelated foods, as demonstrated for LTPs of maize and peach. A class of food allergens of well recognized clinical importance is that of seed storage 2S albumins. They have been identified in the most diffused edible seeds and nuts, such as mustard, sesame, Brazil nut, walnut and peanut. In particular, a strong correlation between IgE-binding to these proteins and food-induced anaphylaxis has been demonstrated for Brazil nut and sesame seeds. PMID:11298008

  19. Class III viral membrane fusion proteins

    PubMed Central

    Backovic, Marija

    2010-01-01

    SUMMARY Accumulating structural studies of viral fusion glycoproteins have revealed unanticipated structural relationships between unrelated virus families and allowed the grouping of these membrane fusogens into three distinct classes. Here we review the newly identified group of class III viral fusion proteins, whose members include fusion proteins from rhabdoviruses, herpesviruses and baculoviruses. While clearly related in structure, the class III viral fusion proteins exhibit distinct structural features in their architectures as well as in their membrane-interacting fusion loops, which are likely related to their virus-specific differences in cellular entry. Further study of the similarities and differences in the class III viral fusion glycoproteins may provide greater insights into protein:membrane interactions that are key to promoting efficient bilayer fusion during virus entry. PMID:19356922

  20. Purification, identification and preliminary crystallographic studies of a 2S albumin seed protein from Lens culinaris

    SciTech Connect

    Gupta, Pankaj; Gaur, Vineet; Salunke, Dinakar M.

    2008-08-01

    A 2S albumin from L. culinaris was purified and crystallized and preliminary crystallographic studies were carried out. Lens culinaris (lentil) is a widely consumed high-protein-content leguminous crop. A 2S albumin protein (26.5 kDa) has been identified using NH{sub 2}-terminal sequencing from a 90% ammonium sulfate saturation fraction of total L. culinaris seed protein extract. The NH{sub 2}-terminal sequence shows very high homology to PA2, an allergy-related protein from Pisum sativum. The 2S albumin protein was purified using a combination of size-exclusion and ion-exchange chromatography. Crystals of the 2S seed albumin obtained using the hanging-drop vapour-diffusion method diffracted to 2.5 Å resolution and were indexed in space group P4{sub 1} (or P4{sub 3}), with unit-cell parameters a = b = 78.6, c = 135.2 Å.

  1. Glass transitions in aqueous solutions of protein (bovine serum albumin).

    PubMed

    Shinyashiki, Naoki; Yamamoto, Wataru; Yokoyama, Ayame; Yoshinari, Takeo; Yagihara, Shin; Kita, Rio; Ngai, K L; Capaccioli, Simone

    2009-10-29

    Measurements by adiabatic calorimetry of heat capacities and enthalpy relaxation rates of a 20% (w/w) aqueous solution of bovine serum albumin (BSA) by Kawai, Suzuki, and Oguni [Biophys. J. 2006, 90, 3732] have found several enthalpy relaxations at long times indicating different processes undergoing glass transitions. In a quenched sample, one enthalpy relaxation at around 110 K and another over a wide temperature range (120-190 K) were observed. In a sample annealed at 200-240 K after quenching, three separated enthalpy relaxations at 110, 135, and above 180 K were observed. Dynamics of processes probed by adiabatic calorimetric data are limited to long times on the order of 10(3) s. A fuller understanding of the processes can be gained by probing the dynamics over a wider time/frequency range. Toward this goal, we performed broadband dielectric measurements of BSA-water mixtures at various BSA concentrations over a wide frequency range of thirteen decades from 2 mHz to 1.8 GHz at temperatures from 80 to 270 K. Three relevant relaxation processes were detected. For relaxation times equal to 100 s, the three processes are centered approximately at 110, 135, and 200 K, in good agreement with those observed by adiabatic calorimetry. We have made the following interpretation of the molecular origins of the three processes. The fastest relaxation process having relaxation time of 100 or 1000 s at ca. 110 K is due to the secondary relaxation of uncrystallized water (UCW) in the hydration shell. The intermediate relaxation process with 100 s relaxation time at ca. 135 K is due to ice. The slowest relaxation process having relaxation time of 100 s at ca. 200 K is interpreted to originate from local chain conformation fluctuations of protein slaved by water. Experimental evidence supporting these interpretations include the change of temperature dependence of the relaxation time of the UCW at approximately T(gBSA) approximately = 200 K, the glass transition temperature of

  2. Expression and purification of GST fusion proteins.

    PubMed

    Harper, S; Speicher, D W

    2001-05-01

    An increasingly common strategy for expressing proteins and large peptides in prokaryotic systems is to express the protein of interest connected to a "tag" that provides the basis for rapid high-affinity purification. This unit describes the expression and purification of fusion proteins containing the 26-kDa glutathione-S-transferase protein as well as methods for cleaving the affinity tag and repurifying the target protein. Advantages of this popular fusion protein system include high protein yields, high-affinity one-step protein purification of the fusion protein, existence of several alternative protease cleavage sites for removing the affinity tag when required, and ease of removal of the cleaved affinity tag. PMID:18429193

  3. Crystal structures of MBP fusion proteins.

    PubMed

    Waugh, David S

    2016-03-01

    Although chaperone-assisted protein crystallization remains a comparatively rare undertaking, the number of crystal structures of polypeptides fused to maltose-binding protein (MBP) that have been deposited in the Protein Data Bank (PDB) has grown dramatically during the past decade. Altogether, 102 fusion protein structures were detected by Basic Local Alignment Search Tool (BLAST) analysis. Collectively, these structures comprise a range of sizes, space groups, and resolutions that are typical of the PDB as a whole. While most of these MBP fusion proteins were equipped with short inter-domain linkers to increase their rigidity, fusion proteins with long linkers have also been crystallized. In some cases, surface entropy reduction mutations in MBP appear to have facilitated the formation of crystals. A comparison of the structures of fused and unfused proteins, where both are available, reveals that MBP-mediated structural distortions are very rare. PMID:26682969

  4. Role of sequence and structure of the Hendra fusion protein fusion peptide in membrane fusion.

    PubMed

    Smith, Everett Clinton; Gregory, Sonia M; Tamm, Lukas K; Creamer, Trevor P; Dutch, Rebecca Ellis

    2012-08-24

    Viral fusion proteins are intriguing molecular machines that undergo drastic conformational changes to facilitate virus-cell membrane fusion. During fusion a hydrophobic region of the protein, termed the fusion peptide (FP), is inserted into the target host cell membrane, with subsequent conformational changes culminating in membrane merger. Class I fusion proteins contain FPs between 20 and 30 amino acids in length that are highly conserved within viral families but not between. To examine the sequence dependence of the Hendra virus (HeV) fusion (F) protein FP, the first eight amino acids were mutated first as double, then single, alanine mutants. Mutation of highly conserved glycine residues resulted in inefficient F protein expression and processing, whereas substitution of valine residues resulted in hypofusogenic F proteins despite wild-type surface expression levels. Synthetic peptides corresponding to a portion of the HeV F FP were shown to adopt an α-helical secondary structure in dodecylphosphocholine micelles and small unilamellar vesicles using circular dichroism spectroscopy. Interestingly, peptides containing point mutations that promote lower levels of cell-cell fusion within the context of the whole F protein were less α-helical and induced less membrane disorder in model membranes. These data represent the first extensive structure-function relationship of any paramyxovirus FP and demonstrate that the HeV F FP and potentially other paramyxovirus FPs likely require an α-helical structure for efficient membrane disordering and fusion. PMID:22761418

  5. Protein-protein binding before and after photo-modification of albumin

    NASA Astrophysics Data System (ADS)

    Rozinek, Sarah C.; Glickman, Randolph D.; Thomas, Robert J.; Brancaleon, Lorenzo

    2016-03-01

    Bioeffects of directed-optical-energy encompass a wide range of applications. One aspect of photochemical interactions involves irradiating a photosensitizer with visible light in order to induce protein unfolding and consequent changes in function. In the past, irradiation of several dye-protein combinations has revealed effects on protein structure. Beta lactoglobulin, human serum albumin (HSA) and tubulin have all been photo-modified with meso-tetrakis(4- sulfonatophenyl)porphyrin (TSPP) bound, but only in the case of tubulin has binding caused a verified loss of biological function (loss of ability to form microtubules) as a result of this light-induced structural change. The current work questions if the photo-induced structural changes that occur to HSA, are sufficient to disable its biological function of binding to osteonectin. The albumin-binding protein, osteonectin, is about half the molecular weight of HSA, so the two proteins and their bound product can be separated and quantified by size exclusion high performance liquid chromatography. TSPP was first bound to HSA and irradiated, photo-modifying the structure of HSA. Then native HSA or photo-modified HSA (both with TSPP bound) were compared, to assess loss in HSA's innate binding ability as a result of light-induced structure modification.

  6. Affinity purification of proteins binding to GST fusion proteins.

    PubMed

    Swaffield, J C; Johnston, S A

    2001-05-01

    This unit describes the use of proteins fused to glutathione-S-transferase (GST fusion proteins) to affinity purify other proteins, a technique also known as GST pulldown purification. The describes a strategy in which a GST fusion protein is bound to agarose affinity beads and the complex is then used to assay the binding of a specific test protein that has been labeled with [35S]methionine by in vitro translation. However, this method can be adapted for use with other types of fusion proteins; for example, His6, biotin tags, or maltose-binding protein fusions (MBP), and these may offer particular advantages. A describes preparation of an E. coli extract that is added to the reaction mixture with purified test protein to reduce nonspecific binding. PMID:18265191

  7. Continuous versus intermittent exercise effects on urinary excretion of albumin and total protein.

    PubMed

    Montelpare, W J; Klentrou, P; Thoden, J

    2002-09-01

    Several studies have reported post-exercise increases of urinary concentrations of plasma proteins. However, under normal conditions, through mechanisms of size and electrical charge selection, the kidney restricts the clearance of molecules as large as albumin. Post-exercise increases in albuminuria occur following the physiological stress of intense exercise, most likely as a result of the exercise induced blood acidity changes which lead to a change in the arrangement of the albumin molecule, and subsequently the filtration characteristics of the glomerular capillary wall. The purpose of the present study was therefore to determine the extent to which different types of exercise could induce a transient condition of post-exercise increases in the urinary output of total protein and albumin. All 14 males, who agreed to participate in the study, performed a continuous and an intermittent cycling protocol on a stationary bicycle ergometer. The results showed that: a) intermittent exercise had a greater influence than continuous exercise on the total output of urine albumin, and of urine total protein; b) concentrations of blood pH and blood lactate, were associated with changes in the clearance of urine albumin and urine total protein. Post-exercise proteinuria response seems to be transient and therefore renal trauma is not suspected at the early stages of observation. Furthermore, these results indicate that the kidney undergoes distinct physiological adjustments during exercise, and that these adjustments are relative to the intensity of the exercise stress. PMID:12413038

  8. Study of the effect of total serum protein and albumin concentrations on canine fructosamine concentration.

    PubMed Central

    Loste, A; Marca, M C

    1999-01-01

    The relationship among serum fructosamine concentration and total serum protein and albumin concentrations were evaluated in healthy and sick dogs (diabetics and dogs with insulinoma were not included). Fructosamine was determined using a commercial colorimetric nitroblue tetrazolium method applied to the Technicon RA-500 (Bayer). Serum fructosamine concentration was not correlated to total protein in normoproteinemic (r = 0.03) and hyperproteinemic dogs (r = 0.29), but there was a high correlation (r = 0.73) in hypoproteinemic dogs. Similar comparison between serum fructosamine and albumin concentrations showed middle correlation (r = 0.49) in normoalbuminemic dogs and high degree of correlation (r = 0.67) in hypoalbuminemic dogs. These results showed the importance of recognizing serum glucose concentration as well as total serum protein and albumin concentrations in the assay of canine serum fructosamine concentration. PMID:10369572

  9. A kinetic and structural study of two-step aggregation and fusion of neutral phospholipid vesicles promoted by serum albumin at low pH.

    PubMed

    Schenkman, S; Soares de Araujo, P; Sesso, A; Quina, F H; Chaimovich, H

    1981-03-01

    The addition of bovine serum albumin (BSA) to 25 +/- 5 nm diameter single bilayer phosphatidylcholine (PC) vesicles (SBV) (pH 3.5) gives rise to readily visible transient turbidity. Studies of this system, employing a series of techniques, including time-dependent turbidity changes, membrane filtration, centrifugation, Sepharose chromatography and freeze fracture electron microscopy demonstrated that the process involves aggregation and fusion of the vesicles. At least three distinct time-dependent steps have been characterized: (1) the rapid initial formation (in approx. 5 min) of large aggregates (responsible for the visible turbidity) composed of SBV interconnected by BSA in its F form. The formation of these aggregates may be reversed by raising the pH or adding excess BSA to the system at this stage; (2) spontaneous collapse of these large aggregates, in an irreversible step, to form a heterogeneous population of vesicles; (3) fusion produces as the final product of the process, a relatively homogeneous population of larger (50 +/- 10 nm diamter) vesicles. This system serves as a convenient and simple model system for the detailed study of protein-mediated aggregation and fusion of membranes at the molecular level. PMID:7194154

  10. The effects of Hespan on serum and lymphatic albumin, globulin, and coagulant protein.

    PubMed Central

    Lucas, C E; Denis, R; Ledgerwood, A M; Grabow, D

    1988-01-01

    The effects of hydroxyethyl starch (Hespan) resuscitation on serum and lymphatic proteins following hemorrhagic shock were studied in 34 splenectomized dogs. Following shock, five randomly assigned treatment groups received the shed blood plus 50 mL/kg of salt solution (RL) or RL with varying concentrations (0.22-1.5 gm/kg) of Hespan. Each dog received 50 ml/kg/d of the test solution for three days after shock. Prothrombin time, partial thromboplastin time, thrombin time, total serum protein, albumin, globulin, and coagulant protein activity of fibrinogen, prothrombin, and factor VIII were measured before shock, at the end of shock, following resuscitation, and on day 3; thoracic duct lymph values were obtained on day 3. Hespan-supplemented resuscitation lowered all serum proteins including albumin, globulin and coagulant proteins; concomitantly, the lymph protein rose after Hespan resuscitation. This decrease in serum proteins and rise in lymph proteins parallels similar results after albumin resuscitation in man and animals and suggests that Hespan induces an oncotically controlled extravascular protein relocation. Further studies on the significance of these findings need to be conducted. PMID:2451485

  11. Fluorescent sensors based on bacterial fusion proteins

    NASA Astrophysics Data System (ADS)

    Prats Mateu, Batirtze; Kainz, Birgit; Pum, Dietmar; Sleytr, Uwe B.; Toca-Herrera, José L.

    2014-06-01

    Fluorescence proteins are widely used as markers for biomedical and technological purposes. Therefore, the aim of this project was to create a fluorescent sensor, based in the green and cyan fluorescent protein, using bacterial S-layers proteins as scaffold for the fluorescent tag. We report the cloning, expression and purification of three S-layer fluorescent proteins: SgsE-EGFP, SgsE-ECFP and SgsE-13aa-ECFP, this last containing a 13-amino acid rigid linker. The pH dependence of the fluorescence intensity of the S-layer fusion proteins, monitored by fluorescence spectroscopy, showed that the ECFP tag was more stable than EGFP. Furthermore, the fluorescent fusion proteins were reassembled on silica particles modified with cationic and anionic polyelectrolytes. Zeta potential measurements confirmed the particle coatings and indicated their colloidal stability. Flow cytometry and fluorescence microscopy showed that the fluorescence of the fusion proteins was pH dependent and sensitive to the underlying polyelectrolyte coating. This might suggest that the fluorescent tag is not completely exposed to the bulk media as an independent moiety. Finally, it was found out that viscosity enhanced the fluorescence intensity of the three fluorescent S-layer proteins.

  12. Serum Albumin Stimulates Protein Kinase G-dependent Microneme Secretion in Toxoplasma gondii.

    PubMed

    Brown, Kevin M; Lourido, Sebastian; Sibley, L David

    2016-04-29

    Microneme secretion is essential for motility, invasion, and egress in apicomplexan parasites. Although previous studies indicate that Ca(2+) and cGMP control microneme secretion, little is known about how these pathways are naturally activated. Here we have developed genetically encoded indicators for Ca(2+) and microneme secretion to better define the signaling pathways that regulate these processes in Toxoplasma gondii We found that microneme secretion was triggered in vitro by exposure to a single host protein, serum albumin. The natural agonist serum albumin induced microneme secretion in a protein kinase G-dependent manner that correlated with increased cGMP levels. Surprisingly, serum albumin acted independently of elevated Ca(2+) and yet it was augmented by artificial agonists that raise Ca(2+), such as ethanol. Furthermore, although ethanol elevated intracellular Ca(2+), it alone was unable to trigger secretion without the presence of serum or serum albumin. This dichotomy was recapitulated by zaprinast, a phosphodiesterase inhibitor that elevated cGMP and separately increased Ca(2+) in a protein kinase G-independent manner leading to microneme secretion. Taken together, these findings reveal that microneme secretion is centrally controlled by protein kinase G and that this pathway is further augmented by elevation of intracellular Ca(2.) PMID:26933037

  13. Fusion proteins useful for producing pinene

    DOEpatents

    Peralta-Yahya, Pamela P.; Keasling, Jay D

    2016-06-28

    The present invention provides for a modified host cell comprising a heterologous pinene synthase (PS), or enzymatically active fragment or variant thereof, and optionally a geranyl pyrophosphate synthase (GPPS), or enzymatically active fragment or variant thereof, or a fusion protein comprising: (a) a PS and (b) a GPPS linked by a linker.

  14. Solution structure of RicC3, a 2S albumin storage protein from Ricinus communis.

    PubMed

    Pantoja-Uceda, David; Bruix, Marta; Giménez-Gallego, Guillermo; Rico, Manuel; Santoro, Jorge

    2003-12-01

    The three-dimensional structure in aqueous solution of recombinant (15)N labeled RicC3, a 2S albumin protein from the seeds of castor bean (Ricinus communis), has been determined by NMR methods. The computed structures were based on 1564 upper limit distance constraints derived from NOE cross-correlation intensities measured in the 2D-NOESY and 3D-HSQC-NOESY experiments, 70 phi torsion angle constraints obtained from (3)J(HNH)(alpha) couplings measured in the HNHA experiment, and 30 psi torsion angle constraints derived from (3)J(H)(alpha)(Ni+1) couplings measured in the HNHB experiment. The computed structures showed a RMSD radius of 0.64 A for the structural core. The resulting structure consists of five amphipatic helices arranged in a right-handed super helix, a folding motif first observed in nonspecific lipid transfer proteins. Different than the latter, RicC3 does have not an internal cavity, a fact that can be related to the exchange in the pairing of disulfide bridges in the segment.CXC. Previous attempts to determine high resolution structures of a 2S albumin protein by either X-ray crystallography or NMR methods failed because of the heterogeneity of the protein prepared from natural sources. Both 2S albumins and nonspecific lipid transfer proteins belong to the prolamine superfamily, some of whose members are food allergens. The solution structure for recombinant RicC3 determined here is a suitable representative structure for the broad family of seed 2S albumin proteins, which may help to establish meaningful relationships between structure and allergenicity. RicC3 is also the peptidic component of the immunomodulator Inmunoferon, a widely used pharmaceutical product, and its structure is expected to help understand its pharmaceutical activity. PMID:14636051

  15. The Structural Characterization of Tumor Fusion Genes and Proteins.

    PubMed

    Wang, Dandan; Li, Daixi; Qin, Guangrong; Zhang, Wen; Ouyang, Jian; Zhang, Menghuan; Xie, Lu

    2015-01-01

    Chromosomal translocation, which generates fusion proteins in blood tumor or solid tumor, is considered as one of the major causes leading to cancer. Recent studies suggested that the disordered fragments in a fusion protein might contribute to its carcinogenicity. Here, we investigated the sequence feature near the breakpoints in the fusion partner genes, the structure features of breakpoints in fusion proteins, and the posttranslational modification preference in the fusion proteins. Results show that the breakpoints in the fusion partner genes have both sequence preference and structural preference. At the sequence level, nucleotide combination AG is preferred before the breakpoint and GG is preferred at the breakpoint. At the structural level, the breakpoints in the fusion proteins prefer to be located in the disordered regions. Further analysis suggests the phosphorylation sites at serine, threonine, and the methylation sites at arginine are enriched in disordered regions of the fusion proteins. Using EML4-ALK as an example, we further explained how the fusion protein leads to the protein disorder and contributes to its carcinogenicity. The sequence and structural features of the fusion proteins may help the scientific community to predict novel breakpoints in fusion genes and better understand the structure and function of fusion proteins. PMID:26347798

  16. The Structural Characterization of Tumor Fusion Genes and Proteins

    PubMed Central

    Wang, Dandan; Li, Daixi; Qin, Guangrong; Zhang, Wen; Ouyang, Jian; Zhang, Menghuan; Xie, Lu

    2015-01-01

    Chromosomal translocation, which generates fusion proteins in blood tumor or solid tumor, is considered as one of the major causes leading to cancer. Recent studies suggested that the disordered fragments in a fusion protein might contribute to its carcinogenicity. Here, we investigated the sequence feature near the breakpoints in the fusion partner genes, the structure features of breakpoints in fusion proteins, and the posttranslational modification preference in the fusion proteins. Results show that the breakpoints in the fusion partner genes have both sequence preference and structural preference. At the sequence level, nucleotide combination AG is preferred before the breakpoint and GG is preferred at the breakpoint. At the structural level, the breakpoints in the fusion proteins prefer to be located in the disordered regions. Further analysis suggests the phosphorylation sites at serine, threonine, and the methylation sites at arginine are enriched in disordered regions of the fusion proteins. Using EML4-ALK as an example, we further explained how the fusion protein leads to the protein disorder and contributes to its carcinogenicity. The sequence and structural features of the fusion proteins may help the scientific community to predict novel breakpoints in fusion genes and better understand the structure and function of fusion proteins. PMID:26347798

  17. Trans-splicing Into Highly Abundant Albumin Transcripts for Production of Therapeutic Proteins In Vivo

    PubMed Central

    Wang, Jun; Mansfield, S Gary; Cote, Colette A; Jiang, Ping Du; Weng, Ke; Amar, Marcelo JA; Brewer, Bryan H; Remaley, Alan T; McGarrity, Gerard J; Garcia-Blanco, Mariano A; Puttaraju, M

    2008-01-01

    Spliceosome-mediated RNA trans-splicing has emerged as an exciting mode of RNA therapy. Here we describe a novel trans-splicing strategy, which targets highly abundant pre-mRNAs, to produce therapeutic proteins in vivo. First, we used a pre-trans-splicing molecule (PTM) that mediated trans-splicing of human apolipoprotein A-I (hapoA-I) into the highly abundant mouse albumin exon 1. Hydrodynamic tail vein injection of the hapoA-I PTM plasmid in mice followed by analysis of the chimeric transcripts and protein, confirmed accurate and efficient trans-splicing into albumin pre-mRNA and production of hapoA-I protein. The versatility of this approach was demonstrated by producing functional human papillomavirus type-16 E7 (HPV16-E7) single-chain antibody in C57BL/6 mice and functional factor VIII (FVIII) and phenotypic correction in hemophilia A mice. Altogether, these studies demonstrate that trans-splicing to highly abundant albumin transcripts can be used as a general platform to produce therapeutic proteins in vivo. PMID:19066600

  18. Relation of plasma calcium to total protein and albumin in African grey (Psittacus erithacus) and Amazon (Amazona spp.) parrots.

    PubMed

    Lumeij, J T

    1990-10-01

    A significant correlation was found between total calcium and albumin concentration in the plasma of 70 African grey parrots (r=0.37; P<0.05). A correlation formula for plasma calcium concentration in the African grey parrot was derived on the basis of the concentration of albumin: Adjusted Ca (mmol/1) = Ca (mmol/1) - 0.015 Albumin (g/1) + 0.4. About 14% of the variability in calcium was attributable to the change in the concentration of plasma albumin concentration (R2=0.137). The correlation between calcium and total protein in African greys and between calcium and albumin and calcium and total protein in Amazons was not significant. PMID:18679980

  19. Structures and Mechanisms of Viral Membrane Fusion Proteins

    PubMed Central

    White, Judith M.; Delos, Sue E.; Brecher, Matthew; Schornberg, Kathryn

    2009-01-01

    Recent work has identified three distinct classes of viral membrane fusion proteins based on structural criteria. In addition, there are at least four distinct mechanisms by which viral fusion proteins can be triggered to undergo fusion-inducing conformational changes. Viral fusion proteins also contain different types of fusion peptides and vary in their reliance on accessory proteins. These differing features combine to yield a rich diversity of fusion proteins. Yet despite this staggering diversity, all characterized viral fusion proteins convert from a fusion-competent state (dimers or trimers, depending on the class) to a membrane-embedded homotrimeric prehairpin, and then to a trimer-of-hairpins that brings the fusion peptide, attached to the target membrane, and the transmembrane domain, attached to the viral membrane, into close proximity thereby facilitating the union of viral and target membranes. During these conformational conversions, the fusion proteins induce membranes to progress through stages of close apposition, hemifusion, and then the formation of small, and finally large, fusion pores. Clearly, highly divergent proteins have converged on the same overall strategy to mediate fusion, an essential step in the life cycle of every enveloped virus. PMID:18568847

  20. Construction of Lasso Peptide Fusion Proteins.

    PubMed

    Zong, Chuhan; Maksimov, Mikhail O; Link, A James

    2016-01-15

    Lasso peptides are a family of ribosomally synthesized and post-translationally modified peptides (RiPPs) typified by an isopeptide-bonded macrocycle between the peptide N-terminus and an aspartate or glutamate side chain. The C-terminal portion of the peptide threads through the N-terminal macrocycle to give the characteristic lasso fold. Because of the inherent stability, both proteolytic and often thermal, of lasso peptides, we became interested in whether proteins could be fused to the free C-terminus of lasso peptides. Here, we demonstrate fusion of two model proteins, the artificial leucine zipper A1 and the superfolder variant of GFP, to the C-terminus of the lasso peptide astexin-1. Successful lasso cyclization of the N-terminus of these fusion proteins requires a flexible linker in between the C-terminus of the lasso peptide and the N-terminus of the protein of interest. The ability to fuse lasso peptides to a protein of interest is an important step toward phage and bacterial display systems for the high-throughput screening of lasso peptide libraries for new functions. PMID:26492187

  1. Albumin-Like Protein is the Major Protein Constituent of Luminal Fluid in the Human Endolymphatic Sac

    PubMed Central

    Kim, Sung Huhn; Kim, Un-Kyoung; Lee, Won-Sang; Bok, Jinwoong; Song, Jung-Whan; Seong, Je Kyung; Choi, Jae Young

    2011-01-01

    The endolymphatic sac (ES) is an inner ear organ that is connected to the cochleo-vestibular system through the endolymphatic duct. The luminal fluid of the ES contains a much higher concentration of proteins than any other compartment of the inner ear. This high protein concentration likely contributes to inner ear fluid volume regulation by creating an osmotic gradient between the ES lumen and the interstitial fluid. We characterized the protein profile of the ES luminal fluid of patients (n = 11) with enlarged vestibular aqueducts (EVA) by proteomics. In addition, we investigated differences in the protein profiles between patients with recent hearing deterioration and patients without hearing deterioration. The mean total protein concentration of the luminal fluid was 554.7±94.6 mg/dl. A total of 58 out of 517 spots detected by 2-DE were analyzed by MALDI-TOF MS. The protein profile of the luminal fluid was different from the profile of plasma. Proteins identified from 29 of the spots were also present in the MARC-filtered human plasma; however, the proteins identified from the other 25 spots were not detected in the MARC-filtered human plasma. The most abundant protein in the luminal fluid was albumin-like proteins, but most of them were not detected in MARC-filtered human plasma. The concentration of albumin-like proteins was higher in samples from patients without recent hearing deterioration than in patients with recent hearing deterioration. Consequently, the protein of ES luminal fluid is likely to be originated from both the plasma and the inner ear and considering that inner ear fluid volumes increase abnormally in patients with EVA following recent hearing deterioration, it is tempting to speculate that albumin-like proteins may be involved in the regulation of inner ear fluid volume through creation of an osmotic gradient during pathological conditions such as endolymphatic hydrops. PMID:21738753

  2. Determination of protein-ligand binding affinity by NMR: observations from serum albumin model systems.

    PubMed

    Fielding, Lee; Rutherford, Samantha; Fletcher, Dan

    2005-06-01

    The usefulness of bovine serum albumin (BSA) as a model protein for testing NMR methods for the study of protein-ligand interactions is discussed. Isothermal titration calorimetry established the binding affinity and stoichiometry of the specific binding site for L-tryptophan, D-tryptophan, naproxen, ibuprofen, salicylic acid and warfarin. The binding affinities of the same ligands determined by NMR methods are universally weaker (larger KD). This is because the NMR methods are susceptible to interference from additional non-specific binding. The L-tryptophan-BSA and naproxen-BSA systems were the best behaved model systems. PMID:15816062

  3. Effect of auranofin on plasma fibronectin, C reactive protein, and albumin levels in arthritic rats.

    PubMed Central

    Connolly, K M; Stecher, V J; Pruden, D J

    1988-01-01

    Auranofin, a member of a class of compounds with disease modifying activity, was given to arthritic rats to determine if it could reverse the abnormal plasma concentrations of fibronectin (Fn), C reactive protein (CRP), and albumin, which were unaffected by treatment with non-steroidal anti-inflammatory drugs (NSAIDs). When auranofin was orally administered for two weeks to adjuvant induced arthritic rats it significantly inhibited swelling of the injected and non-injected paws at doses of 3 and 10 mg/kg. Rocket electroimmunoassay measurement of plasma proteins in normal, arthritic, and auranofin treated arthritic rats indicated that auranofin at 10 mg/kg significantly decreased (by 77%) the abnormally high concentration of arthritic rat plasma Fn, though it had no effect on Fn concentrations when administered to normal rats. CRP, which was raised approximately twofold above normal in arthritic rats, was reduced by 56% after treatment of arthritic rats with auranofin at 10 mg/kg, though CRP concentrations in normal rats were unaffected by auranofin treatment. Depressed albumin concentrations in arthritic rats were significantly enhanced (by 30%) by dosing with 10 mg/kg of auranofin. At the 3 mg/kg dose, auranofin did not significantly change plasma concentrations of Fn, CRP, and albumin in arthritic rats. At a dose of 10 mg/kg, however, auranofin, in addition to inhibiting chronic systemic paw inflammation, also altered abnormal concentrations of plasma Fn, CRP, and albumin in the adjuvant arthritic rat, thus distinguishing auranofin from standard NSAIDs we have previously tested. PMID:3260094

  4. High Mobility Group Box Protein 1 Boosts Endothelial Albumin Transcytosis through the RAGE/Src/Caveolin-1 Pathway.

    PubMed

    Shang, Dan; Peng, Tao; Gou, Shanmiao; Li, Yiqing; Wu, Heshui; Wang, Chunyou; Yang, Zhiyong

    2016-01-01

    High-mobility group box protein 1 (HMGB1), an inflammatory mediator, has been reported to destroy cell-cell junctions, resulting in vascular endothelial hyperpermeability. Here, we report that HMGB1 increases the endothelial transcytosis of albumin. In mouse lung vascular endothelial cells (MLVECs), HMGB1 at a concentration of 500 ng/ml or less did not harm cell-cell junctions but rapidly induced endothelial hyperpermeability to (125)I-albumin. HMGB1 induced an increase in (125)I-albumin and AlexaFluor 488-labeled albumin internalization in endocytosis assays. Depletion of receptor for advanced glycation end products (RAGE), but not TLR2 or TLR4, suppressed HMGB1-induced albumin transcytosis and endocytosis. Genetic and pharmacological destruction of lipid rafts significantly inhibited HMGB1-induced albumin endocytosis and transcytosis. HMGB1 induced the rapid phosphorylation of caveolin (Cav)-1 and Src. Either RAGE gene silencing or soluble RAGE suppressed Cav-1 Tyr14 phosphorylation and Src Tyr418 phosphorylation. The Src inhibitor 4-amino-5-(4-chlorophenyl)-7-(t-butyl) pyrazolo[3,4-d] pyrimidine (PP2) blocked HMGB1-induced Cav-1 Tyr14 phosphorylation. PP2 and overexpression of Cav-1 with a T14F mutation significantly inhibited HMGB1-induced transcytosis and albumin endocytosis. Our findings suggest that HMGB1 induces the transcytosis of albumin via RAGE-dependent Src phosphorylation and Cav-1 phosphorylation. These studies revealed a new mechanism of HMGB1-induced endothelial hyperpermeability. PMID:27572515

  5. High Mobility Group Box Protein 1 Boosts Endothelial Albumin Transcytosis through the RAGE/Src/Caveolin-1 Pathway

    PubMed Central

    Shang, Dan; Peng, Tao; Gou, Shanmiao; Li, Yiqing; Wu, Heshui; Wang, Chunyou; Yang, Zhiyong

    2016-01-01

    High-mobility group box protein 1 (HMGB1), an inflammatory mediator, has been reported to destroy cell-cell junctions, resulting in vascular endothelial hyperpermeability. Here, we report that HMGB1 increases the endothelial transcytosis of albumin. In mouse lung vascular endothelial cells (MLVECs), HMGB1 at a concentration of 500 ng/ml or less did not harm cell-cell junctions but rapidly induced endothelial hyperpermeability to 125I-albumin. HMGB1 induced an increase in 125I-albumin and AlexaFluor 488-labeled albumin internalization in endocytosis assays. Depletion of receptor for advanced glycation end products (RAGE), but not TLR2 or TLR4, suppressed HMGB1-induced albumin transcytosis and endocytosis. Genetic and pharmacological destruction of lipid rafts significantly inhibited HMGB1-induced albumin endocytosis and transcytosis. HMGB1 induced the rapid phosphorylation of caveolin (Cav)-1 and Src. Either RAGE gene silencing or soluble RAGE suppressed Cav-1 Tyr14 phosphorylation and Src Tyr418 phosphorylation. The Src inhibitor 4-amino-5-(4-chlorophenyl)-7-(t-butyl) pyrazolo[3,4-d] pyrimidine (PP2) blocked HMGB1-induced Cav-1 Tyr14 phosphorylation. PP2 and overexpression of Cav-1 with a T14F mutation significantly inhibited HMGB1-induced transcytosis and albumin endocytosis. Our findings suggest that HMGB1 induces the transcytosis of albumin via RAGE-dependent Src phosphorylation and Cav-1 phosphorylation. These studies revealed a new mechanism of HMGB1-induced endothelial hyperpermeability. PMID:27572515

  6. SUMO fusion technology for difficult-to-express proteins.

    PubMed

    Butt, Tauseef R; Edavettal, Suzanne C; Hall, John P; Mattern, Michael R

    2005-09-01

    The demands of structural and functional genomics for large quantities of soluble, properly folded proteins in heterologous hosts have been aided by advancements in the field of protein production and purification. Escherichia coli, the preferred host for recombinant protein expression, presents many challenges which must be surmounted in order to over-express heterologous proteins. These challenges include the proteolytic degradation of target proteins, protein misfolding, poor solubility, and the necessity for good purification methodologies. Gene fusion technologies have been able to improve heterologous expression by overcoming many of these challenges. The ability of gene fusions to improve expression, solubility, purification, and decrease proteolytic degradation will be discussed in this review. The main disadvantage, cleaving the protein fusion, will also be addressed. Focus will be given to the newly described SUMO fusion system and the improvements that this technology has advanced over traditional gene fusion systems. PMID:16084395

  7. Exocytotic fusion pores are composed of both lipids and proteins

    PubMed Central

    Bao, Huan; Goldschen-Ohm, Marcel; Jeggle, Pia; Chanda, Baron; Edwardson, J Michael; Chapman, Edwin R

    2016-01-01

    During exocytosis, fusion pores form the first aqueous connection that allows escape of neurotransmitters and hormones from secretory vesicles. Although it is well established that SNARE proteins catalyze fusion, the structure and composition of fusion pores remain unknown. Here, we exploited the rigid framework and defined size of nanodiscs to interrogate the properties of reconstituted fusion pores, using the neurotransmitter glutamate as a content-mixing marker. Efficient Ca2+-stimulated bilayer fusion, and glutamate release, occurred with approximately two molecules of mouse synaptobrevin 2 reconstituted into ~6-nm nanodiscs. The transmembrane domains of SNARE proteins assumed distinct roles in lipid mixing versus content release and were exposed to polar solvent during fusion. Additionally, tryptophan substitutions at specific positions in these transmembrane domains decreased glutamate flux. Together, these findings indicate that the fusion pore is a hybrid structure composed of both lipids and proteins. PMID:26656855

  8. Interaction of lipid vesicle with silver nanoparticle-serum albumin protein corona

    NASA Astrophysics Data System (ADS)

    Chen, Ran; Choudhary, Poonam; Schurr, Ryan N.; Bhattacharya, Priyanka; Brown, Jared M.; Chun Ke, Pu

    2012-01-01

    The physical interaction between a lipid vesicle and a silver nanoparticle (AgNP)-human serum albumin (HSA) protein "corona" has been examined. Specifically, the binding of AgNPs and HSA was analyzed by spectrophotometry, and the induced conformational changes of the HSA were inferred from circular dichroism spectroscopy. The fluidity of the vesicle, a model system for mimicking cell membrane, was found to increase with the increased exposure to AgNP-HSA corona, though less pronounced compared to that induced by AgNPs alone. This study offers additional information for understanding the role of physical forces in nanoparticle-cell interaction and has implications for nanomedicine and nanotoxicology.

  9. Interaction of bovine serum albumin protein with self assembled monolayer of mercaptoundecanoic acid

    NASA Astrophysics Data System (ADS)

    Poonia, Monika; Agarwal, Hitesh; Manjuladevi, V.; Gupta, R. K.

    2016-05-01

    Detection of proteins and other biomolecules in liquid phase is the essence for the design of a biosensor. The sensitivity of a sensor can be enhanced by the appropriate functionalization of the sensing area so as to establish the molecular specific interaction. In the present work, we have studied the interaction of bovine serum albumin (BSA) protein with a chemically functionalized surface using a quartz crystal microbalance (QCM). The gold-coated quartz crystals (AT-cut/5 MHz) were functionalized by forming self-assembled monolayer (SAM) of 11-Mercaptoundecanoic acid (MUA). The adsorption characteristics of BSA onto SAM of MUA on quartz crystal are reported. BSA showed the highest affinity for SAM of MUA as compared to pure gold surface. The SAM of MUA provides carboxylated surface which enhances not only the adsorption of the BSA protein but also a very stable BSA-MUA complex in the liquid phase.

  10. Ellipsometric studies of synthetic albumin-binding chitosan-derivatives and selected blood plasma proteins

    NASA Astrophysics Data System (ADS)

    Sarkar, Sabyasachi

    This dissertation summarizes work on the synthesis of chitosan-derivatives and the development of ellipsometric methods to characterize materials of biological origin. Albumin-binding chitosan-derivatives were synthesized via addition reactions that involve amine groups naturally present in chitosan. These surfaces were shown to have an affinity towards human serum albumin via ELISA, UV spectroscopy and SDS PAGE. Modified surfaces were characterized with IR ellipsometry at various stages of their synthesis using appropriate optical models. It was found that spin cast chitosan films were anisotropic in nature. All optical models used for characterizing chitosan-derivatives were thus anisotropic. Chemical signal dependence on molecular structure and composition was illustrated via IR spectroscopic ellipsometry (IRSE). An anisotropic optical model of an ensemble of Lorentz oscillators were used to approximate material behavior. The presence of acetic acid in spin-cast non-neutralized chitosan samples was thus shown. IRSE application to biomaterials was also demonstrated by performing a step-wise chemical characterizations during synthesis stages. Protein adsorbed from single protein solutions on these modified surfaces was monitored by visible in-situ variable wavelength ellipsometry. Based on adsorption profiles obtained from single protein adsorption onto silicon surfaces, lumped parameter kinetic models were developed. These models were used to fit experimental data of immunoglobulin-G of different concentrations and approximate conformational changes in fibrinogen adsorption. Biomaterial characterization by ellipsometry was further extended to include characterization of individual protein solutions in the IR range. Proteins in an aqueous environment were characterized by attenuated total internal reflection (ATR) IR ellipsometry using a ZnSe prism. Parameterized dielectric functions were created for individual proteins using Lorentz oscillators. These

  11. Protein adsorption on low temperature isotropic carbon. III. Isotherms, competitivity, desorption and exchange of human albumin and fibrinogen.

    PubMed

    Feng, L; Andrade, J D

    1994-04-01

    In this paper we consider the adsorption of albumin and fibrinogen on low temperature isotropic carbon (LTIC). A subsequent paper considers the adsorption of other plasma proteins [Feng L, Andrade JD, Colloids and Surfaces (in press)]. Carbon fragments and silica plates were used as adsorbents. Adsorption was carried out by incubating the adsorbents in solutions of 125I-labelled and unlabelled proteins (single component system), or with buffer-diluted human plasma (multicomponent system). Adsorbed proteins then underwent displacement by buffer, by single protein solutions or by dilute plasma. Results show that the LTIC substrate adsorbs a large amount of proteins before saturation, which may be due to multilayer adsorption. LTIC also irreversibly holds adsorbed proteins against the exchange agents used; little adsorbed proteins can be displaced, even after a very short adsorption time. There is no preferential adsorption for either albumin or fibrinogen on LTIC from their binary solutions, suggesting that both proteins have high affinities for the surface. Such strong interactions between LTIC and proteins are not attributed to electrostatic interactions. On the other hand, protein adsorption on the silica surface is selective and reversible, with a much higher affinity for fibrinogen than albumin and an even higher affinity for some other plasma proteins. The paper also discusses the effect of sequential protein addition to a solution on the surface concentration and suppression of adsorption of both proteins in the presence of other plasma proteins. A very important conclusion is that the LTIC surface is very active towards proteins adsorption. PMID:8061122

  12. Modulation of membrane fusion by calcium-binding proteins.

    PubMed Central

    Hong, K; Düzgüneş, N; Papahadjopoulos, D

    1982-01-01

    The effects of several Ca2+-binding proteins (calmodulin, prothrombin, and synexin) on the kinetics of Ca2+-induced membrane fusion were examined. Membrane fusion was assayed by following the mixing of aqueous contents of phospholipid vesicles. Calmodulin inhibited slightly the fusion of phospholipid vesicles. Bovine prothrombin and its proteolytic fragment 1 had a strong inhibitory effect on fusion. Depending on the phospholipid composition, synexin could either facilitate or inhibit Ca2+-induced fusion of vesicles. The effects of synexin were Ca2+ specific. 10 microM Ca2+ was sufficient to induce fusion of vesicles composed of phosphatidic acid/phosphatidylethanolamine (1:3) in the presence of synexin and 1 mM Mg2+. We propose that synexin may be involved in intracellular membrane fusion events mediated by Ca2+, such as exocytosis, and discuss possible mechanisms facilitating fusion. PMID:6459804

  13. Albumin and multiple sclerosis.

    PubMed

    LeVine, Steven M

    2016-01-01

    Leakage of the blood-brain barrier (BBB) is a common pathological feature in multiple sclerosis (MS). Following a breach of the BBB, albumin, the most abundant protein in plasma, gains access to CNS tissue where it is exposed to an inflammatory milieu and tissue damage, e.g., demyelination. Once in the CNS, albumin can participate in protective mechanisms. For example, due to its high concentration and molecular properties, albumin becomes a target for oxidation and nitration reactions. Furthermore, albumin binds metals and heme thereby limiting their ability to produce reactive oxygen and reactive nitrogen species. Albumin also has the potential to worsen disease. Similar to pathogenic processes that occur during epilepsy, extravasated albumin could induce the expression of proinflammatory cytokines and affect the ability of astrocytes to maintain potassium homeostasis thereby possibly making neurons more vulnerable to glutamate exicitotoxicity, which is thought to be a pathogenic mechanism in MS. The albumin quotient, albumin in cerebrospinal fluid (CSF)/albumin in serum, is used as a measure of blood-CSF barrier dysfunction in MS, but it may be inaccurate since albumin levels in the CSF can be influenced by multiple factors including: 1) albumin becomes proteolytically cleaved during disease, 2) extravasated albumin is taken up by macrophages, microglia, and astrocytes, and 3) the location of BBB damage affects the entry of extravasated albumin into ventricular CSF. A discussion of the roles that albumin performs during MS is put forth. PMID:27067000

  14. Protein-driven membrane stresses in fusion and fission

    PubMed Central

    Kozlov, Michael M.; McMahon, Harvey T.; Chernomordik, Leonid V.

    2013-01-01

    Cellular membranes undergo continuous remodeling. Exocytosis and endocytosis, mitochondrial fusion and fission, entry of enveloped viruses into host cellsand release of the newly assembled virions, cell-to-cell fusion and cell division, and budding and fusion of transport carriers all proceed via topologically similar, but oppositely ordered, membrane rearrangements. The biophysical similarities and differences between membrane fusion and fission become more evident if we disregard the accompanying biological processes and consider only remodeling of the lipid bilayer. The forces that determine the bilayer propensity to undergo fusion or fission come from proteins and inmost cases from membrane-bound proteins. In this review, we consider the mechanistic principles underlying the fusion and fission reactions and discuss the current hypotheses on how specific proteins act in the two types of membrane remodeling. PMID:20638285

  15. Structural characterization of Mumps virus fusion protein core

    SciTech Connect

    Liu Yueyong; Xu Yanhui; Lou Zhiyong; Zhu Jieqing; Hu Xuebo; Gao, George F.; Qiu Bingsheng . E-mail: Qiubs@sun.im.ac.cn; Rao Zihe . E-mail: raozh@xtal.tsinghua.edu.cn; Tien, Po . E-mail: tienpo@sun.im.ac.cn

    2006-09-29

    The fusion proteins of enveloped viruses mediating the fusion between the viral and cellular membranes comprise two discontinuous heptad repeat (HR) domains located at the ectodomain of the enveloped glycoproteins. The crystal structure of the fusion protein core of Mumps virus (MuV) was determined at 2.2 A resolution. The complex is a six-helix bundle in which three HR1 peptides form a central highly hydrophobic coiled-coil and three HR2 peptides pack against the hydrophobic grooves on the surface of central coiled-coil in an oblique antiparallel manner. Fusion core of MuV, like those of simian virus 5 and human respiratory syncytium virus, forms typical 3-4-4-4-3 spacing. The similar charecterization in HR1 regions, as well as the existence of O-X-O motif in extended regions of HR2 helix, suggests a basic rule for the formation of the fusion core of viral fusion proteins.

  16. Protein bodies from the cotyledons of Cytisus scoparius L. (Link). Ultrastructure, isolation, and subunit composition of albumin, legumin and vicilin.

    PubMed

    Citharel, L; Citharel, J

    1985-09-01

    The structure of protein bodies differs in the upper and lower parts of the cotyledons of mature seeds of Cytisus scoparius L. The palisade-mesophyll cells contain essentially homogeneous protein bodies, without globoids, but the protein bodies of the spongy-mesophyll cells are heterogeneous, with numerous globoids. Albumins, legumins and vicilins were selectively extracted from isolated protein bodies and their subunits separated by SDS-PAGE, under non-reducing and reducing conditions. PMID:24241309

  17. INITIAL SIZE AND DYNAMICS OF VIRAL FUSION PORES ARE A FUNCTION OF THE FUSION PROTEIN MEDIATING MEMBRANE FUSION

    PubMed Central

    Plonsky, I.; Kingsley, D. H.; Rashtian, A.; Blank, P.S.; Zimmerberg, J.

    2013-01-01

    To investigate the role of the fusogenic protein in the initial size and dynamics of the pore that widens to finalize membrane fusion, two different fusion proteins expressed in the same cell line were investigated: the major glycoprotein of baculovirus Autographa californica (GP64) and the hemaggluttinin of influenza X31 (HA). The host Sf9 cells expressing these viral proteins, irrespective of protein species, fused to human red blood cells (RBC) upon acidification of the medium. High time resolution electrophysiological study of fusion pore conductance revealed fundamental differences in a) the initial pore conductance (pores created by HA were smaller than those created by GP64), b) the ability of pores to flicker (only HA-mediated pores flickered), and c) the time required for pore formation (HA-mediated pores took much longer to form following acidification). Thus 1) HA and GP64 have divergent electrophysiological phenotypes even when they fuse identical membranes, and 2) fusion proteins play a crucial role in determining initial fusion pore characteristics. The structure of the initial fusion pore detected by electrical conductance measurements is sensitive to the nature of the fusion protein. PMID:18208404

  18. Fusion proteins as alternate crystallization paths to difficult structure problems

    NASA Technical Reports Server (NTRS)

    Carter, Daniel C.; Rueker, Florian; Ho, Joseph X.; Lim, Kap; Keeling, Kim; Gilliland, Gary; Ji, Xinhua

    1994-01-01

    The three-dimensional structure of a peptide fusion product with glutathione transferase from Schistosoma japonicum (SjGST) has been solved by crystallographic methods to 2.5 A resolution. Peptides or proteins can be fused to SjGST and expressed in a plasmid for rapid synthesis in Escherichia coli. Fusion proteins created by this commercial method can be purified rapidly by chromatography on immobilized glutathione. The potential utility of using SjGST fusion proteins as alternate paths to the crystallization and structure determination of proteins is demonstrated.

  19. Pharmacokinetics of Peptide-Fc fusion proteins.

    PubMed

    Wu, Benjamin; Sun, Yu-Nien

    2014-01-01

    Peptide-Fc fusion proteins (or peptibodies) are chimeric proteins generated by fusing a biologically active peptide with the Fc-domain of immunoglobulin G. In this review, we describe recent studies that have evaluated the absorption, distribution, metabolism, and excretion characteristics of peptibodies. Key features of the pharmacokinetics of peptibodies include their extended half-life due to recycling by the neonatal Fc receptor (FcRn), a substantial contribution by renal excretion to total clearance and, for certain peptibodies, target-mediated drug disposition. The prolonged half-life of peptibodies permits less-frequent dose administration compared with small therapeutic peptides, thereby supporting patient convenience and compliance. Hence, a considerable number of peptibodies are currently in preclinical and clinical development. Investigation of the metabolism (biotransformation) of biologics is an evolving area of research: ligand-binding mass spectrometry techniques have been employed for the characterization of the peptibody romiplostim, providing a new approach to evaluation of the degradation products of biologics. Pharmacokinetic/pharmacodynamic modeling and simulation techniques have been used to predict the pharmacokinetics of peptibodies which can inform clinical decision-making, particularly selection of dosing regimens. This integrated review highlights the distinct pharmacokinetic characteristics of peptibodies and their influence on the drug development process for this emerging family of therapeutics. PMID:24285510

  20. Membrane fusion of Semliki Forest virus involves homotrimers of the fusion protein.

    PubMed Central

    Wahlberg, J M; Bron, R; Wilschut, J; Garoff, H

    1992-01-01

    Infection of cells with enveloped viruses is accomplished through membrane fusion. The binding and fusion processes are mediated by the spike proteins in the envelope of the virus particle and usually involve a series of conformational changes in these proteins. We have studied the low-pH-mediated fusion process of the alphavirus Semliki Forest virus (SFV). The spike protein of SFV is composed of three copies of the protein heterodimer E2E1. This structure is resistant to solubilization in mild detergents such as Nonidet P-40 (NP40). We have recently shown that the spike structure is reorganized during virus entry into acidic endosomes (J. M. Wahlberg and H. Garoff, J. Cell Biol. 116:339-348, 1992). The original NP40-resistant heterodimer is dissociated, and the E1 subunits form new NP40-resistant protein oligomers. Here, we show that the new oligomer is represented by an E1 trimer. From studies that use an in vitro assay for fusion of SFV with liposomes, we show that the E1 trimer is efficiently expressed during virus-mediated membrane fusion. Time course studies show that both E1 trimer formation and fusion are fast processes, occurring in seconds. It was also possible to inhibit virus binding and fusion with a monoclonal antibody directed toward the trimeric E1. These results give support for a model in which the E1 trimeric structure is involved in the SFV-mediated fusion reaction. Images PMID:1433520

  1. Superior serum half life of albumin tagged TNF ligands

    SciTech Connect

    Mueller, Nicole; Schneider, Britta; Pfizenmaier, Klaus; Wajant, Harald

    2010-06-11

    Due to their immune stimulating and apoptosis inducing properties, ligands of the TNF family attract increasing interest as therapeutic proteins. A general limitation of in vivo applications of recombinant soluble TNF ligands is their notoriously rapid clearance from circulation. To improve the serum half life of the TNF family members TNF, TWEAK and TRAIL, we genetically fused soluble variants of these molecules to human serum albumin (HSA). The serum albumin-TNF ligand fusion proteins were found to be of similar bioactivity as the corresponding HSA-less counterparts. Upon intravenous injection (i.v.), serum half life of HSA-TNF ligand fusion proteins, as determined by ELISA, was around 15 h as compared to approximately 1 h for all of the recombinant control TNF ligands without HSA domain. Moreover, serum samples collected 6 or 24 h after i.v. injection still contained high TNF ligand bioactivity, demonstrating that there is only limited degradation/inactivation of circulating HSA-TNF ligand fusion proteins in vivo. In a xenotransplantation model, significantly less of the HSA-TRAIL fusion protein compared to the respective control TRAIL protein was required to achieve inhibition of tumor growth indicating that the increased half life of HSA-TNF ligand fusion proteins translates into better therapeutic action in vivo. In conclusion, our data suggest that genetic fusion to serum albumin is a powerful and generally applicable mean to improve bioavailability and in vivo activity of TNF ligands.

  2. SAFE Software and FED Database to Uncover Protein-Protein Interactions using Gene Fusion Analysis.

    PubMed

    Tsagrasoulis, Dimosthenis; Danos, Vasilis; Kissa, Maria; Trimpalis, Philip; Koumandou, V Lila; Karagouni, Amalia D; Tsakalidis, Athanasios; Kossida, Sophia

    2012-01-01

    Domain Fusion Analysis takes advantage of the fact that certain proteins in a given proteome A, are found to have statistically significant similarity with two separate proteins in another proteome B. In other words, the result of a fusion event between two separate proteins in proteome B is a specific full-length protein in proteome A. In such a case, it can be safely concluded that the protein pair has a common biological function or even interacts physically. In this paper, we present the Fusion Events Database (FED), a database for the maintenance and retrieval of fusion data both in prokaryotic and eukaryotic organisms and the Software for the Analysis of Fusion Events (SAFE), a computational platform implemented for the automated detection, filtering and visualization of fusion events (both available at: http://www.bioacademy.gr/bioinformatics/projects/ProteinFusion/index.htm). Finally, we analyze the proteomes of three microorganisms using these tools in order to demonstrate their functionality. PMID:22267904

  3. Interaction of lipid vesicle with silver nanoparticle-serum albumin protein corona

    PubMed Central

    Chen, Ran; Choudhary, Poonam; Schurr, Ryan N.; Bhattacharya, Priyanka; Brown, Jared M.; Chun Ke, Pu

    2012-01-01

    The physical interaction between a lipid vesicle and a silver nanoparticle (AgNP)-human serum albumin (HSA) protein “corona” has been examined. Specifically, the binding of AgNPs and HSA was analyzed by spectrophotometry, and the induced conformational changes of the HSA were inferred from circular dichroism spectroscopy. The fluidity of the vesicle, a model system for mimicking cell membrane, was found to increase with the increased exposure to AgNP-HSA corona, though less pronounced compared to that induced by AgNPs alone. This study offers additional information for understanding the role of physical forces in nanoparticle-cell interaction and has implications for nanomedicine and nanotoxicology. PMID:22271932

  4. Structural aspects of a protein-surfactant assembly: native and reduced States of human serum albumin.

    PubMed

    Anand, Uttam; Ray, Sutapa; Ghosh, Subhadip; Banerjee, Rajat; Mukherjee, Saptarshi

    2015-04-01

    The inherently present seventeen disulfide bonds of the circulatory protein, human serum albumin (HSA) provide the necessary structural stability. Various spectroscopic approaches were used to investigate the effect of reduction of these disulfide bonds and its binding with the anionic surfactant, sodium dodecyl sulfate (SDS). Based on several spectroscopic analyses, our investigations highlight the following interesting aspects: (1) HSA on reduction loses not only its tertiary structure but also a significant amount of secondary structure as well. However, the reduced state of the protein is not like the molten-globule, (2) this structural loss of the protein due to reduction is more prominent than that caused by higher SDS concentrations alone and can certainly be attributed to the role of disulfide bonds, (3) lower surfactant concentrations provide marginal structural rigidity to the native state of the protein, whereas, higher concentrations of SDS induces secondary structure to the reduced state of HSA, (4) the binding of SDS with both the native and reduced states of HSA, occurred in three distinct stages which was followed by a saturation stage. However, the nature of such binding is different for both the states as investigated by using the Stern-Volmer equations and estimating the thermodynamic parameters. Besides, in contrast to the native state, the reduced state of HSA shows that the lone tryptophan residue gets more buried. However, there occurs a sudden decrement in the lifetime of the tryptophan and the hydrodynamic diameter increases by twofold. PMID:25821118

  5. Rho GTPase activity modulates paramyxovirus fusion protein-mediated cell-cell fusion

    SciTech Connect

    Schowalter, Rachel M.; Wurth, Mark A.; Aguilar, Hector C.; Lee, Benhur; Moncman, Carole L.; McCann, Richard O.; Dutch, Rebecca Ellis . E-mail: rdutc2@uky.edu

    2006-07-05

    The paramyxovirus fusion protein (F) promotes fusion of the viral envelope with the plasma membrane of target cells as well as cell-cell fusion. The plasma membrane is closely associated with the actin cytoskeleton, but the role of actin dynamics in paramyxovirus F-mediated membrane fusion is unclear. We examined cell-cell fusion promoted by two different paramyxovirus F proteins in three cell types in the presence of constitutively active Rho family GTPases, major cellular coordinators of actin dynamics. Reporter gene and syncytia assays demonstrated that expression of either Rac1{sup V12} or Cdc42{sup V12} could increase cell-cell fusion promoted by the Hendra or SV5 glycoproteins, though the effect was dependent on the cell type expressing the viral glycoproteins. In contrast, RhoA{sup L63} decreased cell-cell fusion promoted by Hendra glycoproteins but had little affect on SV5 F-mediated fusion. Also, data suggested that GTPase activation in the viral glycoprotein-containing cell was primarily responsible for changes in fusion. Additionally, we found that activated Cdc42 promoted nuclear rearrangement in syncytia.

  6. Role of Serum Interleukin 6, Albumin and C-Reactive Protein in COPD Patients

    PubMed Central

    Emami Ardestani, Mohammad

    2015-01-01

    Background: Chronic obstructive pulmonary disease (COPD) is a non-specific inflammation, which involves the airways, lung parenchyma and pulmonary vessels. The inflammation causes the activation of inflammatory cells and the release of various inflammatory mediators such as interleukin-8 (IL-8), IL-6 and tumor necoris factor alpha (TNF-a). The purpose of the present study was to measure serum IL-6, C-reactive protein (CRP) (as a positive phase reactant) and albumin level (as a negative phase reactant) in COPD patients (only due to cigarette smoking not bio-mass), non COPD smokers and healthy subjects using enzyme-linked immunosorbent assay (ELISA); we compared the differences in inflammatory factors among groups. Materials and Methods: A total of 180 males were enrolled in this study and divided into three equal groups. The first group was 60 smokers who had COPD. The second group included 60 smokers without COPD and the third group consisted of people who were not smokers and did not have COPD; 5 mL of venous blood was taken from all participants and it was collected in a test tube containing anticoagulant and then centrifuged at 3000 rpm for 10 minutes. Serum was separated and used to measure the amount of IL-6, CRP and albumin. Spirometry was performed according to the criteria set by the American Thoracic Society. Results: The mean serum level of IL-6 was 83.2±7.5 pg/mL in group I, 54.9±24.3 pg/mL in group II and 46.9±10.4 pg/mL in group III. There was a significant difference among the three groups (P<0.001). The mean serum level of CRP was 28.9±14.9 mg/dL in the first group, 19.9±8.5 mg/dL in the second group and 4.2±2.3 mg/dL in the third group (P=0.02). But by controlling the confounding effects of age, this difference was not significant (P=0.49). The mean serum level of albumin was I 4.1±0.57 mg/dL in group I, 4.3±0.56 mg/dL in group II and 4.1±0.53 mg/dL in group III. There was no significant difference among the three groups in this regard (P=0

  7. The dengue virus type 2 envelope protein fusion peptide is essential for membrane fusion

    SciTech Connect

    Huang, Claire Y.-H.; Butrapet, Siritorn; Moss, Kelly J.; Childers, Thomas; Erb, Steven M.; Calvert, Amanda E.; Silengo, Shawn J.; Kinney, Richard M.; Blair, Carol D.; Roehrig, John T.

    2010-01-20

    The flaviviral envelope (E) protein directs virus-mediated membrane fusion. To investigate membrane fusion as a requirement for virus growth, we introduced 27 unique mutations into the fusion peptide of an infectious cDNA clone of dengue 2 virus and recovered seven stable mutant viruses. The fusion efficiency of the mutants was impaired, demonstrating for the first time the requirement for specific FP AAs in optimal fusion. Mutant viruses exhibited different growth kinetics and/or genetic stabilities in different cell types and adult mosquitoes. Virus particles could be recovered following RNA transfection of cells with four lethal mutants; however, recovered viruses could not re-infect cells. These viruses could enter cells, but internalized virus appeared to be retained in endosomal compartments of infected cells, thus suggesting a fusion blockade. Mutations of the FP also resulted in reduced virus reactivity with flavivirus group-reactive antibodies, confirming earlier reports using virus-like particles.

  8. Association of a high normalized protein catabolic rate and low serum albumin level with carpal tunnel syndrome in hemodialysis patients.

    PubMed

    Huang, Wen-Hung; Hsu, Ching-Wei; Weng, Cheng-Hao; Yen, Tzung-Hai; Lin, Jui-Hsiang; Lee, Meng

    2016-06-01

    Carpal tunnel syndrome (CTS) is the most common mononeuropathy in patients with end-stage renal disease (ESRD). The association between chronic inflammation and CTS in hemodialysis (HD) patients has rarely been investigated. HD patients with a high normalized protein catabolic rate (nPCR) and low serum albumin level likely have adequate nutrition and inflammation. In this study, we assume that a low serum albumin level and high nPCR is associated with CTS in HD patients. We recruited 866 maintenance hemodialysis (MHD) patients and divided them into 4 groups according to their nPCR and serum albumin levels: (1) nPCR <1.2 g/kg/d and serum albumin level <4 g/dL; (2) nPCR ≥1.2 g/kg/d and serum albumin level <4 g/dL; (3) nPCR <1.2 g/kg/d and serum albumin level ≥4 g/dL; and (4) nPCR ≥1.2 g/kg/d and serum albumin level ≥4 g/dL. After adjustment for related variables, HD duration and nPCR ≥1.2 g/kg/d and serum albumin level <4 g/dL were positively correlated with CTS. By calculating the area under the receiver-operating characteristic curve, we calculated that the nPCR and HD duration cut-off points for obtaining the most favorable Youden index were 1.29 g/kg/d and 7.5 years, respectively. Advance multivariate logistic regression analysis revealed that in MHD patients, nPCR ≥1.29 g/kg/d and serum albumin <4 g/dL, and also HD duration >7.5 years were associated with CTS. A high nPCR and low serum albumin level, which likely reflect adequate nutrition and inflammation, were associated with CTS in MHD patients. PMID:27368039

  9. Association of a high normalized protein catabolic rate and low serum albumin level with carpal tunnel syndrome in hemodialysis patients

    PubMed Central

    Huang, Wen-Hung; Hsu, Ching-Wei; Weng, Cheng-Hao; Yen, Tzung-Hai; Lin, Jui-Hsiang; Lee, Meng

    2016-01-01

    Abstract Carpal tunnel syndrome (CTS) is the most common mononeuropathy in patients with end-stage renal disease (ESRD). The association between chronic inflammation and CTS in hemodialysis (HD) patients has rarely been investigated. HD patients with a high normalized protein catabolic rate (nPCR) and low serum albumin level likely have adequate nutrition and inflammation. In this study, we assume that a low serum albumin level and high nPCR is associated with CTS in HD patients. We recruited 866 maintenance hemodialysis (MHD) patients and divided them into 4 groups according to their nPCR and serum albumin levels: (1) nPCR <1.2 g/kg/d and serum albumin level <4 g/dL; (2) nPCR ≥1.2 g/kg/d and serum albumin level <4 g/dL; (3) nPCR <1.2 g/kg/d and serum albumin level ≥4 g/dL; and (4) nPCR ≥1.2 g/kg/d and serum albumin level ≥4 g/dL. After adjustment for related variables, HD duration and nPCR ≥1.2 g/kg/d and serum albumin level <4 g/dL were positively correlated with CTS. By calculating the area under the receiver-operating characteristic curve, we calculated that the nPCR and HD duration cut-off points for obtaining the most favorable Youden index were 1.29 g/kg/d and 7.5 years, respectively. Advance multivariate logistic regression analysis revealed that in MHD patients, nPCR ≥1.29 g/kg/d and serum albumin <4 g/dL, and also HD duration >7.5 years were associated with CTS. A high nPCR and low serum albumin level, which likely reflect adequate nutrition and inflammation, were associated with CTS in MHD patients. PMID:27368039

  10. How Could SNARE Proteins Open a Fusion Pore?

    PubMed Central

    Fang, Qinghua

    2014-01-01

    The SNARE (Soluble NSF Attachment protein REceptor) complex, which in mammalian neurosecretory cells is composed of the proteins synaptobrevin 2 (also called VAMP2), syntaxin, and SNAP-25, plays a key role in vesicle fusion. In this review, we discuss the hypothesis that, in neurosecretory cells, fusion pore formation is directly accomplished by a conformational change in the SNARE complex via movement of the transmembrane domains. PMID:24985331

  11. The study of a light-activated albumin protein solder to bond layers of porcine small intestinal submucosa.

    PubMed

    Ware, Mark H; Buckley, Christine A

    2003-01-01

    This study investigated the feasibility of bonding layers of porcine small intestinal submucosa (SIS, Cook Biotech, Inc.) with a light-activated protein solder. SIS is an acellular, collagen-based extracellular matrix material that is approximately 100 microns thick. The solder consists of bovine serum albumin and indocyanine green dye (ICG) in deionized water. The solder is activated by an 808 nm diode laser, which denatures the albumin, causing the albumin to bond with the collagen of the tissue. The predictable absorption and thermal energy diffusion rates of ICG increase the chances of reproducible results. To determine the optimal condition for laser soldering SIS, the following parameters were varied: albumin concentration (from 30-45% (w/v) in increments of 5%), the concentration of ICG (from 0.5-2.0 mg/ml H2O) and the irradiance of the laser (10-64 W/cm2). While many of the solder compositions and laser irradiance combinations resulted in no bonding, a solder composition of 45% albumin, ICG concentration of 0.5 mg/ml H2O, and a laser irradiance of 21 W/cm2 did produce a bond between two pieces of SIS. The average shear strength of this bond was 29.5 +/- 17.1 kPa (n = 14). This compares favorably to our previous work using fibrin glue as an adhesive, in which the average shear strength was 27 +/- 15.8 kPa (n = 40). PMID:12724859

  12. Recombinant baculovirus vectors expressing glutathione-S-transferase fusion proteins.

    PubMed

    Davies, A H; Jowett, J B; Jones, I M

    1993-08-01

    Recombinant baculoviruses are a popular means of producing heterologous protein in eukaryotic cells. Purification of recombinant proteins away from the insect cell background can, however, remain an obstacle for many developments. Recently, prokaryotic fusion protein expression systems have been developed allowing single-step purification of the heterologous protein and specific proteolytic cleavage of the affinity tag moiety from the desired antigen. Here we report the introduction of these attributes to the baculovirus system. "Baculo-GEX" vectors enable baculovirus production of fusion proteins with the above advantages, but in a eukaryotic post-translational processing environment. Glutathione-S-transferase (GST) fusions are stable cytoplasmic proteins in insect cells and may therefore be released by sonication alone, avoiding the solubility problems and detergent requirements of bacterial systems. Thus large amounts of authentic antigen may be purified in a single, non-denaturing step. PMID:7763917

  13. Grafting of bovine serum albumin proteins on plasma-modified polymers for potential application in tissue engineering

    NASA Astrophysics Data System (ADS)

    Kasálková, Nikola Slepičková; Slepička, Petr; Kolská, Zdeňka; Hodačová, Petra; Kučková, Štěpánka; Švorčík, Václav

    2014-04-01

    In this work, an influence of bovine serum albumin proteins grafting on the surface properties of plasma-treated polyethylene and poly- l-lactic acid was studied. The interaction of the vascular smooth muscle cells with the modified polymer surface was determined. The surface properties were characterized by X-ray photoelectron spectroscopy, atomic force microscopy, nano-LC-ESI-Q-TOF mass spectrometry, electrokinetic analysis, and goniometry. One of the motivations for this work is the idea that by the interaction of the cell with substrate surface, the proteins will form an interlayer between the cell and the substrate. It was proven that when interacting with the plasma-treated high-density polyethylene and poly- l-lactic acid, the bovine serum albumin protein is grafted on the polymer surface. Since the proteins are bonded to the substrate surface, they can stimulate cell adhesion and proliferation.

  14. Chromatography of carbon nanotubes separated albumin from other serum proteins: a method for direct analysis of their interactions.

    PubMed

    Kuboki, Yoshinori; Koshikawa, Takamitu; Takita, Hiroko; Fujisawa, Ryuichi; Lee, Min-ho; Abe, Shigeaki; Akasaka, Tsukasa; Uo, Motohiro; Watari, Fumio; Sammons, Rachel

    2010-08-01

    Chromatography technology was employed to clarify the mechanism of interaction between multi-wall carbon nanotubes (MWCNT) and proteins. A column (16x100 mm) was packed with purified MWCNT, and various proteins were eluted with phosphate buffered saline (PBS) with and without gradient systems. It was found that albumin in bovine serum was eluted immediately from the column without any adsorption to MWCNT. Conversely, the non-albumin proteins, including a protein of 85 kDa molecular mass and a group of proteins with molecular masses higher than 115 kDa, exhibited considerably high affinity towards MWCNT. A sample of pure bovine serum albumin was also eluted immediately from the column, while lysozyme did not elute as a peak with PBS, but eluted with 0.6 M NaCl. Fundamentally, carbon nanotubes are devoid of any electrical charge. Therefore, other forces including the hydrogen bonds, hydrophilic interactions, and van der Waals forces were most probably responsible for the differential elution behaviors. In conclusion, this chromatographic method provided a simple and direct analysis of the interactions between carbon nanotubes and the various proteins. PMID:20610879

  15. Immunological Properties of Hepatitis B Core Antigen Fusion Proteins

    NASA Astrophysics Data System (ADS)

    Francis, Michael J.; Hastings, Gillian Z.; Brown, Alan L.; Grace, Ken G.; Rowlands, David J.; Brown, Fred; Clarke, Berwyn E.

    1990-04-01

    The immunogenicity of a 19 amino acid peptide from foot-and-mouth disease virus has previously been shown to approach that of the inactivated virus from which it was derived after multimeric particulate presentation as an N-terminal fusion with hepatitis B core antigen. In this report we demonstrate that rhinovirus peptide-hepatitis B core antigen fusion proteins are 10-fold more immunogenic than peptide coupled to keyhole limpet hemocyanin and 100-fold more immunogenic than uncoupled peptide with an added helper T-cell epitope. The fusion proteins can be readily administered without adjuvant or with adjuvants acceptable for human and veterinary application and can elicit a response after nasal or oral dosing. The fusion proteins can also act as T-cell-independent antigens. These properties provide further support for their suitability as presentation systems for "foreign" epitopes in the development of vaccines.

  16. Impact of albumin on drug delivery--new applications on the horizon.

    PubMed

    Elsadek, Bakheet; Kratz, Felix

    2012-01-10

    Over the past decades, albumin has emerged as a versatile carrier for therapeutic and diagnostic agents, primarily for diagnosing and treating diabetes, cancer, rheumatoid arthritis and infectious diseases. Market approved products include fatty acid derivatives of human insulin or the glucagon-like-1 peptide (Levemir(®) and Victoza(®)) for treating diabetes, the taxol albumin nanoparticle Abraxane(®) for treating metastatic breast cancer which is also under clinical investigation in further tumor indications, and (99m)Tc-aggregated albumin (Nanocoll(®) and Albures(®)) for diagnosing cancer and rheumatoid arthritis as well as for lymphoscintigraphy. In addition, an increasing number of albumin-based or albumin-binding drugs are in clinical trials such as antibody fusion proteins (MM-111) for treating HER2/neu positive breast cancer (phase I), a camelid albumin-binding nanobody anti-HSA-anti-TNF-α (ATN-103) in phase II studies for treating rheumatoid arthritis, an antidiabetic Exendin-4 analog bound to recombinant human albumin (phase I/II), a fluorescein-labeled albumin conjugate (AFL)-human serum albumin for visualizing the malignant borders of brain tumors for improved surgical resection, and finally an albumin-binding prodrug of doxorubicin (INNO-206) entering phase II studies against sarcoma and gastric cancer. In the preclinical setting, novel approaches include attaching peptides with high-affinity for albumin to antibody fragments, the exploitation of albumin-binding gadolinium contrast agents for magnetic resonance imaging, and physical or covalent attachment of antiviral, antibacterial, and anticancer drugs to albumin that are permanently or transiently attached to human serum albumin (HSA) or act as albumin-binding prodrugs. This review gives an overview of the expanding field of preclinical and clinical drug applications and developments that use albumin as a protein carrier to improve the pharmacokinetic profile of the drug or to target the drug

  17. Size-dependent interaction of silica nanoparticles with lysozyme and bovine serum albumin proteins.

    PubMed

    Yadav, Indresh; Aswal, Vinod K; Kohlbrecher, Joachim

    2016-05-01

    The interaction of three different sized (diameter 10, 18, and 28 nm) anionic silica nanoparticles with two model proteins-cationic lysozyme [molecular weight (MW) 14.7 kDa)] and anionic bovine serum albumin (BSA) (MW 66.4 kDa) has been studied by UV-vis spectroscopy, dynamic light scattering (DLS), and small-angle neutron scattering (SANS). The adsorption behavior of proteins on the nanoparticles, measured by UV-vis spectroscopy, is found to be very different for lysozyme and BSA. Lysozyme adsorbs strongly on the nanoparticles and shows exponential behavior as a function of lysozyme concentration irrespective of the nanoparticle size. The total amount of adsorbed lysozyme, as governed by the surface-to-volume ratio, increases on lowering the size of the nanoparticles for a fixed volume fraction of the nanoparticles. On the other hand, BSA does not show any adsorption for all the different sizes of the nanoparticles. Despite having different interactions, both proteins induce similar phase behavior where the nanoparticle-protein system transforms from one phase (clear) to two phase (turbid) as a function of protein concentration. The phase behavior is modified towards the lower concentrations for both proteins with increasing the nanoparticle size. DLS suggests that the phase behavior arises as a result of the nanoparticles' aggregation on the addition of proteins. The size-dependent modifications in the interaction potential, responsible for the phase behavior, have been determined by SANS data as modeled using the two-Yukawa potential accounting for the repulsive and attractive interactions in the systems. The protein-induced interaction between the nanoparticles is found to be short-range attraction for lysozyme and long-range attraction for BSA. The magnitude of attractive interaction irrespective of protein type is enhanced with increase in the size of the nanoparticles. The total (attractive+repulsive) potential leading to two-phase formation is found to be

  18. In vivo genome editing of the albumin locus as a platform for protein replacement therapy.

    PubMed

    Sharma, Rajiv; Anguela, Xavier M; Doyon, Yannick; Wechsler, Thomas; DeKelver, Russell C; Sproul, Scott; Paschon, David E; Miller, Jeffrey C; Davidson, Robert J; Shivak, David; Zhou, Shangzhen; Rieders, Julianne; Gregory, Philip D; Holmes, Michael C; Rebar, Edward J; High, Katherine A

    2015-10-01

    Site-specific genome editing provides a promising approach for achieving long-term, stable therapeutic gene expression. Genome editing has been successfully applied in a variety of preclinical models, generally focused on targeting the diseased locus itself; however, limited targeting efficiency or insufficient expression from the endogenous promoter may impede the translation of these approaches, particularly if the desired editing event does not confer a selective growth advantage. Here we report a general strategy for liver-directed protein replacement therapies that addresses these issues: zinc finger nuclease (ZFN) -mediated site-specific integration of therapeutic transgenes within the albumin gene. By using adeno-associated viral (AAV) vector delivery in vivo, we achieved long-term expression of human factors VIII and IX (hFVIII and hFIX) in mouse models of hemophilia A and B at therapeutic levels. By using the same targeting reagents in wild-type mice, lysosomal enzymes were expressed that are deficient in Fabry and Gaucher diseases and in Hurler and Hunter syndromes. The establishment of a universal nuclease-based platform for secreted protein production would represent a critical advance in the development of safe, permanent, and functional cures for diverse genetic and nongenetic diseases. PMID:26297739

  19. Human SUMO fusion systems enhance protein expression and solubility.

    PubMed

    Wang, Zhongyuan; Li, Haolong; Guan, Wei; Ling, Haili; Wang, Zhiyong; Mu, Tianyang; Shuler, Franklin D; Fang, Xuexun

    2010-10-01

    A major challenge associated with recombinant protein production in Escherichia coli is generation of large quantities of soluble, functional protein. Yeast SUMO (small ubiquitin-related modifier), has been shown to enhance heterologous protein expression and solubility as fusion tag, however, the effects of human SUMOs on protein expression have not been investigated. Here we describe the use of human SUMO1 and SUMO2 as a useful gene fusion technology. Human SUMO1 and SUMO2 fusion expression vectors were constructed and tested in His-tag and ubiquitin fusion expression systems. Two difficult-to-express model proteins, matrix metalloprotease-13 (MMP13) and enhanced green fluorescence protein (eGFP) were fused to the C-terminus of the human SUMO1 and SUMO2 expression vectors. These constructs were expressed in E. coli and evaluation of MMP13 and eGFP expression and solubility was conducted. We found that both SUMO1 and SUMO2 had the ability to enhance the solubility of MMP13 and eGFP, with the SUMO2 tag having a more significant effect. Since fusion tags produce varying quantities of soluble proteins, we assessed the effect of SUMO2 coupled with ubiquitin (Ub). SUMO2-ubiquitin and ubiquitin-SUMO2 fusion expression plasmids were constructed with eGFP as a passenger protein. Following expression in E. coli, both plasmids could improve eGFP expression and solubility similar to the SUMO2 fusion and better than the ubiquitin fusion. The sequential order of SUMO2 and ubiquitin had little effect on expression and solubility of eGFP. Purification of eGFP from the gene fusion product, SUMO2-ubiquitin-eGFP, involved cleavage by a deubiquitinase (Usp2-cc) and Ni-Sepharose column chromatography. The eGFP protein was purified to high homogeneity. In summary, human SUMO1 and SUMO2 are useful gene fusion technologies enhancing the expression, solubility and purification of model heterologous proteins. PMID:20457256

  20. The kinetics of ER fusion protein activation in vivo

    PubMed Central

    Wilson, Catherine H.; Gamper, Ivonne; Perfetto, Alessandra; Auw, Jeremy; Littlewood, Trevor D.; Evan, Gerard I.

    2014-01-01

    Reversibly switchable proteins are powerful tools with which to explore protein function in vitro and in vivo. For example, the activity of many proteins fused to the hormone-binding domain of the modified estrogen receptor (ERTAM) can be regulated by provision or removal of 4-hydroxytamoxifen (4-OHT). Despite the widespread use of ERTAM fusions in vivo, inadequate data are available as to the most efficacious routes for systemic tamoxifen delivery. In this study, we have used two well-characterised ERTAM fusion proteins, both reversibly activated by 4-OHT, to compare the effectiveness and kinetics of 4-OHT delivery in mice in vivo by either tamoxifen in food or by intraperitoneal injection. Our data indicate that dietary tamoxifen offers an effective, facile and ethically preferable means for long term activation of ERTAM fusion proteins in vivo. PMID:24662815

  1. (Na+ + K+)-ATPase Is a Target for Phosphoinositide 3-Kinase/Protein Kinase B and Protein Kinase C Pathways Triggered by Albumin*

    PubMed Central

    Peruchetti, Diogo B.; Pinheiro, Ana Acacia S.; Landgraf, Sharon S.; Wengert, Mira; Takiya, Christina M.; Guggino, William B.; Caruso-Neves, Celso

    2011-01-01

    In recent decades, evidence has confirmed the crucial role of albumin in the progression of renal disease. However, the possible role of signaling pathways triggered by physiologic concentrations of albumin in the modulation of proximal tubule (PT) sodium reabsorption has not been considered. In the present work, we have shown that a physiologic concentration of albumin increases the expression of the α1 subunit of (Na+ + K+)-ATPase in LLC-PK1 cells leading to an increase in enzyme activity. This process involves the sequential activation of PI3K/protein kinase B and protein kinase C pathways promoting inhibition of protein kinase A. This integrative network is inhibited when albumin concentration is increased, similar to renal disease, leading to a decrease in the α1 subunit of (Na+ + K+)-ATPase expression. Together, the results indicate that variation in albumin concentration in PT cells has an important effect on PT sodium reabsorption and, consequently, on renal sodium excretion. PMID:22057272

  2. Evaluation of capillary zone electrophoresis for the determination of protein composition in therapeutic immunoglobulins and human albumins.

    PubMed

    Christians, Stefan; van Treel, Nadine Denise; Bieniara, Gabriele; Eulig-Wien, Annika; Hanschmann, Kay-Martin; Giess, Siegfried

    2016-07-01

    Capillary zone electrophoresis (CZE) provides an alternative means of separating native proteins on the basis of their inherent electrophoretic mobilities. The major advantage of CZE is the quantification by UV detection, circumventing the drawbacks of staining and densitometry in the case of gel electrophoresis methods. The data of this validation study showed that CZE is a reliable assay for the determination of protein composition in therapeutic preparations of human albumin and human polyclonal immunoglobulins. Data obtained by CZE are in line with "historical" data obtained by the compendial method, provided that peak integration is performed without time correction. The focus here was to establish a rapid and reliable test to substitute the current gel based zone electrophoresis techniques for the control of protein composition of human immunoglobulins or albumins in the European Pharmacopoeia. We believe that the more advanced and modern CZE method described here is a very good alternative to the procedures currently described in the relevant monographs. PMID:27156142

  3. Size-dependent interaction of silica nanoparticles with lysozyme and bovine serum albumin proteins

    NASA Astrophysics Data System (ADS)

    Yadav, Indresh; Aswal, Vinod K.; Kohlbrecher, Joachim

    2016-05-01

    The interaction of three different sized (diameter 10, 18, and 28 nm) anionic silica nanoparticles with two model proteins—cationic lysozyme [molecular weight (MW) 14.7 kDa)] and anionic bovine serum albumin (BSA) (MW 66.4 kDa) has been studied by UV-vis spectroscopy, dynamic light scattering (DLS), and small-angle neutron scattering (SANS). The adsorption behavior of proteins on the nanoparticles, measured by UV-vis spectroscopy, is found to be very different for lysozyme and BSA. Lysozyme adsorbs strongly on the nanoparticles and shows exponential behavior as a function of lysozyme concentration irrespective of the nanoparticle size. The total amount of adsorbed lysozyme, as governed by the surface-to-volume ratio, increases on lowering the size of the nanoparticles for a fixed volume fraction of the nanoparticles. On the other hand, BSA does not show any adsorption for all the different sizes of the nanoparticles. Despite having different interactions, both proteins induce similar phase behavior where the nanoparticle-protein system transforms from one phase (clear) to two phase (turbid) as a function of protein concentration. The phase behavior is modified towards the lower concentrations for both proteins with increasing the nanoparticle size. DLS suggests that the phase behavior arises as a result of the nanoparticles' aggregation on the addition of proteins. The size-dependent modifications in the interaction potential, responsible for the phase behavior, have been determined by SANS data as modeled using the two-Yukawa potential accounting for the repulsive and attractive interactions in the systems. The protein-induced interaction between the nanoparticles is found to be short-range attraction for lysozyme and long-range attraction for BSA. The magnitude of attractive interaction irrespective of protein type is enhanced with increase in the size of the nanoparticles. The total (attractive+repulsive) potential leading to two-phase formation is found to be

  4. Albumin-coated SPIONs: an experimental and theoretical evaluation of protein conformation, binding affinity and competition with serum proteins.

    PubMed

    Yu, Siming; Perálvarez-Marín, Alex; Minelli, Caterina; Faraudo, Jordi; Roig, Anna; Laromaine, Anna

    2016-08-14

    The variety of nanoparticles (NPs) used in biological applications is increasing and the study of their interaction with biological media is becoming more important. Proteins are commonly the first biomolecules that NPs encounter when they interact with biological systems either in vitro or in vivo. Among NPs, super-paramagnetic iron oxide nanoparticles (SPIONs) show great promise for medicine. In this work, we study in detail the formation, composition, and structure of a monolayer of bovine serum albumin (BSA) on SPIONs. We determine, both by molecular simulations and experimentally, that ten molecules of BSA form a monolayer around the outside of the SPIONs and their binding strength to the SPIONs is about 3.5 × 10(-4) M, ten times higher than the adsorption of fetal bovine serum (FBS) on the same SPIONs. We elucidate a strong electrostatic interaction between BSA and the SPIONs, although the secondary structure of the protein is not affected. We present data that supports the strong binding of the BSA monolayer on SPIONs and the properties of the BSA layer as a protein-resistant coating. We believe that a complete understanding of the behavior and morphology of BSA-SPIONs and how the protein interacts with SPIONs is crucial for improving NP surface design and expanding the potential applications of SPIONs in nanomedicine. PMID:27241081

  5. Fc-fusion proteins: new developments and future perspectives

    PubMed Central

    Czajkowsky, Daniel M; Hu, Jun; Shao, Zhifeng; Pleass, Richard J

    2012-01-01

    Since the first description in 1989 of CD4-Fc-fusion antagonists that inhibit human immune deficiency virus entry into T cells, Fc-fusion proteins have been intensely investigated for their effectiveness to curb a range of pathologies, with several notable recent successes coming to market. These promising outcomes have stimulated the development of novel approaches to improve their efficacy and safety, while also broadening their clinical remit to other uses such as vaccines and intravenous immunoglobulin therapy. This increased attention has also led to non-clinical applications of Fc-fusions, such as affinity reagents in microarray devices. Here we discuss recent results and more generally applicable strategies to improve Fc-fusion proteins for each application, with particular attention to the newer, less charted areas. PMID:22837174

  6. ANALYSIS OF DRUG-PROTEIN BINDING BY ULTRAFAST AFFINITY CHROMATOGRAPHY USING IMMOBILIZED HUMAN SERUM ALBUMIN

    PubMed Central

    Mallik, Rangan; Yoo, Michelle J.; Briscoe, Chad J.; Hage, David S.

    2010-01-01

    Human serum albumin (HSA) was explored for use as a stationary phase and ligand in affinity microcolumns for the ultrafast extraction of free drug fractions and the use of this information for the analysis of drug-protein binding. Warfarin, imipramine, and ibuprofen were used as model analytes in this study. It was found that greater than 95% extraction of all these drugs could be achieved in as little as 250 ms on HSA microcolumns. The retained drug fraction was then eluted from the same column under isocratic conditions, giving elution in less than 40 s when working at 4.5 mL/min. The chromatographic behavior of this system gave a good fit with that predicted by computer simulations based on a reversible, saturable model for the binding of an injected drug with immobilized HSA. The free fractions measured by this method were found to be comparable to those determined by ultrafiltration, and equilibrium constants estimated by this approach gave good agreement with literature values. Advantages of this method include its speed and the relatively low cost of microcolumns that contain HSA. The ability of HSA to bind many types of drugs also creates the possibility of using the same affinity microcolumn to study and measure the free fractions for a variety of pharmaceutical agents. These properties make this technique appealing for use in drug binding studies and in the high-throughput screening of new drug candidates. PMID:20227701

  7. Regulation of Paramyxovirus Fusion Activation: the Hemagglutinin-Neuraminidase Protein Stabilizes the Fusion Protein in a Pretriggered State

    PubMed Central

    Salah, Zuhair W.; Gui, Long; DeVito, Ilaria; Jurgens, Eric M.; Lu, Hong; Yokoyama, Christine C.; Palermo, Laura M.; Lee, Kelly K.

    2012-01-01

    The hemagglutinin (HA)-neuraminidase protein (HN) of paramyxoviruses carries out three discrete activities, each of which affects the ability of HN to promote viral fusion and entry: receptor binding, receptor cleaving (neuraminidase), and triggering of the fusion protein. Binding of HN to its sialic acid receptor on a target cell triggers its activation of the fusion protein (F), which then inserts into the target cell and mediates the membrane fusion that initiates infection. We provide new evidence for a fourth function of HN: stabilization of the F protein in its pretriggered state before activation. Influenza virus hemagglutinin protein (uncleaved HA) was used as a nonspecific binding protein to tether F-expressing cells to target cells, and heat was used to activate F, indicating that the prefusion state of F can be triggered to initiate structural rearrangement and fusion by temperature. HN expression along with uncleaved HA and F enhances the F activation if HN is permitted to engage the receptor. However, if HN is prevented from engaging the receptor by the use of a small compound, temperature-induced F activation is curtailed. The results indicate that HN helps stabilize the prefusion state of F, and analysis of a stalk domain mutant HN reveals that the stalk domain of HN mediates the F-stabilization effect. PMID:22993149

  8. Fatty acid-binding site environments of serum vitamin D-binding protein and albumin are different

    PubMed Central

    Swamy, Narasimha; Ray, Rahul

    2008-01-01

    Vitamin D-binding protein (DBP) and albumin (ALB) are abundant serum proteins and both possess high-affinity binding for saturated and unsaturated fatty acids. However, certain differences exist. We surmised that in cases where serum albumin level is low, DBP presumably can act as a transporter of fatty acids. To explore this possibility we synthesized several alkylating derivatives of 14C-palmitic acid to probe the fatty acid binding pockets of DBP and ALB. We observed that N-ethyl-5-phenylisooxazolium-3′-sulfonate-ester (WRK ester) of 14C-palmitic acid specifically labeled DBP; but p-nitrophenyl- and N-hydroxysuccinimidyl-esters failed to do so. However, p-nitrophenyl ester of 14C-palmitic acid specifically labeled bovine ALB, indicating that the micro-environment of the fatty acid-binding domains of DBP and ALB may be different; and DBP may not replace ALB as a transporter of fatty acids. PMID:18374965

  9. Exploring the interaction between picoplatin and human serum albumin: The effects on protein structure and activity.

    PubMed

    Wang, Yanqing; Wu, Peirong; Zhou, Xinchun; Zhang, Hongmei; Qiu, Ligan; Cao, Jian

    2016-09-01

    For the first time, the effects of picoplatin on the structure and esterase-like catalytic activity of human serum albumin (HSA) have been investigated by spectroscopic approaches and molecular modeling. The circular dichroism (CD) spectral examinations indicated that the binding of picoplatin with HSA induced a slight decrease of a-helix content of protein and unfolded the constituent polypeptides of the protein. The synchronous fluorescence and three-dimensional fluorescence spectral methods were used to estimate the effect of picoplatin on the micro-environmental changes of the Trp and Tyr residues of HSA, indicating that the micro-environment around the Tyr and Trp residue is partly disturbed by picoplatin. UV-vis absorption spectral result indicated the formation of the ground state complex between picoplatin with HSA. The ANS binding assay indicated the existence of competitive combination of picoplatin and ANS with HSA. The studies on the effects of picoplatin on the binding of HSA with bilirubin and heme showed that picoplatin binding caused a change of angle between two chromophores of bound bilirubin and the binding site of picoplatin does not locate in subdomain IB in HSA that bound with heme. The molecular modeling results showed that picoplatin binds to the connection between domain I and domain II by hydrophobic, hydrogen bonds, and van der Waals forces. In addition, HSA maintains most of its esterase activity in the presence of picoplatin. The investigations on how picoplatin interacts with HSA are important for the understanding of the anticancer mechanism and toxicity of platinum-based anticancer drug. PMID:27484966

  10. Protections of bovine serum albumin protein from damage on functionalized graphene-based electrodes by flavonoids.

    PubMed

    Sun, Bolu; Gou, Yuqiang; Xue, Zhiyuan; Zheng, Xiaoping; Ma, Yuling; Hu, Fangdi; Zhao, Wanghong

    2016-05-01

    A sensitive electrochemical sensor based on bovine serum albumin (BSA)/poly (diallyldimethylammonium chloride) (PDDA) functionalized graphene nanosheets (PDDA-G) composite film modified glassy carbon electrode (BSA/PDDA-G/GCE) had been developed to investigate the oxidative protein damage and protections of protein from damage by flavonoids. The performance of this sensor was remarkably improved due to excellent electrical conductivity, strong adsorptive ability, and large effective surface area of PDDA-G. The BSA/PDDA-G/GCE displayed the greatest degree of BSA oxidation damage at 40min incubation time and in the pH5.0 Fenton reagent system (12.5mM FeSO4, 50mM H2O2). The antioxidant activities of four flavonoids had been compared by fabricated sensor based on the relative peak current ratio of SWV, because flavonoids prevented BSA damage caused by Fenton reagent and affected the BSA signal in a solution containing Co(bpy)3(3+). The sensor was characterized by cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS), and scanning electron microscopy (SEM). UV-vis spectrophotometry and FTIR were also used to investigate the generation of hydroxyl radical and BSA damage, respectively. On the basis of results from electrochemical methods, the order of the antioxidant activities of flavonoids is as follows: (+)-catechin>kaempferol>apigenin>naringenin. A novel, direct SWV analytical method for detection of BSA damage and assessment of the antioxidant activities of four flavonoids was developed and this electrochemical method provided a simple, inexpensive and rapid detection of BSA damage and evaluation of the antioxidant activities of samples. PMID:26952415

  11. Delivery of Therapeutic Proteins as Secretable TAT Fusion Products

    PubMed Central

    Flinterman, Marcella; Farzaneh, Farzin; Habib, Nagy; Malik, Farooq; Gäken, Joop; Tavassoli, Mahvash

    2008-01-01

    The trans-acting activator of transcription (TAT) protein transduction domain (PTD) mediates the transduction of peptides and proteins into target cells. The TAT-PTD has an important potential as a tool for the delivery of therapeutic agents. The production of TAT fusion proteins in bacteria, however, is problematic because of protein insolubility and the absence of eukaryotic post-translational modification. An attractive alternative, both for in vitro protein production and for in vivo applications, is the use of higher eukaryotic cells for secretion of TAT fusion proteins. However, the ubiquitous expression of furin endoprotease (PACE or SPC1) in the Golgi/endoplasmic reticulum, and the presence of furin recognition sequences within TAT-PTD, results in the cleavage and loss of the TAT-PTD domain during its secretory transition through the endoplasmic reticulum and Golgi. In this study, we show the development of a synthetic TATκ-PTD in which mutation of the furin recognition sequences, but retention of protein transduction activity, allows secretion of recombinant proteins, followed by successful uptake of the modified protein, by the target cells. This system was used to successfully secrete marker protein, green fluorescent protein (GFP), and apoptin, a protein with tumor-specific cytotoxicity. Detection of GFP, phosphorylation, and induction of cell death by TATκ-GFP-apoptin indicated that the secreted proteins were functional in target cells. This novel strategy therefore has important potential for the efficient delivery of therapeutic proteins. PMID:19050698

  12. Subcellular localization of transiently expressed fluorescent fusion proteins.

    PubMed

    Collings, David A

    2013-01-01

    The recent and massive expansion in plant genomics data has generated a large number of gene sequences for which two seemingly simple questions need to be answered: where do the proteins encoded by these genes localize in cells, and what do they do? One widespread approach to answering the localization question has been to use particle bombardment to transiently express unknown proteins tagged with green fluorescent protein (GFP) or its numerous derivatives. Confocal fluorescence microscopy is then used to monitor the localization of the fluorescent protein as it hitches a ride through the cell. The subcellular localization of the fusion protein, if not immediately apparent, can then be determined by comparison to localizations generated by fluorescent protein fusions to known signalling sequences and proteins, or by direct comparison with fluorescent dyes. This review aims to be a tour guide for researchers wanting to travel this hitch-hiker's path, and for reviewers and readers who wish to understand their travel reports. It will describe some of the technology available for visualizing protein localizations, and some of the experimental approaches for optimizing and confirming localizations generated by particle bombardment in onion epidermal cells, the most commonly used experimental system. As the non-conservation of signal sequences in heterologous expression systems such as onion, and consequent mis-targeting of fusion proteins, is always a potential problem, the epidermal cells of the Argenteum mutant of pea are proposed as a model system. PMID:23996319

  13. Albumin-coated SPIONs: an experimental and theoretical evaluation of protein conformation, binding affinity and competition with serum proteins

    NASA Astrophysics Data System (ADS)

    Yu, Siming; Perálvarez-Marín, Alex; Minelli, Caterina; Faraudo, Jordi; Roig, Anna; Laromaine, Anna

    2016-07-01

    The variety of nanoparticles (NPs) used in biological applications is increasing and the study of their interaction with biological media is becoming more important. Proteins are commonly the first biomolecules that NPs encounter when they interact with biological systems either in vitro or in vivo. Among NPs, super-paramagnetic iron oxide nanoparticles (SPIONs) show great promise for medicine. In this work, we study in detail the formation, composition, and structure of a monolayer of bovine serum albumin (BSA) on SPIONs. We determine, both by molecular simulations and experimentally, that ten molecules of BSA form a monolayer around the outside of the SPIONs and their binding strength to the SPIONs is about 3.5 × 10-4 M, ten times higher than the adsorption of fetal bovine serum (FBS) on the same SPIONs. We elucidate a strong electrostatic interaction between BSA and the SPIONs, although the secondary structure of the protein is not affected. We present data that supports the strong binding of the BSA monolayer on SPIONs and the properties of the BSA layer as a protein-resistant coating. We believe that a complete understanding of the behavior and morphology of BSA-SPIONs and how the protein interacts with SPIONs is crucial for improving NP surface design and expanding the potential applications of SPIONs in nanomedicine.The variety of nanoparticles (NPs) used in biological applications is increasing and the study of their interaction with biological media is becoming more important. Proteins are commonly the first biomolecules that NPs encounter when they interact with biological systems either in vitro or in vivo. Among NPs, super-paramagnetic iron oxide nanoparticles (SPIONs) show great promise for medicine. In this work, we study in detail the formation, composition, and structure of a monolayer of bovine serum albumin (BSA) on SPIONs. We determine, both by molecular simulations and experimentally, that ten molecules of BSA form a monolayer around the

  14. The cytoplasmic domain of the gamete membrane fusion protein HAP2 targets the protein to the fusion site in Chlamydomonas and regulates the fusion reaction

    PubMed Central

    Liu, Yanjie; Pei, Jimin; Grishin, Nick; Snell, William J.

    2015-01-01

    Cell-cell fusion between gametes is a defining step during development of eukaryotes, yet we know little about the cellular and molecular mechanisms of the gamete membrane fusion reaction. HAP2 is the sole gamete-specific protein in any system that is broadly conserved and shown by gene disruption to be essential for gamete fusion. The wide evolutionary distribution of HAP2 (also known as GCS1) indicates it was present in the last eukaryotic common ancestor and, therefore, dissecting its molecular properties should provide new insights into fundamental features of fertilization. HAP2 acts at a step after membrane adhesion, presumably directly in the merger of the lipid bilayers. Here, we use the unicellular alga Chlamydomonas to characterize contributions of key regions of HAP2 to protein location and function. We report that mutation of three strongly conserved residues in the ectodomain has no effect on targeting or fusion, although short deletions that include those residues block surface expression and fusion. Furthermore, HAP2 lacking a 237-residue segment of the cytoplasmic region is expressed at the cell surface, but fails to localize at the apical membrane patch specialized for fusion and fails to rescue fusion. Finally, we provide evidence that the ancient HAP2 contained a juxta-membrane, multi-cysteine motif in its cytoplasmic region, and that mutation of a cysteine dyad in this motif preserves protein localization, but substantially impairs HAP2 fusion activity. Thus, the ectodomain of HAP2 is essential for its surface expression, and the cytoplasmic region targets HAP2 to the site of fusion and regulates the fusion reaction. PMID:25655701

  15. Crystal structures of fusion proteins with large-affinity tags.

    PubMed

    Smyth, Douglas R; Mrozkiewicz, Marek K; McGrath, William J; Listwan, Pawel; Kobe, Bostjan

    2003-07-01

    The fusion of a protein of interest to a large-affinity tag, such as the maltose-binding protein (MBP), thioredoxin (TRX), or glutathione-S-transferase (GST), can be advantageous in terms of increased expression, enhanced solubility, protection from proteolysis, improved folding, and protein purification via affinity chromatography. Unfortunately, crystal growth is hindered by the conformational heterogeneity induced by the fusion tag, requiring that the tag is removed by a potentially problematic cleavage step. The first three crystal structures of fusion proteins with large-affinity tags have been reported recently. All three structures used a novel strategy to rigidly fuse the protein of interest to MBP via a short three- to five-amino acid spacer. This strategy has the potential to aid structure determination of proteins that present particular experimental challenges and are not conducive to more conventional crystallization strategies (e.g., membrane proteins). Structural genomics initiatives may also benefit from this approach as a way to crystallize problematic proteins of significant interest. PMID:12824478

  16. Glutaraldehyde mediated conjugation of amino-coated magnetic nanoparticles with albumin protein for nanothermotherapy.

    PubMed

    Zhao, Lingyun; Yang, Bing; Dai, Xiaochen; Wang, Xiaowen; Gao, Fuping; Zhang, Xiaodong; Tang, Jintian

    2010-11-01

    A novel bioconjugation of amino saline capped Fe3O4 magnetic nanoparticles (MNPs) with bovine serum albumin (BSA) was developed by applying glutaraldehyde as activator. Briefly, Fe3O4 MNs were synthesized by the chemical co-precipitation method. Surface modification of the prepared MNPs was performed by employing amino saline as the coating agent. Glutaraldehyde was further applied as an activation agent through which BSA was conjugated to the amino-coated MNPs. The structure of the BSA-MNs was confirmed by FTIR analysis. Physico-chemical characterizations of the BSA-MNPs, such as surface morphology, surface charge and magnetic properties were investigated by Transmission Electron Microscopy (TEM), zeta-Potential and Vibrating Sample Magnetometer (VSM), etc. Magnetic inductive heating characteristics of the BSA-MNPs were analyzed by exposing the MNPs suspension (magnetic fluid) under alternative magnetic field (AMF). The results demonstrate that BSA was successfully conjugated with amino-coated MNs mediated through glutaraldehyde activation. The nanoparticles were spherical shaped with approximately 10 nm diameter. Possessing ideal magnetic inductive heating characteristics, which can generate very rapid and efficient heating while upon AMF exposure, BSA-MNPs can be applied as a novel candidature for magnetic nanothermotherapy for cancer treatment. In vitro cytotoxicity study on the human hepatocellular liver carcinoma cells (HepG-2) indicates that BSA-MNP is an efficient agent for cancer nanothermotherapy with satisfied biocompatibility, as rare cytotoxicity was observed in the absence of AMF. Moreover, our investigation provides a methodology for fabrication protein conjugated MNPs, for instance monoclonal antibody conjugated MNPs for targeting cancer nanothermotherapy. PMID:21137877

  17. Characterization of the comparative drug binding to intra- (liver fatty acid binding protein) and extra- (human serum albumin) cellular proteins.

    PubMed

    Rowland, Andrew; Hallifax, David; Nussio, Matthew R; Shapter, Joseph G; Mackenzie, Peter I; Brian Houston, J; Knights, Kathleen M; Miners, John O

    2015-01-01

    1. This study compared the extent, affinity, and kinetics of drug binding to human serum albumin (HSA) and liver fatty acid binding protein (LFABP) using ultrafiltration and surface plasmon resonance (SPR). 2. Binding of basic and neutral drugs to both HSA and LFABP was typically negligible. Binding of acidic drugs ranged from minor (fu > 0.8) to extensive (fu < 0.1). Of the compounds screened, the highest binding to both HSA and LFABP was observed for the acidic drugs torsemide and sulfinpyrazone, and for β-estradiol (a polar, neutral compound). 3. The extent of binding of acidic drugs to HSA was up to 40% greater than binding to LFABP. SPR experiments demonstrated comparable kinetics and affinity for the binding of representative acidic drugs (naproxen, sulfinpyrazone, and torsemide) to HSA and LFABP. 4. Simulations based on in vitro kinetic constants derived from SPR experiments and a rapid equilibrium model were undertaken to examine the impact of binding characteristics on compartmental drug distribution. Simulations provided mechanistic confirmation that equilibration of intracellular unbound drug with the extracellular unbound drug is attained rapidly in the absence of active transport mechanisms for drugs bound moderately or extensively to HSA and LFABP. PMID:25801059

  18. Production of recombinant oxytocin through sulfitolysis of inteincontaining fusion protein.

    PubMed

    Esipov, Roman S; Stepanenko, Vasily N; Chupova, Larisa A; Miroshnikov, Anatoly I

    2012-05-01

    An artificial gene consisting of seven copies of an oxytocinoyl-lysine encoding sequence arranged in a tandem was synthesized and inserted downstream of the SspDnaB intein gene in a pTWIN1 plasmid. The corresponding fusion protein Dnab-7oxy contained 16 cysteine residues and formed inclusion bodies when expressed in E. coli. The standard protocol involving solubilization of the fusion protein and its autocatalytic cleavage on a chitin resin was not effective because of a very low yield of the cleavage reaction. Attempts to perform a refolding of the intein part of the fusion protein in solution were also unsuccessful because of a high level of protein aggregation. Sulfitolysis of cysteine residues is known to increase a solubility of proteins and peptides. Therefore we suggested a one-step approach that combines solubilization of inclusion bodies and sulfitolysis of a hybrid protein. The fusion protein was completely reduced and solubilized in 8M urea at pH 9.0 in the presence of sodium sulfite and sodium tetrathionate. The sulfitized protein was loaded onto a chitin column, an efficient cleavage was induced by a pH shift from 9.0 to 6.5, and seven successively connected oxytocinoyl- lysine units were released. The heptamer was subjected to trypsinolysis yielding sulfitized monomers of oxytocinoyllysine. Oxytocinoyl-lysine was refolded as described previously and treated by carboxypeptidase B to form the oxytocinic acid. The target oxytocin amide was then synthesized via methyl ester intermediate. Using this approach 6 mg of recombinant oxytocin can be obtained from 1 g of biomass. PMID:22316308

  19. Structural changes of envelope proteins during alphavirus fusion

    SciTech Connect

    Li, Long; Jose, Joyce; Xiang, Ye; Kuhn, Richard J.; Rossmann, Michael G.

    2010-12-08

    Alphaviruses are enveloped RNA viruses that have a diameter of about 700 {angstrom} and can be lethal human pathogens. Entry of virus into host cells by endocytosis is controlled by two envelope glycoproteins, E1 and E2. The E2-E1 heterodimers form 80 trimeric spikes on the icosahedral virus surface, 60 with quasi-three-fold symmetry and 20 coincident with the icosahedral three-fold axes arranged with T = 4 quasi-symmetry. The E1 glycoprotein has a hydrophobic fusion loop at one end and is responsible for membrane fusion. The E2 protein is responsible for receptor binding and protects the fusion loop at neutral pH. The lower pH in the endosome induces the virions to undergo an irreversible conformational change in which E2 and E1 dissociate and E1 forms homotrimers, triggering fusion of the viral membrane with the endosomal membrane and then releasing the viral genome into the cytoplasm. Here we report the structure of an alphavirus spike, crystallized at low pH, representing an intermediate in the fusion process and clarifying the maturation process. The trimer of E2-E1 in the crystal structure is similar to the spikes in the neutral pH virus except that the E2 middle region is disordered, exposing the fusion loop. The amino- and carboxy-terminal domains of E2 each form immunoglobulin-like folds, consistent with the receptor attachment properties of E2.

  20. Molecular Principles of Gene Fusion Mediated Rewiring of Protein Interaction Networks in Cancer.

    PubMed

    Latysheva, Natasha S; Oates, Matt E; Maddox, Louis; Flock, Tilman; Gough, Julian; Buljan, Marija; Weatheritt, Robert J; Babu, M Madan

    2016-08-18

    Gene fusions are common cancer-causing mutations, but the molecular principles by which fusion protein products affect interaction networks and cause disease are not well understood. Here, we perform an integrative analysis of the structural, interactomic, and regulatory properties of thousands of putative fusion proteins. We demonstrate that genes that form fusions (i.e., parent genes) tend to be highly connected hub genes, whose protein products are enriched in structured and disordered interaction-mediating features. Fusion often results in the loss of these parental features and the depletion of regulatory sites such as post-translational modifications. Fusion products disproportionately connect proteins that did not previously interact in the protein interaction network. In this manner, fusion products can escape cellular regulation and constitutively rewire protein interaction networks. We suggest that the deregulation of central, interaction-prone proteins may represent a widespread mechanism by which fusion proteins alter the topology of cellular signaling pathways and promote cancer. PMID:27540857

  1. Structure of Serum Albumin

    NASA Technical Reports Server (NTRS)

    Carter, Daniel C.; Ho, Joseph X.

    1994-01-01

    Because of its availability, low cost, stability, and unusual ligand-binding properties, serum albumin has been one of the mst extensively studied and applied proteins in biochemistry. However, as a protein, albumin is far from typical, and the widespread interest in and application of albumin have not been balanced by an understanding of its molecular structure. Indeed, for more than 30 years structural information was surmised based solely on techniques such as hydrodynamics, low-angle X-ray scattering, and predictive methods.

  2. Evaluation of Ischemia-Modified Albumin, Malondialdehyde, and Advanced Oxidative Protein Products as Markers of Vascular Injury in Diabetic Nephropathy

    PubMed Central

    Ahmad, Afzal; Manjrekar, Poornima; Yadav, Charu; Agarwal, Ashish; Srikantiah, Rukmini Mysore; Hegde, Anupama

    2016-01-01

    AIM This study aimed at evaluation of ischemia-modified albumin (IMA), malondialdehyde (MDA), and advanced oxidative protein products (AOPP) as markers of vascular injury in diabetic nephropathy (DN) with derivation of cutoff values for the same. MATERIALS AND METHODS Study population comprised 60 diabetes patients and 30 controls, with diabetes patients further categorized into three groups based on urine albumin/creatinine ratio (UACR) of <30 mg/g (diabetes without microalbuminuria), 30–300 mg/g (early DN), and >300 mg/g of creatinine (overt DN). Serum IMA, MDA, and AOPP were estimated by enzyme-linked immunosorbent assay; HbA1c, serum creatinine, urine albumin, and urine creatinine were estimated using automated analyzers. Statistical analysis was done using analysis of variance, Pearson’s correlation coefficient, and receiver-operating characteristic curve. RESULTS A statistically significant difference was found in the levels of IMA among patients with early DN (154 ng/mL), diabetes without nephropathy (109.4 ng/mL), and healthy controls (45.7 ng/mL), with highest levels in early DN cases. Similar increase was seen in AOPP as well. A significant correlation was observed between IMA and UACR in diabetes without nephropathy (r = 0.448). CONCLUSION The present study postulates serum IMA as a novel biomarker for the assessment of disease progression in diabetes even before microalbuminuria, and a cutoff point ≥99 ng/mL can be used for detection of early DN. PMID:27158221

  3. Oligomerization and toxicity of A{beta} fusion proteins

    SciTech Connect

    Caine, Joanne M.; Bharadwaj, Prashant R.; Sankovich, Sonia E.; Ciccotosto, Giuseppe D.; Streltsov, Victor A.; Varghese, Jose

    2011-06-10

    Highlights: {yields} We expressed amyloid-{beta} (A{beta}) peptide as a soluble maltose binding protein fusion (MBP-A{beta}42 and MBP-A{beta}16). {yields} The full length A{beta} peptide fusion, MBP-A{beta}42, forms oligomeric species as determined by SDS-PAGE gels, gel filtration and DLS. {yields} The MBP-A{beta}42, but not MBP-A{beta}16 or MBP alone, is toxic to both yeast and mammalian cells as determined by toxicity assays. -- Abstract: This study has found that the Maltose binding protein A{beta}42 fusion protein (MBP-A{beta}42) forms soluble oligomers while the shorter MBP-A{beta}16 fusion and control MBP did not. MBP-A{beta}42, but neither MBP-A{beta}16 nor control MBP, was toxic in a dose-dependent manner in both yeast and primary cortical neuronal cells. This study demonstrates the potential utility of MBP-A{beta}42 as a reagent for drug screening assays in yeast and neuronal cell cultures and as a candidate for further A{beta}42 characterization.

  4. Fusion protein technologies for biopharmaceuticals: Applications and challenges

    PubMed Central

    Berger, Sven; Lowe, Peter; Tesar, Michael

    2015-01-01

    Stefan R. Schmidt consolidates the hugely diverse field of fusion proteins and their application in the creation of biopharmaceuticals. The text is replete with case studies and clinical data that inform and intrigue the reader as to the myriad possibilities available when considering the creation of a fusion protein. This valuable text will serve the novice as a broad introduction or the seasoned professional as a thorough review of the state of the art. The first marketed therapeutic recombinant protein was human insulin (Humulin® R). Its approval in 1982 was followed by other such products, including erythropoietin (EPO), interferon (IFN), and tissue plasminogen activator (tPa). Since the 1980s, the number and general availability of recombinant products that replace natural proteins harvested from animal or human sources has increased considerably. Following the initial success, researchers started de novo designs of therapeutic proteins that do not occur in nature. The first of these new drugs to be approved was etanercept (Enbrel®), a fusion portion containing a section of the tumor necrosis factor (TNF) receptor fused to the Fc portion of human IgG1.

  5. Site-selective protein immobilization by covalent modification of GST fusion proteins.

    PubMed

    Zhou, Yiqing; Guo, Tianlin; Tang, Guanghui; Wu, Hui; Wong, Nai-Kei; Pan, Zhengying

    2014-11-19

    The immobilization of functional proteins onto solid supports using affinity tags is an attractive approach in recent development of protein microarray technologies. Among the commonly used fusion protein tags, glutathione S-transferase (GST) proteins have been indispensable tools for protein-protein interaction studies and have extensive applications in recombinant protein purification and reversible protein immobilization. Here, by utilizing pyrimidine-based small-molecule probes with a sulfonyl fluoride reactive group, we report a novel and general approach for site-selective immobilization of Schistosoma japonicum GST (sjGST) fusion proteins through irreversible and specific covalent modification of the tyrosine-111 residue of the sjGST tag. As demonstrated by sjGST-tagged eGFP and sjGST-tagged kinase activity assays, this immobilization approach offers the advantages of high immobilization efficiency and excellent retention of protein structure and activity. PMID:25340706

  6. The role of human bone morphogenetic proteins in spinal fusion.

    PubMed

    Zlotolow, D A; Vaccaro, A R; Salamon, M L; Albert, T J

    2000-01-01

    The attainment of a stable arthrodesis is critical to the successful management of some types of spinal disorders. Autologous iliac-crest bone graft has been the most commonly utilized substance associated with predictable healing in spinal fusion applications. Although alternative graft substances exist, these have not been shown to be as uniformly effective in achieving spinal fusion. Because of the morbidity associated with bone autograft harvest, there is increasing interest in alternative graft substances and especially in the osteoinductive abilities of bone morphogenetic proteins (BMPs). Several animal models have demonstrated that BMP-containing allograft or synthetic carrier medium is as effective as or superior to autograft bone in promoting spinal fusion. Furthermore, the limited number of human trials utilizing BMPs to treat nonunions in the appendicular skeleton indicate that the results found in animal models will be reproducible in the clinical setting. PMID:10666648

  7. The actin cytoskeleton inhibits pore expansion during PIV5 fusion protein-promoted cell-cell fusion

    SciTech Connect

    Wurth, Mark A.; Schowalter, Rachel M.; Smith, Everett Clinton; Moncman, Carole L.; Ellis Dutch, Rebecca; McCann, Richard O.

    2010-08-15

    Paramyxovirus fusion (F) proteins promote both virus-cell fusion, required for viral entry, and cell-cell fusion, resulting in syncytia formation. We used the F-actin stabilizing drug, jasplakinolide, and the G-actin sequestrant, latrunculin A, to examine the role of actin dynamics in cell-cell fusion mediated by the parainfluenza virus 5 (PIV5) F protein. Jasplakinolide treatment caused a dose-dependent increase in cell-cell fusion as measured by both syncytia and reporter gene assays, and latrunculin A treatment also resulted in fusion stimulation. Treatment with jasplakinolide or latrunculin A partially rescued a fusion pore opening defect caused by deletion of the PIV5 F protein cytoplasmic tail, but these drugs had no effect on fusion inhibited at earlier stages by either temperature arrest or by a PIV5 heptad repeat peptide. These data suggest that the cortical actin cytoskeleton is an important regulator of fusion pore enlargement, an energetically costly stage of viral fusion protein-mediated membrane merger.

  8. Measles Virus Fusion Protein: Structure, Function and Inhibition

    PubMed Central

    Plattet, Philippe; Alves, Lisa; Herren, Michael; Aguilar, Hector C.

    2016-01-01

    Measles virus (MeV), a highly contagious member of the Paramyxoviridae family, causes measles in humans. The Paramyxoviridae family of negative single-stranded enveloped viruses includes several important human and animal pathogens, with MeV causing approximately 120,000 deaths annually. MeV and canine distemper virus (CDV)-mediated diseases can be prevented by vaccination. However, sub-optimal vaccine delivery continues to foster MeV outbreaks. Post-exposure prophylaxis with antivirals has been proposed as a novel strategy to complement vaccination programs by filling herd immunity gaps. Recent research has shown that membrane fusion induced by the morbillivirus glycoproteins is the first critical step for viral entry and infection, and determines cell pathology and disease outcome. Our molecular understanding of morbillivirus-associated membrane fusion has greatly progressed towards the feasibility to control this process by treating the fusion glycoprotein with inhibitory molecules. Current approaches to develop anti-membrane fusion drugs and our knowledge on drug resistance mechanisms strongly suggest that combined therapies will be a prerequisite. Thus, discovery of additional anti-fusion and/or anti-attachment protein small-molecule compounds may eventually translate into realistic therapeutic options. PMID:27110811

  9. Measles Virus Fusion Protein: Structure, Function and Inhibition.

    PubMed

    Plattet, Philippe; Alves, Lisa; Herren, Michael; Aguilar, Hector C

    2016-04-01

    Measles virus (MeV), a highly contagious member of the Paramyxoviridae family, causes measles in humans. The Paramyxoviridae family of negative single-stranded enveloped viruses includes several important human and animal pathogens, with MeV causing approximately 120,000 deaths annually. MeV and canine distemper virus (CDV)-mediated diseases can be prevented by vaccination. However, sub-optimal vaccine delivery continues to foster MeV outbreaks. Post-exposure prophylaxis with antivirals has been proposed as a novel strategy to complement vaccination programs by filling herd immunity gaps. Recent research has shown that membrane fusion induced by the morbillivirus glycoproteins is the first critical step for viral entry and infection, and determines cell pathology and disease outcome. Our molecular understanding of morbillivirus-associated membrane fusion has greatly progressed towards the feasibility to control this process by treating the fusion glycoprotein with inhibitory molecules. Current approaches to develop anti-membrane fusion drugs and our knowledge on drug resistance mechanisms strongly suggest that combined therapies will be a prerequisite. Thus, discovery of additional anti-fusion and/or anti-attachment protein small-molecule compounds may eventually translate into realistic therapeutic options. PMID:27110811

  10. Expression, purification and characterization of a recombinant Tat47-57-Oct4 fusion protein in Pichia pastoris.

    PubMed

    Wang, Haotian; Zhang, Xinmin; Kong, Ning; Wei, Anhui; Zhang, Yanhong; Ma, Jie; Zhou, Yulai; Yan, Weiqun

    2014-02-01

    The transcription factor, Oct-4, is involved in the self-renewal of undifferentiated embryonic stem cells, and is also significant in the reprogramming process and in the development of tumors. In the present study, the fusion protein, Tat47‑57-Oct4, was secreted by the signal peptide of human serum albumin in Pichia pastoris under the control of alcohol oxidase promoter 1. The yield of recombinant Tat47‑57-Oct4 fusion protein was ~210 mg/l. Following pilot‑scale fermentation, Tat47‑57-Oct4 was purified by ammonium sulfate precipitation, Vivaflow 200 ultrafiltration and SP Sepharose fast flow chromatography in order to obtain 95.6% purity. Immunofluorescence analysis validated the ability of Tat47‑57-Oct4 to cross the cell membrane. The results demonstrated that the experimental procedure developed in the present study could produce large quantities of active Tat47‑57-Oct4 fusion protein from P. pastoris. PMID:24336974

  11. Design of a bifunctional fusion protein for ovarian cancer drug delivery: single-chain anti-CA125 core-streptavidin fusion protein.

    PubMed

    Wang, Welson Wen-Shang; Das, Dipankar; McQuarrie, Stephen A; Suresh, Mavanur R

    2007-03-01

    We have developed a universal ovarian cancer cell targeting vehicle that can deliver biotinylated therapeutic drugs. A single-chain antibody variable domain (scFv) that recognizes the CA125 antigen of ovarian cancer cells was fused with a core-streptavidin domain (core-streptavidin-VL-VH and VL-VH-core-streptavidin orientations) using recombinant DNA technology and then expressed in Escherichia coli using the T7 expression system. The bifunctional fusion protein (bfFp) was expressed in a shaker flask culture, extracted from the periplasmic soluble protein, and affinity purified using an IMAC column. The two distinct activities (biotin binding and anti-CA125) of the bfFp were demonstrated using ELISA, Western blot and confocal laser-scanning microscopy (CLSM). The ELISA method utilized human NIH OVCAR-3 cells along with biotinylated bovine serum albumin (B-BSA) or biotinylated liposomes, whereas, the Western blot involved probing with B-BSA. The CLSM study has shown specificity in binding to the OVCAR-3 cell-line. ELISA and Western blot studies have confirmed the bifunctional activity and specificity. In the presence of bfFp, there was enhanced binding of biotinylated antigen and liposome to OVCAR-3 cells. In contrast, the control EMT6 cells, which do not express the CA125 antigen, showed minimal binding of the bfFp. Consequently, bfFp based targeting of biotinylated therapeutic drugs, proteins, liposomes, or nanoparticles could be an alternative, convenient method to deliver effective therapy to ovarian cancer patients. Peritoneal infusion of the bfFp-therapeutic complex could also be effective in locally targeting the most common site of metastatic spread. PMID:17257818

  12. Phycobiliprotein fusion proteins: versatile intensely fluorescent constructs

    NASA Astrophysics Data System (ADS)

    Glazer, Alexander N.; Cai, Yuping A.; Tooley, Aaron J.

    2004-06-01

    Since 1982, phycobiliproteins have served as fluorescent labels in a wide variety of cell and molecule analyses. The exceptional spectroscopic properties of these labels include very high absorbance coefficients and quantum yields, and large Stokes shifts. The spectroscopic diversity of these reagents is restricted to a subset of naturally occurring phycobiliproteins with stable assembly states in vitro, whose target specificity is generated by chemical conjugation to proteins or small molecules. The latter step generates heterogeneity. These limitations have been overcome by expressing various recombinant phycobiliprotein constructs in the cyanobacterium Anabaena sp. PCC7120. Modular recombinant phycobiliprotein-based labels were constructed with some or all of the following features (a) an affinity purification tag; (b) a stable oligomerization domain (to maintain stable higher order assemblies of the phycobiliprotein monomers at very low protein concentration); (c) a biospecific recognition domain. Such phycobiliprotein constructs are readily purified from crude cell extracts by affinity chromatography and used directly as fluorescent labels. To generate constructs for intracellular in vivo labeling, the entire pathways for the biosynthesis of the His-tagged holo- α (phycocyanobilin-bearing) subunit of phycocyanin (emission max. 641 nm) and of the His-tagged holo-α (phycobiliviolin-bearing) subunit of phycoerythrocyanin (emission max. 582 nm) were reconstituted in Escherichia coli.

  13. Interaction study on bovine serum albumin physically binding to silver nanoparticles: Evolution from discrete conjugates to protein coronas

    NASA Astrophysics Data System (ADS)

    Guo, Jun; Zhong, Ruibo; Li, Wanrong; Liu, Yushuang; Bai, Zhijun; Yin, Jun; Liu, Jingran; Gong, Pei; Zhao, Xinmin; Zhang, Feng

    2015-12-01

    The nanostructures formed by inorganic nanoparticles together with organic molecules especially biomolecules have attracted increasing attention from both industries and researching fields due to their unique hybrid properties. In this paper, we systemically studied the interactions between amphiphilic polymer coated silver nanoparticles and bovine serum albumins by employing the fluorescence quenching approach in combination with the Stern-Volmer and Hill equations. The binding affinity was determined to 1.30 × 107 M-1 and the interaction was spontaneously driven by mainly the van der Waals force and hydrogen-bond mediated interactions, and negatively cooperative from the point of view of thermodynamics. With the non-uniform coating of amphiphilic polymer, the silver nanoparticles can form protein coronas which can become discrete protein-nanoparticle conjugates when controlling their molar ratios of mixing. The protein's conformational changes upon binding nanoparticles was also studied by using the three-dimensional fluorescence spectroscopy.

  14. Using Fluorescent Protein Fusions to Study Protein Subcellular Localization and Dynamics in Plant Cells.

    PubMed

    Cui, Yong; Gao, Caiji; Zhao, Qiong; Jiang, Liwen

    2016-01-01

    Studies of protein subcellular localization and dynamics are helpful in understanding the cellular functions of proteins in an organism. In the past decade, the use of green fluorescent protein (GFP) as a fusion tag has dramatically extended our knowledge in this field. Transient expression and stable transformation of GFP-tagged proteins have been wildly used to study protein localization in vivo in different systems. Although GFP-based tags provide a fast and convenient way to characterize protein properties in living cells, several reports have demonstrated that GFP fusions might not accurately reflect the localization of the native protein as GFP tags may alter the protein properties. To facilitate proper usage of GFP tags in plant cell biology study, we describe detailed protocols to identify possible inhibitory effects of fluorescent tags on protein subcellular localization and to determine if a fluorescently tagged protein is localized to the correct subcellular compartment. Using Arabidopsis Endomembrane protein 12 (EMP12) as an example, we first show the possible inhibitory effect of GFP tags on proper protein localization and then describe the immunofluorescence labeling method to verify the correct localization of GFP fusion proteins. Next, a method is presented using the ImageJ program with the Pearson-Spearman correlation (PSC) colocalization plug-in for statistical quantification of colocalization ratios of two fluorophores. Finally we provide a detailed method for protein dynamics studies using spinning disk confocal microscopy in Arabidopsis cells. PMID:27515077

  15. GS-5806 Inhibits Pre- to Postfusion Conformational Changes of the Respiratory Syncytial Virus Fusion Protein

    PubMed Central

    Xing, Weimei; Niedziela-Majka, Anita; Wong, Jinny S.; Hung, Magdeleine; Brendza, Katherine M.; Perron, Michel; Jordan, Robert; Sperandio, David; Liu, Xiaohong; Mackman, Richard; Sakowicz, Roman

    2015-01-01

    GS-5806 is a small-molecule inhibitor of human respiratory syncytial virus fusion protein-mediated viral entry. During viral entry, the fusion protein undergoes major conformational changes, resulting in fusion of the viral envelope with the host cell membrane. This process is reproduced in vitro using a purified, truncated respiratory syncytial virus (RSV) fusion protein. GS-5806 blocked these conformational changes, suggesting a possible mechanism for antiviral activity. PMID:26324264

  16. GS-5806 inhibits pre- to postfusion conformational changes of the respiratory syncytial virus fusion protein.

    PubMed

    Samuel, Dharmaraj; Xing, Weimei; Niedziela-Majka, Anita; Wong, Jinny S; Hung, Magdeleine; Brendza, Katherine M; Perron, Michel; Jordan, Robert; Sperandio, David; Liu, Xiaohong; Mackman, Richard; Sakowicz, Roman

    2015-11-01

    GS-5806 is a small-molecule inhibitor of human respiratory syncytial virus fusion protein-mediated viral entry. During viral entry, the fusion protein undergoes major conformational changes, resulting in fusion of the viral envelope with the host cell membrane. This process is reproduced in vitro using a purified, truncated respiratory syncytial virus (RSV) fusion protein. GS-5806 blocked these conformational changes, suggesting a possible mechanism for antiviral activity. PMID:26324264

  17. In-vivo delivery of therapeutic proteins by genetically-modified cells: comparison of organoids and human serum albumin alginate-coated beads.

    PubMed

    Shinya, E; Dervillez, X; Edwards-Lévy, F; Duret, V; Brisson, E; Ylisastigui, L; Lévy, M C; Cohen, J H; Klatzmann, D

    1999-12-01

    We have designed a self-assembling multimeric soluble CD4 molecule by inserting the C-terminal fragment of the alpha chain of human C4-binding protein (C4bp alpha) at the C-terminal end of human soluble CD4 genes. This CD4-C4bp alpha fusion protein (sMulti-CD4) and two other reference molecules, a fusion protein of human serum albumin (HSA) and the first two domains of CD4 (HSA-CD4) and monomeric soluble CD4 (sMono-CD4), were delivered in vivo by genetically modified 293 cells. These cells were implanted in mice as organoids and also encapsulated in HSA alginate-coated beads. sMulti-CD4 showed an apparent molecular weight of about 300-350 kDa, in accordance with a possible heptamer formula. sMulti-CD4 produced either in cell culture or in vivo in mice appeared to be a better invitro inhibitor of HIV infection than sMono-CD4. Plasma levels of sMulti-CD4, HSA-CD4, and sMono-CD4 reached approximately 2,300, 2,700, and 170 ng/mL, respectively, 13 weeks after in-vivo organoid implantation, which had formed tumours at that time. This suggests that the plasma half-life of sMulti-CD4 is much longer than that of sMono-CD4. The 293 xenogeneic cells encapsulated in HSA alginate-coated beads remained alive and kept secreting sMono-CD4 or HSA-CD4 continuously at significant levels for 18 weeks in nude mice, without tumour formation. When implanted in immunocompetent Balb/c mice, they were rejected two to three weeks after implantation. In contrast, encapsulated BL4 hybridoma cells remained alive and kept secreting BL4 anti-CD4 mAb for at least four weeks in Balb/c mice. These results suggest the clinical potential of the C4bp-multimerizing system, which could improve both the biological activity and the poor in-vivo pharmacokinetic performance of a monomeric functional protein like soluble CD4. These data also show that a systemic delivery of therapeutic proteins, including immunoglobulins, can be obtained by the in-vivo implantation of engineered allogeneic cells encapsulated

  18. Accelerated Nucleation of Hydroxyapatite Using an Engineered Hydrophobin Fusion Protein.

    PubMed

    Melcher, Melanie; Facey, Sandra J; Henkes, Thorsten M; Subkowski, Thomas; Hauer, Bernhard

    2016-05-01

    Calcium phosphate mineralization is of particular interest in dental repair. A biomimetic approach using proteins or peptides is a highly promising way to reconstruct eroded teeth. In this study, the screening of several proteins is described for their binding and nucleating activities toward hydroxyapatite. Out of 27 tested candidates, only two hydrophobin fusion proteins showed binding abilities to hydroxyapatite in a mouthwash formulation and an increased nucleation in artificial saliva. Using a semirational approach, one of the two candidates (DEWA_5), a fusion protein consisting of a truncated section of the Bacillus subtilis synthase YaaD, the Aspergillus nidulans hydrophobin DEWA, and the rationally designed peptide P11-4 described in the literature, could be further engineered toward a faster mineral formation. The variants DEWA_5a (40aaYaaD-SDSDSD-DEWA) and DEWA_5b (40aaYaaD-RDRDRD-DEWA) were able to enhance the nucleation activity without losing the ability to form hydroxyapatite. In the case of variant DEWA_5b, an additional increase in the binding toward hydroxyapatite could be achieved. Especially with the variant DEWA_5a, the protein engineering of the rationally designed peptide sequence resulted in a resemblance of an amino acid motif that is found in nature. The engineered peptide resembles the amino acid motif in dentin phosphoprotein, one of the major proteins involved in dentinogenesis. PMID:27010648

  19. Nanodisc-cell fusion: control of fusion pore nucleation and lifetimes by SNARE protein transmembrane domains.

    PubMed

    Wu, Zhenyong; Auclair, Sarah M; Bello, Oscar; Vennekate, Wensi; Dudzinski, Natasha R; Krishnakumar, Shyam S; Karatekin, Erdem

    2016-01-01

    The initial, nanometer-sized connection between the plasma membrane and a hormone- or neurotransmitter-filled vesicle -the fusion pore- can flicker open and closed repeatedly before dilating or resealing irreversibly. Pore dynamics determine release and vesicle recycling kinetics, but pore properties are poorly known because biochemically defined single-pore assays are lacking. We isolated single flickering pores connecting v-SNARE-reconstituted nanodiscs to cells ectopically expressing cognate, "flipped" t-SNAREs. Conductance through single, voltage-clamped fusion pores directly reported sub-millisecond pore dynamics. Pore currents fluctuated, transiently returned to baseline multiple times, and disappeared ~6 s after initial opening, as if the fusion pore fluctuated in size, flickered, and resealed. We found that interactions between v- and t-SNARE transmembrane domains (TMDs) promote, but are not essential for pore nucleation. Surprisingly, TMD modifications designed to disrupt v- and t-SNARE TMD zippering prolonged pore lifetimes dramatically. We propose that the post-fusion geometry of the proteins contribute to pore stability. PMID:27264104

  20. Nanodisc-cell fusion: control of fusion pore nucleation and lifetimes by SNARE protein transmembrane domains

    PubMed Central

    Wu, Zhenyong; Auclair, Sarah M.; Bello, Oscar; Vennekate, Wensi; Dudzinski, Natasha R.; Krishnakumar, Shyam S.; Karatekin, Erdem

    2016-01-01

    The initial, nanometer-sized connection between the plasma membrane and a hormone- or neurotransmitter-filled vesicle –the fusion pore– can flicker open and closed repeatedly before dilating or resealing irreversibly. Pore dynamics determine release and vesicle recycling kinetics, but pore properties are poorly known because biochemically defined single-pore assays are lacking. We isolated single flickering pores connecting v-SNARE-reconstituted nanodiscs to cells ectopically expressing cognate, “flipped” t-SNAREs. Conductance through single, voltage-clamped fusion pores directly reported sub-millisecond pore dynamics. Pore currents fluctuated, transiently returned to baseline multiple times, and disappeared ~6 s after initial opening, as if the fusion pore fluctuated in size, flickered, and resealed. We found that interactions between v- and t-SNARE transmembrane domains (TMDs) promote, but are not essential for pore nucleation. Surprisingly, TMD modifications designed to disrupt v- and t-SNARE TMD zippering prolonged pore lifetimes dramatically. We propose that the post-fusion geometry of the proteins contribute to pore stability. PMID:27264104

  1. Post-translational control of protein function with light using a LOV-intein fusion protein.

    PubMed

    Jones, D C; Mistry, I N; Tavassoli, A

    2016-04-01

    Methods for the post-translational control of protein function with light hold much value as tools in cell biology. To this end, we report a fusion protein that consists of DnaE split-inteins, flanking the light sensitive LOV2 domain of Avena sativa. The resulting chimera combines the activities of these two unrelated proteins to enable controlled formation of a functional protein via upregulation of intein splicing with blue light in bacterial and human cells. PMID:26940144

  2. Protein accumulation and rumen stability of wheat γ-gliadin fusion proteins in tobacco and alfalfa.

    PubMed

    Sun, Xiaodong; Chi-Ham, Cecilia L; Cohen-Davidyan, Tamar; DeBen, Christopher; Getachow, Girma; DePeters, Edward; Putnam, Daniel; Bennett, Alan

    2015-09-01

    The nutritional value of various crops can be improved by engineering plants to produce high levels of proteins. For example, because methionine deficiency limits the protein quality of Medicago Sativa (alfalfa) forage, producing alfalfa plants that accumulate high levels of a methionine-rich protein could increase the nutritional value of that crop. We used three strategies in designing methionine-rich recombinant proteins that could accumulate to high levels in plants and thereby serve as candidates for improving the protein quality of alfalfa forage. In tobacco, two fusion proteins, γ-gliadin-δ-zein and γ-δ-zein, as well as δ-zein co-expressed with β-zein, all formed protein bodies. However, the γ-gliadin-δ-zein fusion protein accumulated to the highest level, representing up to 1.5% of total soluble protein (TSP) in one transformant. In alfalfa, γ-gliadin-δ-zein accumulated to 0.2% of TSP, and in an in vitro rumen digestion assay, γ-gliadin-δ-zein was more resistant to microbial degradation than Rubisco. Additionally, although it did not form protein bodies, a γ-gliadin-GFP fusion protein accumulated to much higher levels, 7% of TSP, than a recombinant protein comprised of an ER localization signal fused to GFP in tobacco. Based on our results, we conclude that γ-gliadin-δ-zein is a potential candidate protein to use for enhancing methionine levels in plants and for improving rumen stability of forage protein. γ-gliadin fusion proteins may provide a general platform for increasing the accumulation of recombinant proteins in transgenic plants. PMID:25659597

  3. The antifungal properties of a 2S albumin-homologous protein from passion fruit seeds involve plasma membrane permeabilization and ultrastructural alterations in yeast cells.

    PubMed

    Agizzio, Ana Paula; Da Cunha, Maura; Carvalho, André O; Oliveira, Marco Antônio; Ribeiro, Suzanna F F; Gomes, Valdirene M

    2006-10-01

    Different types of antimicrobial proteins were purified from plant seeds, including chitinases, β-1,3-glucanases, defensins, thionins, lipid transfer proteins and 2S albumins. It has become clear that these groups of proteins play an important role in the protection of plants from microbial infection. Recent results from our laboratory have shown that the defense-related proteins from passion fruit seeds, named Pf1 and Pf2 (which show sequence homology with 2S albumins), inhibit fungal growth and glucose-stimulated acidification of the medium by Saccharomyces cerevisiae cells. The aim of this study was to determine whether 2S albumins from passion fruit seeds induce plasma membrane permeabilization and cause morphological alterations in yeast cells. Initially, we used an assay based on the uptake of SYTOX Green, an organic compound that fluoresces upon interaction with nucleic acids and penetrates cells with compromised plasma membranes, to investigate membrane permeabilization in S. cerevisiae cells. When viewed with a confocal laser microscope, S. cervisiae cells showed strong SYTOX Green fluorescence in the cytosol, especially in the nuclei. 2S albumins also inhibited glucose-stimulated acidification of the medium by S. cerevisiae cells, which indicates a probable impairment of fungal metabolism. The microscopical analysis of the yeast cells treated with 2S albumins demonstrated several morphological alterations in cell shape, cell surface, cell wall and bud formation, as well as in the organization of intracellular organelles. PMID:25193649

  4. Spectroscopic investigations of the interactions of tramadol hydrochloride and 5-azacytidine drugs with human serum albumin and human hemoglobin proteins.

    PubMed

    Tunç, Sibel; Cetinkaya, Ahmet; Duman, Osman

    2013-03-01

    The interactions of tramadol hydrochloride (THC) and 5-azacytidine (AZA) drugs with human serum albumin (HSA) and human hemoglobin (HMG) proteins were investigated by fluorescence, UV absorption and circular dichroism (CD) spectroscopy at pH 7.4 and different temperatures. The UV absorption spectra and the fluorescence quenching of HSA and HMG proteins indicated the formation of HSA-THC and HMG-THC complexes via static quenching mechanism. AZA did not interact with HSA and HMG proteins. It was found that the formation of HMG-THC complex was stronger than that of HSA-THC complex. The stability of HSA-THC and HMG-THC complexes decreased with increasing temperature. The number of binding site was found as one for HSA-THC and HMG-THC systems. Negative enthalpy change (ΔH) and Gibbs free energy change (ΔG) and positive entropy change (ΔS) values were obtained for these systems. The binding of THC-HSA and HMG proteins was spontaneous and exothermic. In addition, electrostatic interactions between protein and drug molecules played an important role in the binding processes. The results of CD analysis revealed that the addition of THC led to a significant conformational change in the secondary structure of HSA protein, on the contrary to HMG protein. PMID:23428887

  5. Trimeric transmembrane domain interactions in paramyxovirus fusion proteins: roles in protein folding, stability, and function.

    PubMed

    Smith, Everett Clinton; Smith, Stacy E; Carter, James R; Webb, Stacy R; Gibson, Kathleen M; Hellman, Lance M; Fried, Michael G; Dutch, Rebecca Ellis

    2013-12-13

    Paramyxovirus fusion (F) proteins promote membrane fusion between the viral envelope and host cell membranes, a critical early step in viral infection. Although mutational analyses have indicated that transmembrane (TM) domain residues can affect folding or function of viral fusion proteins, direct analysis of TM-TM interactions has proved challenging. To directly assess TM interactions, the oligomeric state of purified chimeric proteins containing the Staphylococcal nuclease (SN) protein linked to the TM segments from three paramyxovirus F proteins was analyzed by sedimentation equilibrium analysis in detergent and buffer conditions that allowed density matching. A monomer-trimer equilibrium best fit was found for all three SN-TM constructs tested, and similar fits were obtained with peptides corresponding to just the TM region of two different paramyxovirus F proteins. These findings demonstrate for the first time that class I viral fusion protein TM domains can self-associate as trimeric complexes in the absence of the rest of the protein. Glycine residues have been implicated in TM helix interactions, so the effect of mutations at Hendra F Gly-508 was assessed in the context of the whole F protein. Mutations G508I or G508L resulted in decreased cell surface expression of the fusogenic form, consistent with decreased stability of the prefusion form of the protein. Sedimentation equilibrium analysis of TM domains containing these mutations gave higher relative association constants, suggesting altered TM-TM interactions. Overall, these results suggest that trimeric TM interactions are important driving forces for protein folding, stability and membrane fusion promotion. PMID:24178297

  6. Denaturation of human serum albumin under the action of cetyltrimethylammonium bromide according to fluorescence polarization data of protein

    NASA Astrophysics Data System (ADS)

    Vlasova, I. M.; Zhuravleva, V. V.; Saletskii, A. M.

    2012-03-01

    Denaturation of human serum albumin (HSA) under the action of cationic detergent cetyltrimethylammonium bromide (CTAB) is studied at different pH values by estimating the rotational diffusion of protein via fluorescence polarization. The degree of polarization of HSA tryptophan fluorescence, the rotational relaxation time, the rotational diffusion coefficient and the effective Einstein radius of the HSA molecules in solutions with different CTAB concentrations at different pH values are determined. The obtained rotational diffusion parameters of the HSA molecules show that under the action of CTAB, HSA denaturation has a one-stage character and proceeds more intensely and effectively at pH values higher than the p I value of protein (4.7).

  7. Conformational changes of bovine plasma albumin prior to the salting-out of protein in concentrated salt solution.

    PubMed

    Sogami, M; Inouye, H; Nagaoka, S; Era, S

    1982-09-01

    By working at very low protein concentration (ca. 0.003%), it is possible to measure tryptophyl fluorescence intensity at 350 nm (F350) of bovine plasma albumin (BPA) as a function of pH under precipitating conditions (acidic concentrated salt solutions). Under such conditions, distinct changes in F350 were seen before the starting of precipitation of BPA and no further changes in F350 over the precipitating pH range. Comparison of pH-profiles monitored by F350 with those by solubility in the presence of various salts at various concentrations indicated that the change of solubility is observed after definite changes in conformation of the protein. PMID:7129758

  8. Decoding signals for membrane protein assembly using alkaline phosphatase fusions.

    PubMed Central

    McGovern, K; Ehrmann, M; Beckwith, J

    1991-01-01

    We have used genetic methods to investigate the role of the different domains of a bacterial cytoplasmic membrane protein, MalF, in determining its topology. This was done by analyzing the effects of MalF topology of deleting various domains of the protein using MalF-alkaline phosphatase fusion proteins. Our results show that the cytoplasmic domains of the protein are the pre-eminent topogenic signals. These domains contain information that determines their cytoplasmic location and, thus, the orientation of the membrane spanning segments surrounding them. Periplasmic domains do not appear to have equivalent information specifying their location and membrane spanning segments do not contain information defining their orientation in the membrane. The strength of cytoplasmic domains as topogenic signals varies, correlated with the density of positively charged amino acids within them. Images PMID:1915262

  9. Canine distemper virus envelope protein interactions modulated by hydrophobic residues in the fusion protein globular head.

    PubMed

    Avila, Mislay; Khosravi, Mojtaba; Alves, Lisa; Ader-Ebert, Nadine; Bringolf, Fanny; Zurbriggen, Andreas; Plemper, Richard K; Plattet, Philippe

    2015-01-15

    Membrane fusion for morbillivirus cell entry relies on critical interactions between the viral fusion (F) and attachment (H) envelope glycoproteins. Through extensive mutagenesis of an F cavity recently proposed to contribute to F's interaction with the H protein, we identified two neighboring hydrophobic residues responsible for severe F-to-H binding and fusion-triggering deficiencies when they were mutated in combination. Since both residues reside on one side of the F cavity, the data suggest that H binds the F globular head domain sideways. PMID:25355896

  10. Protein function prediction based on data fusion and functional interrelationship.

    PubMed

    Meng, Jun; Wekesa, Jael-Sanyanda; Shi, Guan-Li; Luan, Yu-Shi

    2016-04-01

    One of the challenging tasks of bioinformatics is to predict more accurate and confident protein functions from genomics and proteomics datasets. Computational approaches use a variety of high throughput experimental data, such as protein-protein interaction (PPI), protein sequences and phylogenetic profiles, to predict protein functions. This paper presents a method that uses transductive multi-label learning algorithm by integrating multiple data sources for classification. Multiple proteomics datasets are integrated to make inferences about functions of unknown proteins and use a directed bi-relational graph to assign labels to unannotated proteins. Our method, bi-relational graph based transductive multi-label function annotation (Bi-TMF) uses functional correlation and topological PPI network properties on both the training and testing datasets to predict protein functions through data fusion of the individual kernel result. The main purpose of our proposed method is to enhance the performance of classifier integration for protein function prediction algorithms. Experimental results demonstrate the effectiveness and efficiency of Bi-TMF on multi-sources datasets in yeast, human and mouse benchmarks. Bi-TMF outperforms other recently proposed methods. PMID:26869536

  11. Moringa oleifera aqueous leaf extract inhibits reducing monosaccharide-induced protein glycation and oxidation of bovine serum albumin.

    PubMed

    Nunthanawanich, Pornpimon; Sompong, Weerachat; Sirikwanpong, Sukrit; Mäkynen, Kittana; Adisakwattana, Sirichai; Dahlan, Winai; Ngamukote, Sathaporn

    2016-01-01

    Advanced glycation end products (AGEs) play an important factor for pathophysiology of diabetes and its complications. Moringa oleifera is one of the medicinal plants that have anti-hyperglycemic activity. However, anti-glycation property of Moringa oleifera leaf extract on the different types of reducing monosaccharides-induced protein glycation has not been investigated. Therefore, the aim of this study was to examine the protective effect of Moringa oleifera aqueous leaf extract (MOE) on reducing sugars-induced protein glycation and protein oxidation. Total phenolic content of MOE was measured using the Folin-Ciocalteu method. Bovine serum albumin was incubated with 0.5 M of reducing sugars (glucose or fructose) with or without MOE (0.5-2.0 mg/mL) for 1, 2, 3 and 4 weeks. The results found that total phenolic content was 38.56 ± 1.50 mg gallic acid equivalents/g dry extract. The formation of fluorescent and non-fluorescent AGEs [N (ε)-(carboxymethyl) lysine (CML)] and the level of fructosamine were determined to indicate protein glycation, whereas the level of protein carbonyl content and thiol group were examined for protein oxidation. MOE (0.5-2.0 mg/mL) significantly inhibited the formation of fluorescent, N (ε)-CML and markedly decreased fructosamine level (P < 0.05). Moreover, MOE significantly prevented protein oxidation manifested by reducing protein carbonyl and the depletion of protein thiol in a dose-dependent manner (P < 0.05). Thus, the findings indicated that polyphenols containing in MOE have high potential for decreasing protein glycation and protein oxidation that may delay or prevent AGE-related diabetic complications. PMID:27468399

  12. Fibroblast adhesion to recombinant tropoelastin expressed as a protein A-fusion protein.

    PubMed Central

    Grosso, L E; Parks, W C; Wu, L J; Mecham, R P

    1991-01-01

    A bovine tropoelastin cDNA encoding exons 15-36 that includes the elastin-receptor binding site was expressed in Escherichia coli as a fusion protein with Protein A from Staphylococcus aureus. After isolation of the fusion protein by affinity chromatography on Ig-Sepharose, the tropoelastin domain was separated from plasmid-pR1T2T-encoded Protein A (Protein A') by CNBr cleavage. Cell-adhesion assays demonstrated specific adhesion to the recombinant tropoelastin. Furthermore, the data indicate that interactions involving the bovine elastin receptor mediate nuchalligament fibroblast adhesion to the recombinant protein. In agreement with earlier studies of fibroblast chemotaxis to bovine tropoelastin, nuchal-ligament fibroblast adhesion demonstrated developmental regulation of the elastin receptor. Images Fig. 2. Fig. 3. PMID:1996952

  13. Interaction of singlet oxygen with bovine serum albumin and the role of the protein nano-compartmentalization.

    PubMed

    Giménez, Rodrigo E; Vargová, Veronika; Rey, Valentina; Turbay, M Beatriz Espeche; Abatedaga, Inés; Morán Vieyra, Faustino E; Paz Zanini, Verónica I; Mecchia Ortiz, Juan H; Katz, Néstor E; Ostatná, Veronika; Borsarelli, Claudio D

    2016-05-01

    Singlet molecular oxygen ((1)O2) contributes to protein damage triggering biophysical and biochemical changes that can be related with aging and oxidative stress. Serum albumins, such as bovine serum albumin (BSA), are abundant proteins in blood plasma with different biological functions. This paper presents a kinetic and spectroscopic study of the (1)O2-mediated oxidation of BSA using the tris(2,2'-bipyridine)ruthenium(II) cation [Ru(bpy)3](2+) as sensitizer. BSA quenches efficiently (1)O2 with a total (chemical+physical interaction) rate constant kt(BSA)=7.3(±0.4)×10(8)M(-1)s(-1), where the chemical pathway represented 37% of the interaction. This efficient quenching by BSA indicates the participation of several reactive residues. MALDI-TOF MS analysis of intact BSA confirmed that after oxidation by (1)O2, the mass protein increased the equivalent of 13 oxygen atoms. Time-resolved emission spectra analysis of BSA established that Trp residues were oxidized to N'-formylkynurenine, being the solvent-accessible W134 preferentially oxidized by (1)O2 as compared with the buried W213. MS confirmed oxidation of at least two Tyr residues to form dihydroxyphenylalanine, with a global reactivity towards (1)O2 six-times lower than for Trp residues. Despite the lack of MS evidences, kinetic and chemical analysis also suggested that residues other than Trp and Tyr, e.g. Met, must react with (1)O2. Modeling of the 3D-structure of BSA indicated that the oxidation pattern involves a random distribution of (1)O2 into BSA; allowing also the interaction of (1)O2 with buried residues by its diffusion from the bulk solvent through interconnected internal hydrophilic and hydrophobic grooves. PMID:26898504

  14. Identification of a plastid protein involved in vesicle fusion and/or membrane protein translocation.

    PubMed Central

    Hugueney, P; Bouvier, F; Badillo, A; d'Harlingue, A; Kuntz, M; Camara, B

    1995-01-01

    Structural evidence has accumulated suggesting that fusion and/or translocation factors are involved in plastid membrane biogenesis. To test this hypothesis, we have developed an in vitro system in which the extent of fusion and/or translocation is monitored by the conversion of the xanthophyll epoxide (antheraxanthin) into the red ketocarotenoid (capsanthin). Only chromoplast membrane vesicles from red pepper fruits (Capsicum annuum) contain the required enzyme. Vesicles prepared from the mutant yellow cultivar are devoid of this enzyme and accumulate antheraxanthin. The fusion and/or translocation activity is characterized by complementation due to the synthesis of capsanthin and the parallel decrease of antheraxanthin when the two types of vesicles are incubated together in the presence of plastid stroma. We show that the extent of conversion is dependent upon an ATP-requiring protein that is sensitive to N-ethylmaleimide. Further purification and immunological analysis have revealed that the active factor, designated plastid fusion and/or translocation factor (Pftf), resides in a protein of 72 kDa. cDNA cloning revealed that mature Pftf has significant homology to yeast and animal (NSF) or bacterial (Ftsh) proteins involved in vesicle fusion or membrane protein translocation. Images Fig. 1 Fig. 3 Fig. 4 PMID:7777561

  15. Electrolyte effect on the phase behavior of silica nanoparticles with lysozyme and bovine-serum-albumin proteins

    NASA Astrophysics Data System (ADS)

    Yadav, Indresh; Aswal, V. K.; Kohlbrecher, J.

    2015-05-01

    Small-angle neutron scattering (SANS) and dynamic light scattering (DLS) studies have been carried out to investigate the effect of an electrolyte on the phase behavior of anionic silica nanoparticles with two globular proteins—cationic lysozyme [molecular weight (MW) 14.7 kDa] and anionic bovine serum albumin (MW 66.4 kDa). The results are compared with our earlier published work on similar systems without any electrolyte [I. Yadav, S. Kumar, V. K. Aswal, and J. Kohlbrecher, Phys. Rev. E 89, 032304 (2014), 10.1103/PhysRevE.89.032304]. Both the nanoparticle-protein systems transform to two phase at lower concentration of protein in the presence of an electrolyte. The autocorrelation function in DLS suggests that the diffusion coefficient (D) of a nanoparticle-protein system decreases in approaching two phase with the increase in protein concentration. This variation in D can be attributed to increase in attractive interaction and/or overall increase in the size. Further, these two contributions (interaction and structure) are determined from the SANS data. The changes in the phase behavior of nanoparticle-protein systems in the presence of an electrolyte are explained in terms of modifications in both the repulsive and attractive components of interaction between nanoparticles. In a two-phase system individual silica nanoparticles coexist along with their fractal aggregates.

  16. A fluorescence-based high throughput assay for the determination of small molecule–human serum albumin protein binding

    PubMed Central

    McCallum, Megan M.; Pawlak, Alan J.; Shadrick, William R.; Simeonov, Anton; Jadhav, Ajit; Yasgar, Adam; Maloney, David J.; Arnold, Leggy A.

    2014-01-01

    Herein, we describe the development of a fluorescence-based high throughput assay to determine the small molecule binding towards human serum albumin (HSA). This innovative competition assay is based on the use of a novel fluorescent small molecule Red Mega 500 with unique spectroscopic and binding properties. The commercially available probe displays a large fluorescence intensity difference between the protein-bound and protein-unbound state. The competition of small molecules for HSA binding in the presence of probe resulted in low fluorescence intensities. The assay was evaluated with the LOPAC small molecule library of 1280 compounds identifying known high protein binders. The small molecule competition of HSA–Red Mega 500 binding was saturable at higher compound concentrations and exhibited IC50 values between 3–24 μM. The compound affinity towards HSA was confirmed by isothermal titration calorimetry indicating that the new protein binding assay is a valid high throughput assay to determine plasma protein binding. PMID:24390461

  17. Affinity of rosmarinic acid to human serum albumin and its effect on protein conformation stability.

    PubMed

    Peng, Xin; Wang, Xiangchao; Qi, Wei; Su, Rongxin; He, Zhimin

    2016-02-01

    Rosmarinic acid (RA) is a natural polyphenol contained in many aromatic plants with promising biological activities. The interaction between RA and human serum albumin (HSA) was investigated by multi-spectroscopic, electrochemistry, molecular docking and molecular dynamics simulation methods. The fluorescence emission of HSA was quenched by RA through a combined static and dynamic quenching mechanism, but the static quenching was the major constituent. Fluorescence experiments suggested that RA was bound to HSA with moderately strong binding affinity through hydrophobic interaction. The probable binding location of RA was located near site I of HSA. Additionally, as shown by the Fourier transform infrared (FT-IR) and circular dichroism (CD) spectra, RA can result in conformational and structural alterations of HSA. Furthermore, the molecular dynamics studies were used to investigate the stability of the HSA and HSA-RA system. Altogether, the results can provide an important insight for the applications of RA in the food industry. PMID:26304336

  18. Pre-association of polynuclear platinum anticancer agents on a protein, human serum albumin. Implications for drug design†

    PubMed Central

    Montero, Eva I.; Benedetti, Brad T.; Mangrum, John B.; Oehlsen, Michael J.; Qu, Yun; Farrell, Nicholas P.

    2009-01-01

    The interactions of polynuclear platinum complexes with human serum albumin were studied. The compounds examined were the “non-covalent” analogs of the trinuclear BBR3464 as well as the dinuclear spermidine-bridged compounds differing in only the presence or absence of a central -NH2-+ (BBR3571 and analogs). Thus, closely-related compounds could be compared. Evidence for pre-association, presumably through electrostatic and hydrogen-bonding, was obtained from fluorescence and circular dichroism spectroscopy and Electrospray Ionization Mass Spectrometry (ESI-MS). In the case of those compounds containing Pt-Cl bonds, further reaction took place presumably through displacement by sulfur nucleophiles. The implications for protein pre-association and plasma stability of polynuclear platinum compounds are discussed. PMID:17992278

  19. Novel channel enzyme fusion proteins confer arsenate resistance.

    PubMed

    Wu, Binghua; Song, Jie; Beitz, Eric

    2010-12-17

    Steady exposure to environmental arsenic has led to the evolution of vital cellular detoxification mechanisms. Under aerobic conditions, a two-step process appears most common among microorganisms involving reduction of predominant, oxidized arsenate (H(2)As(V)O(4)(-)/HAs(V)O(4)(2-)) to arsenite (As(III)(OH)(3)) by a cytosolic enzyme (ArsC; Escherichia coli type arsenate reductase) and subsequent extrusion via ArsB (E. coli type arsenite transporter)/ACR3 (yeast type arsenite transporter). Here, we describe novel fusion proteins consisting of an aquaglyceroporin-derived arsenite channel with a C-terminal arsenate reductase domain of phosphotyrosine-phosphatase origin, providing transposable, single gene-encoded arsenate resistance. The fusion occurred in actinobacteria from soil, Frankia alni, and marine environments, Salinispora tropica; Mycobacterium tuberculosis encodes an analogous ACR3-ArsC fusion. Mutations rendered the aquaglyceroporin channel more polar resulting in lower glycerol permeability and enhanced arsenite selectivity. The arsenate reductase domain couples to thioredoxin and can complement arsenate-sensitive yeast strains. A second isoform with a nonfunctional channel may use the mycothiol/mycoredoxin cofactor pool. These channel enzymes constitute prototypes of a novel concept in metabolism in which a substrate is generated and compartmentalized by the same molecule. Immediate diffusion maintains the dynamic equilibrium and prevents toxic accumulation of metabolites in an energy-saving fashion. PMID:20947511

  20. Novel Channel Enzyme Fusion Proteins Confer Arsenate Resistance*

    PubMed Central

    Wu, Binghua; Song, Jie; Beitz, Eric

    2010-01-01

    Steady exposure to environmental arsenic has led to the evolution of vital cellular detoxification mechanisms. Under aerobic conditions, a two-step process appears most common among microorganisms involving reduction of predominant, oxidized arsenate (H2AsVO4−/HAsVO42−) to arsenite (AsIII(OH)3) by a cytosolic enzyme (ArsC; Escherichia coli type arsenate reductase) and subsequent extrusion via ArsB (E. coli type arsenite transporter)/ACR3 (yeast type arsenite transporter). Here, we describe novel fusion proteins consisting of an aquaglyceroporin-derived arsenite channel with a C-terminal arsenate reductase domain of phosphotyrosine-phosphatase origin, providing transposable, single gene-encoded arsenate resistance. The fusion occurred in actinobacteria from soil, Frankia alni, and marine environments, Salinispora tropica; Mycobacterium tuberculosis encodes an analogous ACR3-ArsC fusion. Mutations rendered the aquaglyceroporin channel more polar resulting in lower glycerol permeability and enhanced arsenite selectivity. The arsenate reductase domain couples to thioredoxin and can complement arsenate-sensitive yeast strains. A second isoform with a nonfunctional channel may use the mycothiol/mycoredoxin cofactor pool. These channel enzymes constitute prototypes of a novel concept in metabolism in which a substrate is generated and compartmentalized by the same molecule. Immediate diffusion maintains the dynamic equilibrium and prevents toxic accumulation of metabolites in an energy-saving fashion. PMID:20947511

  1. Localization of a Region in the Fusion Protein of Avian Metapneumovirus That Modulates Cell-Cell Fusion

    PubMed Central

    Wei, Yongwei; Feng, Kurtis; Yao, Xiangjie; Cai, Hui; Li, Junan; Mirza, Anne M.; Iorio, Ronald M.

    2012-01-01

    The genus Metapneumovirus within the subfamily Pneumovirinae of the family Paramyxoviridae includes two members, human metapneumovirus (hMPV) and avian metapneumovirus (aMPV), causing respiratory tract infections in humans and birds, respectively. Paramyxoviruses enter host cells by fusing the viral envelope with a host cell membrane. Membrane fusion of hMPV appears to be unique, in that fusion of some hMPV strains requires low pH. Here, we show that the fusion (F) proteins of aMPV promote fusion in the absence of the attachment protein and low pH is not required. Furthermore, there are notable differences in cell-cell fusion among aMPV subtypes. Trypsin was required for cell-cell fusion induced by subtype B but not subtypes A and C. The F protein of aMPV subtype A was highly fusogenic, whereas those from subtypes B and C were not. By construction and evaluation of chimeric F proteins composed of domains from the F proteins of subtypes A and B, we localized a region composed of amino acid residues 170 to 338 in the F protein that is responsible for the hyperfusogenic phenotype of the F from subtype A. Further mutagenesis analysis revealed that residues R295, G297, and K323 in this region collectively contributed to the hyperfusogenicity. Taken together, we have identified a region in the aMPV F protein that modulates the extent of membrane fusion. A model for fusion consistent with these data is presented. PMID:22915815

  2. Morbillivirus and henipavirus attachment protein cytoplasmic domains differently affect protein expression, fusion support and particle assembly.

    PubMed

    Sawatsky, Bevan; Bente, Dennis A; Czub, Markus; von Messling, Veronika

    2016-05-01

    The amino-terminal cytoplasmic domains of paramyxovirus attachment glycoproteins include trafficking signals that influence protein processing and cell surface expression. To characterize the role of the cytoplasmic domain in protein expression, fusion support and particle assembly in more detail, we constructed chimeric Nipah virus (NiV) glycoprotein (G) and canine distemper virus (CDV) haemagglutinin (H) proteins carrying the respective heterologous cytoplasmic domain, as well as a series of mutants with progressive deletions in this domain. CDV H retained fusion function and was normally expressed on the cell surface with a heterologous cytoplasmic domain, while the expression and fusion support of NiV G was dramatically decreased when its cytoplasmic domain was replaced with that of CDV H. The cell surface expression and fusion support functions of CDV H were relatively insensitive to cytoplasmic domain deletions, while short deletions in the corresponding region of NiV G dramatically decreased both. In addition, the first 10 residues of the CDV H cytoplasmic domain strongly influence its incorporation into virus-like particles formed by the CDV matrix (M) protein, while the co-expression of NiV M with NiV G had no significant effect on incorporation of G into particles. The cytoplasmic domains of both the CDV H and NiV G proteins thus contribute differently to the virus life cycle. PMID:26813519

  3. Blot overlays with 32P-labeled fusion proteins.

    PubMed

    Zhao, Z; Lim, L; Manser, E

    2001-07-01

    Proteins labeled with 32P can be used as sensitive "prime" in blot overlays to detect binding proteins or domains. Small G-protein Ras can bind GTP with extremely high affinity (Kd approximately 10(-11)-10(-12) M) in the presence of Mg2+. We have taken advantage of this property of Ras to develop a vector that expresses proteins of interest such as glutathione S-transferase (GST)/Ras fusion proteins for noncovalent labeling with [gamma-32P]GTP. The labeling efficiency of this method is >60% and involves a single short incubation step. We have previously identified several binding proteins for the second SH3 domain of the adaptor Nck using this method. Here we illustrate the overlay method using the GST/Ras system and compare results with the SH3 domain labeled by phosphorylation with [gamma-32P]ATP. Both methods are similarly specific and sensitive; however, we show that signals are dependent primarily on GST-mediated probe dimerization. These dimeric probes allow a more stable probe-target complex similar to immunoglobulin interactions, thus significantly improving the sensitivity of the technique. PMID:11403569

  4. Covalent attachment of metal chelates to proteins:the stability in vivo and in vitro of the conjugate of albumin with a chelate of 111indium.

    PubMed Central

    Meares, C F; Goodwin, D A; Leung, C S; Girgis, A Y; Silvester, D J; Nunn, A D; Lavender, P J

    1976-01-01

    Human serum albumin has been conjugated to 1-(p-bnezenediazonium)-(ethylenedinitrilo)tetraacetic acid, a powerful chelating agent, and radioactive 111indium ions have been added specifically to the chelating groups. The product, with a specific radioactivity of about 1 mCi/mg of protein, was employed as a radiotracer in scintillation scanning studies with human volunteers. Results show that 48 hr after injection, practically all of the label remains attached to albumin. This is confirmed by electrophoresis of serum proteins; 7 days after injection, 85% of the radioactivity in the serum is still in the albumin fraction. These observations agree with in vitro studies of the labeled albumin in human serum, where loss of the metal ion from the chelating group to the protein transferrin amounts to less than 3% after 1 week and less than 5% after 2 weeks. Measurements of the distribution of label in mice up to 23 days after injection suggest that metabolism of the labeled protein does not lead to binding of indium ions by transferrin. The binding of indium and other metal ions by transferrin has previously posed a major impediment to the use of metal chelates for in vivo diagnostic procedures. Demonstration of the kinetic inertness of the chelate in these experiments suggests the use of related chelates as physical probes of biological systems. Images PMID:825856

  5. Albumin Test

    MedlinePlus

    ... to a variety of conditions in addition to malnutrition , a decrease in albumin needs to be evaluated ... can also be seen in inflammation , shock, and malnutrition . They may be seen with conditions in which ...

  6. Pooled-matrix protein interaction screens using Barcode Fusion Genetics.

    PubMed

    Yachie, Nozomu; Petsalaki, Evangelia; Mellor, Joseph C; Weile, Jochen; Jacob, Yves; Verby, Marta; Ozturk, Sedide B; Li, Siyang; Cote, Atina G; Mosca, Roberto; Knapp, Jennifer J; Ko, Minjeong; Yu, Analyn; Gebbia, Marinella; Sahni, Nidhi; Yi, Song; Tyagi, Tanya; Sheykhkarimli, Dayag; Roth, Jonathan F; Wong, Cassandra; Musa, Louai; Snider, Jamie; Liu, Yi-Chun; Yu, Haiyuan; Braun, Pascal; Stagljar, Igor; Hao, Tong; Calderwood, Michael A; Pelletier, Laurence; Aloy, Patrick; Hill, David E; Vidal, Marc; Roth, Frederick P

    2016-01-01

    High-throughput binary protein interaction mapping is continuing to extend our understanding of cellular function and disease mechanisms. However, we remain one or two orders of magnitude away from a complete interaction map for humans and other major model organisms. Completion will require screening at substantially larger scales with many complementary assays, requiring further efficiency gains in proteome-scale interaction mapping. Here, we report Barcode Fusion Genetics-Yeast Two-Hybrid (BFG-Y2H), by which a full matrix of protein pairs can be screened in a single multiplexed strain pool. BFG-Y2H uses Cre recombination to fuse DNA barcodes from distinct plasmids, generating chimeric protein-pair barcodes that can be quantified via next-generation sequencing. We applied BFG-Y2H to four different matrices ranging in scale from ~25 K to 2.5 M protein pairs. The results show that BFG-Y2H increases the efficiency of protein matrix screening, with quality that is on par with state-of-the-art Y2H methods. PMID:27107012

  7. Albuminated Glycoenzymes: Enzyme Stabilization through Orthogonal Attachment of a Single-Layered Protein Shell around a Central Glycoenzyme Core.

    PubMed

    Ritter, Dustin W; Newton, Jared M; Roberts, Jason R; McShane, Michael J

    2016-05-18

    Here we demonstrate an approach to stabilize enzymes through the orthogonal covalent attachment of albumin on the single-enzyme level. Albuminated glycoenzymes (AGs) based upon glucose oxidase and catalase from Aspergillus niger were prepared in this manner. Gel filtration chromatography and dynamic light scattering support modification, with an increase in hydrodynamic radius of ca. 60% upon albumination. Both AGs demonstrate a marked resistance to aggregation during heating to 90 °C, but this effect is more profound in albuminated catalase. The functional characteristics of albuminated glucose oxidase vary considerably with exposure type. The AG's thermal inactivation is reduced more than 25 times compared to native glucose oxidase, and moderate stabilization is observed with one month storage at 37 °C. However, albumination has no effect on operational stability of glucose oxidase. PMID:27111632

  8. Identification of Domains on the Fusion (F) Protein Trimer That Influence the Hemagglutinin-Neuraminidase Specificity of the F Protein in Mediating Cell-Cell Fusion

    PubMed Central

    Tsurudome, Masato; Ito, Morihiro; Nishio, Machiko; Nakahashi, Mito; Kawano, Mitsuo; Komada, Hiroshi; Nosaka, Tetsuya; Ito, Yasuhiko

    2011-01-01

    For most paramyxoviruses, virus type-specific interaction between fusion (F) protein and attachment protein (hemagglutinin-neuraminidase [HN], hemagglutinin [H], or glycoprotein [G]) is a prerequisite for mediating virus-cell fusion and cell-cell fusion. Our previous cell-cell fusion assay using the chimeric F proteins of human parainfluenza virus 2 (HPIV2) and simian virus 41 (SV41) suggested that the middle region of the HPIV2 F protein contains the site(s) that determines its specificity for the HPIV2 HN protein. In the present study, we further investigated the sites of the F protein that could be critical for determining the HN protein specificity. By analyzing the reported structure of the F protein of parainfluenza virus 5 (PIV5), we found that four major domains (M1, M2, M3, and M4) and five minor domains (A to E) in the middle region of the PIV5 F protein were exposed on the trimer surface. We then replaced these domains with the SV41 F counterparts individually or in combination and examined whether the resulting chimeras could mediate cell-cell fusion when coexpressed with the SV41 HN protein. The results showed that a chimera designated M(1+2), which harbored SV41 F-derived domains M1 and M2, mediated cell-cell fusion with the coexpressed SV41 HN protein, suggesting that these domains are involved in determining the HN protein specificity. Intriguingly, another chimera which harbored the SV41 F-derived domain B in addition to domains M1 and M2 showed increased specificity for the SV41 HN protein compared to that of M(1+2), although it was capable of mediating cell-cell fusion by itself. PMID:21270148

  9. Protein adsorption and cell adhesion on nanoscale bioactive coatings formed from poly(ethylene glycol) and albumin microgels

    PubMed Central

    Scott, Evan A.; Nichols, Michael D.; Cordova, Lee H.; George, Brandon J.; Jun, Young-Shin; Elbert, Donald L.

    2008-01-01

    Late-term thrombosis on drug-eluting stents is an emerging problem that might be addressed using extremely thin, biologically-active hydrogel coatings. We report a dip-coating strategy to covalently link poly(ethylene glycol) (PEG) to substrates, producing coatings with <≈100 nm thickness. Gelation of PEG-octavinylsulfone with amines in either bovine serum albumin (BSA) or PEG-octaamine was monitored by dynamic light scattering (DLS), revealing the presence of microgels before macrogelation. NMR also revealed extremely high end group conversions prior to macrogelation, consistent with the formation of highly crosslinked microgels and deviation from Flory-Stockmayer theory. Before macrogelation, the reacting solutions were diluted and incubated with nucleophile-functionalized surfaces. Using optical waveguide lightmode spectroscopy (OWLS) and quartz crystal microbalance with dissipation (QCM-D), we identified a highly hydrated, protein-resistant layer with a thickness of approximately 75 nm. Atomic force microscopy in buffered water revealed the presence of coalesced spheres of various sizes but with diameters less than about 100 nm. Microgel-coated glass or poly(ethylene terephthalate) exhibited reduced protein adsorption and cell adhesion. Cellular interactions with the surface could be controlled by using different proteins to cap unreacted vinylsulfone groups within the coating. PMID:18771802

  10. Rapid Screening of Drug-Protein Binding Using High-Performance Affinity Chromatography with Columns Containing Immobilized Human Serum Albumin

    PubMed Central

    Li, Ying-Fei; Zhang, Xiao-Qiong; Hu, Wei-Yu; Li, Zheng; Liu, Ping-Xia; Zhang, Zhen-Qing

    2013-01-01

    For drug candidates, a plasma protein binding (PPB) more than 90% is more meaningful and deserves further investigation in development. In the study, a high-performance liquid chromatography method employing column containing immobilized human serum albumin (HSA) to screen in vitro PPB of leading compounds was established and successfully applied to tested compounds. Good correlation (a coefficient correlation of 0.96) was attained between the reciprocal values (X) of experimentally obtained retention time of reference compounds eluted through HSA column and the reported PPB values (Y) with a correlation equation of Y = 92.03 − 97.01X. The method was successfully applied to six test compounds, and the result was confirmed by the conventional ultrafiltration technique, and both yielded equal results. However, due to the particular protein immobilized to column, the method cannot be applied for all compounds and should be exploited judiciously based on the value of the logarithmic measure of the acid dissociation constant (pKa) as per the requirement. If α1-acid glycoprotein and other plasma proteins could be immobilized like HSA with their actual ratio in plasma to column simultaneously, the result attained using immobilized column may be more accurate, and the method could be applied to more compounds without pKa limitation. PMID:23607050

  11. A promiscuous biotin ligase fusion protein identifies proximal and interacting proteins in mammalian cells.

    PubMed

    Roux, Kyle J; Kim, Dae In; Raida, Manfred; Burke, Brian

    2012-03-19

    We have developed a new technique for proximity-dependent labeling of proteins in eukaryotic cells. Named BioID for proximity-dependent biotin identification, this approach is based on fusion of a promiscuous Escherichia coli biotin protein ligase to a targeting protein. BioID features proximity-dependent biotinylation of proteins that are near-neighbors of the fusion protein. Biotinylated proteins may be isolated by affinity capture and identified by mass spectrometry. We apply BioID to lamin-A (LaA), a well-characterized intermediate filament protein that is a constituent of the nuclear lamina, an important structural element of the nuclear envelope (NE). We identify multiple proteins that associate with and/or are proximate to LaA in vivo. The most abundant of these include known interactors of LaA that are localized to the NE, as well as a new NE-associated protein named SLAP75. Our results suggest BioID is a useful and generally applicable method to screen for both interacting and neighboring proteins in their native cellular environment. PMID:22412018

  12. Significance of functional hepatic resection rate calculated using 3D CT/99mTc-galactosyl human serum albumin single-photon emission computed tomography fusion imaging

    PubMed Central

    Tsuruga, Yosuke; Kamiyama, Toshiya; Kamachi, Hirofumi; Shimada, Shingo; Wakayama, Kenji; Orimo, Tatsuya; Kakisaka, Tatsuhiko; Yokoo, Hideki; Taketomi, Akinobu

    2016-01-01

    AIM: To evaluate the usefulness of the functional hepatic resection rate (FHRR) calculated using 3D computed tomography (CT)/99mTc-galactosyl-human serum albumin (GSA) single-photon emission computed tomography (SPECT) fusion imaging for surgical decision making. METHODS: We enrolled 57 patients who underwent bi- or trisectionectomy at our institution between October 2013 and March 2015. Of these, 26 patients presented with hepatocellular carcinoma, 12 with hilar cholangiocarcinoma, six with intrahepatic cholangiocarcinoma, four with liver metastasis, and nine with other diseases. All patients preoperatively underwent three-phase dynamic multidetector CT and 99mTc-GSA scintigraphy. We compared the parenchymal hepatic resection rate (PHRR) with the FHRR, which was defined as the resection volume counts per total liver volume counts on 3D CT/99mTc-GSA SPECT fusion images. RESULTS: In total, 50 patients underwent bisectionectomy and seven underwent trisectionectomy. Biliary reconstruction was performed in 15 patients, including hepatopancreatoduodenectomy in two. FHRR and PHRR were 38.6 ± 19.9 and 44.5 ± 16.0, respectively; FHRR was strongly correlated with PHRR. The regression coefficient for FHRR on PHRR was 1.16 (P < 0.0001). The ratio of FHRR to PHRR for patients with preoperative therapies (transcatheter arterial chemoembolization, radiation, radiofrequency ablation, etc.), large tumors with a volume of > 1000 mL, and/or macroscopic vascular invasion was significantly smaller than that for patients without these factors (0.73 ± 0.19 vs 0.82 ± 0.18, P < 0.05). Postoperative hyperbilirubinemia was observed in six patients. Major morbidities (Clavien-Dindo grade ≥ 3) occurred in 17 patients (29.8%). There was no case of surgery-related death. CONCLUSION: Our results suggest that FHRR is an important deciding factor for major hepatectomy, because FHRR and PHRR may be discrepant owing to insufficient hepatic inflow and congestion in patients with preoperative

  13. Multifunctionality of Acidulated Serum Albumin on Inhibiting Zn²⁺-Mediated Amyloid β-Protein Fibrillogenesis and Cytotoxicity.

    PubMed

    Xie, Baolong; Dong, Xiaoyan; Wang, Yongjian; Sun, Yan

    2015-07-01

    Fibrillogenesis of amyloid β-proteins (Aβ) mediated by transition-metal ions such as Zn(2+) in neuronal cells plays a causative role in Alzheimer's disease. Hence, it is highly desired to design multifunctional agents capable of inhibiting Aβ aggregation and modulating metal-Aβ species. In this study, we fabricated acidulated human serum albumin (A-HSA) as a multifunctional agent for binding Zn(2+) and modulating Zn(2+)-mediated Aβ fibrillogenesis and cytotoxicity. On average, 19.5 diglycolic anhydrides were modified onto the surface of human serum albumin (HSA). It was confirmed that A-HSA kept the stability and biocompatibility of native HSA. Moreover, it could inhibit Aβ42 fibrillogenesis and change the pathway of Zn(2+)-mediated Aβ42 aggregation, as demonstrated by extensive biophysical assays. In addition, upon incubation with A-HSA, the cytotoxicity presented by Zn(2+)-Aβ42 aggregates was significantly mitigated in living cells. The results showed that A-HSA had much stronger inhibitory effect on Zn(2+)-mediated Aβ42 fibrillogenesis and cytotoxicity than equimolar HSA. Isothermal titration calorimetry and stopped-flow fluorescence measurements were then performed to investigate the working mechanism of A-HSA. The studies showed that the A-HSA surface, with more negative charges, not only had stronger affinity for Zn(2+) but also might decrease the binding affinity of Aβ42 for Zn(2+). Moreover, hydrophobic binding and electrostatic repulsion could work simultaneously on the bound Aβ42 on the A-HSA surface. As a result, Aβ42 conformations could be stretched, which avoided the formation of toxic Zn(2+)-Aβ42 aggregates. The research thus revealed that A-HSA is a multifunctional agent capable of altering the pathway of Zn(2+)-mediated Aβ42 aggregation and greatly mitigating the amyloid cytotoxicity. PMID:26070334

  14. Albumin stimulates p44/p42 extracellular-signal-regulated mitogen-activated protein kinase in opossum kidney proximal tubular cells.

    PubMed

    Dixon, R; Brunskill, N J

    2000-03-01

    The presence of protein in the urine of patients with renal disease is an adverse prognostic feature. It has therefore been suggested that proteinuria per se may be responsible for the development of renal tubulo-interstitial scarring and fibrosis, and disturbances in tubular cell growth and proliferation. We have used the opossum kidney proximal tubular cell line to investigate the effects of albumin on cell growth. The effect of albumin on cell proliferation was investigated by cell counting and measurement of [(3)H]thymidine incorporation. We studied the effect of recombinant human albumin on the activity of p44/p42 extracellular-signal-regulated mitogen-activated protein kinase (MAP kinase ) using an in vitro kinase assay, and immunoblotting with antibodies against active extracellular-signal-regulated kinase (ERK). The effects of the ERK inhibitor PD98059 were also examined. Recombinant human albumin was found to stimulate proliferation of opossum kidney cells in a dose-dependent manner, with maximal stimulation at a concentration of 1 mg/ml. In addition, recombinant human albumin activated ERK in a time-dependent (maximal after 5 min) and dose-dependent (maximal at 1 mg/ml) fashion. These effects on cell proliferation and ERK activity were inhibited by PD98059, and were not reproduced by ovalbumin or mannitol. The data therefore indicate that albumin is able to stimulate growth and proliferation of proximal tubular cells that is dependent on the ERK family of MAP kinases. The potential importance of this pathway in the development of renal disease is discussed. PMID:10677388

  15. The MARVEL domain protein, Singles Bar, is required for progression past the pre-fusion complex stage of myoblast fusion

    PubMed Central

    Estrada, Beatriz; Maeland, Anne D.; Gisselbrecht, Stephen S.; Bloor, James W.; Brown, Nicholas H.; Michelson, Alan M.

    2007-01-01

    Summary Multinucleated myotubes develop by the sequential fusion of individual myoblasts. Using a convergence of genomic and classical genetic approaches, we have discovered a novel gene, singles bar (sing), that is essential for myoblast fusion. sing encodes a small multipass transmembrane protein containing a MARVEL domain, which is found in vertebrate proteins involved in processes such as tight junction formation and vesicle trafficking where—as in myoblast fusion—membrane apposition occurs. sing is expressed in both founder cells and fusion competent myoblasts preceding and during myoblast fusion. Examination of embryos injected with double-stranded sing RNA or embryos homozygous for ethane methyl sulfonate-induced sing alleles revealed an identical phenotype: replacement of multinucleated myofibers by groups of single, myosin-expressing myoblasts at a stage when formation of the mature muscle pattern is complete in wild-type embryos. Unfused sing mutant myoblasts form clusters, suggesting that early recognition and adhesion of these cells is unimpaired. To further investigate this phenotype, we undertook electron microscopic ultrastructural studies of fusing myoblasts in both sing and wild-type embryos. These experiments revealed that more sing mutant myoblasts than wild-type contain pre-fusion complexes, which are characterized by electron-dense vesicles paired on either side of the fusing plasma membranes. In contrast, embryos mutant for another muscle fusion gene, blown fuse (blow), have a normal number of such complexes. Together, these results lead to the hypothesis that sing acts at a step distinct from that of blow, and that sing is required on both founder cell and fusion-competent myoblast membranes to allow progression past the pre-fusion complex stage of myoblast fusion, possibly by mediating fusion of the electron-dense vesicles to the plasma membrane. PMID:17537424

  16. Correlation Between C-reactive Protein and Non-enzymatic Antioxidants (Albumin, Ferritin, Uric Acid and Bilirubin) in Hemodialysis Patients

    PubMed Central

    Beciragic, Amela; Resic, Halima; Prohic, Nejra; Karamehic, Jasenko; Smajlovic, Ajdin; Masnic, Fahrudin; Ajanovic, Selma; Coric, Aida

    2015-01-01

    Introduction: Increased levels of C-Reactive Protein are found in 30-60% on hemodialysis patients and it is closely associated with the progression of atherosclerosis, cardiovascular morbidity and mortality. Non enzymatic antioxidants are antioxidants which primarily retain potentially dangerous ions of iron and copper in their inactive form and thereby prevent its participation in the production of free radicals. Aim: The aim of the study was to examine the relationship of CRP and non enzymatic antioxidants (albumin, ferritin, uric acid and bilirubin) i.e. examine the importance of CRP as a serum biomarker in assessing the condition of inflammation and its relationship to antioxidant protection in patients on hemodialysis. Methods: The study was cross-sectional, clinical, comparative and descriptive. The study involved 100 patients (non diabetic) on chronic hemodialysis. The control group consisted of 50 subjects without subjective and objective indicators of chronic renal disease. In all patients, the concentration of CRP as well as concentrations of non enzymatic antioxidants were determined. Results: In the group of hemodialysis patients 60% were men and 40% women. The average age of hemodialysis patients was 54.13 ± 11.8 years and the average age of the control group 41.72 ± 9.8 years. The average duration of hemodialysis treatment was 91.42 ± 76.2 months. In the group of hemodialysis patients statistically significant, negative linear correlation was determined between the concentration of CRP in and albumin concentration (rho = -0.251, p = 0.012) as well as negative, statistics insignificant, linear correlation between serum CRP and the concentration of uric acid (r = -0.077, p = 0.448). Furthermore, the positive, linear correlation was determined between serum CRP and ferritin (r = 0.159, p = 0.114) and positive linear correlation between CRP and total serum bilirubin (r = 0.121, p = 0.230). In the control group was determined a statistically significant

  17. Biocompatible Size-Defined Dendrimer-Albumin Binding Protein Hybrid Materials as a Versatile Platform for Biomedical Applications.

    PubMed

    Maly, Jan; Stanek, Ondrej; Frolik, Jan; Maly, Marek; Ennen, Franka; Appelhans, Dietmar; Semeradtova, Alena; Wrobel, Dominika; Stofik, Marcel; Knapova, Tereza; Kuchar, Milan; Stastna, Lucie Cervenkova; Cermak, Jan; Sebo, Peter; Maly, Petr

    2016-04-01

    For the design of a biohybrid structure as a ligand-tailored drug delivery system (DDS), it is highly sophisticated to fabricate a DDS based on smoothly controllable conjugation steps. This article reports on the synthesis and the characterization of biohybrid conjugates based on noncovalent conjugation between a multivalent biotinylated and PEGylated poly(amido amine) (PAMAM) dendrimer and a tetrameric streptavidin-small protein binding scaffold. This protein binding scaffold (SA-ABDwt) possesses nM affinity toward human serum albumin (HSA). Thus, well-defined biohybrid structures, finalized by binding of one or two HSA molecules, are available at each conjugation step in a controlled molar ratio. Overall, these biohybrid assemblies can be used for (i) a controlled modification of dendrimers with the HSA molecules to increase their blood-circulation half-life and passive accumulation in tumor; (ii) rendering dendrimers a specific affinity to various ligands based on mutated ABD domain, thus replacing tedious dendrimer-antibody covalent coupling and purification procedures. PMID:26748571

  18. Mutant Fusion Proteins with Enhanced Fusion Activity Promote Measles Virus Spread in Human Neuronal Cells and Brains of Suckling Hamsters

    PubMed Central

    Shirogane, Yuta; Suzuki, Satoshi O.; Ikegame, Satoshi; Koga, Ritsuko

    2013-01-01

    Subacute sclerosing panencephalitis (SSPE) is a fatal degenerative disease caused by persistent measles virus (MV) infection in the central nervous system (CNS). From the genetic study of MV isolates obtained from SSPE patients, it is thought that defects of the matrix (M) protein play a crucial role in MV pathogenicity in the CNS. In this study, we report several notable mutations in the extracellular domain of the MV fusion (F) protein, including those found in multiple SSPE strains. The F proteins with these mutations induced syncytium formation in cells lacking SLAM and nectin 4 (receptors used by wild-type MV), including human neuronal cell lines, when expressed together with the attachment protein hemagglutinin. Moreover, recombinant viruses with these mutations exhibited neurovirulence in suckling hamsters, unlike the parental wild-type MV, and the mortality correlated with their fusion activity. In contrast, the recombinant MV lacking the M protein did not induce syncytia in cells lacking SLAM and nectin 4, although it formed larger syncytia in cells with either of the receptors. Since human neuronal cells are mainly SLAM and nectin 4 negative, fusion-enhancing mutations in the extracellular domain of the F protein may greatly contribute to MV spread via cell-to-cell fusion in the CNS, regardless of defects of the M protein. PMID:23255801

  19. Human serum albumin-mimetic chromatography based hexadecyltrimethylammonium bromide as a novel direct probe for protein binding of acidic drugs.

    PubMed

    Salary, Mina; Hadjmohammadi, Mohammadreza

    2015-10-10

    Human serum albumin (HSA) is the most important drug carrier in humans mainly binding acidic drugs. Negatively charged compounds bind more strongly to HSA than it would be expected from their lipophilicity alone. With the development of new acidic drugs, there is a high need for rapid and simple protein binding screening technologies. Biopartitioning micellar chromatography (BMC) is a mode of micellar liquid chromatography, which can be used as an in vitro system to model the biopartitioning process of drugs when there are no active processes. In this study, a new kind of BMC using hexadecyltrimethylammonium bromide (CTAB) as micellar mobile phases was used for the prediction of protein binding of acidic drugs based on the similar property of CTAB micelles to HSA. The use of BMC is simple, reproducible and can provide key information about the pharmacological behavior of drugs such as protein binding properties of new compounds during the drug discovery process. The relationships between the MLC retention data of a heterogeneous set of 17 acidic and neutral drugs and their plasma protein binding parameter were studied and second-order polynomial models obtained in two different concentrations (0.07 and 0.09M) of CTAB. However, the developed models are only being able to distinguish between strongly and weakly binding drugs. Also, the developed models were characterized by both the descriptive and predictive ability (R(2)=0.885, RCV(2)=0.838 and R(2)=0.898, RCV(2)=0.859 for 0.07 and 0.09M CTAB, respectively). The application of the developed model to a prediction set demonstrated that the model was also reliable with good predictive accuracy. PMID:25988296

  20. The interaction of human serum albumin with selected lanthanide and actinide ions: Binding affinities, protein unfolding and conformational changes.

    PubMed

    Ali, Manjoor; Kumar, Amit; Kumar, Mukesh; Pandey, Badri N

    2016-04-01

    Human serum albumin (HSA), the most abundant soluble protein in blood plays critical roles in transportation of biomolecules and maintenance of osmotic pressure. In view of increasing applications of lanthanides- and actinides-based materials in nuclear energy, space, industries and medical applications, the risk of exposure with these metal ions is a growing concern for human health. In present study, binding interaction of actinides/lanthanides [thorium: Th(IV), uranium: U(VI), lanthanum: La(III), cerium: Ce(III) and (IV)] with HSA and its structural consequences have been investigated. Ultraviolet-visible, Fourier transform-infrared, Raman, Fluorescence and Circular dichroism spectroscopic techniques were applied to study the site of metal ions interaction, binding affinity determination and the effect of metal ions on protein unfolding and HSA conformation. Results showed that these metal ions interacted with carbonyl (CO..:)/amide(N..-H) groups and induced exposure of aromatic residues of HSA. The fluorescence analysis indicated that the actinide binding altered the microenvironment around Trp214 in the subdomain IIA. Binding affinity of U(VI) to HSA was slightly higher than that of Th(IV). Actinides and Ce(IV) altered the secondary conformation of HSA with a significant decrease of α-helix and an increase of β-sheet, turn and random coil structures, indicating a partial unfolding of HSA. A correlation was observed between metal ion's ability to alter HSA conformation and protein unfolding. Both cationic effects and coordination ability of metal ions seemed to determine the consequences of their interaction with HSA. Present study improves our understanding about the protein interaction of these heavy ions and their impact on its secondary structure. In addition, binding characteristics may have important implications for the development of rational antidote for the medical management of health effects of actinides and lanthanides. PMID:26821345

  1. Matrix metalloproteinase-mediation of tumor targeting human recombinant tumor necrosis factor-α fusion protein.

    PubMed

    Ren, Hui; Shao, Xin; Zeng, Liang; Wang, Fa; Huang, Di-Nan; Hou, Gan

    2015-08-01

    The aim of the present study was to use genetic engineering in order to establish an efficient tumor necrosis factor (TNF)-α fusion protein with low toxicity, which may be used to target tumors. Four types of matrix metalloproteinase (MMP)-mediated tumor targeting human recombinant TNF-α (rhTNF-α) fusion protein vectors were constructed. These were subsequently introduced into Escherichia coli. rhTNF-α fusion protein with a glutathione S-transferase (GST)-tag was purified using GST resin affinity chromatography, and GST-tags were digested using factor Xa. The cytotoxic effects of the fusion protein on L929 cells were determined using MTT assays. At a concentration of 1 pM, the GST-tagged fusion protein exerted no cytotoxic effects on the cells, compared with the negative control cells (P=0.975>0.05). However, at a concentration of 1000 pM, the deblocking fusion protein exerted greater cytotoxic effects on L929 cells, compared with positive control cells (P<0.05). Treatment with the fusion protein also induced cell apoptosis in the nasopharyngeal cancer cell line, CNE-2Z, which secretes high levels of MMP-1. In conclusion, the results of the present study suggested that MMP-mediated rhTNF-α fusion protein induces CNE-2Z cells apoptosis. rhTNF-α exhibits high efficacy and tumor cell targeting capability, with low toxicity effects on healthy cells. PMID:25891416

  2. Using data fusion for scoring reliability of protein-protein interactions.

    PubMed

    Vazifedoost, Alireza; Rahgozar, Maseud; Moshiri, Behzad; Sadeghi, Mehdi; Chua, Hon Nian; Ng, See Kiong; Wong, Limsoon

    2014-08-01

    Protein-protein interactions (PPIs) are important for understanding the cellular mechanisms of biological functions, but the reliability of PPIs extracted by high-throughput assays is known to be low. To address this, many current methods use multiple evidence from different sources of information to compute reliability scores for such PPIs. However, they often combine the evidence without taking into account the uncertainty of the evidence values, potential dependencies between the information sources used and missing values from some information sources. We propose to formulate the task of scoring PPIs using multiple information sources as a multi-criteria decision making problem that can be solved using data fusion to model potential interactions between the multiple information sources. Using data fusion, the amount of contribution from each information source can be proportioned accordingly to systematically score the reliability of PPIs. Our experimental results showed that the reliability scores assigned by our data fusion method can effectively classify highly reliable PPIs from multiple information sources, with substantial improvement in scoring over conventional approach such as the Adjust CD-Distance approach. In addition, the underlying interactions between the information sources used, as well as their relative importance, can also be determined with our data fusion approach. We also showed that such knowledge can be used to effectively handle missing values from information sources. PMID:25152039

  3. Measles virus attachment proteins with impaired ability to bind CD46 interact more efficiently with the homologous fusion protein

    SciTech Connect

    Corey, Elizabeth A.; Iorio, Ronald M.

    2009-01-05

    Fusion promotion by measles virus (MV) depends on an interaction between the hemagglutinin (H) and fusion (F) glycoproteins. Amino acid substitutions in MV H that drastically reduce hemagglutinating activity result in an increase in the amount of H (primarily the 74 kDa isoform) detectable in a complex with F at the cell surface. This is in direct contrast to the loss of the ability to detect a complex between the fusion protein of Newcastle disease virus and most attachment proteins that lack receptor binding activity. These opposing results provide support for the existence of different mechanisms for the regulation of fusion by these two paramyxoviruses.

  4. Premature Activation of the Paramyxovirus Fusion Protein before Target Cell Attachment with Corruption of the Viral Fusion Machinery*

    PubMed Central

    Farzan, Shohreh F.; Palermo, Laura M.; Yokoyama, Christine C.; Orefice, Gianmarco; Fornabaio, Micaela; Sarkar, Aurijit; Kellogg, Glen E.; Greengard, Olga; Porotto, Matteo; Moscona, Anne

    2011-01-01

    Paramyxoviruses, including the childhood pathogen human parainfluenza virus type 3, enter host cells by fusion of the viral and target cell membranes. This fusion results from the concerted action of its two envelope glycoproteins, the hemagglutinin-neuraminidase (HN) and the fusion protein (F). The receptor-bound HN triggers F to undergo conformational changes that render it competent to mediate fusion of the viral and cellular membranes. We proposed that, if the fusion process could be activated prematurely before the virion reaches the target host cell, infection could be prevented. We identified a small molecule that inhibits paramyxovirus entry into target cells and prevents infection. We show here that this compound works by an interaction with HN that results in F-activation prior to receptor binding. The fusion process is thereby prematurely activated, preventing fusion of the viral membrane with target cells and precluding viral entry. This first evidence that activation of a paramyxovirus F can be specifically induced before the virus contacts its target cell suggests a new strategy with broad implications for the design of antiviral agents. PMID:21799008

  5. G-protein coupled receptor BAI3 promotes myoblast fusion in vertebrates.

    PubMed

    Hamoud, Noumeira; Tran, Viviane; Croteau, Louis-Philippe; Kania, Artur; Côté, Jean-François

    2014-03-11

    Muscle fibers form as a result of myoblast fusion, yet the cell surface receptors regulating this process are unknown in vertebrates. In Drosophila, myoblast fusion involves the activation of the Rac pathway by the guanine nucleotide exchange factor Myoblast City and its scaffolding protein ELMO, downstream of cell-surface cell-adhesion receptors. We previously showed that the mammalian ortholog of Myoblast City, DOCK1, functions in an evolutionarily conserved manner to promote myoblast fusion in mice. In search for regulators of myoblast fusion, we identified the G-protein coupled receptor brain-specific angiogenesis inhibitor (BAI3) as a cell surface protein that interacts with ELMO. In cultured cells, BAI3 or ELMO1/2 loss of function severely impaired myoblast fusion without affecting differentiation and cannot be rescued by reexpression of BAI3 mutants deficient in ELMO binding. The related BAI protein family member, BAI1, is functionally distinct from BAI3, because it cannot rescue the myoblast fusion defects caused by the loss of BAI3 function. Finally, embryonic muscle precursor expression of a BAI3 mutant unable to bind ELMO was sufficient to block myoblast fusion in vivo. Collectively, our findings provide a role for BAI3 in the relay of extracellular fusion signals to their intracellular effectors, identifying it as an essential transmembrane protein for embryonic vertebrate myoblast fusion. PMID:24567399

  6. Antigenicity of Recombinant Maltose Binding Protein-Mycobacterium avium subsp. paratuberculosis Fusion Proteins with and without Factor Xa Cleaving

    PubMed Central

    Begg, Douglas J.; Purdie, Auriol C.; Bannantine, John P.; Whittington, Richard J.

    2013-01-01

    Mycobacterium avium subsp. paratuberculosis causes Johne's disease (JD) in ruminants. Proteomic studies have shown that M. avium subsp. paratuberculosis expresses certain proteins when exposed to in vitro physiological stress conditions similar to the conditions experienced within a host during natural infection. Such proteins are hypothesized to be expressed in vivo, are recognized by the host immune system, and may be of potential use in the diagnosis of JD. In this study, 50 recombinant maltose binding protein (MBP)-M. avium subsp. paratuberculosis fusion proteins were evaluated using serum samples from sheep infected with M. avium subsp. paratuberculosis, and 29 (58%) were found to be antigenic. Among 50 fusion proteins, 10 were evaluated in MBP fusion and factor Xa-cleaved forms. A total of 31 proteins (62%) were found to be antigenic in either MBP fusion or factor Xa-cleaved forms. Antigenicity after cleavage and removal of the MBP tag was marginally enhanced. PMID:24132604

  7. Protein body formation in stable transgenic tobacco expressing elastin-like polypeptide and hydrophobin fusion proteins

    PubMed Central

    2013-01-01

    Background Plants are recognized as an efficient and inexpensive system to produce valuable recombinant proteins. Two different strategies have been commonly used for the expression of recombinant proteins in plants: transient expression mediated by Agrobacterium; or stable transformation of the plant genome. However, the use of plants as bioreactors still faces two main limitations: low accumulation levels of some recombinant proteins and lack of efficient purification methods. Elastin-like polypeptide (ELP), hydrophobin I (HFBI) and Zera® are three fusion partners found to increase the accumulation levels of recombinant proteins and induce the formation of protein bodies (PBs) in leaves when targeted to the endoplasmic reticulum (ER) in transient expression assays. In this study the effects of ELP and HFBI fusion tags on recombinant protein accumulation levels and PB formation was examined in stable transgenic Nicotiana tabacum. Results The accumulation of recombinant protein and PB formation was evaluated in two cultivars of Nicotiana tabacum transformed with green fluorescent protein (GFP) fused to ELP or HFBI, both targeted and retrieved to the ER. The ELP and HFBI tags increased the accumulation of the recombinant protein and induced the formation of PBs in leaves of stable transgenic plants from both cultivars. Furthermore, these tags induced the formation of PBs in a concentration-dependent manner, where a specific level of recombinant protein accumulation was required for PBs to appear. Moreover, agro-infiltration of plants accumulating low levels of recombinant protein with p19, a suppressor of post-transcriptional gene silencing (PTGS), increased accumulation levels in four independent transgenic lines, suggesting that PTGS might have caused the low accumulation levels in these plants. Conclusion The use of ELP and HFBI tags as fusion partners in stable transgenic plants of tobacco is feasible and promising. In a constitutive environment, these tags

  8. Analysis of an engineered Salmonella flagellar fusion protein, FliR-FlhB.

    PubMed

    Van Arnam, John S; McMurry, Jonathan L; Kihara, May; Macnab, Robert M

    2004-04-01

    Salmonella FliR and FlhB are membrane proteins necessary for flagellar export. In Clostridium a fliR-flhB fusion gene exists. We constructed a similar Salmonella fusion gene which is able to complement fliR, flhB, and fliR flhB null strains. Western blotting revealed that the FliR-FlhB fusion protein retains the FlhB protein's cleavage properties. We conclude that the FliR and FlhB proteins are physically associated in the wild-type Salmonella basal body, probably in a 1:1 ratio. PMID:15060055

  9. Synthesis of nano-bioactive glass-ceramic powders and its in vitro bioactivity study in bovine serum albumin protein

    NASA Astrophysics Data System (ADS)

    Nabian, Nima; Jahanshahi, Mohsen; Rabiee, Sayed Mahmood

    2011-07-01

    Bioactive glasses and ceramics have proved to be able to chemically bond to living bone due to the formation of an apatite-like layer on its surface. The aim of this work was preparation and characterization of bioactive glass-ceramic by sol-gel method. Nano-bioglass-ceramic material was crushed into powder and its bioactivity was examined in vitro with respect to the ability of hydroxyapatite layer to form on the surface as a result of contact with bovine serum albumin (BSA) protein. The obtained nano-bioactive glass-ceramic was analyzed before and after contact with BSA solution. This study used scanning electron microscope (SEM), transmission electron microscope (TEM), X-ray powder diffraction (XRD) and Fourier transform infrared spectroscopy (FTIR) analysis to examine its morphology, crystallinity and composition. The TEM images showed that the NBG particles size were 10-40 nm. Bioactivity of nanopowder was confirmed by SEM and XRD due to the presence of a rich bone-like apatite layer. Therefore, this nano-BSA-bioglass-ceramic composite material is promising for medical applications such as bone substitutes and drug carriers.

  10. Functional analysis of the transmembrane domain in paramyxovirus F protein-mediated membrane fusion

    PubMed Central

    Bissonnette, Mei Lin Z.; Donald, Jason E.; DeGrado, William F.; Jardetzky, Theodore S.; Lamb, Robert A.

    2009-01-01

    To enter cells enveloped viruses use fusion-mediating glycoproteins to facilitate the merger of the viral and host cell membranes. These glycoproteins undergo large-scale irreversible refolding during membrane fusion. The paramyxovirus parainfluenza virus 5 (PIV5) mediates membrane merger through its fusion protein (F). The transmembrane (TM) domains of viral fusion proteins are typically required for fusion. The TM domain of F is particularly interesting in that it is potentially unusually long; multiple calculations suggest a TM helix length between 25 and 48 residues. Oxidative cross-linking of single cysteine substitutions indicates the F TM trimer forms a helical bundle within the membrane. To assess the functional role of the PIV5 F protein TM domain, alanine scanning mutagenesis was performed. Two residues located in the outer leaflet of the bilayer are critical for fusion. Multiple amino acid substitutions at these positions indicate the physical properties of the side chain play a critical role in supporting or blocking fusion. Analysis of intermediate steps in F protein refolding indicated that the mutants were not trapped at the open stalk intermediate or the prehairpin intermediate. Incorporation of a known F protein destabilizing mutation that causes a hyperfusogenic phenotype restored fusion activity to the mutants. Further, altering the curvature of the lipid bilayer by addition of oleic acid promoted fusion of the F protein mutants. In aggregate, these data indicate that the TM domain plays a functional role in fusion beyond merely anchoring the protein in the viral envelope and that it can affect the structures and steady-state concentrations of the various conformational intermediates en route to the final postfusion state. We suggest that the unusual length of this TM helix might allow it to serve as a template for formation of or specifically stabilize the lipid stalk intermediate in fusion. PMID:19121325

  11. Aequorin fusion proteins as bioluminescent tracers for competitive immunoassays

    NASA Astrophysics Data System (ADS)

    Mirasoli, Mara; Michelini, Elisa; Deo, Sapna K.; Dikici, Emre; Roda, Aldo; Daunert, Sylvia

    2004-06-01

    The use of bio- and chemiluminescence for the development of quantitative binding assays offers undoubted advantages over other detection systems, such as spectrophotometry, fluorescence, or radioactivity. Indeed, bio- and chemiluminescence detection provides similar, or even better, sensitivity and detectability than radioisotopes, while avoiding the problems of health hazards, waste disposal, and instability associated with the use of radioisotopes. Among bioluminescent labels, the calcium-activated photoprotein aequorin, originally isolated from Aequorea victoria and today available as a recombinant product, is characterized by very high detectability, down to attomole levels. It has been used as a bioluminescent label for developing a variety of highly sensitive immunoassays, using various analyte-aequorin conjugation strategies. When the analyte is a protein or a peptide, genetic engineering techniques can be used to produce protein fusions where the analyte is in-frame fused with aequorin, thus producing homogeneous one-to-one conjugation products, available in virtually unlimited amount. Various assays were developed using this strategy: a short review of the most interesting applications is presented, as well as the cloning, purification and initial characterization of an endothelin-1-aequorin conjugate suitable for developing a competitive immunoassay for endothelin-1, a potent vasoconstrictor peptide, involved in hypertension.

  12. Graphene Biosensor Programming with Genetically Engineered Fusion Protein Monolayers.

    PubMed

    Soikkeli, Miika; Kurppa, Katri; Kainlauri, Markku; Arpiainen, Sanna; Paananen, Arja; Gunnarsson, David; Joensuu, Jussi J; Laaksonen, Päivi; Prunnila, Mika; Linder, Markus B; Ahopelto, Jouni

    2016-03-01

    We demonstrate a label-free biosensor concept based on specific receptor modules, which provide immobilization and selectivity to the desired analyte molecules, and on charge sensing with a graphene field effect transistor. The receptor modules are fusion proteins in which small hydrophobin proteins act as the anchor to immobilize the receptor moiety. The functionalization of the graphene sensor is a single-step process based on directed self-assembly of the receptor modules on a hydrophobic surface. The modules are produced separately in fungi or plants and purified before use. The modules form a dense and well-oriented monolayer on the graphene transistor channel and the receptor module monolayer can be removed, and a new module monolayer with a different selectivity can be assembled in situ. The receptor module monolayers survive drying, showing that the functionalized devices can be stored and have a reasonable shelf life. The sensor is tested with small charged peptides and large immunoglobulin molecules. The measured sensitivities are in the femtomolar range, and the response is relatively fast, of the order of one second. PMID:26960769

  13. LAMP proteins are required for fusion of lysosomes with phagosomes.

    PubMed

    Huynh, Kassidy K; Eskelinen, Eeva-Liisa; Scott, Cameron C; Malevanets, Anatoly; Saftig, Paul; Grinstein, Sergio

    2007-01-24

    Lysosome-associated membrane proteins 1 and 2 (LAMP-1 and LAMP-2) are delivered to phagosomes during the maturation process. We used cells from LAMP-deficient mice to analyze the role of these proteins in phagosome maturation. Macrophages from LAMP-1- or LAMP-2-deficient mice displayed normal fusion of lysosomes with phagosomes. Because ablation of both the lamp-1 and lamp-2 genes yields an embryonic-lethal phenotype, we were unable to study macrophages from double knockouts. Instead, we reconstituted phagocytosis in murine embryonic fibroblasts (MEFs) by transfection of FcgammaIIA receptors. Phagosomes formed by FcgammaIIA-transfected MEFs obtained from LAMP-1- or LAMP-2- deficient mice acquired lysosomal markers. Remarkably, although FcgammaIIA-transfected MEFs from double-deficient mice ingested particles normally, phagosomal maturation was arrested. LAMP-1 and LAMP-2 double-deficient phagosomes acquired Rab5 and accumulated phosphatidylinositol 3-phosphate, but failed to recruit Rab7 and did not fuse with lysosomes. We attribute the deficiency to impaired organellar motility along microtubules. Time-lapse cinematography revealed that late endosomes/lysosomes as well as phagosomes lacking LAMP-1 and LAMP-2 had reduced ability to move toward the microtubule-organizing center, likely precluding their interaction with each other. PMID:17245426

  14. Generation of New M2e-HA2 Fusion Chimeric Peptide to Development of a Recombinant Fusion Protein Vaccine

    PubMed Central

    Ameghi, Ali; Baradaran, Behzad; Aghaiypour, Khosrow; Barzegar, Abolfazl; Pilehvar-Soltanahmadi, Yones; Moghadampour, Masood; Taghizadeh, Morteza; Zarghami, Nosratollah

    2015-01-01

    Purpose: The purpose was to design a new construction containing influenza virus (H1N1) M2e gene and HA2 gene by bioinformatics approach, cloning the construct in to Escherichia coli and produce M2e-HA2 peptide. Methods: The procedure was done by virus cultivation in SPF eggs, hemagglutination assay (HA), RNA isolation, RT-PCR, primers designed (DNAMAN 4 and Oligo7), virtual fusion construction translation (ExPASy), N-Glycosylated sites prediction (Ensemblegly-Iowa), complete open reading frame (ORF), stop codon studied (NCBI ORF Finder), rare codon determination (GenScript), Solvent accessibility of epitopes (Swiss-PdbViewer), antigenic sites prediction (Protean), fusion PCR of M2e-HA2 gene, sequence analysis, nested PCR, gel electrophoresis, double digestion of pET22b(+) plasmid and the fusion construct, ligation of them, transformation of the ligated vector (pET22b-M2e-HA2) to E.coli (BL21), mass culture the cloned bacterium ,induction the expression by isopropyl-beta-D-thiogalactopyranoside (IPTG), sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), purification the fusion peptide by Ni-NTA column, western blot to verify the purification. Results: In this study we developed a new approach for fusion of Influenza virus M2e (96 nucleotides) and HA2 (663 nucleotides) genes based on fusion PCR strategy and produced a fused fragment with 793 nucleotides. The construct was successfully cloned and expressed. Conclusion: This construct is a 261 amino acid chimeric fusion peptide with about 30 KD molecular weight. According on the latest information; this is the first case of expression and purification M2e-HA2 fusion chimeric peptide, which could be used for development of a recombinant M2e-HA2 fusion protein vaccine. PMID:26793615

  15. Surface density of the Hendra G protein modulates Hendra F protein-promoted membrane fusion: Role for Hendra G protein trafficking and degradation

    SciTech Connect

    Whitman, Shannon D.; Dutch, Rebecca Ellis . E-mail: rdutc2@uky.edu

    2007-07-05

    Hendra virus, like most paramyxoviruses, requires both a fusion (F) and attachment (G) protein for promotion of cell-cell fusion. Recent studies determined that Hendra F is proteolytically processed by the cellular protease cathepsin L after endocytosis. This unique cathepsin L processing results in a small percentage of Hendra F on the cell surface. To determine how the surface densities of the two Hendra glycoproteins affect fusion promotion, we performed experiments that varied the levels of glycoproteins expressed in transfected cells. Using two different fusion assays, we found a marked increase in fusion when expression of the Hendra G protein was increased, with a 1:1 molar ratio of Hendra F:G on the cell surface resulting in optimal membrane fusion. Our results also showed that Hendra G protein levels are modulated by both more rapid protein turnover and slower protein trafficking than is seen for Hendra F.

  16. The Human Metapneumovirus Fusion Protein Mediates Entry via an Interaction with RGD-Binding Integrins

    PubMed Central

    Cox, Reagan G.; Livesay, S. Brent; Johnson, Monika; Ohi, Melanie D.

    2012-01-01

    Paramyxoviruses use a specialized fusion protein to merge the viral envelope with cell membranes and initiate infection. Most paramyxoviruses require the interaction of two viral proteins to enter cells; an attachment protein binds cell surface receptors, leading to the activation of a fusion (F) protein that fuses the viral envelope and host cell plasma membrane. In contrast, human metapneumovirus (HMPV) expressing only the F protein is replication competent, suggesting a primary role for HMPV F in attachment and fusion. We previously identified an invariant arginine-glycine-aspartate (RGD) motif in the HMPV F protein and showed that the RGD-binding integrin αVβ1-promoted HMPV infection. Here we show that both HMPV F-mediated binding and virus entry depend upon multiple RGD-binding integrins and that HMPV F can mediate binding and fusion in the absence of the viral attachment (G) protein. The invariant F-RGD motif is critical for infection, as an F-RAE virus was profoundly impaired. Further, F-integrin binding is required for productive viral RNA transcription, indicating that RGD-binding integrins serve as receptors for the HMPV fusion protein. Thus, HMPV F is triggered to induce virus-cell fusion by interactions with cellular receptors in a manner that is independent of the viral G protein. These results suggest a stepwise mechanism of HMPV entry mediated by the F protein through its interactions with cellular receptors, including RGD-binding integrins. PMID:22933271

  17. Revealing Surface Waters on an Antifreeze Protein by Fusion Protein Crystallography Combined with Molecular Dynamic Simulations.

    PubMed

    Sun, Tianjun; Gauthier, Sherry Y; Campbell, Robert L; Davies, Peter L

    2015-10-01

    Antifreeze proteins (AFPs) adsorb to ice through an extensive, flat, relatively hydrophobic surface. It has been suggested that this ice-binding site (IBS) organizes surface waters into an ice-like clathrate arrangement that matches and fuses to the quasi-liquid layer on the ice surface. On cooling, these waters join the ice lattice and freeze the AFP to its ligand. Evidence for the generality of this binding mechanism is limited because AFPs tend to crystallize with their IBS as a preferred protein-protein contact surface, which displaces some bound waters. Type III AFP is a 7 kDa globular protein with an IBS made up two adjacent surfaces. In the crystal structure of the most active isoform (QAE1), the part of the IBS that docks to the primary prism plane of ice is partially exposed to solvent and has clathrate waters present that match this plane of ice. The adjacent IBS, which matches the pyramidal plane of ice, is involved in protein-protein crystal contacts with few surface waters. Here we have changed the protein-protein contacts in the ice-binding region by crystallizing a fusion of QAE1 to maltose-binding protein. In this 1.9 Å structure, the IBS that fits the pyramidal plane of ice is exposed to solvent. By combining crystallography data with MD simulations, the surface waters on both sides of the IBS were revealed and match well with the target ice planes. The waters on the pyramidal plane IBS were loosely constrained, which might explain why other isoforms of type III AFP that lack the prism plane IBS are less active than QAE1. The AFP fusion crystallization method can potentially be used to force the exposure to solvent of the IBS on other AFPs to reveal the locations of key surface waters. PMID:26371748

  18. Viral fusion protein transmembrane domain adopts β-strand structure to facilitate membrane topological changes for virus–cell fusion

    PubMed Central

    Yao, Hongwei; Lee, Michelle W.; Waring, Alan J.; Wong, Gerard C. L.; Hong, Mei

    2015-01-01

    The C-terminal transmembrane domain (TMD) of viral fusion proteins such as HIV gp41 and influenza hemagglutinin (HA) is traditionally viewed as a passive α-helical anchor of the protein to the virus envelope during its merger with the cell membrane. The conformation, dynamics, and lipid interaction of these fusion protein TMDs have so far eluded high-resolution structure characterization because of their highly hydrophobic nature. Using magic-angle-spinning solid-state NMR spectroscopy, we show that the TMD of the parainfluenza virus 5 (PIV5) fusion protein adopts lipid-dependent conformations and interactions with the membrane and water. In phosphatidylcholine (PC) and phosphatidylglycerol (PG) membranes, the TMD is predominantly α-helical, but in phosphatidylethanolamine (PE) membranes, the TMD changes significantly to the β-strand conformation. Measured order parameters indicate that the strand segments are immobilized and thus oligomerized. 31P NMR spectra and small-angle X-ray scattering (SAXS) data show that this β-strand–rich conformation converts the PE membrane to a bicontinuous cubic phase, which is rich in negative Gaussian curvature that is characteristic of hemifusion intermediates and fusion pores. 1H-31P 2D correlation spectra and 2H spectra show that the PE membrane with or without the TMD is much less hydrated than PC and PG membranes, suggesting that the TMD works with the natural dehydration tendency of PE to facilitate membrane merger. These results suggest a new viral-fusion model in which the TMD actively promotes membrane topological changes during fusion using the β-strand as the fusogenic conformation. PMID:26283363

  19. Expression and purification of recombinant antibody formats and antibody fusion proteins.

    PubMed

    Siegemund, Martin; Richter, Fabian; Seifert, Oliver; Unverdorben, Felix; Kontermann, Roland E

    2014-01-01

    In the laboratory-scale production of antibody fragments or antibody fusion proteins, it is often difficult to keep track on the most suitable affinity tags for protein purification from either prokaryotic or eukaryotic host systems. Here, we describe how such recombinant proteins derived from Escherichia coli lysates as well as HEK293 cell culture supernatants are purified by IMAC and by different affinity chromatography methods based on fusions to FLAG-tag, Strep-tag, and Fc domains. PMID:24515473

  20. [The significance of low levels of total proteins, albumins, globulins and complement factors in ascitic fluid and the development of spontaneous bacterial peritonitis in patients with liver cirrhosis].

    PubMed

    Ljubicić, N; Bilić, A; Babić, Z; Roić, D; Banić, M

    1992-01-01

    Spontaneous bacterial peritonitis is one of the most common complications of ascitic fluid in patients with liver cirrhosis. The aim of this study was to investigate the role of total protein, albumin, globulin and complement ascitic fluid concentrations in development of spontaneous bacterial peritonitis in patients with liver cirrhosis. In patients with liver cirrhosis and spontaneous bacterial peritonitis (n = 8) the ascitic fluid total protein, albumin and globulin concentrations were significantly lower than in patients with sterile ascites (n = 11) (p < 0.01). The ascitic fluid complement C3 and C4 concentrations were significantly lower in patients with spontaneous bacterial peritonitis than in patients with sterile ascites (9.1 +/- 3.1 mg/dL to 22.9 +/- 17.4 mg/dL, p < 0.01; 3.8 +/- 5.9 mg/dL to 8.2 +/- 5.9 mg/dL, p < 0.01, respectively). The ascites total protein, albumin, globulin and complement concentrations in cirrhotic patients with spontaneous bacterial peritonitis were significantly lower than in patients with sterile ascites demonstrating the importance of those factors in ascitic fluid defense against secondary bacterial infection. PMID:1343119

  1. Development of new fusion proteins for visualizing amyloid-β oligomers in vivo

    PubMed Central

    Ochiishi, Tomoyo; Doi, Motomichi; Yamasaki, Kazuhiko; Hirose, Keiko; Kitamura, Akira; Urabe, Takao; Hattori, Nobutaka; Kinjo, Masataka; Ebihara, Tatsuhiko; Shimura, Hideki

    2016-01-01

    The intracellular accumulation of amyloid-β (Aβ) oligomers critically contributes to disease progression in Alzheimer’s disease (AD) and can be the potential target of AD therapy. Direct observation of molecular dynamics of Aβ oligomers in vivo is key for drug discovery research, however, it has been challenging because Aβ aggregation inhibits the fluorescence from fusion proteins. Here, we developed Aβ1-42-GFP fusion proteins that are oligomerized and visualize their dynamics inside cells even when aggregated. We examined the aggregation states of Aβ-GFP fusion proteins using several methods and confirmed that they did not assemble into fibrils, but instead formed oligomers in vitro and in live cells. By arranging the length of the liker between Aβ and GFP, we generated two fusion proteins with “a long-linker” and “a short-linker”, and revealed that the aggregation property of fusion proteins can be evaluated by measuring fluorescence intensities using rat primary culture neurons transfected with Aβ-GFP plasmids and Aβ-GFP transgenic C. elegans. We found that Aβ-GFP fusion proteins induced cell death in COS7 cells. These results suggested that novel Aβ-GFP fusion proteins could be utilized for studying the physiological functions of Aβ oligomers in living cells and animals, and for drug screening by analyzing Aβ toxicity. PMID:26982553

  2. Development of new fusion proteins for visualizing amyloid-β oligomers in vivo.

    PubMed

    Ochiishi, Tomoyo; Doi, Motomichi; Yamasaki, Kazuhiko; Hirose, Keiko; Kitamura, Akira; Urabe, Takao; Hattori, Nobutaka; Kinjo, Masataka; Ebihara, Tatsuhiko; Shimura, Hideki

    2016-01-01

    The intracellular accumulation of amyloid-β (Aβ) oligomers critically contributes to disease progression in Alzheimer's disease (AD) and can be the potential target of AD therapy. Direct observation of molecular dynamics of Aβ oligomers in vivo is key for drug discovery research, however, it has been challenging because Aβ aggregation inhibits the fluorescence from fusion proteins. Here, we developed Aβ1-42-GFP fusion proteins that are oligomerized and visualize their dynamics inside cells even when aggregated. We examined the aggregation states of Aβ-GFP fusion proteins using several methods and confirmed that they did not assemble into fibrils, but instead formed oligomers in vitro and in live cells. By arranging the length of the liker between Aβ and GFP, we generated two fusion proteins with "a long-linker" and "a short-linker", and revealed that the aggregation property of fusion proteins can be evaluated by measuring fluorescence intensities using rat primary culture neurons transfected with Aβ-GFP plasmids and Aβ-GFP transgenic C. elegans. We found that Aβ-GFP fusion proteins induced cell death in COS7 cells. These results suggested that novel Aβ-GFP fusion proteins could be utilized for studying the physiological functions of Aβ oligomers in living cells and animals, and for drug screening by analyzing Aβ toxicity. PMID:26982553

  3. Generation of monoclonal antibodies specific of the postfusion conformation of the Pneumovirinae fusion (F) protein.

    PubMed

    Rodríguez, Laura; Olmedillas, Eduardo; Mas, Vicente; Vázquez, Mónica; Cano, Olga; Terrón, María C; Luque, Daniel; Palomo, Concepción; Melero, José A

    2015-11-01

    Paramyxovirus entry into cells requires fusion of the viral and cell membranes mediated by one of the major virus glycoproteins, the fusion (F) glycoprotein which transits from a metastable pre-fusion conformation to a highly stable post-fusion structure during the membrane fusion process. F protein refolding involves large conformational changes of the protein trimer. One of these changes results in assembly of two heptad repeat sequences (HRA and HRB) from each protomer into a six-helix bundle (6HB) motif. To assist in distinguishing pre- and post-fusion conformations of the Pneumovirinae F proteins, and as extension of previous work (Palomo et al., 2014), a general strategy was designed to obtain polyclonal and particularly monoclonal antibodies specific of the 6HB motif of the Pneumovirinae fusion protein. The antibodies reported here should assist in the characterization of the structural changes that the F protein of human metapneumovirus or respiratory syncytial virus experiences during the process of membrane fusion. PMID:26275682

  4. E protein silencing by the leukemogenic AML1-ETO fusion protein.

    PubMed

    Zhang, Jinsong; Kalkum, Markus; Yamamura, Soichiro; Chait, Brian T; Roeder, Robert G

    2004-08-27

    The AML1-ETO fusion protein, generated by the t(8;21) chromosomal translocation, is causally involved in nearly 15% of acute myeloid leukemia (AML) cases. This study shows that AML1-ETO, as well as ETO, inhibits transcriptional activation by E proteins through stable interactions that preclude recruitment of p300/CREB-binding protein (CBP) coactivators. These interactions are mediated by a conserved ETO TAF4 homology domain and a 17-amino acid p300/CBP and ETO target motif within AD1 activation domains of E proteins. In t(8;21) leukemic cells, very stable interactions between AML1-ETO and E proteins underlie a t(8;21) translocation-specific silencing of E protein function through an aberrant cofactor exchange mechanism. These studies identify E proteins as AML1-ETO targets whose dysregulation may be important for t(8;21) leukemogenesis, as well as an E protein silencing mechanism that is distinct from that associated with differentiation-inhibitory proteins. PMID:15333839

  5. High level expression of peptides and proteins using cytochrome b5 as a fusion host.

    PubMed

    Mitra, Ashima; Chakrabarti, Kalyan Sundar; Shahul Hameed, M S; Srinivas, Kalyan V; Senthil Kumar, Ganesan; Sarma, Siddhartha P

    2005-05-01

    A novel fusion protein system based on the highly soluble heme-binding domain of cytochrome b5 has been designed. The ability of cytochrome b5 to increase the levels of expression and solubility of target proteins has been tested by expressing several proteins and peptides, viz., alpha hemoglobin stabilizing protein, the regulatory subunits of acetohydroxy acid synthase I (ilvM) and II (ilvN), the carboxy terminal domains of mouse neuronal kinesin and pantothenate synthatase, two peptide toxins from cone snails, and the inactivation gate from the brain voltage gated sodium channel, NaV1.2. The fusion protein system has been designed to incorporate protease cleavage sites for commonly used proteases, viz., enterokinase, Factor Xa, and Tobacco etch virus protease. Accumulation of expressed protein as a function of time may be visually ascertained by the fact that the cells take on a bright red color during the course of induction. In all the cases tested so far, the fusion protein accumulates in the soluble fraction to high levels. A novel purification protocol has been designed to purify the fusion proteins using metal affinity chromatography, without the need of a hexahistidine-tag. Mass spectral analysis has shown that the fusion proteins are of full length. CD studies have shown that the solubilized fusion proteins are structured. The proteins of interest may be cleaved from the parent protein by either chemical or enzymatic means. The results presented here demonstrate the versatility of the cytochrome b5 based fusion system for the production of peptides and small proteins (<15 kDa). PMID:15802225

  6. Hyper-Enhanced Production of Foreign Recombinant Protein by Fusion with the Partial Polyhedrin of Nucleopolyhedrovirus

    PubMed Central

    Bae, Sung Min; Kim, Hee Jung; Lee, Jun Beom; Choi, Jae Bang; Shin, Tae Young; Koo, Hyun Na; Choi, Jae Young; Lee, Kwang Sik; Je, Yeon Ho; Jin, Byung Rae; Yoo, Sung Sik; Woo, Soo Dong

    2013-01-01

    To enhance the production efficiency of foreign protein in baculovirus expression systems, the effects of polyhedrin fragments were investigated by fusion expressing them with the enhanced green fluorescent protein (EGFP). Recombinant viruses were generated to express EGFP fused with polyhedrin fragments based on the previously reported minimal region for self-assembly and the KRKK nuclear localization signal (NLS). Fusion expressions with polyhedrin amino acids 19 to 110 and 32 to 110 lead to localization of recombinant protein into the nucleus and mediate its assembly. The marked increase of EGFP by these fusion expressions was confirmed through protein and fluorescence intensity analyses. The importance of nuclear localization for enhanced production was shown by the mutation of the NLS within the fused polyhedrin fragment. In addition, when the polyhedrin fragment fused with EGFP was not localized in the nucleus, some fragments increased the production of protein. Among these fragments, some degradation of only the fused polyhedrin was observed in the fusion of amino acids 19 to 85 and 32 to 85. The fusion of amino acids 32 to 85 may be more useful for the enhanced and intact production of recombinant protein. The production of E2 protein, which is a major antigen of classical swine fever virus, was dramatically increased by fusion expression with polyhedrin amino acids 19 to 110, and its preliminary immunogenicity was verified using experimental guinea pigs. This study suggests a new option for higher expression of useful foreign recombinant protein by using the partial polyhedrin in baculovirus. PMID:23593321

  7. Kits and methods of detection using cellulose binding domain fusion proteins

    DOEpatents

    Shoseyov, O.; Yosef, K.

    1998-04-14

    A cellulose binding domain (CBD) having a high affinity for crystalline cellulose and chitin is disclosed, along with methods for the molecular cloning and recombinant production. Fusion products comprising the CBD and a second protein are likewise described. A wide range of applications are contemplated for both the CBD and the fusion products, including drug delivery, affinity separations, and diagnostic techniques. 16 figs.

  8. Kits and methods of detection using cellulose binding domain fusion proteins

    DOEpatents

    Shoseyov, Oded

    1998-01-01

    A cellulose binding domain (CBD) having a high affinity for crystalline cellulose and chitin is disclosed, along with methods for the molecular cloning and recombinant production thereof. Fusion products comprising the CBD and a second protein are likewise described. A wide range of applications are contemplated for both the CBD and the fusion products, including drug delivery, affinity separations, and diagnostic techniques.

  9. Conformational stability of a model protein (bovine serum albumin) during primary emulsification process of PLGA microspheres synthesis.

    PubMed

    Kang, Feirong; Singh, Jagdish

    2003-07-01

    The goal of this study was to investigate the conformational stability of a model protein, bovine serum albumin (BSA), during the primary emulsification process of poly(D,L-lactide-co-glycolide) (PLGA) microspheres preparation. Differential scanning calorimeter (DSC) was utilized to assess the conformational structure of BSA during primary emulsification in the presence and absence of PLGA. Three excipients [i.e. mannitol, hydroxypropyl-beta-cyclodextrin (HP-beta-CD) and sodium dodecyl sulfate (SDS)] were investigated for their stabilizing effect on BSA during emulsification process. The DSC profile of intact BSA was best fitted by a non-2-state model with two peaks, which have midpoint temperatures (T(m1), 60.9 +/- 0.4 degrees C and T(m2), 66.4 +/- 1.0 degrees C), respectively, and a total calorimetric enthalpy Delta H(tot) of 599 +/- 42 kJ/mol. After emulsifying BSA aqueous solution with methylene chloride, an additional apparent peak at a higher temperature was observed. The T(m) of this peak was 77.4 +/- 0.8 degrees C. HP-beta-CD was able to suppress the occurrence of an additional peak, whereas mannitol failed. SDS increased the thermal stability of BSA dramatically. Furthermore, HP-beta-CD increased BSA recovery from 72 +/- 8% to 89 +/- 7% after extraction from w/o in the presence of PLGA. These results provided evidence that HP-beta-CD could be a promising excipient for conformational stability of BSA during synthesis of PLGA microspheres. PMID:12818819

  10. Antigen presentation of detergent free glutamate decarboxylase (GAD65) is affected by human serum albumin as carrier protein

    PubMed Central

    Steed, Jordan; Gilliam, Lisa K.; Harris, Robert A.; Lernmark, Åke; Hampe, Christiane S.

    2008-01-01

    1. Summary The smaller isoform of glutamate decarboxylase (GAD65) is a major autoantigen in type 1 diabetes (TID). Its hydrophobic character requires detergent to keep the protein in solution, which complicates studies of antigen processing and presentation. In this study an attempt was made to replace detergent with human serum albumin (HSA) for in vitro antigen presentation. Different preparations of recombinant human GAD65 complexed with HSA were incubated with Priess B cells (HLA DRB1*0401) and antigen presentation was tested with HLA DRB1*0401-restricted and epitope-specific T33.1 (GAD65 epitope 274-286) and T35 (GAD65 epitope 115-127) T cell hybridomas. Specific epitope recognition by T33.1 (274-286) and T35 (115-127) cells varied between the different GAD65/HSA preparations, and a reverse pattern of antigen presentation were detected by the two hybridoma. The HSA-specific T-cell hybridoma 17.9 response to the different GAD65/HSA preparations followed the same pattern as that observed for the T33.1 cells. The content of immunoreactive GAD65 measured with four GAD65 antibodies indicated that the lowest GAD65 concentration resulted in the highest 274-286, but the lowest 115-127 presentation. This suggests that HSA-GAD65 complexes qualitatively affect the epitope specificity of GAD65 presentation. HSA may enhance the 274-286 epitope presentation, while suppressing the 115-127 epitope. PMID:18353353

  11. Role of protein disulfide isomerase and other thiol-reactive proteins in HIV-1 envelope protein-mediated fusion

    SciTech Connect

    Ou Wu . E-mail: wou@niaid.nih.gov; Silver, Jonathan . E-mail: jsilver@nih.gov

    2006-07-05

    Cell-surface protein disulfide isomerase (PDI) has been proposed to promote disulfide bond rearrangements in HIV-1 envelope protein (Env) that accompany Env-mediated fusion. We evaluated the role of PDI in ways that have not been previously tested by downregulating PDI with siRNA and by overexpressing wild-type or variant forms of PDI in transiently and stably transfected cells. These manipulations, as well as treatment with anti-PDI antibodies, had only small effects on infection or cell fusion mediated by NL4-3 or AD8 strains of HIV-1. However, the cell-surface thiol-reactive reagent 5, 5'-dithiobis(2-nitrobenzoic acid) (DTNB) had a much stronger inhibitory effect in our system, suggesting that cell-surface thiol-containing molecules other than PDI, acting alone or in concert, have a greater effect than PDI on HIV-1 Env-mediated fusion. We evaluated one such candidate, thioredoxin, a PDI family member reported to reduce a labile disulfide bond in CD4. We found that the ability of thioredoxin to reduce the disulfide bond in CD4 is enhanced in the presence of HIV-1 Env gp120 and that thioredoxin also reduces disulfide bonds in gp120 directly in the absence of CD4. We discuss the implications of these observations for identification of molecules involved in disulfide rearrangements in Env during fusion.

  12. An evolved Mxe GyrA intein for enhanced production of fusion proteins.

    PubMed

    Marshall, Carrie J; Grosskopf, Vanessa A; Moehling, Taylor J; Tillotson, Benjamin J; Wiepz, Gregory J; Abbott, Nicholas L; Raines, Ronald T; Shusta, Eric V

    2015-02-20

    Expressing antibodies as fusions to the non-self-cleaving Mxe GyrA intein enables site-specific, carboxy-terminal chemical modification of the antibodies by expressed protein ligation (EPL). Bacterial antibody-intein fusion protein expression platforms typically yield insoluble inclusion bodies that require refolding to obtain active antibody-intein fusion proteins. Previously, we demonstrated that it was possible to employ yeast surface display to express properly folded single-chain antibody (scFv)-intein fusions, therefore permitting the direct small-scale chemical functionalization of scFvs. Here, directed evolution of the Mxe GyrA intein was performed to improve both the display and secretion levels of scFv-intein fusion proteins from yeast. The engineered intein was shown to increase the yeast display levels of eight different scFvs by up to 3-fold. Additionally, scFv- and green fluorescent protein (GFP)-intein fusion proteins can be secreted from yeast, and while fusion of the scFvs to the wild-type intein resulted in low expression levels, the engineered intein increased scFv-intein production levels by up to 30-fold. The secreted scFv- and GFP-intein fusion proteins retained their respective binding and fluorescent activities, and upon intein release, EPL resulted in carboxy-terminal azide functionalization of the target proteins. The azide-functionalized scFvs and GFP were subsequently employed in a copper-free, strain-promoted click reaction to site-specifically immobilize the proteins on surfaces, and it was demonstrated that the functionalized, immobilized scFvs retained their antigen binding specificity. Taken together, the evolved yeast intein platform provides a robust alternative to bacterial intein expression systems. PMID:25384269

  13. Structure of the Newcastle disease virus F protein in the post-fusion conformation

    SciTech Connect

    Swanson, Kurt; Wen, Xiaolin; Leser, George P.; Paterson, Reay G.; Lamb, Robert A.; Jardetzky, Theodore S.

    2010-11-17

    The paramyxovirus F protein is a class I viral membrane fusion protein which undergoes a significant refolding transition during virus entry. Previous studies of the Newcastle disease virus, human parainfluenza virus 3 and parainfluenza virus 5 F proteins revealed differences in the pre- and post-fusion structures. The NDV Queensland (Q) F structure lacked structural elements observed in the other two structures, which are key to the refolding and fusogenic activity of F. Here we present the NDV Australia-Victoria (AV) F protein post-fusion structure and provide EM evidence for its folding to a pre-fusion form. The NDV AV F structure contains heptad repeat elements missing in the previous NDV Q F structure, forming a post-fusion six-helix bundle (6HB) similar to the post-fusion hPIV3 F structure. Electrostatic and temperature factor analysis of the F structures points to regions of these proteins that may be functionally important in their membrane fusion activity.

  14. A minichaperone-based fusion system for producing insoluble proteins in soluble stable forms.

    PubMed

    Sharapova, Olga A; Yurkova, Maria S; Fedorov, Alexey N

    2016-02-01

    We have developed a fusion system for reliable production of insoluble hydrophobic proteins in soluble stable forms. A carrier is thermophilic minichaperone, GroEL apical domain (GrAD), a 15 kDa monomer able to bind diverse protein substrates. The Met-less variant of GrAD has been made for further convenient use of Met-specific CNBr chemical cleavage, if desired. The Met-less GrAD retained stability and solubility of the original protein. Target polypeptides can be fused to either C-terminus or N-terminus of GrAD. The system has been tested with two unrelated insoluble proteins fused to the C-terminus of GrAD. One of the proteins was also fused to GrAD N-terminus. The fusions formed inclusion bodies at 25°C and above and were partly soluble only at lower expression temperatures. Most importantly, however, after denaturation in urea, all fusions without exception were completely renatured in soluble stable forms that safely survived freezing-thawing as well as lyophilization. All fusions for both tested target proteins retained solubility at high concentrations for days. Functional analysis revealed that a target protein may retain functionality in the fusion. Convenience features include potential thermostability of GrAD fusions, capacity for chemical and enzymatic cleavage of a target and His6 tag for purification. PMID:26612097

  15. Immune sensitization to methylene diphenyl diisocyanate (MDI) resulting from skin exposure: albumin as a carrier protein connecting skin exposure to subsequent respiratory responses

    PubMed Central

    2011-01-01

    Background Methylene diphenyl diisocyanate (MDI), a reactive chemical used for commercial polyurethane production, is a well-recognized cause of occupational asthma. The major focus of disease prevention efforts to date has been respiratory tract exposure; however, skin exposure may also be an important route for inducing immune sensitization, which may promote subsequent airway inflammatory responses. We developed a murine model to investigate pathogenic mechanisms by which MDI skin exposure might promote subsequent immune responses, including respiratory tract inflammation. Methods Mice exposed via the skin to varying doses (0.1-10% w/v) of MDI diluted in acetone/olive oil were subsequently evaluated for MDI immune sensitization. Serum levels of MDI-specific IgG and IgE were measured by enzyme-linked immunosorbant assay (ELISA), while respiratory tract inflammation, induced by intranasal delivery of MDI-mouse albumin conjugates, was evaluated based on bronchoalveolar lavage (BAL). Autologous serum IgG from "skin only" exposed mice was used to detect and guide the purification/identification of skin proteins antigenically modified by MDI exposure in vivo. Results Skin exposure to MDI resulted in specific antibody production and promoted subsequent respiratory tract inflammation in animals challenged intranasally with MDI-mouse albumin conjugates. The degree of (secondary) respiratory tract inflammation and eosinophilia depended upon the (primary) skin exposure dose, and was maximal in mice exposed to 1% MDI, but paradoxically limited in mice receiving 10-fold higher doses (e.g. 10% MDI). The major antigenically-modified protein at the local MDI skin exposure site was identified as albumin, and demonstrated biophysical changes consistent with MDI conjugation. Conclusions MDI skin exposure can induce MDI-specific immune sensitivity and promote subsequent respiratory tract inflammatory responses and thus, may play an important role in MDI asthma pathogenesis. MDI

  16. Fusion tags for protein solubility, purification and immunogenicity in Escherichia coli: the novel Fh8 system

    PubMed Central

    Costa, Sofia; Almeida, André; Castro, António; Domingues, Lucília

    2014-01-01

    Proteins are now widely produced in diverse microbial cell factories. The Escherichia coli is still the dominant host for recombinant protein production but, as a bacterial cell, it also has its issues: the aggregation of foreign proteins into insoluble inclusion bodies is perhaps the main limiting factor of the E. coli expression system. Conversely, E. coli benefits of cost, ease of use and scale make it essential to design new approaches directed for improved recombinant protein production in this host cell. With the aid of genetic and protein engineering novel tailored-made strategies can be designed to suit user or process requirements. Gene fusion technology has been widely used for the improvement of soluble protein production and/or purification in E. coli, and for increasing peptide’s immunogenicity as well. New fusion partners are constantly emerging and complementing the traditional solutions, as for instance, the Fh8 fusion tag that has been recently studied and ranked among the best solubility enhancer partners. In this review, we provide an overview of current strategies to improve recombinant protein production in E. coli, including the key factors for successful protein production, highlighting soluble protein production, and a comprehensive summary of the latest available and traditionally used gene fusion technologies. A special emphasis is given to the recently discovered Fh8 fusion system that can be used for soluble protein production, purification, and immunogenicity in E. coli. The number of existing fusion tags will probably increase in the next few years, and efforts should be taken to better understand how fusion tags act in E. coli. This knowledge will undoubtedly drive the development of new tailored-made tools for protein production in this bacterial system. PMID:24600443

  17. [Preparation and the biological effect of fusion protein GLP-1-exendin-4/ IgG4(Fc) fusion protein as long acting GLP-1 receptor agonist].

    PubMed

    Zheng, Yun-cheng

    2015-12-01

    GLP-1 has a variety of anti-diabetic effects. However, native GLP-1 is not suitable for treatment of diabetes due to its short half-life (t½, 2-5 min). Exendin-4 is a polypeptide isolated from lizard saliva, which can bind to GLP-1 receptor, produce physiological effects similar to GLP-1, t½ up to 2.5 h, therefore, we developed a long-lasting GLP-1 receptor agonists and GLP-1-exendin-4 fusion IgG4 Fc [GLP-1-exendin-4/ IgG4(Fc)]. We constructed the eukaryotic expression vector of human GLP-1-exendin-4/IgG4(Fc)-pOptiVEC- TOPO by gene recombination technique and expressed the fusion protein human GLP-1-IgG4 (Fc) in CHO/DG44 cells. The fusion protein stimulated the INS-1 cells secretion of insulin, GLP-1, exendin-4 and fusion protein in CD1 mice pharmacokinetic experiments, as well as GLP-1, exendin-4 and fusion protein did anti-diabetic effect on streptozotocin induced mice. Results demonstrated that the GLP-1-exendin-4/IgG4(Fc) positive CHO/DG44 clones were chosen and the media from these positive clones. Western blotting showed that one protein band was found to match well with the predicted relative molecular mass of human GLP-1-exendin-4/IgG4(Fc). Insulin RIA showed that GLP-1-exendin-4/IgG4(Fc) dose-dependently stimulated insulin secretion from INS-1 cells. Pharmacokinetic studies in CD1 mice showed that with intraperitoneal injection (ip), the fusion protein peaked at 30 min in circulation and maintained a plateau for 200 h. Natural biological half-life of exendin-4 was (1.39 ± 0.28) h, GLP-1 in vivo t½ 4 min, indicating that fusion protein has long-lasting effects on the modulation of glucose homeostasis. GLP-1-exendin-4/IgG4(Fc) was found to be effective in reducing the incidence of diabetes in multiple-low-dose streptozotocin-induced diabetes in mice, longer duration of the biological activity of the fusion protein. The biological activity was significantly higher than that of GLP-1 and exendin-4. GLP-1-exendin-4/IgG4(Fc) has good anti-diabetic activity

  18. Structure of the uncleaved ectodomain of the paramyxovirus (hPIV3) fusion protein

    SciTech Connect

    Yin, Hsien-Sheng; Paterson, Reay G.; Wen, Xiaolin; Lamb, Robert A.; Jardetzky, Theodore S.

    2010-03-08

    Class I viral fusion proteins share common mechanistic and structural features but little sequence similarity. Structural insights into the protein conformational changes associated with membrane fusion are based largely on studies of the influenza virus hemagglutinin in pre- and postfusion conformations. Here, we present the crystal structure of the secreted, uncleaved ectodomain of the paramyxovirus, human parainfluenza virus 3 fusion (F) protein, a member of the class I viral fusion protein group. The secreted human parainfluenza virus 3 F forms a trimer with distinct head, neck, and stalk regions. Unexpectedly, the structure reveals a six-helix bundle associated with the postfusion form of F, suggesting that the anchor-minus ectodomain adopts a conformation largely similar to the postfusion state. The transmembrane anchor domains of F may therefore profoundly influence the folding energetics that establish and maintain a metastable, prefusion state.

  19. Structure of the uncleaved ectodomain of the paramyxovirus (hPIV3) fusion protein

    PubMed Central

    Yin, Hsien-Sheng; Paterson, Reay G.; Wen, Xiaolin; Lamb, Robert A.; Jardetzky, Theodore S.

    2005-01-01

    Class I viral fusion proteins share common mechanistic and structural features but little sequence similarity. Structural insights into the protein conformational changes associated with membrane fusion are based largely on studies of the influenza virus hemagglutinin in pre- and postfusion conformations. Here, we present the crystal structure of the secreted, uncleaved ectodomain of the paramyxovirus, human parainfluenza virus 3 fusion (F) protein, a member of the class I viral fusion protein group. The secreted human parainfluenza virus 3 F forms a trimer with distinct head, neck, and stalk regions. Unexpectedly, the structure reveals a six-helix bundle associated with the postfusion form of F, suggesting that the anchor-minus ectodomain adopts a conformation largely similar to the postfusion state. The transmembrane anchor domains of F may therefore profoundly influence the folding energetics that establish and maintain a metastable, prefusion state. PMID:15964978

  20. ALBUMIN CAUSES INCREASED MYOSIN LIGHT CHAIN KINASE EXPRESSION IN ASTROCYTES VIA P38 MITOGEN ACTIVATED PROTEIN KINASE

    PubMed Central

    Rossi, Janet L.; Ralay Ranaivo, Hantamala; Patel, Fatima; Chrzaszcz, MaryAnn; Venkatesan, Charu; Wainwright, Mark S.

    2011-01-01

    Myosin light chain kinase (MLCK) plays an important role in the reorganization of the cytoskeleton leading to disruption of vascular barrier integrity in multiple organs including the blood brain barrier (BBB) after traumatic brain injury (TBI). MLCK has been linked to transforming growth factor (TGF) and rho kinase signaling pathways, but the mechanisms regulating MLCK expression following TBI are not well understood. Albumin leaks into the brain parenchyma following TBI, activates glia and has been linked to TGF-β receptor signaling. We investigated the role of albumin in the increase in MLCK in astrocytes and the signaling pathways involved in this increase. Following midline closed-skull TBI in mice, there was a significant increase in MLCK-immunoreactive (IR) cells and albumin extravasation, which was prevented by treatment with the MLCK inhibitor ML-7. Using immunohistochemical methods, we identified the MCLK-IR cells as astrocytes. In primary astrocytes, exposure to albumin increased both isoforms of MLCK, 130 and 210. Inhibition of the TGF-β receptor partially prevented the albumin-induced increase in both isoforms, which was not prevented by inhibition of smad3. Inhibition of p38 MAPK, but not ERK, JNK or rho kinase also prevented this increase. These results are further evidence of a role of MCLK in the mechanisms of BBB compromise following TBI, and identify astrocytes as a cell type, in addition to endothelium in the BBB which express MLCK. These findings implicate albumin, acting through p38 MAPK, in a novel mechanism by which activation of MLCK following TBI may lead to compromise of the BBB. PMID:21360574

  1. Analysis of drug-protein interactions by high-performance affinity chromatography: interactions of sulfonylurea drugs with normal and glycated human serum albumin.

    PubMed

    Matsuda, Ryan; Anguizola, Jeanethe; Hoy, Krina S; Hage, David S

    2015-01-01

    High-performance affinity chromatography (HPAC) is a type of liquid chromatography that has seen growing use as a tool for the study of drug-protein interactions. This report describes how HPAC can be used to provide information on the number of binding sites, equilibrium constants, and changes in binding that can occur during drug-protein interactions. This approach will be illustrated through recent data that have been obtained by HPAC for the binding of sulfonylurea drugs and other solutes to the protein human serum albumin (HSA), and especially to forms of this protein that have been modified by non-enzymatic glycation. The theory and use of both frontal analysis and zonal elution competition studies in such work will be discussed. Various practical aspects of these experiments will be presented, as well as factors to consider in the extension of these methods to other drugs and proteins or additional types of biological interactions. PMID:25749961

  2. The heads of the measles virus attachment protein move to transmit the fusion-triggering signal

    PubMed Central

    Navaratnarajah, Chanakha K; Oezguen, Numan; Rupp, Levi; Kay, Leah; Leonard, Vincent HJ; Braun, Werner; Cattaneo, Roberto

    2010-01-01

    The measles virus entry system, constituted of attachment (hemagglutinin, H) and fusion proteins, operates based on a variety of natural and targeted receptors. However, the mechanism triggering fusion of the viral envelope with the plasma membrane is not understood. Here we tested a model considering that the two heads of an H-dimer, which are covalently linked at their base, after binding two receptor molecules, move relative to each other to transmit the fusion-triggering signal. Indeed, stabilizing the H-dimer interface by additional inter-molecular disulfide bonds prevented membrane fusion, an effect reversed by a reducing agent. Moreover, a membrane-anchored designated receptor efficiently triggered fusion, provided it engaged the H-dimer at locations proximal to where the natural receptors bind, and distal to the H-dimer interface. We discuss how receptors may force H-heads to switch partners and transmit the fusion-triggering signal. PMID:21217701

  3. Photooxidation of Tryptophan and Tyrosine Residues in Human Serum Albumin Sensitized by Pterin: A Model for Globular Protein Photodamage in Skin.

    PubMed

    Reid, Lara O; Roman, Ernesto A; Thomas, Andrés H; Dántola, M Laura

    2016-08-30

    Human serum albumin (HSA) is the most abundant protein in the circulatory system. Oxidized albumin was identified in the skin of patients suffering from vitiligo, a depigmentation disorder in which the protection against ultraviolet (UV) radiation fails because of the lack of melanin. Oxidized pterins, efficient photosensitizers under UV-A irradiation, accumulate in the skin affected by vitiligo. In this work, we have investigated the ability of pterin (Ptr), the parent compound of oxidized pterins, to induce structural and chemical changes in HSA under UV-A irradiation. Our results showed that Ptr is able to photoinduce oxidation of the protein in at least two amino acid residues: tryptophan (Trp) and tyrosine (Tyr). HSA undergoes oligomerization, yielding protein structures whose molecular weight increases with irradiation time. The protein cross-linking, due to the formation of dimers of Tyr, does not significantly affect the secondary and tertiary structures of HSA. Trp is consumed in the photosensitized process, and N-formylkynurenine was identified as one of its oxidation products. The photosensitization of HSA takes place via a purely dynamic process, which involves the triplet excited state of Ptr. The results presented in this work suggest that protein photodamage mediated by endogenous photosensitizers can significantly contribute to the harmful effects of UV-A radiation on the human skin. PMID:27500308

  4. Fragments of Target Cells are Internalized into Retroviral Envelope Protein-Expressing Cells during Cell-Cell Fusion by Endocytosis

    PubMed Central

    Izumida, Mai; Kamiyama, Haruka; Suematsu, Takashi; Honda, Eri; Koizumi, Yosuke; Yasui, Kiyoshi; Hayashi, Hideki; Ariyoshi, Koya; Kubo, Yoshinao

    2016-01-01

    Retroviruses enter into host cells by fusion between viral and host cell membranes. Retroviral envelope glycoprotein (Env) induces the membrane fusion, and also mediates cell-cell fusion. There are two types of cell-cell fusions induced by the Env protein. Fusion-from-within is induced by fusion between viral fusogenic Env protein-expressing cells and susceptible cells, and virions induce fusion-from-without by fusion between adjacent cells. Although entry of ecotropic murine leukemia virus (E-MLV) requires host cell endocytosis, the involvement of endocytosis in cell fusion is unclear. By fluorescent microscopic analysis of the fusion-from-within, we found that fragments of target cells are internalized into Env-expressing cells. Treatment of the Env-expressing cells with an endocytosis inhibitor more significantly inhibited the cell fusion than that of the target cells, indicating that endocytosis in Env-expressing cells is required for the cell fusion. The endocytosis inhibitor also attenuated the fusion-from-without. Electron microscopic analysis suggested that the membrane fusion resulting in fusion-from-within initiates in endocytic membrane dents. This study shows that two types of the viral cell fusion both require endocytosis, and provides the cascade of fusion-from-within. PMID:26834711

  5. Cofilin-induced cooperative conformational changes of actin subunits revealed using cofilin-actin fusion protein

    PubMed Central

    Umeki, Nobuhisa; Hirose, Keiko; Uyeda, Taro Q. P.

    2016-01-01

    To investigate cooperative conformational changes of actin filaments induced by cofilin binding, we engineered a fusion protein made of Dictyostelium cofilin and actin. The filaments of the fusion protein were functionally similar to actin filaments bound with cofilin in that they did not bind rhodamine-phalloidin, had quenched fluorescence of pyrene attached to Cys374 and showed enhanced susceptibility of the DNase loop to cleavage by subtilisin. Quantitative analyses of copolymers made of different ratios of the fusion protein and control actin further demonstrated that the fusion protein affects the structure of multiple neighboring actin subunits in copolymers. Based on these and other recent related studies, we propose a mechanism by which conformational changes induced by cofilin binding is propagated unidirectionally to the pointed ends of the filaments, and cofilin clusters grow unidirectionally to the pointed ends following this path. Interestingly, the fusion protein was unable to copolymerize with control actin at pH 6.5 and low ionic strength, suggesting that the structural difference between the actin moiety in the fusion protein and control actin is pH-sensitive. PMID:26842224

  6. Construction of three different recombinant scorpion fusion proteins with bifunctional activity.

    PubMed

    Cui, Y; Guo, G L; Liu, Y F; Mao, Y Z; Zhang, R; Wu, C F; Zhang, J H

    2011-06-01

    This is the first report of three different fusion proteins with an antitumor-analgesic peptide obtained from Chinese scorpion Buthus martensii Karsch (BmKAGAP). The fusion proteins were constructed in the form of chimeric toxins, aiming to obtain bifunctional analgesic and antitumor activity. The fusion proteins consisted of luteinizing hormone-releasing hormone (LHRH), three different types of flexible linkers (L1, Ser-Ser-His-His-His-His-His-His-Ser-Ser-Gly-Leu-Val-Pro-Arg-Gly-Ser-His-Met; L2, Gly-Gly-Gly-Ser-Gly-Gly-Gly-Ser; L3, Ser-Gly-Gly-Ser-Gly-Gly-Ser-Gly-Gly-Gly-Ser-Ser-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser), and BmKAGAP. The genes coding three fusion proteins were cloned and expressed in E. coli in soluble form. Following two successive column chromatographic separations, purified fusion proteins were obtained. These fusion proteins exhibited analgesic activity in mice and were cytotoxic to a hepatocellular carcinoma cell line Hep3B. PMID:21793303

  7. Towards reconstitution of membrane fusion mediated by SNAREs and other synaptic proteins

    PubMed Central

    Brunger, Axel T.; Cipriano, Daniel J.; Diao, Jiajie

    2015-01-01

    Abstract Proteoliposomes have been widely used for in vitro studies of membrane fusion mediated by synaptic proteins. Initially, such studies were made with large unsynchronized ensembles of vesicles. Such ensemble assays limited the insights into the SNARE-mediated fusion mechanism that could be obtained from them. Single particle microscopy experiments can alleviate many of these limitations but they pose significant technical challenges. Here we summarize various approaches that have enabled studies of fusion mediated by SNAREs and other synaptic proteins at a single-particle level. Currently available methods are described and their advantages and limitations are discussed. PMID:25788028

  8. Cell-based analysis of Chikungunya virus E1 protein in membrane fusion

    PubMed Central

    2012-01-01

    Background Chikungunya fever is a pandemic disease caused by the mosquito-borne Chikungunya virus (CHIKV). E1 glycoprotein mediation of viral membrane fusion during CHIKV infection is a crucial step in the release of viral genome into the host cytoplasm for replication. How the E1 structure determines membrane fusion and whether other CHIKV structural proteins participate in E1 fusion activity remain largely unexplored. Methods A bicistronic baculovirus expression system to produce recombinant baculoviruses for cell-based assay was used. Sf21 insect cells infected by recombinant baculoviruses bearing wild type or single-amino-acid substitution of CHIKV E1 and EGFP (enhanced green fluorescence protein) were employed to investigate the roles of four E1 amino acid residues (G91, V178, A226, and H230) in membrane fusion activity. Results Western blot analysis revealed that the E1 expression level and surface features in wild type and mutant substituted cells were similar. However, cell fusion assay found that those cells infected by CHIKV E1-H230A mutant baculovirus showed little fusion activity, and those bearing CHIKV E1-G91D mutant completely lost the ability to induce cell-cell fusion. Cells infected by recombinant baculoviruses of CHIKV E1-A226V and E1-V178A mutants exhibited the same membrane fusion capability as wild type. Although the E1 expression level of cells bearing monomeric-E1-based constructs (expressing E1 only) was greater than that of cells bearing 26S-based constructs (expressing all structural proteins), the sizes of syncytial cells induced by infection of baculoviruses containing 26S-based constructs were larger than those from infections having monomeric-E1 constructs, suggesting that other viral structure proteins participate or regulate E1 fusion activity. Furthermore, membrane fusion in cells infected by baculovirus bearing the A226V mutation constructs exhibited increased cholesterol-dependences and lower pH thresholds. Cells bearing the V178

  9. Intrinsic activity of human immunodeficiency virus type 1 protease heterologous fusion proteins in mammalian cells.

    PubMed

    Arrigo, S J; Haines, J K; Huffman, K M

    1995-01-01

    We have generated various mammalian expression constructs that produce fusion proteins of human immunodeficiency virus type 1 (HIV-1) protease (PR) with the HIV-1 Nef protein. The expression of these proteins is inducible by the HIV-1 Tat protein. High-level expression of proteolytically active PR was produced from PR imbedded into Nef coding sequences, flanked by PR cleavage sites. The fusion protein was cleaved nearly to completion and did not exhibit the regulated processing that is seen with the virally encoded PR. No cytotoxic effect of PR expression was detected. The self-cleavage of PR could be inhibited by a specific inhibitor of HIV-1 PR (U75875). Elimination of the aminoterminal PR cleavage site did not have a measurable effect on cleavage of the precursor fusion protein. The cleaved fusion proteins appeared to be extremely unstable in the transfected cells. These findings demonstrate the intrinsic activity of HIV-1 PR in mammalian cells, in the context of a heterologous fusion protein. PMID:7832989

  10. SUMO fusions and SUMO-specific protease for efficient expression and purification of proteins.

    PubMed

    Malakhov, Michael P; Mattern, Michael R; Malakhova, Oxana A; Drinker, Mark; Weeks, Stephen D; Butt, Tauseef R

    2004-01-01

    SUMO (small ubiquitin-related modifier) modulates protein structure and function by covalently binding to the lysine side chains of the target proteins. Yeast cells contain two SUMO proteases, Ulp1 and Ulp2, that cleave sumoylated proteins in the cell. Ulp1 (SUMO protease 1) processes the SUMO precursor to its mature form and also de-conjugates SUMO from side chain lysines of target proteins. Here we demonstrate that attachment of SUMO to the N-terminus of under-expressed proteins dramatically enhances their expression in E. coli. SUMO protease 1 was able to cleave a variety of SUMO fusions robustly and with impeccable specificity. Purified recombinant SUMO-GFPs were efficiently cleaved when any amino acid, except proline, was in the+1 position of the cleavage site. The enzyme was active over a broad range of buffer and temperature conditions. Purification of certain recombinant proteins is accomplished by production of Ub-fusions from which Ub can be subsequently removed by de-ubiquitinating enzymes (DUBs). However, DUBs are unstable enzymes that are difficult to produce and inexpensive DUBs are not available commercially. Our findings demonstrate that SUMO protease 1/SUMO-fusion system may be preferable to DUB/Ub-fusion. Enhanced expression and solubility of proteins fused to SUMO combined with broad specificity and highly efficient cleavage properties of the SUMO protease 1 indicates that SUMO-fusion technology will become a useful tool in purification of proteins and peptides. PMID:15263846

  11. Copper inhibits activated protein C: protective effect of human albumin and an analogue of its high-affinity copper-binding site, d-DAHK.

    PubMed

    Bar-Or, David; Rael, Leonard T; Winkler, James V; Yukl, Richard L; Thomas, Gregory W; Shimonkevitz, Richard P

    2002-02-01

    Activated protein C (APC) is useful in the treatment of sepsis. Ischemia and acidosis, which often accompany sepsis, cause the release of copper from loosely bound sites. We investigated (i) whether physiological concentrations of copper inhibit APC anticoagulant activity and (ii) if any copper-induced APC inhibition is reversible by human serum albumin (HSA) or a high-affinity copper-binding analogue of the human albumin N-terminus, d-Asp-d-Ala-d-His-d-Lys (d-DAHK). APC activity after 30 min of incubation with CuCl2 (10 microM) was decreased 26% below baseline. HSA, both alone and when combined with various ratios of CuCl2, increased APC activity significantly above baseline. d-DAHK alone and 2:1 and 4:1 ratios of d-DAHK:CuCl2 also increased APC activity. APC contained 1.4 microM copper, which helps explain the increased APC activity with HSA and d-DAHK alone. These in vitro results indicate that copper inhibits APC activity and that albumin and d-DAHK reverse the copper-induced APC deactivation. PMID:11820775

  12. Assessment of protein domain fusions in human protein interaction networks prediction: application to the human kinetochore model.

    PubMed

    Morilla, Ian; Lees, Jon G; Reid, Adam J; Orengo, Christine; Ranea, Juan A G

    2010-12-31

    In order to understand how biological systems function it is necessary to determine the interactions and associations between proteins. Some proteins, involved in a common biological process and encoded by separate genes in one organism, can be found fused within a single protein chain in other organisms. By detecting these triplets, a functional relationship can be established between the unfused proteins. Here we use a domain fusion prediction method to predict these protein interactions for the human interactome. We observed that gene fusion events are more related to physical interaction between proteins than to other weaker functional relationships such as participation in a common biological pathway. These results suggest that domain fusion is an appropriate method for predicting protein complexes. The most reliable fused domain predictions were used to build protein-protein interaction (PPI) networks. These predicted PPI network models showed the same topological features as real biological networks and different features from random behaviour. We built the PPI domain fusion sub-network model of the human kinetochore and observed that the majority of the predicted interactions have not yet been experimentally characterised in the publicly available PPI repositories. The study of the human kinetochore domain fusion sub-network reveals undiscovered kinetochore proteins with presumably relevant functions, such as hubs with many connections in the kinetochore sub-network. These results suggest that experimentally hidden regions in the predicted PPI networks contain key functional elements, associated with important functional areas, still undiscovered in the human interactome. Until novel experiments shed light on these hidden regions; domain fusion predictions provide a valuable approach for exploring them. PMID:20851221

  13. Polymeric human Fc-fusion proteins with modified effector functions

    NASA Astrophysics Data System (ADS)

    Mekhaiel, David N. A.; Czajkowsky, Daniel M.; Andersen, Jan Terje; Shi, Jianguo; El-Faham, Marwa; Doenhoff, Michael; McIntosh, Richard S.; Sandlie, Inger; He, Jianfeng; Hu, Jun; Shao, Zhifeng; Pleass, Richard J.

    2011-10-01

    The success of Fc-fusion bio-therapeutics has spurred the development of other Fc-fusion products for treating and/or vaccinating against a range of diseases. We describe a method to modulate their function by converting them into well-defined stable polymers. This strategy resulted in cylindrical hexameric structures revealed by tapping mode atomic force microscopy (AFM). Polymeric Fc-fusions were significantly less immunogenic than their dimeric or monomeric counterparts, a result partly owing to their reduced ability to interact with critical Fc-receptors. However, in the absence of the fusion partner, polymeric IgG1-Fc molecules were capable of binding selectively to FcγRs, with significantly increased affinity owing to their increased valency, suggesting that these reagents may prove of immediate utility in the development of well-defined replacements for intravenous immunoglobulin (IVIG) therapy. Overall, these findings establish an effective IgG Fc-fusion based polymeric platform with which the therapeutic and vaccination applications of Fc-fusion immune-complexes can now be explored.

  14. Rapamycin-induced oligomer formation system of FRB-FKBP fusion proteins.

    PubMed

    Inobe, Tomonao; Nukina, Nobuyuki

    2016-07-01

    Most proteins form larger protein complexes and perform multiple functions in the cell. Thus, artificial regulation of protein complex formation controls the cellular functions that involve protein complexes. Although several artificial dimerization systems have already been used for numerous applications in biomedical research, cellular protein complexes form not only simple dimers but also larger oligomers. In this study, we showed that fusion proteins comprising the induced heterodimer formation proteins FRB and FKBP formed various oligomers upon addition of rapamycin. By adjusting the configuration of fusion proteins, we succeeded in generating an inducible tetramer formation system. Proteins of interest also formed tetramers by fusing to the inducible tetramer formation system, which exhibits its utility in a broad range of biological applications. PMID:26777239

  15. Fusion

    NASA Astrophysics Data System (ADS)

    Herman, Robin

    1990-10-01

    The book abounds with fascinating anecdotes about fusion's rocky path: the spurious claim by Argentine dictator Juan Peron in 1951 that his country had built a working fusion reactor, the rush by the United States to drop secrecy and publicize its fusion work as a propaganda offensive after the Russian success with Sputnik; the fortune Penthouse magazine publisher Bob Guccione sank into an unconventional fusion device, the skepticism that met an assertion by two University of Utah chemists in 1989 that they had created "cold fusion" in a bottle. Aimed at a general audience, the book describes the scientific basis of controlled fusion--the fusing of atomic nuclei, under conditions hotter than the sun, to release energy. Using personal recollections of scientists involved, it traces the history of this little-known international race that began during the Cold War in secret laboratories in the United States, Great Britain and the Soviet Union, and evolved into an astonishingly open collaboration between East and West.

  16. Polyclonal and monoclonal antibodies specific for the six-helix bundle of the human respiratory syncytial virus fusion glycoprotein as probes of the protein post-fusion conformation

    SciTech Connect

    Palomo, Concepción; Mas, Vicente; Vázquez, Mónica; Cano, Olga; Luque, Daniel; Terrón, María C.; Calder, Lesley J.; Melero, José A.

    2014-07-15

    Human respiratory syncytial virus (hRSV) has two major surface glycoproteins (G and F) anchored in the lipid envelope. Membrane fusion promoted by hRSV{sub F} occurs via refolding from a pre-fusion form to a highly stable post-fusion state involving large conformational changes of the F trimer. One of these changes results in assembly of two heptad repeat sequences (HRA and HRB) into a six-helix bundle (6HB) motif. To assist in distinguishing pre- and post-fusion conformations of hRSV{sub F}, we have prepared polyclonal (α-6HB) and monoclonal (R145) rabbit antibodies specific for the 6HB. Among other applications, these antibodies were used to explore the requirements of 6HB formation by isolated protein segments or peptides and by truncated mutants of the F protein. Site-directed mutagenesis and electron microscopy located the R145 epitope in the post-fusion hRSV{sub F} at a site distantly located from previously mapped epitopes, extending the repertoire of antibodies that can decorate the F molecule. - Highlights: • Antibodies specific for post-fusion respiratory syncytial virus fusion protein are described. • Polyclonal antibodies were obtained in rabbit inoculated with chimeric heptad repeats. • Antibody binding required assembly of a six-helix bundle in the post-fusion protein. • A monoclonal antibody with similar structural requirements is also described. • Binding of this antibody to the post-fusion protein was visualized by electron microscopy.

  17. Enhanced expression of rabies virus surface G-protein in Escherichia coli using SUMO fusion.

    PubMed

    Singh, Ankit; Yadav, Dinesh; Rai, Krishan Mohan; Srivastava, Meenal; Verma, Praveen C; Singh, Pradhyumna K; Tuli, Rakesh

    2012-01-01

    Fusion systems are known to increase the expression of difficult to express recombinant proteins in soluble form to facilitate their purification. Rabies glycoprotein was also tough to express at sufficient level in soluble form in both E. coli and plant. The present work was aimed to over-express and purify this membrane protein from soluble extract of E. coli. Fusion of Small Ubiqutin like Modifier (SUMO) with rabies glycoprotein increased ~1.5 fold higher expression and ~3.0 fold solubility in comparison to non-fused in E. coli. The SUMO fusion also simplified the purification process. Previously engineered rabies glycoprotein gene in tobacco plants provides complete protection to mice, but the expression was very low for purification. Our finding demonstrated that the SUMO-fusion was useful for enhancing expression and solubility of the membrane protein and again proves to be a good alternative technology for applications in biomedical and pharmaceutical research. PMID:22134654

  18. Enhanced protein expression in mammalian cells using engineered SUMO fusions: secreted phospholipase A2.

    PubMed

    Peroutka, Raymond J; Elshourbagy, Nabil; Piech, Tara; Butt, Tauseef R

    2008-09-01

    SUMOylation, the covalent attachment of SUMO (small ubiquitin-like modifier), is a eukaryotic post-translational event that has been demonstrated to play a critical role in several biological processes. When used as an N-terminal tag or fusion partner, SUMO has been shown to enhance functional protein production significantly by improving folding, solubility, and stability. We have engineered several SUMOs and, through their fusion, developed a system for enhancing the expression and secretion of complex proteins. To demonstrate the fidelity of this fusion technology, secreted phospholipase A(2) proteins (sPLA(2)) were produced using HEK-293T and CHO-K1 cells. Five mouse sPLA(2) homologs were expressed and secreted in mammalian cell cultures using SUMO or SUMO-derived, N-terminal fusion partners. Mean and median increases of 43- and 18-fold, respectively, were obtained using novel SUMO mutants that are resistant to digestion by endogenous deSUMOylases. PMID:18539905

  19. Mitochondrial Fusion and ERK Activity Regulate Steroidogenic Acute Regulatory Protein Localization in Mitochondria

    PubMed Central

    Duarte, Alejandra; Castillo, Ana Fernanda; Podestá, Ernesto J.; Poderoso, Cecilia

    2014-01-01

    The rate-limiting step in the biosynthesis of steroid hormones, known as the transfer of cholesterol from the outer to the inner mitochondrial membrane, is facilitated by StAR, the Steroidogenic Acute Regulatory protein. We have described that mitochondrial ERK1/2 phosphorylates StAR and that mitochondrial fusion, through the up-regulation of a fusion protein Mitofusin 2, is essential during steroidogenesis. Here, we demonstrate that mitochondrial StAR together with mitochondrial active ERK and PKA are necessary for maximal steroid production. Phosphorylation of StAR by ERK is required for the maintenance of this protein in mitochondria, observed by means of over-expression of a StAR variant lacking the ERK phosphorylation residue. Mitochondrial fusion regulates StAR levels in mitochondria after hormone stimulation. In this study, Mitofusin 2 knockdown and mitochondrial fusion inhibition in MA-10 Leydig cells diminished StAR mRNA levels and concomitantly mitochondrial StAR protein. Together our results unveil the requirement of mitochondrial fusion in the regulation of the localization and mRNA abundance of StAR. We here establish the relevance of mitochondrial phosphorylation events in the correct localization of this key protein to exert its action in specialized cells. These discoveries highlight the importance of mitochondrial fusion and ERK phosphorylation in cholesterol transport by means of directing StAR to the outer mitochondrial membrane to achieve a large number of steroid molecules per unit of StAR. PMID:24945345

  20. Composites containing albumin protein or cyanoacrylate adhesives and biodegradable scaffolds: II. In vivo wound closure study in a rat model

    NASA Astrophysics Data System (ADS)

    McNally-Heintzelman, Karen M.; Heintzelman, Douglas L.; Duffy, Mark T.; Bloom, Jeffrey N.; Soller, Eric C.; Gilmour, Travis M.; Hoffman, Grant T.; Edward, Deepak

    2004-07-01

    Our Scaffold-Enhanced Biological Adhesive (SEBA) system was investigated as an alternative to sutures or adhesives alone for repair of wounds. Two scaffold materials were investigated: (i) a synthetic biodegradable material fabricated from poly(L-lactic-co-glycolic acid); and (ii) a biologic material, small intestinal submucosa, manufactured by Cook BioTech. Two adhesive materials were also investigated: (i) a biologic adhesive composed of 50%(w/v) bovine serum albumin solder and 0.5mg/ml indocyanine green dye mixed in deionized water, and activated with an 808-nm diode laser; and (ii) Ethicon"s Dermabond, a 2-octyl-cyanoacrylate. The tensile strength and time-to-failure of skin incisions repaired in vivo in a rat model were measured at seven days postoperative. Incisions closed by protein solder alone, by Dermabond alone, or by suture, were also tested for comparison. The tensile strength of repairs formed using the SEBA system were 50% to 65% stronger than repairs formed by suture or either adhesive alone, with significantly less variations within each experimental group (average standard deviations of 15% for SEBA versus 38% for suture and 28% for adhesive alone). In addition, the time-to-failure curves showed a longevity not previously seen with the suture or adhesive alone techniques. The SEBA system acts to keep the dermis in tight apposition during the critical early phase of wound healing when tissue gaps are bridged by scar and granulation tissue. It has the property of being more flexible than either of the adhesives alone and may allow the apposed edges to move in conjunction with each other as a unit for a longer period of time and over a greater range of stresses than adhesives alone. This permits more rapid healing and establishment of integrity since the microgaps between the dermis edges are significantly reduced. By the time the scaffolds are sloughed from the wound site, there is greater strength and healing than that produced by adhesive alone or

  1. Enhanced protein expression in the baculovirus/insect cell system using engineered SUMO fusions.

    PubMed

    Liu, Li; Spurrier, Joshua; Butt, Tauseef R; Strickler, James E

    2008-11-01

    Recombinant protein expression in insect cells varies greatly from protein to protein. A fusion tag that is not only a tool for detection and purification, but also enhances expression and/or solubility would greatly facilitate both structure/function studies and therapeutic protein production. We have shown that fusion of SUMO (small ubiquitin-related modifier) to several test proteins leads to enhanced expression levels in Escherichia coli. In eukaryotic expression systems, however, the SUMO tag could be cleaved by endogenous desumoylase. In order to adapt SUMO-fusion technology to these systems, we have developed an alternative SUMO-derived tag, designated SUMOstar, which is not processed by native SUMO proteases. In the present study, we tested the SUMOstar tag in a baculovirus/insect cell system with several proteins, i.e. mouse UBP43, human tryptase beta II, USP4, USP15, and GFP. Our results demonstrate that fusion to SUMOstar enhanced protein expression levels at least 4-fold compared to either the native or His(6)-tagged proteins. We isolated active SUMOstar tagged UBP43, USP4, USP15, and GFP. Tryptase was active following cleavage with a SUMOstar specific protease. The SUMOstar system will make significant impact in difficult-to-express proteins and especially to those proteins that require the native N-terminal residue for function. PMID:18713650

  2. Connecting two proteins using a fusion alpha helix stabilized by a chemical cross linker

    NASA Astrophysics Data System (ADS)

    Jeong, Woo Hyeon; Lee, Haerim; Song, Dong Hyun; Eom, Jae-Hoon; Kim, Sun Chang; Lee, Hee-Seung; Lee, Hayyoung; Lee, Jie-Oh

    2016-03-01

    Building a sophisticated protein nano-assembly requires a method for linking protein components in a predictable and stable structure. Most of the cross linkers available have flexible spacers. Because of this, the linked hybrids have significant structural flexibility and the relative structure between their two components is largely unpredictable. Here we describe a method of connecting two proteins via a `fusion α helix' formed by joining two pre-existing helices into a single extended helix. Because simple ligation of two helices does not guarantee the formation of a continuous helix, we used EY-CBS, a synthetic cross linker that has been shown to react selectively with cysteines in α-helices, to stabilize the connecting helix. Formation and stabilization of the fusion helix was confirmed by determining the crystal structures of the fusion proteins with and without bound EY-CBS. Our method should be widely applicable for linking protein building blocks to generate predictable structures.

  3. Continuous Flow Separation of Hydrophobin Fusion Proteins from Plant Cell Culture Extract.

    PubMed

    Reuter, Lauri J; Conley, Andrew J; Joensuu, Jussi J

    2016-01-01

    Fusion to fungal hydrophobins has proven to be a useful tool to enhance accumulation and recovery of recombinant proteins in plants. Aqueous two-phase separation (ATPS) is an attractive system to capture hydrophobin fusion proteins from plant extracts. The process can simultaneously purify and concentrate target protein with minimal background. ATPS avoids the use of chromatographic column steps, can be carried out in a short time frame, and is amenable to industrial-scale protein purification. A drawback of performing ATPS in large volumes is the lengthy time required for phase separation; however, this can be avoided by incorporating continuous systems, which are often preferred by the processing industry. This method chapter illustrates the capture of GFP-HFBI hydrophobin fusion protein from BY-2 plant cell suspension extract using a semi-continuous ATPS method. PMID:26614291

  4. Connecting two proteins using a fusion alpha helix stabilized by a chemical cross linker

    PubMed Central

    Jeong, Woo Hyeon; Lee, Haerim; Song, Dong Hyun; Eom, Jae-Hoon; Kim, Sun Chang; Lee, Hee-Seung; Lee, Hayyoung; Lee, Jie-Oh

    2016-01-01

    Building a sophisticated protein nano-assembly requires a method for linking protein components in a predictable and stable structure. Most of the cross linkers available have flexible spacers. Because of this, the linked hybrids have significant structural flexibility and the relative structure between their two components is largely unpredictable. Here we describe a method of connecting two proteins via a ‘fusion α helix' formed by joining two pre-existing helices into a single extended helix. Because simple ligation of two helices does not guarantee the formation of a continuous helix, we used EY-CBS, a synthetic cross linker that has been shown to react selectively with cysteines in α-helices, to stabilize the connecting helix. Formation and stabilization of the fusion helix was confirmed by determining the crystal structures of the fusion proteins with and without bound EY-CBS. Our method should be widely applicable for linking protein building blocks to generate predictable structures. PMID:26980593

  5. β2 Adrenergic Receptor Fluorescent Protein Fusions Traffic to the Plasma Membrane and Retain Functionality

    PubMed Central

    Bubnell, Jaclyn; Pfister, Patrick; Sapar, Maria L.; Rogers, Matthew E.; Feinstein, Paul

    2013-01-01

    Green fluorescent protein (GFP) has proven useful for the study of protein interactions and dynamics for the last twenty years. A variety of new fluorescent proteins have been developed that expand the use of available excitation spectra. We have undertaken an analysis of seven of the most useful fluorescent proteins (XFPs), Cerulean (and mCerulean3), Teal, GFP, Venus, mCherry and TagRFP657, as fusions to the archetypal G-protein coupled receptor, the β2 adrenergic receptor (β2AR). We have characterized these β2AR::XFP fusions in respect to membrane trafficking and G-protein activation. We noticed that in the mouse neural cell line, OP 6, that membrane bound β2AR::XFP fusions robustly localized in the filopodia identical to gap::XFP fusions. All β2AR::XFP fusions show responses indistinguishable from each other and the non-fused form after isoprenaline exposure. Our results provide a platform by which G-protein coupled receptors can be dissected for their functionality. PMID:24086401

  6. Polybasic KKR Motif in the Cytoplasmic Tail of Nipah Virus Fusion Protein Modulates Membrane Fusion by Inside-Out Signaling▿

    PubMed Central

    Aguilar, Hector C.; Matreyek, Kenneth A.; Choi, Daniel Y.; Filone, Claire Marie; Young, Sophia; Lee, Benhur

    2007-01-01

    The cytoplasmic tails of the envelope proteins from multiple viruses are known to contain determinants that affect their fusogenic capacities. Here we report that specific residues in the cytoplasmic tail of the Nipah virus fusion protein (NiV-F) modulate its fusogenic activity. Truncation of the cytoplasmic tail of NiV-F greatly inhibited cell-cell fusion. Deletion and alanine scan analysis identified a tribasic KKR motif in the membrane-adjacent region as important for modulating cell-cell fusion. The K1A mutation increased fusion 5.5-fold, while the K2A and R3A mutations decreased fusion 3- to 5-fold. These results were corroborated in a reverse-pseudotyped viral entry assay, where receptor-pseudotyped reporter virus was used to infect cells expressing wild-type or mutant NiV envelope glycoproteins. Differential monoclonal antibody binding data indicated that hyper- or hypofusogenic mutations in the KKR motif affected the ectodomain conformation of NiV-F, which in turn resulted in faster or slower six-helix bundle formation, respectively. However, we also present evidence that the hypofusogenic phenotypes of the K2A and R3A mutants were effected via distinct mechanisms. Interestingly, the K2A mutant was also markedly excluded from lipid rafts, where ∼20% of wild-type F and the other mutants can be found. Finally, we found a strong negative correlation between the relative fusogenic capacities of these cytoplasmic-tail mutants and the avidities of NiV-F and NiV-G interactions (P = 0.007, r2 = 0.82). In toto, our data suggest that inside-out signaling by specific residues in the cytoplasmic tail of NiV-F can modulate its fusogenicity by multiple distinct mechanisms. PMID:17301148

  7. A Humanized Mouse Model to Study Human Albumin and Albumin Conjugates Pharmacokinetics.

    PubMed

    Low, Benjamin E; Wiles, Michael V

    2016-01-01

    Albumin is a large, highly abundant protein circulating in the blood stream which is regulated and actively recycled via the neonatal Fc receptor (FcRn). In humans this results in serum albumin having an exceptional long half-life of ~21 days. Some time ago it was realized that these intrinsic properties could be harnessed and albumin could be used as a privileged drug delivery vehicle. However, active development of albumin based therapeutics has been hampered by the lack of economic, relevant experimental models which can accurately recapitulate human albumin metabolism and pharmacokinetics. In mice for example, introduced human albumin is not recycled and is catabolized rapidly. This is mainly due to the failure of mouse FcRn to bind human albumin consequently, human albumin has a half-life of only 2-3 days in mice. To overcome this we developed and characterized a humanized mouse model which is null for mouse FcRn and mouse albumin, but is transgenic for, and expressing functional human FcRn. Published data clearly demonstrate that upon injection of human albumin into this model animal that it accurately recapitulates human albumin FcRn dependent serum recycling, with human albumin now having a half-life ~24 days, closely mimicking that observed in humans. In this practical review we briefly review this model and outline its use for pharmacokinetic studies of human albumin. PMID:27150087

  8. The novel Fh8 and H fusion partners for soluble protein expression in Escherichia coli: a comparison with the traditional gene fusion technology.

    PubMed

    Costa, Sofia J; Almeida, André; Castro, António; Domingues, Lucília; Besir, Hüseyin

    2013-08-01

    The Escherichia coli host system is an advantageous choice for simple and inexpensive recombinant protein production but it still presents bottlenecks at expressing soluble proteins from other organisms. Several efforts have been taken to overcome E. coli limitations, including the use of fusion partners that improve protein expression and solubility. New fusion technologies are emerging to complement the traditional solutions. This work evaluates two novel fusion partners, the Fh8 tag (8 kDa) and the H tag (1 kDa), as solubility enhancing tags in E. coli and their comparison to commonly used fusion partners. A broad range comparison was conducted in a small-scale screening and subsequently scaled-up. Six difficult-to-express target proteins (RVS167, SPO14, YPK1, YPK2, Frutalin and CP12) were fused to eight fusion tags (His, Trx, GST, MBP, NusA, SUMO, H and Fh8). The resulting protein expression and solubility levels were evaluated by sodium dodecyl sulfate polyacrylamide gel electrophoresis before and after protein purification and after tag removal. The Fh8 partner improved protein expression and solubility as the well-known Trx, NusA or MBP fusion partners. The H partner did not function as a solubility tag. Cleaved proteins from Fh8 fusions were soluble and obtained in similar or higher amounts than proteins from the cleavage of other partners as Trx, NusA or MBP. The Fh8 fusion tag therefore acts as an effective solubility enhancer, and its low molecular weight potentially gives it an advantage over larger solubility tags by offering a more reliable assessment of the target protein solubility when expressed as a fusion protein. PMID:23160981

  9. Expression of Leukemia-Associated Nup98 Fusion Proteins Generates an Aberrant Nuclear Envelope Phenotype

    PubMed Central

    Fahrenkrog, Birthe; Martinelli, Valérie; Nilles, Nadine; Fruhmann, Gernot; Chatel, Guillaume; Juge, Sabine; Sauder, Ursula; Di Giacomo, Danika; Mecucci, Cristina; Schwaller, Jürg

    2016-01-01

    Chromosomal translocations involving the nucleoporin NUP98 have been described in several hematopoietic malignancies, in particular acute myeloid leukemia (AML). In the resulting chimeric proteins, Nup98's N-terminal region is fused to the C-terminal region of about 30 different partners, including homeodomain (HD) transcription factors. While transcriptional targets of distinct Nup98 chimeras related to immortalization are relatively well described, little is known about other potential cellular effects of these fusion proteins. By comparing the sub-nuclear localization of a large number of Nup98 fusions with HD and non-HD partners throughout the cell cycle we found that while all Nup98 chimeras were nuclear during interphase, only Nup98-HD fusion proteins exhibited a characteristic speckled appearance. During mitosis, only Nup98-HD fusions were concentrated on chromosomes. Despite the difference in localization, all tested Nup98 chimera provoked morphological alterations in the nuclear envelope (NE), in particular affecting the nuclear lamina and the lamina-associated polypeptide 2α (LAP2α). Importantly, such aberrations were not only observed in transiently transfected HeLa cells but also in mouse bone marrow cells immortalized by Nup98 fusions and in cells derived from leukemia patients harboring Nup98 fusions. Our findings unravel Nup98 fusion-associated NE alterations that may contribute to leukemogenesis. PMID:27031510

  10. Mo-CBP3, an Antifungal Chitin-Binding Protein from Moringa oleifera Seeds, Is a Member of the 2S Albumin Family

    PubMed Central

    Freire, José E. C.; Vasconcelos, Ilka M.; Moreno, Frederico B. M. B.; Batista, Adelina B.; Lobo, Marina D. P.; Pereira, Mirella L.; Lima, João P. M. S.; Almeida, Ricardo V. M.; Sousa, Antônio J. S.; Monteiro-Moreira, Ana C. O.; Oliveira, José T. A.; Grangeiro, Thalles B.

    2015-01-01

    Mo-CBP3 is a chitin-binding protein from M. oleifera seeds that inhibits the germination and mycelial growth of phytopathogenic fungi. This protein is highly thermostable and resistant to pH changes, and therefore may be useful in the development of new antifungal drugs. However, the relationship of MoCBP3 with the known families of carbohydrate-binding domains has not been established. In the present study, full-length cDNAs encoding 4 isoforms of Mo-CBP3 (Mo-CBP3-1, Mo-CBP3-2, Mo-CBP3-3 and Mo-CBP3-4) were cloned from developing seeds. The polypeptides encoded by the Mo-CBP3 cDNAs were predicted to contain 160 (Mo-CBP3-3) and 163 amino acid residues (Mo-CBP3-1, Mo-CBP3-2 and Mo-CBP3-4) with a signal peptide of 20-residues at the N-terminal region. A comparative analysis of the deduced amino acid sequences revealed that Mo-CBP3 is a typical member of the 2S albumin family, as shown by the presence of an eight-cysteine motif, which is a characteristic feature of the prolamin superfamily. Furthermore, mass spectrometry analysis demonstrated that Mo-CBP3 is a mixture of isoforms that correspond to different mRNA products. The identification of Mo-CBP3 as a genuine member of the 2S albumin family reinforces the hypothesis that these seed storage proteins are involved in plant defense. Moreover, the chitin-binding ability of Mo-CBP3 reveals a novel functionality for a typical 2S albumin. PMID:25789746

  11. Selective hydrolysis of milk proteins to facilitate the elimination of the ABBOS epitope of bovine serum albumin and other immunoreactive epitopes.

    PubMed

    Alting, A C; Meijer, R J; van Beresteijn, E C

    1998-08-01

    Milk proteins are hydrolyzed to prevent immunological reactions, but immunoreactive epitopes, including the ABBOS epitope of bovine serum albumin (BSA), can still be detected in commercially available milk protein hydrolysates. We used lactococcal cell-envelope proteinase (CEP) for the hydrolysis of the individual milk proteins and of mixtures thereof, or for the hydrolysis of sodium caseinate (contaminated with whey proteins). CEP exclusively degraded casein, leaving the four major whey proteins intact. This property facilitated the removal of the intact whey proteins from the casein fragments by ultrafiltration. Depending on the molecular mass of the whey protein to be removed, membranes with cutoff values between 3 and 30 kDa were used, resulting in casein hydrolysates free of protein fragments with cross-reactive whey-protein-specific IgE (immunoglobulin E) or ABBOS antibody-binding sites. Even the casein itself was degraded in such a way by CEP that cross-reactive casein-specific IgE antibody-binding sites could be eliminated. The product could find application in infant formulas for therapeutic and preventive treatment of children with cow's milk allergy; in addition, the preventive use of such formulas in children genetically susceptible to the development of insulin-dependent diabetes mellitus (IDDM) should be considered if a relationship between the consumption of BSA and IDDM were to become more apparent. The method is also applicable for preparing casein-free whey protein preparations. PMID:9713762

  12. Trophoblast Cell Fusion and Differentiation Are Mediated by Both the Protein Kinase C and A Pathways

    PubMed Central

    Omata, Waka; Ackerman, William E.; Vandre, Dale D.; Robinson, John M.

    2013-01-01

    The syncytiotrophoblast of the human placenta is an epithelial barrier that interacts with maternal blood and is a key for the transfer of nutrients and other solutes to the developing fetus. The syncytiotrophoblast is a true syncytium and fusion of progenitor cytotrophoblasts is the cardinal event leading to the formation of this layer. BeWo cells are often used as a surrogate for cytotrophoblasts, since they can be induced to fuse, and then express certain differentiation markers associated with trophoblast syncytialization. Dysferlin, a syncytiotrophoblast membrane repair protein, is up-regulated in BeWo cells induced to fuse by treatment with forskolin; this fusion is thought to occur through cAMP/protein kinase A-dependent mechanisms. We hypothesized that dysferlin may also be up-regulated in response to fusion through other pathways. Here, we show that BeWo cells can also be induced to fuse by treatment with an activator of protein kinase C, and that this fusion is accompanied by increased expression of dysferlin. Moreover, a dramatic synergistic increase in dysferlin expression is observed when both the protein kinase A and protein kinase C pathways are activated in BeWo cells. This synergy in fusion is also accompanied by dramatic increases in mRNA for the placental fusion proteins syncytin 1, syncytin 2, as well as dysferlin. Dysferlin, however, was shown to be dispensable for stimulus-induced BeWo cell syncytialization, since dysferlin knockdown lines fused to the same extent as control cells. The classical trophoblast differentiation marker human chorionic gonadotropin was also monitored and changes in the expression closely parallel that of dysferlin in all of the experimental conditions employed. Thus different biochemical markers of trophoblast fusion behave in concert supporting the hypothesis that activation of both protein kinase C and A pathways lead to trophoblastic differentiation. PMID:24236208

  13. Purification of CD47-streptavidin fusion protein from bacterial lysate using biotin-agarose affinity chromatography.

    PubMed

    Salehi, Nasrin; Peng, Ching-An

    2016-07-01

    CD47 is a widely expressed transmembrane glycoprotein that modulates the activity of a plethora of immune cells via its extracellular domain. Therefore, CD47 plays important roles in the regulation of immune responses and may serve as targets for the development of immunotherapeutic agents. To make sure CD47 functionality is intact under the process of protein conjugation, CD47-streptavidin fusion protein was expressed and purified because it can easily bind to biotin-tagged materials via the unique biotin-streptavidin affinity. In this study, gene sequences of CD47 extracellular domain (CD47ECD) and core streptavidin (coreSA) with a total 834 bp were inserted into pET20b plasmid to construct recombinant plasmid encoding CD47-SA fusion gene. After bacteria transformation, the CD47-SA fusion protein was expressed by isopropyl-β-d-thiogalactopyranoside (IPTG) induction. The collected bacteria lysate was loaded on biotinylated agarose to proceed the purification of CD47-SA fusion protein. Due to the unexpected high affinity between biotin and coreSA, standard washing and elution approaches (e.g., varying pH, using biotin, and applying guanidine hydrochloride) reported for biotin-streptavidin affinity chromatography were not able to separate the target fusion protein. Instead, using low concentration of the non-ionic detergent Triton X-100 followed with alkaline buffer could efficiently weaken the binding between biotin and coreSA, thereby eluting out CD47-SA fusion protein from the biotin agarose column. The purified CD47-SA fusion protein was further characterized by molecular biology methods and its antiphagocytic functionality was confirmed by the phagocytosis assay. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:949-958, 2016. PMID:27110670

  14. Antibody-independent Targeted Quantification of TMPRSS2-ERG Fusion Protein Products in Prostate Cancer

    SciTech Connect

    He, Jintang; Sun, Xuefei; Shi, Tujin; Schepmoes, Athena A.; Fillmore, Thomas L.; Petyuk, Vladislav A.; Xie, Fang; Zhao, Rui; Gritsenko, Marina A.; Yang, Feng; Kitabayashi, Naoki; Chae, Sung Suk; Rubin, Mark; Siddiqui, Javed; Wei, John; Chinnaiyan, Arul M.; Qian, Weijun; Smith, Richard D.; Kagan, Jacob; Srivastava, Sudhir; Rodland, Karin D.; Liu, Tao; Camp, David G.

    2014-10-01

    Fusions between the transmembrane protease serine 2 (TMPRSS2) and ETS related gene (ERG) represent one of the most specific biomarkers that define a distinct molecular subtype of prostate cancer. The studies on TMPRSS2-ERG gene fusions have seldom been performed at the protein level, primarily due to the lack of high-quality antibodies or an antibody-independent method that is sufficiently sensitive for detecting the truncated ERG protein products resulting from TMPRSS2-ERG gene fusions and alternative splicing. Herein, we applied a recently developed PRISM (high-pressure high-resolution separations with intelligent selection and multiplexing)-SRM (selected reaction monitoring) strategy for quantifying ERG protein in prostate cancer cell lines and tumors. The highly sensitive PRISM-SRM assays led to confident detection of 6 unique ERG peptides in either the TMPRSS2-ERG positive cell lines or tissues but not in the negative controls, indicating that ERG protein expression is highly correlated with TMPRSS2-ERG gene rearrangements. Significantly, our results demonstrated for the first time that at least two groups of ERG protein isoforms were simultaneously expressed at variable levels in TMPRSS2-ERG positive samples as evidenced by concomitant detection of two mutually exclusive peptides. Three peptides shared across almost all fusion protein products were determined to be the most abundant peptides, and hence can be used as “signature” peptides for detecting ERG overexpression resulting from TMPRSS2-ERG gene fusion. These PRISM-SRM assays provide valuable tools for studying TMPRSS2-ERG gene fusion protein products, thus improving our understanding of the role of TMPRSS2-ERG gene fusion in the biology of prostate cancer.

  15. Use of protein A gene fusions for the analysis of structure-function relationship of the transactivator protein C of bacteriophage Mu.

    PubMed

    De, A; Paul, B D; Ramesh, V; Nagaraja, V

    1997-08-01

    A sensitive dimerization assay for DNA binding proteins has been developed using gene fusion technology. For this purpose, we have engineered a gene fusion using protein A gene of Staphylococcus aureus and C gene, the late gene transactivator of bacteriophage Mu. The C gene was fused to the 3' end of the gene for protein A to generate an A-C fusion. The overexpressed fusion protein was purified in a single step using immunoglobulin affinity chromatography. Purified fusion protein exhibits DNA binding activity as demonstrated by electrophoretic mobility shift assays. When the fusion protein A-C was mixed with C and analyzed for DNA binding, in addition to C and A-C specific complexes, a single intermediate complex comprising of a heterodimer of C and A-C fusion proteins was observed. Further, the protein A moiety in the fusion protein A-C does not contribute to DNA binding as demonstrated by proteolytic cleavage and circular dichroism (CD) analysis. The assay has also been applied to analyze the DNA binding domain of C protein by generating fusions between protein A and N- and C-terminal deletion mutants of C. The results indicate a role for the region towards the carboxy terminal of the protein in DNA binding. The general applicability of this method is discussed. PMID:9415443

  16. Novel fusion of GLP-1 with a domain antibody to serum albumin prolongs protection against myocardial ischemia/reperfusion injury in the rat

    PubMed Central

    2013-01-01

    Background Glucagon-like peptide-1 (GLP-1) and its mimetics reduce infarct size in the setting of acute myocardial ischemia/reperfusion (I/R) injury. However, the short serum half-life of GLP-1 and its mimetics may limit their therapeutic use in acute myocardial ischemia. Domain antibodies to serum albumin (AlbudAbs) have been developed to extend the serum half-life of short lived therapeutic proteins, peptides and small molecules. In this study, we compared the effect of a long acting GLP-1 agonist, DPP-IV resistant GLP-1 (7–36, A8G) fused to an AlbudAb (GAlbudAb), with the effect of the GLP-1 mimetic, exendin-4 (short half-life GLP-1 agonist) on infarct size following acute myocardial I/R injury. Methods Male Sprague–Dawley rats (8-week-old) were treated with vehicle, GAlbudAb or exendin-4. Myocardial ischemia was induced 2 h following the final dose for GAlbudAb and 30 min post the final dose for exendin-4. In a subgroup of animals, the final dose of exendin-4 was administered (1 μg/kg, SC, bid for 2 days) 6 h prior to myocardial ischemia when plasma exendin-4 was at its minimum concentration (Cmin). Myocardial infarct size, area at risk and cardiac function were determined 24 h after myocardial I/R injury. Results GAlbudAb and exendin-4 significantly reduced myocardial infarct size by 28% and 23% respectively, compared to vehicle (both p < 0.01 vs. vehicle) after I/R injury. Moreover, both GAlbudAb and exendin-4 markedly improved post-ischemic cardiac contractile function. Body weight loss and reduced food intake consistent with the activation of GLP-1 receptors was observed in all treatment groups. However, exendin-4 failed to reduce infarct size when administered 6 h prior to myocardial ischemia, suggesting continuous activation of the GLP-1 receptors is needed for cardioprotection. Conclusions Cardioprotection provided by GAlbudAb, a long acting GLP-1 mimetic, following myocardial I/R injury was comparable in magnitude, but more sustained in

  17. Solid-state 13C-NMR spectroscopy of adduction products of 2,5-hexanedione with ribonuclease, albumin, and rat neurofilament protein.

    PubMed

    Yan, B; DeCaprio, A P; Zhu, M; Bank, S

    1996-10-21

    The Paal-Knorr condensation reaction between the gamma-diketone 2,5-hexanedione (2,5-HD) and epsilon-amine moieties of proteins of various molecular weight, including ribonuclease (RNase), bovine serum albumin (BSA) and rat neurofilament (NF), has been investigated by solid-state 13C-NMR spectroscopy. These proteins all reacted with 2,5-HD with the formation of 2,5-dimethylpyrrole (2,5-DMP) derivatives. The size and complexity of the protein affected the rate of formation of 2,5-DMP derivatives. Using the selective reducing reagent NaCNBH3, the Paal-Knorr reaction intermediates were trapped by conversion into amines, which were identified by solid-state NMR spectroscopy. The secondary autoxidation reaction following the formation of 2,5-DMP derivatives was also studied by solid-state NMR spectroscopy. PMID:8950225

  18. The production of antibody fragments and antibody fusion proteins by yeasts and filamentous fungi

    PubMed Central

    Joosten, Vivi; Lokman, Christien; van den Hondel, Cees AMJJ; Punt, Peter J

    2003-01-01

    In this review we will focus on the current status and views concerning the production of antibody fragments and antibody fusion proteins by yeasts and filamentous fungi. We will focus on single-chain antibody fragment production (scFv and VHH) by these lower eukaryotes and the possible applications of these proteins. Also the coupling of fragments to relevant enzymes or other components will be discussed. As an example of the fusion protein strategy, the 'magic bullet' approach for industrial applications, will be highlighted. PMID:12605725

  19. A Betabaculovirus-Encoded gp64 Homolog Codes for a Functional Envelope Fusion Protein

    PubMed Central

    Ardisson-Araújo, Daniel M. P.; Melo, Fernando L.; Clem, Rollie J.; Wolff, José L. C.

    2015-01-01

    The GP64 envelope fusion protein is a hallmark of group I alphabaculoviruses. However, the Diatraea saccharalis granulovirus genome sequence revealed the first betabaculovirus species harboring a gp64 homolog (disa118). In this work, we have shown that this homolog encodes a functional envelope fusion protein and could enable the infection and fusogenic abilities of a gp64-null prototype baculovirus. Therefore, GP64 may complement or may be in the process of replacing F protein activity in this virus lineage. PMID:26537678

  20. A Betabaculovirus-Encoded gp64 Homolog Codes for a Functional Envelope Fusion Protein.

    PubMed

    Ardisson-Araújo, Daniel M P; Melo, Fernando L; Clem, Rollie J; Wolff, José L C; Ribeiro, Bergmann M

    2016-02-01

    The GP64 envelope fusion protein is a hallmark of group I alphabaculoviruses. However, the Diatraea saccharalis granulovirus genome sequence revealed the first betabaculovirus species harboring a gp64 homolog (disa118). In this work, we have shown that this homolog encodes a functional envelope fusion protein and could enable the infection and fusogenic abilities of a gp64-null prototype baculovirus. Therefore, GP64 may complement or may be in the process of replacing F protein activity in this virus lineage. PMID:26537678

  1. PLEKHM1 regulates autophagosome-lysosome fusion through HOPS complex and LC3/GABARAP proteins.

    PubMed

    McEwan, David G; Popovic, Doris; Gubas, Andrea; Terawaki, Seigo; Suzuki, Hironori; Stadel, Daniela; Coxon, Fraser P; Miranda de Stegmann, Diana; Bhogaraju, Sagar; Maddi, Karthik; Kirchof, Anja; Gatti, Evelina; Helfrich, Miep H; Wakatsuki, Soichi; Behrends, Christian; Pierre, Philippe; Dikic, Ivan

    2015-01-01

    The lysosome is the final destination for degradation of endocytic cargo, plasma membrane constituents, and intracellular components sequestered by macroautophagy. Fusion of endosomes and autophagosomes with the lysosome depends on the GTPase Rab7 and the homotypic fusion and protein sorting (HOPS) complex, but adaptor proteins that link endocytic and autophagy pathways with lysosomes are poorly characterized. Herein, we show that Pleckstrin homology domain containing protein family member 1 (PLEKHM1) directly interacts with HOPS complex and contains a LC3-interacting region (LIR) that mediates its binding to autophagosomal membranes. Depletion of PLEKHM1 blocks lysosomal degradation of endocytic (EGFR) cargo and enhances presentation of MHC class I molecules. Moreover, genetic loss of PLEKHM1 impedes autophagy flux upon mTOR inhibition and PLEKHM1 regulates clearance of protein aggregates in an autophagy- and LIR-dependent manner. PLEKHM1 is thus a multivalent endocytic adaptor involved in the lysosome fusion events controlling selective and nonselective autophagy pathways. PMID:25498145

  2. Beyond anchoring: the expanding role of the hendra virus fusion protein transmembrane domain in protein folding, stability, and function.

    PubMed

    Smith, Everett Clinton; Culler, Megan R; Hellman, Lance M; Fried, Michael G; Creamer, Trevor P; Dutch, Rebecca Ellis

    2012-03-01

    While work with viral fusion proteins has demonstrated that the transmembrane domain (TMD) can affect protein folding, stability, and membrane fusion promotion, the mechanism(s) remains poorly understood. TMDs could play a role in fusion promotion through direct TMD-TMD interactions, and we have recently shown that isolated TMDs from three paramyxovirus fusion (F) proteins interact as trimers using sedimentation equilibrium (SE) analysis (E. C. Smith, et al., submitted for publication). Immediately N-terminal to the TMD is heptad repeat B (HRB), which plays critical roles in fusion. Interestingly, addition of HRB decreased the stability of the trimeric TMD-TMD interactions. This result, combined with previous findings that HRB forms a trimeric coiled coil in the prefusion form of the whole protein though HRB peptides fail to stably associate in isolation, suggests that the trimeric TMD-TMD interactions work in concert with elements in the F ectodomain head to stabilize a weak HRB interaction. Thus, changes in TMD-TMD interactions could be important in regulating F triggering and refolding. Alanine insertions between the TMD and HRB demonstrated that spacing between these two regions is important for protein stability while not affecting TMD-TMD interactions. Additional mutagenesis of the C-terminal end of the TMD suggests that β-branched residues within the TMD play a role in membrane fusion, potentially through modulation of TMD-TMD interactions. Our results support a model whereby the C-terminal end of the Hendra virus F TMD is an important regulator of TMD-TMD interactions and show that these interactions help hold HRB in place prior to the triggering of membrane fusion. PMID:22238302

  3. What Characteristics Confer Proteins the Ability to Induce Allergic Responses? IgE Epitope Mapping and Comparison of the Structure of Soybean 2S Albumins and Ara h 2.

    PubMed

    Han, Youngshin; Lin, Jing; Bardina, Ludmilla; Grishina, Galina A; Lee, Chaeyoon; Seo, Won Hee; Sampson, Hugh A

    2016-01-01

    Ara h 2, a peanut 2S albumin, is associated with severe allergic reactions, but a homologous protein, soybean 2S albumin, is not recognized as an important allergen. Structural difference between these proteins might explain this clinical discrepancy. Therefore, we mapped sequential epitopes and compared the structure of Ara h 2, Soy Al 1, and Soy Al 3 (Gly m 8) to confirm whether structural differences account for the discrepancy in clinical responses to these two proteins. Commercially synthesized peptides covering the full length of Ara h 2 and two soybean 2S albumins were analyzed by peptide microarray. Sera from 10 patients with peanut and soybean allergies and seven non-atopic controls were examined. The majority of epitopes in Ara h 2 identified by microarray are consistent with those identified previously. Several regions in the 2S albumins are weakly recognized by individual sera from different patients. A comparison of allergenic epitopes on peanut and soybean proteins suggests that loop-helix type secondary structures and some amino acids with a large side chain including lone electron pair, such as arginine, glutamine, and tyrosine, makes the peptides highly recognizable by the immune system. By utilizing the peptide microarray assay, we mapped IgE epitopes of Ara h 2 and two soybean 2S albumins. The use of peptide microarray mapping and analysis of the epitope characteristics may provide critical information to access the allergenicity of food proteins. PMID:27187334

  4. Construction and evaluation of novel fusion proteins for targeted delivery of micro particles to cellulose surfaces.

    PubMed

    Lewis, William; Keshavarz-Moore, Eli; Windust, John; Bushell, Donna; Parry, Neil

    2006-07-01

    The use of IgG antibodies and fragments has been limited to specific sectors of the biotechnology industry due to the high cost of producing large batches of product necessary for alternative applications. A novel class of Camelid antibodies, known as V(HH) offer a more economical opportunity to meet a wider application in industry. In this study, we report the evaluation of four llama V(HH)-cellulose binding domain fusion proteins displaying varying formats of V(HH) and CBD domains. Proteins were characterized in a targeted particle delivery system as a method of delivering agents such as perfume to laundry in the wash cycle. Fusion proteins were shown to be stable at high pH and in the presence of a detergent base. They were also shown to bind effectively to both the designated antigen, the azo-dye reactive-red 6 (either conjugated to BSA or attached to coacervate microparticles), and cellulose. Binding strength differences were observed between the different fusion protein formats using surface plasmon resonance. The effect of key laundry ingredients was also studied. Combining the fusion proteins and particles into a delivery and deposition study generated clear microscopy evidence for bifunctionality. Confirmation of this was validated by GC-MS analysis of retained fragrance. This research, reporting the construction and characterization of a variety of fusion proteins, illustrates that the single multidomain fusion protein route offers a new technology for successful targeted delivery of encapsulated benefit agents. Furthermore, the potential to modify or select for proteins to recognize a wide range of surfaces is also possible. PMID:16673421

  5. Amadori albumin in diabetic nephropathy.

    PubMed

    Neelofar, Km; Ahmad, Jamal

    2015-01-01

    Nonenzymatic glycation of macromolecules in diabetes mellitus (DM) is accelerated due to persistent hyperglycemia. Reducing sugar such as glucose reacts non enzymatically with free €-amino groups of proteins through series of reactions forming Schiff bases. These bases are converted into Amadori product and further into AGEs. Non enzymatic glycation has the potential to alter the biological, structural and functional properties of macromolecules both in vitro and in vivo. Studies have suggested that amadori as well as AGEs are involved in the micro-macro vascular complications in DM, but most studies have focused on the role of AGEs in vascular complications of diabetes. Recently putative AGE-induced patho-physiology has shifted attention from the possible role of amadori-modified proteins, the predominant form of the glycated proteins in the development of the diabetic complications. Human serum albumin (HSA), the most abundant protein in circulation contains 59 lysine and 23 arginine residues that could, in theory be involved in glycation. Albumin has dual nature, first as a marker of intermediate glycation and second as a causative agent of the damage of tissues. Among the blood proteins, hemoglobin and albumin are the most common proteins that are glycated. HSA with a shorter half life than RBC, appears to be an alternative marker of glycemic control as it can indicate blood glucose status over a short period (2-3 weeks) and being unaffected by RBCs life span and variant haemoglobin, anemia etc which however, affect HbA1c. On the other hand, Amadori albumin may accumulate in the body tissues of the diabetic patients and participate in secondary complications. Amadori-albumin has potential role in diabetic glomerulosclerosis due to long term hyperglycaemia and plays an important role in the pathogenesis of diabetic nephropathy. This review is an approach to compile both the nature of glycated albumin as a damaging agent of tissues and as an intermediate

  6. Podocytes degrade endocytosed albumin primarily in lysosomes.

    PubMed

    Carson, John M; Okamura, Kayo; Wakashin, Hidefumi; McFann, Kim; Dobrinskikh, Evgenia; Kopp, Jeffrey B; Blaine, Judith

    2014-01-01

    Albuminuria is a strong, independent predictor of chronic kidney disease progression. We hypothesize that podocyte processing of albumin via the lysosome may be an important determinant of podocyte injury and loss. A human urine derived podocyte-like epithelial cell (HUPEC) line was used for in vitro experiments. Albumin uptake was quantified by Western blot after loading HUPECs with fluorescein-labeled (FITC) albumin. Co-localization of albumin with lysosomes was determined by confocal microscopy. Albumin degradation was measured by quantifying FITC-albumin abundance in HUPEC lysates by Western blot. Degradation experiments were repeated using HUPECs treated with chloroquine, a lysosome inhibitor, or MG-132, a proteasome inhibitor. Lysosome activity was measured by fluorescence recovery after photo bleaching (FRAP). Cytokine production was measured by ELISA. Cell death was determined by trypan blue staining. In vivo, staining with lysosome-associated membrane protein-1 (LAMP-1) was performed on tissue from a Denys-Drash trangenic mouse model of nephrotic syndrome. HUPECs endocytosed albumin, which co-localized with lysosomes. Choloroquine, but not MG-132, inhibited albumin degradation, indicating that degradation occurs in lysosomes. Cathepsin B activity, measured by FRAP, significantly decreased in HUPECs exposed to albumin (12.5% of activity in controls) and chloroquine (12.8%), and declined further with exposure to albumin plus chloroquine (8.2%, p<0.05). Cytokine production and cell death were significantly increased in HUPECs exposed to albumin and chloroquine alone, and these effects were potentiated by exposure to albumin plus chloroquine. Compared to wild-type mice, glomerular staining of LAMP-1 was significantly increased in Denys-Drash mice and appeared to be most prominent in podocytes. These data suggest lysosomes are involved in the processing of endocytosed albumin in podocytes, and lysosomal dysfunction may contribute to podocyte injury and

  7. Podocytes Degrade Endocytosed Albumin Primarily in Lysosomes

    PubMed Central

    Carson, John M.; Okamura, Kayo; Wakashin, Hidefumi; McFann, Kim; Dobrinskikh, Evgenia; Kopp, Jeffrey B.; Blaine, Judith

    2014-01-01

    Albuminuria is a strong, independent predictor of chronic kidney disease progression. We hypothesize that podocyte processing of albumin via the lysosome may be an important determinant of podocyte injury and loss. A human urine derived podocyte-like epithelial cell (HUPEC) line was used for in vitro experiments. Albumin uptake was quantified by Western blot after loading HUPECs with fluorescein-labeled (FITC) albumin. Co-localization of albumin with lysosomes was determined by confocal microscopy. Albumin degradation was measured by quantifying FITC-albumin abundance in HUPEC lysates by Western blot. Degradation experiments were repeated using HUPECs treated with chloroquine, a lysosome inhibitor, or MG-132, a proteasome inhibitor. Lysosome activity was measured by fluorescence recovery after photo bleaching (FRAP). Cytokine production was measured by ELISA. Cell death was determined by trypan blue staining. In vivo, staining with lysosome-associated membrane protein-1 (LAMP-1) was performed on tissue from a Denys-Drash trangenic mouse model of nephrotic syndrome. HUPECs endocytosed albumin, which co-localized with lysosomes. Choloroquine, but not MG-132, inhibited albumin degradation, indicating that degradation occurs in lysosomes. Cathepsin B activity, measured by FRAP, significantly decreased in HUPECs exposed to albumin (12.5% of activity in controls) and chloroquine (12.8%), and declined further with exposure to albumin plus chloroquine (8.2%, p<0.05). Cytokine production and cell death were significantly increased in HUPECs exposed to albumin and chloroquine alone, and these effects were potentiated by exposure to albumin plus chloroquine. Compared to wild-type mice, glomerular staining of LAMP-1 was significantly increased in Denys-Drash mice and appeared to be most prominent in podocytes. These data suggest lysosomes are involved in the processing of endocytosed albumin in podocytes, and lysosomal dysfunction may contribute to podocyte injury and

  8. Conjugation of poly-L-lysine to albumin and horseradish peroxidase: a novel method of enhancing the cellular uptake of proteins.

    PubMed Central

    Shen, W C; Ryser, H J

    1978-01-01

    The carbodiimide-catalyzed conjugation of a 6700 molecular weight fragment of poly-L-lysine to radiolabeled human serum albumin or to horseradish peroxidase enhances the membrane transport of each protein into cultured mouse fibroblasts approximately 11- and 200-fold, respectively. At least 50% of the peroxidase activity remained after conjugation. Trypsinization and carbamylation of the two conjugates demonstrates that the enhancement of their cellular uptake is related to their poly-L-lysine content. Simple addition to the medium of comparable amounts of free poly-L-lysine has no effect on the transport of either native protein. Addition of poly-L-ornithine (molecular weight 200,000) at 3-30 microgram/ml, a condition known to cause enhancement of 125I-labeled human serum albumin uptake by mouse sarcoma cells, has no visible effect on the cellular uptake of native horseradish peroxidase. The intracellular localization of the enzyme-poly-L-lysine conjugate can be demonstrated cytochemically by either light or transmission electron microscopy. A concentration of conjugate that increases the uptake more than 200-fold does not cause any detectable morphological change suggestive of cell toxicity. Furthermore, because poly-L-lysine is an excellent substrate for intracellular proteolytic enzymes, it can be expected to be broken down and reutilized in the cell. Images PMID:273916

  9. Characterization of the fusion core in zebrafish endogenous retroviral envelope protein

    SciTech Connect

    Shi, Jian; Zhang, Huaidong; Gong, Rui; Xiao, Gengfu

    2015-05-08

    Zebrafish endogenous retrovirus (ZFERV) is the unique endogenous retrovirus in zebrafish, as yet, containing intact open reading frames of its envelope protein gene in zebrafish genome. Similarly, several envelope proteins of endogenous retroviruses in human and other mammalian animal genomes (such as syncytin-1 and 2 in human, syncytin-A and B in mouse) were identified and shown to be functional in induction of cell–cell fusion involved in placental development. ZFERV envelope protein (Env) gene appears to be also functional in vivo because it is expressible. After sequence alignment, we found ZFERV Env shares similar structural profiles with syncytin and other type I viral envelopes, especially in the regions of N- and C-terminal heptad repeats (NHR and CHR) which were crucial for membrane fusion. We expressed the regions of N + C protein in the ZFERV Env (residues 459–567, including predicted NHR and CHR) to characterize the fusion core structure. We found N + C protein could form a stable coiled-coil trimer that consists of three helical NHR regions forming a central trimeric core, and three helical CHR regions packing into the grooves on the surface of the central core. The structural characterization of the fusion core revealed the possible mechanism of fusion mediated by ZFERV Env. These results gave comprehensive explanation of how the ancient virus infects the zebrafish and integrates into the genome million years ago, and showed a rational clue for discovery of physiological significance (e.g., medicate cell–cell fusion). - Highlights: • ZFERV Env shares similar structural profiles with syncytin and other type I viral envelopes. • The fusion core of ZFERV Env forms stable coiled-coil trimer including three NHRs and three CHRs. • The structural mechanism of viral entry mediated by ZFERV Env is disclosed. • The results are helpful for further discovery of physiological function of ZFERV Env in zebrafish.

  10. Small-angle neutron scattering study of differences in phase behavior of silica nanoparticles in the presence of lysozyme and bovine serum albumin proteins

    NASA Astrophysics Data System (ADS)

    Yadav, Indresh; Kumar, Sugam; Aswal, V. K.; Kohlbrecher, J.

    2014-03-01

    The differences in phase behavior of anionic silica nanoparticles (88 Å) in the presence of two globular proteins [cationic lysozyme (molecular weight (MW) 14.7 kD) and anionic bovine serum albumin (BSA) (MW 66.4 kD)] have been studied by small-angle neutron scattering. The measurements were carried out on a fixed concentration (1 wt %) of Ludox silica nanoparticles with varying concentrations of proteins (0-5 wt %) at pH = 7. It is found that, despite having different natures (opposite charges), both proteins can render to the same kind of aggregation of silica nanoparticles. However, the concentration regions over which the aggregation is observed are widely different for the two proteins. Lysozyme with very small amounts (e.g., 0.01 wt %) leads to the aggregation of silica nanoparticles. On the other hand, silica nanoparticles coexist with BSA as independent entities at low protein concentrations and turn to aggregates at high protein concentrations (>1 wt %). In the case of lysozyme, the charge neutralization by the protein on the nanoparticles gives rise to the protein-mediated aggregation of the nanoparticles. The nanoparticle aggregates coexist with unaggregated nanoparticles at low protein concentrations, whereas, they coexist with a free protein at higher protein concentrations. For BSA, the nonadsorbing nature of the protein produces the depletion force that causes the aggregation of the nanoparticles at higher protein concentrations. The evolution of the interaction is modeled by the two Yukawa potential, taking account of both attractive and repulsive terms of the interaction in these systems. The nanoparticle aggregation is found to be governed by the short-range attraction for lysozyme and the long-range attraction for BSA. The aggregates are characterized by the diffusion limited aggregate type of mass fractal morphology.

  11. Expression and purification of SARS coronavirus proteins using SUMO-fusions.

    PubMed

    Zuo, Xun; Mattern, Michael R; Tan, Robin; Li, Shuisen; Hall, John; Sterner, David E; Shoo, Joshua; Tran, Hiep; Lim, Peter; Sarafianos, Stefan G; Kazi, Lubna; Navas-Martin, Sonia; Weiss, Susan R; Butt, Tauseef R

    2005-07-01

    Severe acute respiratory syndrome coronavirus (SARS-CoV) proteins belong to a large group of proteins that is difficult to express in traditional expression systems. The ability to express and purify SARS-CoV proteins in large quantities is critical for basic research and for development of pharmaceutical agents. The work reported here demonstrates: (1) fusion of SUMO (small ubiquitin-related modifier), a 100 amino acid polypeptide, to the N-termini of SARS-CoV proteins dramatically enhances expression in Escherichia coli cells and (2) 6x His-tagged SUMO-fusions facilitate rapid purification of the viral proteins on a large scale. We have exploited the natural chaperoning properties of SUMO to develop an expression system suitable for proteins that cannot be expressed by traditional methodologies. A unique feature of the system is the SUMO tag, which enhances expression, facilitates purification, and can be efficiently cleaved by a SUMO-specific protease to generate native protein with a desired N-terminus. We have purified various SARS-CoV proteins under either native or denaturing conditions. These purified proteins have been used to generate highly specific polyclonal antibodies. Our study suggests that the SUMO-fusion technology will be useful for enhancing expression and purification of the viral proteins for structural and functional studies as well as for therapeutic uses. PMID:15939295

  12. Specific interaction between adenoviral 55-kDa E1B protein and in vivo produced p53 fusion proteins.

    PubMed

    Chumakov, A; Koeffler, H P

    1993-09-15

    Several protein fusion systems have been used in recent years to study protein-protein and DNA-protein interactions. Most of them use bacterially produced proteins which have several inherent disadvantages, notably, the absence of correct post-translational modifications and the frequent insolubility of recombinant proteins. We sought to develop a system to study proteins interacting with the nuclear phosphoprotein p53, which is believed to be a tumor suppressor. To prepare fusions of p53, we developed a convenient system that permits both in vivo and in vitro production and easy affinity purification of peptides and protein fragments as glutathione-transferase fusions. We placed the coding sequence of the Schistosoma japonica glutathione S-transferase (GST) under the control of the strong CMV/T7 promoter and SV40 splice and polyadenylation signals. An extensive polylinker (MCS) at the 3' end of the GST gene is preceded by the sequence encoding the cleavage site of the site-specific protease. We cloned the complete coding sequences of human wild-type p53, as well as p53 mutants representing all four mutational hotspots (codons 141, 175, 248, and 273), into our expression vector. In vitro transcription using the upstream T7 promoter and translation in reticulocyte lysates form an easy way to produce hybrid proteins; affinity purification on a glutathione-agarose column removes proteins that are present in reticulocyte lysates. We have also studied specific in vivo interactions of human p53 with the adenoviral 55-kDa E1B protein by transfecting expression constructs of GST-p53 fusions into human Ad5-transformed 293 cells. PMID:8406015

  13. SNARE and regulatory proteins induce local membrane protrusions to prime docked vesicles for fast calcium-triggered fusion

    PubMed Central

    Bharat, Tanmay A M; Malsam, Jörg; Hagen, Wim J H; Scheutzow, Andrea; Söllner, Thomas H; Briggs, John A G

    2014-01-01

    Synaptic vesicles fuse with the plasma membrane in response to Ca2+ influx, thereby releasing neurotransmitters into the synaptic cleft. The protein machinery that mediates this process, consisting of soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) and regulatory proteins, is well known, but the mechanisms by which these proteins prime synaptic membranes for fusion are debated. In this study, we applied large-scale, automated cryo-electron tomography to image an in vitro system that reconstitutes synaptic fusion. Our findings suggest that upon docking and priming of vesicles for fast Ca2+-triggered fusion, SNARE proteins act in concert with regulatory proteins to induce a local protrusion in the plasma membrane, directed towards the primed vesicle. The SNAREs and regulatory proteins thereby stabilize the membrane in a high-energy state from which the activation energy for fusion is profoundly reduced, allowing synchronous and instantaneous fusion upon release of the complexin clamp. PMID:24493260

  14. Affinity Purification of a Recombinant Protein Expressed as a Fusion with the Maltose-Binding Protein (MBP) Tag

    PubMed Central

    Duong-Ly, Krisna C.; Gabelli, Sandra B.

    2015-01-01

    Expression of fusion proteins such as MBP fusions can be used as a way to improve the solubility of the expressed protein in E. coli (Fox and Waugh, 2003; Nallamsetty et al., 2005; Nallamsetty and Waugh, 2006) and as a way to introduce an affinity purification tag. The protocol that follows was designed by the authors as a first step in the purification of a recombinant protein fused with MBP, using fast protein liquid chromatography (FPLC). Cells should have been thawed, resuspended in binding buffer, and lysed by sonication or microfluidization before mixing with the amylose resin or loading on the column. Slight modifications to this protocol may be made to accommodate both the protein of interest and the availability of equipment. PMID:26096500

  15. Cleaved thioredoxin fusion protein enables the crystallization of poorly soluble ERα in complex with synthetic ligands

    SciTech Connect

    Cura, Vincent; Gangloff, Monique; Eiler, Sylvia; Moras, Dino; Ruff, Marc

    2008-01-01

    A new crystallization strategy: the presence of cleaved thioredoxin fusion is critical for crystallization of the estrogen nuclear receptor ligand binding domain in complex with synthetic ligands. This novel technique should be regarded as an interesting alternative for crystallization of difficult proteins. The ligand-binding domain (LBD) of human oestrogen receptor α was produced in Escherichia coli as a cleavable thioredoxin (Trx) fusion in order to improve solubility. Crystallization trials with either cleaved and purified LBD or with the purified fusion protein both failed to produce crystals. In another attempt, Trx was not removed from the LBD after endoproteolytic cleavage and its presence promoted nucleation and subsequent crystal growth, which allowed the structure determination of two different LBD–ligand–coactivator peptide complexes at 2.3 Å resolution. This technique is likely to be applicable to other low-solubility proteins.

  16. In vivo immobilization of fusion proteins on bioplastics by the novel tag BioF.

    PubMed

    Moldes, Cristina; García, Pedro; García, José L; Prieto, María A

    2004-06-01

    A new protein immobilization and purification system has been developed based on the use of polyhydroxyalkanoates (PHAs, or bioplastics), which are biodegradable polymers accumulated as reserve granules in the cytoplasm of certain bacteria. The N-terminal domain of the PhaF phasin (a PHA-granule-associated protein) from Pseudomonas putida GPo1 was used as a polypeptide tag (BioF) to anchor fusion proteins to PHAs. This tag provides a novel way to immobilize proteins in vivo by using bioplastics as supports. The granules carrying the BioF fusion proteins can be isolated by a simple centrifugation step and used directly for some applications. Moreover, when required, a practically pure preparation of the soluble BioF fusion protein can be obtained by a mild detergent treatment of the granule. The efficiency of this system has been demonstrated by constructing two BioF fusion products, including a functional BioF-beta-galactosidase. This is the first example of an active bioplastic consisting of a biodegradable matrix carrying an active enzyme. PMID:15184113

  17. Cloning, expression, purification and preliminary crystallographic analysis of the RNase HI domain of the Mycobacterium tuberculosis protein Rv2228c as a maltose-binding protein fusion

    PubMed Central

    Watkins, Harriet A.; Baker, Edward N.

    2008-01-01

    The predicted ribonuclease (RNase) HI domain of the open reading frame Rv2228c from Mycobacterium tuberculosis has been cloned as a hexahistidine fusion and a maltose-binding protein (MBP) fusion. Expression was only observed for the MBP-fusion protein, which was purified using amylose affinity chromatography and gel filtration. The RNase HI domain could be cleaved from the MBP-fusion protein by factor Xa digestion, but was very unstable. In contrast, the fusion protein was stable, could be obtained in high yield and gave crystals which diffracted to 2.25 Å resolution. The crystals belong to space group P21 and have unit-cell parameters a = 73.63, b = 101.38, c = 76.09 Å, β = 109.0°. Two fusion-protein molecules of 57 417 Da were present in each asymmetric unit. PMID:18678948

  18. Crystal structure of the membrane fusion protein CusB from Escherichia coli

    PubMed Central

    Su, Chih-Chia; Yang, Feng; Long, Feng; Reyon, Deepak; Routh, Mathew D.; Kuo, Dennis W.; Mokhtari, Adam K.; Van Ornam, Jonathan D.; Rabe, Katherine L.; Hoy, Julie A.; Lee, Young Jin; Rajashankar, Kanagalaghatta R.; Yu, Edward W.

    2009-01-01

    Gram-negative bacteria, such as Escherichia coli, frequently utilize tripartite efflux complexes belonging to the resistance-nodulation-division family to expel diverse toxic compounds from the cell. These systems contain a periplasmic membrane fusion protein that is critical for substrate transport. We here present the x-ray structures of the CusB membrane fusion protein from the copper/silver efflux system of E. coli. This is the first structure of any membrane fusion proteins associated with heavy-metal efflux transporters. CusB bridges the inner membrane efflux pump CusA and outer membrane channel CusC to mediate resistance to Cu+ and Ag+ ions. Two distinct structures of the elongated molecules of CusB were found in the asymmetric unit of a single crystal, which suggests the flexible nature of this protein. Each protomer of CusB can be divided into four different domains, whereby the first three domains are mostly β-strands and the last domain adopts an entirely helical architecture. Unlike other known structures of membrane fusion proteins, the α-helical domain of CusB is folded into a three-helix bundle. This three-helix bundle presumably interacts with the periplasmic domain of CusC. The N and C-termini of CusB form the first β-strand domain, which is found to interact with the periplasmic domain of the CusA efflux pump. Atomic details of how this efflux protein binds Cu+ and Ag+ were revealed by the crystals of the CusB-Cu(I) and CusB-Ag(I) complexes. The structures indicate that CusB consists of multiple binding sites for these metal ions. These findings reveal novel structural features of a membrane fusion protein in the resistance-nodulation-division efflux system, and provide evidence that this protein specifically interacts with transported substrates. PMID:19695261

  19. Cleaved thioredoxin fusion protein enables the crystallization of poorly soluble ERα in complex with synthetic ligands

    PubMed Central

    Cura, Vincent; Gangloff, Monique; Eiler, Sylvia; Moras, Dino; Ruff, Marc

    2008-01-01

    The ligand-binding domain (LBD) of human oestrogen receptor α was produced in Escherichia coli as a cleavable thioredoxin (Trx) fusion in order to improve solubility. Crystallization trials with either cleaved and purified LBD or with the purified fusion protein both failed to produce crystals. In another attempt, Trx was not removed from the LBD after endoproteolytic cleavage and its presence promoted nucleation and subsequent crystal growth, which allowed the structure determination of two different LBD–ligand–coactivator peptide complexes at 2.3 Å resolution. This technique is likely to be applicable to other low-solubility proteins. PMID:18097104

  20. Localized cyclic AMP-dependent protein kinase activity is required for myogenic cell fusion

    SciTech Connect

    Mukai, Atsushi; Hashimoto, Naohiro

    2008-01-15

    Multinucleated myotubes are formed by fusion of mononucleated myogenic progenitor cells (myoblasts) during terminal skeletal muscle differentiation. In addition, myoblasts fuse with myotubes, but terminally differentiated myotubes have not been shown to fuse with each other. We show here that an adenylate cyclase activator, forskolin, and other reagents that elevate intracellular cyclic AMP (cAMP) levels induced cell fusion between small bipolar myotubes in vitro. Then an extra-large myotube, designated a 'myosheet,' was produced by both primary and established mouse myogenic cells. Myotube-to-myotube fusion always occurred between the leading edge of lamellipodia at the polar end of one myotube and the lateral plasma membrane of the other. Forskolin enhanced the formation of lamellipodia where cAMP-dependent protein kinase (PKA) was accumulated. Blocking enzymatic activity or anchoring of PKA suppressed forskolin-enhanced lamellipodium formation and prevented fusion of multinucleated myotubes. Localized PKA activity was also required for fusion of mononucleated myoblasts. The present results suggest that localized PKA plays a pivotal role in the early steps of myogenic cell fusion, such as cell-to-cell contact/recognition through lamellipodium formation. Furthermore, the localized cAMP-PKA pathway might be involved in the specification of the fusion-competent areas of the plasma membrane in lamellipodia of myogenic cells.

  1. Chemokines derived from soluble fusion proteins expressed in Escherichia coli are biologically active

    SciTech Connect

    Magistrelli, Giovanni; Gueneau, Franck; Muslmani, Machadiya; Ravn, Ulla; Kosco-Vilbois, Marie; Fischer, Nicolas . E-mail: nfischer@novimmune.com

    2005-08-26

    Chemokines are a class of low molecular weight proteins that are involved in leukocytes trafficking. Due to their involvement in recruiting immune cells to sites of inflammation, chemokines, and chemokine receptors have become an attractive class of therapeutic targets. However, when expressed in Escherichia coli chemokines are poorly soluble and accumulate in inclusion bodies. Several purification methods have been described but involve time-consuming refolding, buffer exchange, and purification steps that complicate expression of these proteins. Here, we describe a simple and reliable method to express chemokines as fusions to the protein NusA. The fusion proteins were largely found in the soluble fraction and could be readily purified in a single step. Proteolytic cleavage was used to obtain soluble recombinant chemokines that were found to be very active in a novel in vitro chemotaxis assays. This method could be applied to several {alpha} and {beta} human chemokines, suggesting that it is generally applicable to this class of proteins.

  2. Studying protein-reconstituted proteoliposome fusion with content indicators in vitro

    PubMed Central

    Diao, Jiajie; Zhao, Minglei; Zhang, Yunxiang; Kyoung, Minjoung; Brunger, Axel T.

    2015-01-01

    In vitro reconstitution assays are commonly used to study biological membrane fusion. However, to date, most ensemble and single-vesicle experiments involving SNARE proteins have been performed only with lipid-mixing, but not content-mixing indicators. Through simultaneous detection of lipid and small content-mixing indicators, we found that lipid mixing often occurs seconds prior to content mixing, or without any content mixing at all, during a 50-sec observation period, for Ca2+-triggered fusion with SNAREs, full-length synaptotagmin-1, and complexin. Our results illustrate the caveats of commonly used bulk lipid-mixing fusion experiments. We recommend that proteoliposome fusion experiments should always employ content-mixing indicators in addition to, or in place of, lipid-mixing indicators. PMID:23625805

  3. Association of the fusion protein NSF with clathrin-coated vesicle membranes.

    PubMed Central

    Steel, G J; Tagaya, M; Woodman, P G

    1996-01-01

    N-ethylmaleimide-sensitive fusion protein (NSF) is a component of intracellular transport reactions. In order to understand the role of NSF during the fusion of endocytic transport vesicles with the endosome, we have investigated the binding of NSF to purified clathrin-coated vesicle components. First, we have examined whether detergent-solubilized coated vesicle membranes will support formation of NSF-containing 'fusion complexes'. Our results show that these membranes are substantially enriched in components capable of driving formation of these complexes, when compared with membranes from other sources. Secondly, we have analysed coated vesicle preparations for their NSF content. Coated vesicle preparations contain significant amounts of NSF. This was shown to be associated with coated vesicles rather than contaminating membranes by a number of criteria, and was found to be bound in an ATP-independent manner. These findings are discussed in the light of current models for vesicle fusion. Images PMID:8631296

  4. Dicistronic expression of human proinsulin-protein A fusion in tobacco chloroplast.

    PubMed

    Yarbakht, Melina; Jalali-Javaran, Mokhtar; Nikkhah, Maryam; Mohebodini, Mehdi

    2015-01-01

    Different expression systems such as bacteria and mammalian cells have been used to produce pharmaceutical proteins. In recent years, the use of plants as bioreactors offers efficient and economical systems in recombinant protein production. Furthermore, because of the large number of plastid copies in plants, chloroplast engineering functions as an effective method to increase recombinant protein expression. Because the commercially available insulin for treatment does not contain C-peptide, which is of great importance for type 1 diabetic patients, the current study introduces the human proinsulin gene fused with protein A into the tobacco chloroplast genome using the biolistic method. To achieve homoplasmy, three rounds of selection and regeneration of transforming cells were performed on the medium that contained spectinomycin antibiotic and hormones. The PCR analysis indicated the presence of the proinsulin gene in transplastomic plants. The reverse-transcription PCR analysis confirmed the expression of the proinsulin-protein A fusion at the transcription level. Immunoblot assays of leaf-derived protein extracts confirmed that the target gene expression is up to 0.2% of the total soluble protein. Our study showed that protein A fusion is not as efficient as other reported fusions. The transplastomic plants were also confirmed for homoplasmy using Southern blot analysis. PMID:24716841

  5. Malnutrition in Alzheimer’s Disease, Dementia with Lewy Bodies, and Frontotemporal Lobar Degeneration: Comparison Using Serum Albumin, Total Protein, and Hemoglobin Level

    PubMed Central

    Hashimoto, Mamoru; Tanaka, Hibiki; Fujise, Noboru; Matsushita, Masateru; Miyagawa, Yusuke; Hatada, Yutaka; Fukuhara, Ryuji; Hasegawa, Noriko; Todani, Shuji; Matsukuma, Kengo; Kawano, Michiyo; Ikeda, Manabu

    2016-01-01

    Malnutrition among dementia patients is an important issue. However, the biochemical markers of malnutrition have not been well studied in this population. The purpose of this study was to compare biochemical blood markers among patients with Alzheimer’s disease (AD), dementia with Lewy bodies (DLB), and frontotemporal lobar degeneration (FTLD). A total of 339 dementia outpatients and their family caregivers participated in this study. Low serum albumin was 7.2 times more prevalent among patients with DLB and 10.1 times more prevalent among those with FTLD than among those with AD, with adjustment for age. Low hemoglobin was 9.1 times more common in female DLB patients than in female AD patients, with adjustment for age. The levels of biochemical markers were not significantly correlated with cognitive function. Family caregivers of patients with low total protein, low albumin, or low hemoglobin were asked if the patients had loss of weight or appetite; 96.4% reported no loss of weight or appetite. In conclusion, nutritional status was worse in patients with DLB and FTLD than in those with AD. A multidimensional approach, including blood testing, is needed to assess malnutrition in patients with dementia. PMID:27336725

  6. Outcomes for Single-Level Lumbar Fusion: The Role of Bone Morphogenetic Protein

    PubMed Central

    Cahill, Kevin S.; Chi, John H.; Groff, Michael W.; McGuire, Kevin; Afendulis, Christopher C.; Claus, Elizabeth B.

    2011-01-01

    Study Design Retrospective analysis of a population-based insurance claims dataset. Objective To determine the risk of repeat fusion and total costs associated with bone morphogenetic protein (BMP) use in single-level lumbar fusion for degenerative spinal disease. Summary of Background Data The use of BMP has been proposed to reduce overall costs of spinal fusion through prevention of repeat fusion procedures. Although radiographic fusion rates associated with BMP use have been examined in clinical trials, little data exists regarding outcomes associated with BMP use in the general population. Methods Using the MarketScan© claims dataset, 15,862 patients that underwent single-level lumbar fusion from 2003 to 2007 for degenerative disease were identified. Propensity scores were used to match 2,372 patients that underwent fusion with BMP to patients that underwent fusion without BMP. Logistic regression models, Kaplan-Meier estimates, and Cox proportional hazards models were used to examine risk of repeat fusion, length of stay, and 30-day readmission by BMP use. Cost comparisons were evaluated with linear regression models using logarithmic transformed data. Results At one year from surgery, BMP was associated with a 1.1% absolute decrease in the risk of repeat fusion (2.3% with BMP vs 3.4% without BMP, p=.03) and an odds ratio for repeat fusion of 0.66 (95% confidence interval 0.47-0.94) after multivariate adjustment. BMP was also associated with a decreased hazards for long-term repeat fusion (adjusted hazards ratio =0.74, 95% confidence interval 0.58-0.93). Cost analysis indicated that BMP was associated with initial increased costs for the surgical procedure (13.9% adjusted increase, 95% confidence interval 9.9%-17.9%) as well as total one year costs (10.1% adjusted increase, 95% confidence interval 6.2%-14.0%). Conclusions At one year, BMP use was associated with a decreased risk of repeat fusion but also increased healthcare costs. PMID:21311404

  7. Appoptosin interacts with mitochondrial outer-membrane fusion proteins and regulates mitochondrial morphology.

    PubMed

    Zhang, Cuilin; Shi, Zhun; Zhang, Lingzhi; Zhou, Zehua; Zheng, Xiaoyuan; Liu, Guiying; Bu, Guojun; Fraser, Paul E; Xu, Huaxi; Zhang, Yun-Wu

    2016-03-01

    Mitochondrial morphology is regulated by fusion and fission machinery. Impaired mitochondria dynamics cause various diseases, including Alzheimer's disease. Appoptosin (encoded by SLC25A38) is a mitochondrial carrier protein that is located in the mitochondrial inner membrane. Appoptosin overexpression causes overproduction of reactive oxygen species (ROS) and caspase-dependent apoptosis, whereas appoptosin downregulation abolishes β-amyloid-induced mitochondrial fragmentation and neuronal death during Alzheimer's disease. Herein, we found that overexpression of appoptosin resulted in mitochondrial fragmentation in a manner independent of its carrier function, ROS production or caspase activation. Although appoptosin did not affect levels of mitochondrial outer-membrane fusion (MFN1 and MFN2), inner-membrane fusion (OPA1) and fission [DRP1 (also known as DNM1L) and FIS1] proteins, appoptosin interacted with MFN1 and MFN2, as well as with the mitochondrial ubiquitin ligase MITOL (also known as MARCH5) but not OPA1, FIS1 or DRP1. Appoptosin overexpression impaired the interaction between MFN1 and MFN2, and mitochondrial fusion. By contrast, co-expression of MFN1, MITOL and a dominant-negative form of DRP1, DRP1(K38A), partially rescued appoptosin-induced mitochondrial fragmentation and apoptosis, whereas co-expression of FIS1 aggravated appoptosin-induced apoptosis. Together, our results demonstrate that appoptosin can interact with mitochondrial outer-membrane fusion proteins and regulates mitochondrial morphology. PMID:26813789

  8. Design and characterization of novel recombinant listeriolysin O-protamine fusion proteins for enhanced gene delivery.

    PubMed

    Kim, Na Hyung; Provoda, Chester; Lee, Kyung-Dall

    2015-02-01

    To improve the efficiency of gene delivery for effective gene therapy, it is essential that the vector carries functional components that can promote overcoming barriers in various steps leading to the transport of DNA from extracellular to ultimately nuclear compartment. In this study, we designed genetically engineered fusion proteins as a platform to incorporate multiple functionalities in one chimeric protein. Prototypes of such a chimera tested here contain two domains: one that binds to DNA; the other that can facilitate endosomal escape of DNA. The fusion proteins are composed of listeriolysin O (LLO), the endosomolytic pore-forming protein from Listeria monocytogenes, and a 22 amino acid sequence of the DNA-condensing polypeptide protamine (PN), singly or as a pair: LLO-PN and LLO-PNPN. We demonstrate dramatic enhancement of the gene delivery efficiency of protamine-condensed DNA upon incorporation of a small amount of LLO-PN fusion protein and further improvement with LLO-PNPN in vitro using cultured cells. Additionally, the association of anionic liposomes with cationic LLO-PNPN/protamine/DNA complexes, yielding a net negative surface charge, resulted in better in vitro transfection efficiency in the presence of serum. An initial, small set of data in mice indicated that the observed enhancement in gene expression could also be applicable to in vivo gene delivery. This study suggests that incorporation of a recombinant fusion protein with multiple functional components, such as LLO-protamine fusion protein, in a nonviral vector is a promising strategy for various nonviral gene delivery systems. PMID:25521817

  9. Analysis of drug-protein binding using on-line immunoextraction and high-performance affinity microcolumns: Studies with normal and glycated human serum albumin.

    PubMed

    Matsuda, Ryan; Jobe, Donald; Beyersdorf, Jared; Hage, David S

    2015-10-16

    A method combining on-line immunoextraction microcolumns with high-performance affinity chromatography (HPAC) was developed and tested for use in examining drug-protein interactions with normal or modified proteins. Normal human serum albumin (HSA) and glycated HSA were used as model proteins for this work. High-performance immunoextraction microcolumns with sizes of 1.0-2.0 cm × 2.1mm i.d. and containing anti-HSA polyclonal antibodies were developed and tested for their ability to bind normal HSA or glycated HSA. These microcolumns were able to extract up to 82-93% for either type of protein at 0.05-0.10 mL/min and had a binding capacity of 0.34-0.42 nmol HSA for a 1.0 cm × 2.1mm i.d. microcolumn. The immunoextraction microcolumns and their adsorbed proteins were tested for use in various approaches for drug binding studies. Frontal analysis was used with the adsorbed HSA/glycated HSA to measure the overall affinities of these proteins for the drugs warfarin and gliclazide, giving comparable values to those obtained previously using similar protein preparations that had been covalently immobilized within HPAC columns. Zonal elution competition studies with gliclazide were next performed to examine the specific interactions of this drug at Sudlow sites I and II of the adsorbed proteins. These results were also comparable to those noted in prior work with covalently immobilized samples of normal HSA or glycated HSA. These experiments indicated that drug-protein binding studies can be carried out by using on-line immunoextraction microcolumns with HPAC. The same method could be used in the future with clinical samples and other drugs or proteins of interest in pharmaceutical studies or biomedical research. PMID:26381571

  10. Role of spike protein conformational changes in fusion of Semliki Forest virus.

    PubMed

    Justman, J; Klimjack, M R; Kielian, M

    1993-12-01

    The alphavirus Semliki Forest virus (SFV) and a number of other enveloped animal viruses infect cells via a membrane fusion reaction triggered by the low pH within endocytic vesicles. In addition to having a low pH requirement, SFV fusion and infection are also strictly dependent on the presence of cholesterol in the host cell membrane. A number of conformational changes in the SFV spike protein occur following low-pH treatment, including dissociation of the E1-E2 dimer, conformational changes in the E1 and E2 subunits, and oligomerization of E1 to a homotrimer. To allow the ordering of these events, we have compared the kinetics of these conformational changes with those of fusion, using pH treatment near the fusion threshold and low-temperature incubation to slow the fusion reaction. Dimer dissociation, the E1 conformational change, and E1 trimerization all occur prior to the mixing of virus and cell membranes. Studies of cells incubated at 20 degrees C showed that as with virus fusion, E1 trimerization occurred in the endosome before transport to lysosomes. However, unlike the strictly cholesterol-dependent membrane fusion reaction, the E1 homotrimer was produced in vivo during virus uptake by cholesterol-depleted cells or in vitro by low-pH treatment of virus in the presence of artificial liposomes with or without cholesterol. Purified, lipid-free spike protein rosettes were assayed to determine the requirement for virus membrane cholesterol in E1 homotrimer formation. Spike protein rosettes were found to undergo E1 oligomerization upon exposure to low pH and target liposomes and showed an enhancement of oligomerization with cholesterol-containing membranes. The E1 homotrimer may represent a perfusion complex that requires cholesterol to carry out the final coalescence of the viral and target membranes. PMID:8230478

  11. Studies to Prevent Degradation of Recombinant Fc-Fusion Protein Expressed in Mammalian Cell Line and Protein Characterization

    PubMed Central

    Chakrabarti, Sanjukta; Barrow, Colin J.; Kanwar, Rupinder K.; Ramana, Venkata; Kanwar, Jagat R.

    2016-01-01

    Clipping of recombinant proteins is a major issue in animal cell cultures. A recombinant Fc-fusion protein, VEGFR1(D1–D3)-Fc expressed in CHOK1SV GS-KO cells was observed to be undergoing clippings in lab scale cultures. Partial cleaving of expressed protein initiated early on in cell culture and was observed to increase over time in culture and also on storage. In this study, a few parameters were explored in a bid to inhibit clipping in the fusion protein The effects of culture temperature, duration of culture, the addition of an anti-clumping agent, ferric citrate and use of protease inhibitor cocktail on inhibition of proteolysis of the Fc fusion were studied. Lowering of culture temperature from 37 to 30 °C alone appears to be the best solution for reducing protein degradation from the quality, cost and regulatory points of view. The obtained Fc protein was characterized and found to be in its stable folded state, exhibiting a high affinity for its ligand and also biological and functional activities. PMID:27294920

  12. Studies to Prevent Degradation of Recombinant Fc-Fusion Protein Expressed in Mammalian Cell Line and Protein Characterization.

    PubMed

    Chakrabarti, Sanjukta; Barrow, Colin J; Kanwar, Rupinder K; Ramana, Venkata; Kanwar, Jagat R

    2016-01-01

    Clipping of recombinant proteins is a major issue in animal cell cultures. A recombinant Fc-fusion protein, VEGFR1(D1-D3)-Fc expressed in CHOK1SV GS-KO cells was observed to be undergoing clippings in lab scale cultures. Partial cleaving of expressed protein initiated early on in cell culture and was observed to increase over time in culture and also on storage. In this study, a few parameters were explored in a bid to inhibit clipping in the fusion protein The effects of culture temperature, duration of culture, the addition of an anti-clumping agent, ferric citrate and use of protease inhibitor cocktail on inhibition of proteolysis of the Fc fusion were studied. Lowering of culture temperature from 37 to 30 °C alone appears to be the best solution for reducing protein degradation from the quality, cost and regulatory points of view. The obtained Fc protein was characterized and found to be in its stable folded state, exhibiting a high affinity for its ligand and also biological and functional activities. PMID:27294920

  13. Genetically engineered endostatin-lidamycin fusion proteins effectively inhibit tumor growth and metastasis

    PubMed Central

    2013-01-01

    Background Endostatin (ES) inhibits endothelial cell proliferation, migration, invasion, and tube formation. It also shows antiangiogenesis and antitumor activities in several animal models. Endostatin specifically targets tumor vasculature to block tumor growth. Lidamycin (LDM), which consists of an active enediyne chromophore (AE) and a non-covalently bound apo-protein (LDP), is a member of chromoprotein family of antitumor antibiotics with extremely potent cytotoxicity to cancer cells. Therefore, we reasoned that endostatin-lidamycin (ES-LDM) fusion proteins upon energizing with enediyne chromophore may obtain the combined capability targeting tumor vasculature and tumor cell by respective ES and LDM moiety. Methods In this study, we designed and obtained two new endostatin-based fusion proteins, endostatin-LDP (ES-LDP) and LDP-endostatin (LDP-ES). In vitro, the antiangiogenic effect of fusion proteins was determined by the wound healing assay and tube formation assay and the cytotoxicity of their enediyne-energized analogs was evaluated by CCK-8 assay. Tissue microarray was used to analyze the binding affinity of LDP, ES or ES-LDP with specimens of human lung tissue and lung tumor. The in vivo efficacy of the fusion proteins was evaluated with human lung carcinoma PG-BE1 xenograft and the experimental metastasis model of 4T1-luc breast cancer. Results ES-LDP and LDP-ES disrupted the formation of endothelial tube structures and inhibited endothelial cell migration. Evidently, ES-LDP accumulated in the tumor and suppressed tumor growth and metastasis. ES-LDP and ES show higher binding capability than LDP to lung carcinoma; in addition, ES-LDP and ES share similar binding capability. Furthermore, the enediyne-energized fusion protein ES-LDP-AE demonstrated significant efficacy against lung carcinoma xenograft in athymic mice. Conclusions The ES-based fusion protein therapy provides some fundamental information for further drug development. Targeting both tumor

  14. Enhanced SUMOylation of proteins containing a SUMO-interacting motif by SUMO-Ubc9 fusion

    SciTech Connect

    Kim, Eui Tae; Kim, Kyeong Kyu; Matunis, Mike J.; Ahn, Jin-Hyun

    2009-10-09

    Identifying new targets for SUMO and understanding the function of protein SUMOylation are largely limited by low level of SUMOylation. It was found recently that Ubc9, the SUMO E2 conjugating enzyme, is covalently modified by SUMO at a lysine 14 in the N-terminal alpha helix, and that SUMO-modified Ubc9 has enhanced conjugation activity for certain target proteins containing a SUMO-interacting motif (SIM). Here, we show that, compared to intact Ubc9, the SUMO-Ubc9 fusion protein has higher conjugating activity for SIM-containing targets such as Sp100 and human cytomegalovirus IE2. Assays using an IE2 SIM mutant revealed the requirement of SIM for the enhanced IE2 SUMOylation by SUMO-Ubc9. In pull-down assays with cell extracts, the SUMO-Ubc9 fusion protein bound to more diverse cellular proteins and interacted with some SIM-containing proteins with higher affinities than Ubc9. Therefore, the devised SUMO-Ubc9 fusion will be useful for identifying SIM-containing SUMO targets and producing SUMO-modified proteins.

  15. SUMO fusion technology for enhanced protein production in prokaryotic and eukaryotic expression systems.

    PubMed

    Panavas, Tadas; Sanders, Carsten; Butt, Tauseef R

    2009-01-01

    In eukaryotic cells, the reversible attachment of small ubiquitin-like modifier (SUMO) protein is a post-translational modification that has been demonstrated to play an important role in various cellular processes. Moreover, it has been found that SUMO as an N-terminal fusion partner enhances functional protein production in prokaryotic and eukaryotic expression systems, based upon significantly improved protein stability and solubility. Following the expression and purification of the fusion protein, the SUMO-tag can be cleaved by specific (SUMO) proteases via their endopeptidase activity in vitro to generate the desired N-terminus of the released protein partner. In addition to its physiological relevance in eukaryotes, SUMO can, thus, be used as a powerful biotechnological tool for protein expression in prokaryotic and eukaryotic cell systems.In this chapter, we will describe the construction of a fusion protein with the SUMO-tag, its expression in Escherichia coli, and its purification followed by the removal of the SUMO-tag by a SUMO-specific protease in vitro. PMID:19107426

  16. A single vesicle-vesicle fusion assay for in vitro studies of SNAREs and accessory proteins.

    PubMed

    Diao, Jiajie; Ishitsuka, Yuji; Lee, Hanki; Joo, Chirlmin; Su, Zengliu; Syed, Salman; Shin, Yeon-Kyun; Yoon, Tae-Young; Ha, Taekjip

    2012-05-01

    SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins are a highly regulated class of membrane proteins that drive the efficient merger of two distinct lipid bilayers into one interconnected structure. This protocol describes our fluorescence resonance energy transfer (FRET)-based single vesicle-vesicle fusion assays for SNAREs and accessory proteins. Both lipid-mixing (with FRET pairs acting as lipophilic dyes in the membranes) and content-mixing assays (with FRET pairs present on a DNA hairpin that becomes linear via hybridization to a complementary DNA) are described. These assays can be used to detect substages such as docking, hemifusion, and pore expansion and full fusion. The details of flow cell preparation, protein-reconstituted vesicle preparation, data acquisition and analysis are described. These assays can be used to study the roles of various SNARE proteins, accessory proteins and effects of different lipid compositions on specific fusion steps. The total time required to finish one round of this protocol is 3–6 d. PMID:22582418

  17. A single vesicle-vesicle fusion assay for in vitro studies of SNAREs and accessory proteins

    PubMed Central

    Diao, Jiajie; Ishitsuka, Yuji; Lee, Hanki; Joo, Chirlmin; Su, Zengliu; Syed, Salman; Shin, Yeon-Kyun; Yoon, Tae-Young; Ha, Taekjip

    2015-01-01

    SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins are a highly regulated class of membrane proteins that drive the efficient merger of two distinct lipid bilayers into one interconnected structure. This protocol describes our fluorescence resonance energy transfer (FRET)-based single vesicle-vesicle fusion assays for SNAREs and accessory proteins. Both lipid-mixing (with FRET pairs acting as lipophilic dyes in the membranes) and content-mixing assays (with FRET pairs present on a DNA hairpin that becomes linear via hybridization to a complementary DNA) are described. These assays can be used to detect substages such as docking, hemifusion, and pore expansion and full fusion. The details of flow cell preparation, protein-reconstituted vesicle preparation, data acquisition and analysis are described. These assays can be used to study the roles of various SNARE proteins, accessory proteins and effects of different lipid compositions on specific fusion steps. The total time required to finish one round of this protocol is 3–6 d. PMID:22582418

  18. Characterization of the binding of metoprolol tartrate and guaifenesin drugs to human serum albumin and human hemoglobin proteins by fluorescence and circular dichroism spectroscopy.

    PubMed

    Duman, Osman; Tunç, Sibel; Kancı Bozoğlan, Bahar

    2013-07-01

    The interactions of metoprolol tartrate (MPT) and guaifenesin (GF) drugs with human serum albumin (HSA) and human hemoglobin (HMG) proteins at pH 7.4 were studied by fluorescence and circular dichroism (CD) spectroscopy. Drugs quenched the fluorescence spectra of HSA and HMG proteins through a static quenching mechanism. For each protein-drug system, the values of Stern-Volmer quenching constant, bimolecular quenching constant, binding constant and number of binding site on the protein molecules were determined at 288.15, 298.15, 310.15 and 318.15 K. It was found that the binding constants of HSA-MPT and HSA-GF systems were smaller than those of HMG-MPT and HMG-GF systems. For both drugs, the affinity of HMG was much higher than that of HSA. An increase in temperature caused a negative effect on the binding reactions. The number of binding site on blood proteins for MPT and GF drugs was approximately one. Thermodynamic parameters showed that MPT interacted with HSA through electrostatic attraction forces. However, hydrogen bonds and van der Waals forces were the main interaction forces in the formation of HSA-GF, HMG-MPT and HMG-GF complexes. The binding processes between protein and drug molecules were exothermic and spontaneous owing to negative ∆H and ∆G values, respectively. The values of binding distance between protein and drug molecules were calculated from Förster resonance energy transfer theory. It was found from CD analysis that the bindings of MPT and GF drugs to HSA and HMG proteins altered the secondary structure of HSA and HMG proteins. PMID:23471625

  19. Integrin αvβ1 Modulation Affects Subtype B Avian Metapneumovirus Fusion Protein-mediated Cell-Cell Fusion and Virus Infection.

    PubMed

    Yun, Bing-Ling; Guan, Xiao-Lu; Liu, Yong-Zhen; Zhang, Yao; Wang, Yong-Qiang; Qi, Xiao-Le; Cui, Hong-Yu; Liu, Chang-Jun; Zhang, Yan-Ping; Gao, Hong-Lei; Gao, Li; Li, Kai; Gao, Yu-Long; Wang, Xiao-Mei

    2016-07-01

    Avian metapneumovirus (aMPV) fusion (F) protein mediates virus-cell membrane fusion to initiate viral infection, which requires F protein binding to its receptor(s) on the host cell surface. However, the receptor(s) for aMPV F protein is still not identified. All known subtype B aMPV (aMPV/B) F proteins contain a conserved Arg-Asp-Asp (RDD) motif, suggesting that the aMPV/B F protein may mediate membrane fusion via the binding of RDD to integrin. When blocked with integrin-specific peptides, aMPV/B F protein fusogenicity and viral replication were significantly reduced. Specifically we identified integrin αv and/or β1-mediated F protein fusogenicity and viral replication using antibody blocking, small interfering RNAs (siRNAs) knockdown, and overexpression. Additionally, overexpression of integrin αv and β1 in aMPV/B non-permissive cells conferred aMPV/B F protein binding and aMPV/B infection. When RDD was altered to RAE (Arg-Ala-Glu), aMPV/B F protein binding and fusogenic activity were profoundly impaired. These results suggest that integrin αvβ1 is a functional receptor for aMPV/B F protein-mediated membrane fusion and virus infection, which will provide new insights on the fusogenic mechanism and pathogenesis of aMPV. PMID:27226547

  20. Glycated Serum Albumin and AGE Receptors.

    PubMed

    Vetter, Stefan W

    2015-01-01

    In vivo modification of proteins by molecules with reactive carbonyl groups leads to intermediate and advanced glycation end products (AGE). Glucose is a significant glycation reagent due to its high physiological concentration and poorly controlled diabetics show increased albumin glycation. Increased levels of glycated and AGE-modified albumin have been linked to diabetic complications, neurodegeneration, and vascular disease. This review discusses glycated albumin formation, structural consequences of albumin glycation on drug binding, removal of circulating AGE by several scavenger receptors, as well as AGE-induced proinflammatory signaling through activation of the receptor for AGE. Analytical methods for quantitative detection of protein glycation and AGE formation are compared. Finally, the use of glycated albumin as a novel clinical marker to monitor glycemic control is discussed and compared to glycated hemoglobin (HbA1c) as long-term indicator of glycemic status. PMID:26471084

  1. Purification of recombinant EGFP by fusion with L2 (252-273) from ribosomal protein L2 using magnetic particles.

    PubMed

    Li, Junhua; Dong, Yiting; Zhang, Yang; Yang, Yanjun

    2013-02-15

    A basic polypeptide L2 (252-273) derived from Escherichia coli ribosomal protein L2 was used as a purification tag. In order to develop faster, less expensive methods for expression and purification of proteins, the L2 (252-273)-small ubiquitin like modifier (SUMO) fusion expression system was constructed. We comparatively analyzed the adsorption properties of the deleted protein of L2 (L2 (252-273)) on diatomite and superparamagnetic carboxymethyl chitosan nanoparticles. The time required to reach adsorption equilibrium of L2 (252-273) fusion protein on diatomite was shorter than that of L2 (252-273) fusion protein on magnetic particles. The maximum adsorption capacity of L2 (252-273) fusion protein on magnetic particles was about 5 times larger than that of L2 (252-273) fusion protein on diatomite. SUMO was introduced as a specific protease cleavage site between the target protein and the purification tags. The enhanced green fluorescent protein (EGFP) as a model protein was fused with the L2 (252-273)-SUMO fusion protein and purified by a simple method which involves the electrostatic adsorption of L2 (252-273) fusion proteins on superparamagnetic carboxymethyl chitosan nanoparticles and the L2 (252-273)-SUMO fusion partner was removed based on the robust cleavage by the poly lysine tagged SUMO protease. The high purity of tag-free EGFP (>93%) was obtained. Our results preliminary proved that the system was an effective fusion expression system for the production of recombinant proteins in E. coli. PMID:23353812

  2. A multipurpose fusion tag derived from an unstructured and hyperacidic region of the amyloid precursor protein

    PubMed Central

    Sangawa, Takeshi; Tabata, Sanae; Suzuki, Kei; Saheki, Yasushi; Tanaka, Keiji; Takagi, Junichi

    2013-01-01

    Expression and purification of aggregation-prone and disulfide-containing proteins in Escherichia coli remains as a major hurdle for structural and functional analyses of high-value target proteins. Here, we present a novel gene-fusion strategy that greatly simplifies purification and refolding procedure at very low cost using a unique hyperacidic module derived from the human amyloid precursor protein. Fusion with this polypeptide (dubbed FATT for Flag-Acidic-Target Tag) results in near-complete soluble expression of variety of extracellular proteins, which can be directly refolded in the crude bacterial lysate and purified in one-step by anion exchange chromatography. Application of this system enabled preparation of functionally active extracellular enzymes and antibody fragments without the need for condition optimization. PMID:23526492

  3. Downregulation of RUNX1/CBFβ by MLL fusion proteins enhances hematopoietic stem cell self-renewal

    PubMed Central

    Zhao, Xinghui; Chen, Aili; Yan, Xiaomei; Zhang, Yue; He, Fuhong; Hayashi, Yoshihiro; Dong, Yunzhu; Rao, Yalan; Li, Bo; Conway, Rajeana M.; Maiques-Diaz, Alba; Elf, Shannon E.; Huang, Nuomin; Zuber, Johannes; Xiao, Zhijian; Tse, William; Tenen, Daniel G.; Wang, Qianfei; Chen, Wei; Mulloy, James C.; Nimer, Stephen D.

    2014-01-01

    RUNX1/CBFβ (core binding factor [CBF]) is a heterodimeric transcription factor complex that is frequently involved in chromosomal translocations, point mutations, or deletions in acute leukemia. The mixed lineage leukemia (MLL) gene is also frequently involved in chromosomal translocations or partial tandem duplication in acute leukemia. The MLL protein interacts with RUNX1 and prevents RUNX1 from ubiquitin-mediated degradation. RUNX1/CBFβ recruits MLL to regulate downstream target genes. However, the functional consequence of MLL fusions on RUNX1/CBFβ activity has not been fully understood. In this report, we show that MLL fusion proteins and the N-terminal MLL portion of MLL fusions downregulate RUNX1 and CBFβ protein expression via the MLL CXXC domain and flanking regions. We confirmed this finding in Mll-Af9 knock-in mice and human M4/M5 acute myeloid leukemia (AML) cell lines, with or without MLL translocations, showing that MLL translocations cause a hypomorph phenotype of RUNX1/CBFβ. Overexpression of RUNX1 inhibits the development of AML in Mll-Af9 knock-in mice; conversely, further reducing Runx1/Cbfβ levels accelerates MLL-AF9–mediated AML in bone marrow transplantation assays. These data reveal a newly defined negative regulation of RUNX1/CBFβ by MLL fusion proteins and suggest that targeting RUNX1/CBFβ levels may be a potential therapy for MLLs. PMID:24449215

  4. Newcastle disease virus fusion and haemagglutinin-neuraminidase proteins contribute to its macrophage host range

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Newcastle disease virus (NDV) is an avian paramyxovirus that causes significant economic damage to international poultry industry. Different strains of NDV express a wide range of virulence that is primarily dependent on the amino acid sequence of the strain’s fusion (F) protein cleavage site. Two c...

  5. Recombinant albumin monolayers on latex particles.

    PubMed

    Sofińska, Kamila; Adamczyk, Zbigniew; Kujda, Marta; Nattich-Rak, Małgorzata

    2014-01-14

    The adsorption of recombinant human serum albumin (rHSA) on negatively charged polystyrene latex micro-particles was studied at pH 3.5 and the NaCl concentration range of 10(-3) to 0.15 M. The electrophoretic mobility of latex monotonically increased with the albumin concentration in the suspension. The coverage of adsorbed albumin was quantitatively determined using the depletion method, where the residual protein concentration was determined by electrokinetic measurements and AFM imaging. It was shown that albumin adsorption was irreversible. Its maximum coverage on latex varied between 0.7 mg m(-2) for 10(-3) M NaCl to 1.3 mg m(-2) for 0.15 M NaCl. The latter value matches the maximum coverage previously determined for human serum albumin on mica using the streaming potential method. The increase in the maximum coverage was interpreted in terms of reduced electrostatic repulsion among adsorbed molecules. These facts confirm that albumin adsorption at pH 3.5 is governed by electrostatic interactions and proceeds analogously to colloid particle deposition. The stability of albumin monolayers was measured in additional experiments where changes in the latex electrophoretic mobility and the concentration of free albumin in solutions were monitored over prolonged time periods. Based on these experimental data, a robust procedure of preparing albumin monolayers on latex particles of well-controlled coverage and molecule distribution was proposed. PMID:24354916

  6. Human podocytes perform polarized, caveolae-dependent albumin endocytosis

    PubMed Central

    Dobrinskikh, Evgenia; Okamura, Kayo; Kopp, Jeffrey B.; Doctor, R. Brian

    2014-01-01

    The renal glomerulus forms a selective filtration barrier that allows the passage of water, ions, and small solutes into the urinary space while restricting the passage of cells and macromolecules. The three layers of the glomerular filtration barrier include the vascular endothelium, glomerular basement membrane (GBM), and podocyte epithelium. Podocytes are capable of internalizing albumin and are hypothesized to clear proteins that traverse the GBM. The present study followed the fate of FITC-labeled albumin to establish the mechanisms of albumin endocytosis and processing by podocytes. Confocal imaging and total internal reflection fluorescence microscopy of immortalized human podocytes showed FITC-albumin endocytosis occurred preferentially across the basal membrane. Inhibition of clathrin-mediated endocytosis and caveolae-mediated endocytosis demonstrated that the majority of FITC-albumin entered podocytes through caveolae. Once internalized, FITC-albumin colocalized with EEA1 and LAMP1, endocytic markers, and with the neonatal Fc receptor, a marker for transcytosis. After preloading podocytes with FITC-albumin, the majority of loaded FITC-albumin was lost over the subsequent 60 min of incubation. A portion of the loss of albumin occurred via lysosomal degradation as pretreatment with leupeptin, a lysosomal protease inhibitor, partially inhibited the loss of FITC-albumin. Consistent with transcytosis of albumin, preloaded podocytes also progressively released FITC-albumin into the extracellular media. These studies confirm the ability of podocytes to endocytose albumin and provide mechanistic insight into cellular mechanisms and fates of albumin handling in podocytes. PMID:24573386

  7. C-E1 fusion protein synthesized by rubella virus DI RNAs maintained during serial passage

    SciTech Connect

    Tzeng, W.-P.; Frey, Teryl K. . E-mail: tfrey@gsu.edu

    2006-12-20

    Rubella virus (RUB) replicons are derivatives of the RUB infectious cDNA clone that retain the nonstructural open reading frame (NS-ORF) that encodes the replicase proteins but not the structural protein ORF (SP-ORF) that encodes the virion proteins. RUB defective interfering (DI) RNAs contain deletions within the SP-ORF and thus resemble replicons. DI RNAs often retain the 5' end of the capsid protein (C) gene that has been shown to modulate virus-specific RNA synthesis. However, when replicons either with or without the C gene were passaged serially in the presence of wt RUB as a source of the virion proteins, it was found that neither replicon was maintained and DI RNAs were generated. The majority DI RNA species contained in-frame deletions in the SP-ORF leading to a fusion between the 5' end of the C gene and the 3' end of the E1 glycoprotein gene. DI infectious cDNA clones were constructed and transcripts from these DI infectious cDNA clones were maintained during serial passage with wt RUB. The C-E1 fusion protein encoded by the DI RNAs was synthesized and was required for maintenance of the DI RNA during serial passage. This is the first report of a functional novel gene product resulting from deletion during DI RNA generation. Thus far, the role of the C-E1 fusion protein in maintenance of DI RNAs during serial passage remained elusive as it was found that the fusion protein diminished rather than enhanced DI RNA synthesis and was not incorporated into virus particles.

  8. Shank2 Regulates Renal Albumin Endocytosis

    PubMed Central

    Dobrinskikh, Evgenia; Lewis, Linda; Doctor, R Brian; Okamura, Kayo; Lee, Min Goo; Altmann, Christopher; Faubel, Sarah; Kopp, Jeffrey B; Blaine, Judith

    2015-01-01

    Albuminuria is a strong and independent predictor of kidney disease progression but the mechanisms of albumin handling by the kidney remain to be fully defined. Previous studies have shown that podocytes endocytose albumin. Here we demonstrate that Shank2, a large scaffolding protein originally identified at the neuronal postsynaptic density, is expressed in podocytes in vivo and in vitro and plays an important role in albumin endocytosis in podocytes. Knockdown of Shank2 in cultured human podocytes decreased albumin uptake, but the decrease was not statistically significant likely due to residual Shank2 still present in the knockdown podocytes. Complete knockout of Shank2 in podocytes significantly diminished albumin uptake in vitro. Shank2 knockout mice develop proteinuria by 8 weeks of age. To examine albumin handling in vivo in wild-type and Shank2 knockout mice we used multiphoton intravital imaging. While FITC-labeled albumin was rapidly seen in the renal tubules of wild-type mice after injection, little albumin was seen in the tubules of Shank2 knockout mice indicating dysregulated renal albumin trafficking in the Shank2 knockouts. We have previously found that caveolin-1 is required for albumin endocytosis in cultured podocytes. Shank2 knockout mice had significantly decreased expression and altered localization of caveolin-1 in podocytes suggesting that disruption of albumin endocytosis in Shank2 knockouts is mediated via caveolin-1. In summary, we have identified Shank2 as another component of the albumin endocytic pathway in podocytes. PMID:26333830

  9. Transgenic plants expressing ω-ACTX-Hv1a and snowdrop lectin (GNA) fusion protein show enhanced resistance to aphids

    PubMed Central

    Nakasu, Erich Y. T.; Edwards, Martin G.; Fitches, Elaine; Gatehouse, John A.; Gatehouse, Angharad M. R.

    2014-01-01

    Recombinant fusion proteins containing arthropod toxins have been developed as a new class of biopesticides. The recombinant fusion protein Hv1a/GNA, containing the spider venom toxin ω-ACTX-Hv1a linked to snowdrop lectin (GNA) was shown to reduce survival of the peach-potato aphid Myzus persicae when delivered in artificial diet, with survival <10% after 8 days exposure to fusion protein at 1 mg/ml. Although the fusion protein was rapidly degraded by proteases in the insect, Hv1a/GNA oral toxicity to M. persicae was significantly greater than GNA alone. A construct encoding the fusion protein, including the GNA leader sequence, under control of the constitutive CaMV 35S promoter was transformed into Arabidopsis; the resulting plants contained intact fusion protein in leaf tissues at an estimated level of 25.6 ± 4.1 ng/mg FW. Transgenic Arabidopsis expressing Hv1a/GNA induced up to 40% mortality of M. persicae after 7 days exposure in detached leaf bioassays, demonstrating that transgenic plants can deliver fusion proteins to aphids. Grain aphids (Sitobion avenae) were more susceptible than M. persicae to the Hv1a/GNA fusion protein in artificial diet bioassays (LC50 = 0.73 mg/ml after 2 days against LC50 = 1.81 mg/ml for M. persicae), as they were not able to hydrolyze the fusion protein as readily as M. persicae. Expression of this fusion protein in suitable host plants for the grain aphid is likely to confer higher levels of resistance than that shown with the M. persicae/Arabidopsis model system. PMID:25506351

  10. Purification of recombinant protein by cold-coacervation of fusion constructs incorporating resilin-inspired polypeptides.

    PubMed

    Lyons, Russell E; Elvin, Christopher M; Taylor, Karin; Lekieffre, Nicolas; Ramshaw, John A M

    2012-12-01

    Polypeptides containing between 4 and 32 repeats of a resilin-inspired sequence AQTPSSYGAP, derived from the mosquito Anopheles gambiae, have been used as tags on recombinant fusion proteins. These repeating polypeptides were inspired by the repeating structures that are found in resilins and sequence-related proteins from various insects. Unexpectedly, an aqueous solution of a recombinant resilin protein displays an upper critical solution temperature (cold-coacervation) when held on ice, leading to a separation into a protein rich phase, typically exceeding 200 mg/mL, and a protein-poor phase. We show that purification of recombinant proteins by cold-coacervation can be performed when engineered as a fusion partner to a resilin-inspired repeat sequence. In this study, we demonstrate the process by the recombinant expression and purification of enhanced Green fluorescent protein (EGFP) in E. coli. This facile purification system can produce high purity, concentrated protein solutions without the need for affinity chromatography or other time-consuming or expensive purification steps, and that it can be used with other bulk purification steps such as low concentration ammonium sulfate precipitation. Protein purification by cold-coacervation also minimizes the exposure of the target protein to enhanced proteolysis at higher temperature. PMID:22627880

  11. A heme fusion tag for protein affinity purification and quantification

    PubMed Central

    Asher, Wesley B; Bren, Kara L

    2010-01-01

    We report a novel affinity-based purification method for proteins expressed in Escherichia coli that uses the coordination of a heme tag to an l-histidine-immobilized sepharose (HIS) resin. This approach provides an affinity purification tag visible to the eye, facilitating tracking of the protein. We show that azurin and maltose binding protein are readily purified from cell lysate using the heme tag and HIS resin. Mild conditions are used; heme-tagged proteins are bound to the HIS resin in phosphate buffer, pH 7.0, and eluted by adding 200–500 mM imidazole or binding buffer at pH 5 or 8. The HIS resin exhibits a low level of nonspecific binding of untagged cellular proteins for the systems studied here. An additional advantage of the heme tag-HIS method for purification is that the heme tag can be used for protein quantification by using the pyridine hemochrome absorbance method for heme concentration determination. PMID:20665691

  12. In Vitro Enhancement of Carvedilol Glucuronidation by Amiodarone-Mediated Altered Protein Binding in Incubation Mixture of Human Liver Microsomes with Bovine Serum Albumin.

    PubMed

    Sekimoto, Makoto; Takamori, Toru; Nakamura, Saki; Taguchi, Masato

    2016-01-01

    Carvedilol is mainly metabolized in the liver to O-glucuronide (O-Glu). We previously found that the glucuronidation activity of racemic carvedilol in pooled human liver microsomes (HLM) was increased, R-selectively, in the presence of amiodarone. The aim of this study was to clarify the mechanisms for the enhancing effect of amiodarone on R- and S-carvedilol glucuronidation. We evaluated O-Glu formation of R- and S-carvedilol enantiomers in a reaction mixture of HLM including 0.2% bovine serum albumin (BSA). In the absence of amiodarone, glucuronidation activity of R- and S-carvedilol for 25 min was 0.026, and 0.51 pmol/min/mg protein, and that was increased by 6.15 and 1.60-fold in the presence of 50 µM amiodarone, respectively. On the other hand, in the absence of BSA, or when BSA was replaced with human serum albumin, no enhancing effect of amiodarone on glucuronidation activity was observed, suggesting that BSA played a role in the mechanisms for the enhancement of glucuronidation activity. Unbound fraction of S-carvedilol in the reaction mixture was greater than that of R-carvedilol in the absence of amiodarone. Also, the addition of amiodarone caused a greater increase of unbound fraction of R-carvedilol than that of S-carvedilol. These results suggest that the altered protein binding by amiodarone is a key mechanism for R-selective stimulation of carvedilol glucuronidation. PMID:27476943

  13. Construction of a linker library with widely controllable flexibility for fusion protein design.

    PubMed

    Li, Gang; Huang, Ziliang; Zhang, Chong; Dong, Bo-Jun; Guo, Ruo-Hai; Yue, Hong-Wei; Yan, Li-Tang; Xing, Xin-Hui

    2016-01-01

    Flexibility or rigidity of the linker between two fused proteins is an important parameter that affects the function of fusion proteins. In this study, we constructed a linker library with five elementary units based on the combination of the flexible (GGGGS) and the rigid (EAAAK) units. Molecular dynamics (MD) simulation showed that more rigid units in the linkers lead to more helical conformation and hydrogen bonds, and less distance fluctuation between the N- and C-termini of the linker. The diversity of linker flexibility of the linker library was then studied by fluorescence resonance energy transfer (FRET) of cyan fluorescent protein (CFP)-yellow fluorescent protein (YFP) fusion proteins, which showed that there is a wide range of distribution of the FRET efficiency. Dissipative particle dynamics (DPD) simulation of CFP-YFP with different linkers also gave identical results with that of FRET efficiency analysis, and we further found that the combination manner of the linker peptide had a remarkable effect on the orientation of CFP and YFP domains. Our studies demonstrated that the construction of the linker library with the widely controllable flexibility could provide appropriate linkers with the desirable characteristics to engineer the fusion proteins with the expected functions. PMID:26394862

  14. An Efficient Genome-Wide Fusion Partner Screening System for Secretion of Recombinant Proteins in Yeast

    PubMed Central

    Bae, Jung-Hoon; Hyun Sung, Bong; Kim, Hyun-Jin; Park, Soon-Ho; Lim, Kwang-Mook; Kim, Mi-Jin; Lee, Cho-Ryong; Sohn, Jung-Hoon

    2015-01-01

    To produce rarely secreted recombinant proteins in the yeast Saccharomyces cerevisiae, we developed a novel genome-wide optimal translational fusion partner (TFP) screening system that involves recruitment of an optimal secretion signal and fusion partner. A TFP library was constructed from a genomic and truncated cDNA library by using the invertase-based signal sequence trap technique. The efficiency of the system was demonstrated using two rarely secreted proteins, human interleukin (hIL)-2 and hIL-32. Optimal TFPs for secretion of hIL-2 and hIL-32 were easily selected, yielding secretion of these proteins up to hundreds of mg/L. Moreover, numerous uncovered yeast secretion signals and fusion partners were identified, leading to efficient secretion of various recombinant proteins. Selected TFPs were found to be useful for the hypersecretion of other recombinant proteins at yields of up to several g/L. This screening technique could provide new methods for the production of various types of difficult-to-express proteins. PMID:26195161

  15. Recombinant Human Bone Morphogenetic Protein-2 in Posterolateral Spinal Fusion: What's the Right Dose?

    PubMed Central

    Jones, Clifford Barry; Sietsema, Debra Lynn

    2016-01-01

    Study Design Single center retrospective cohort analysis. Purpose The goal was to evaluate the influence of varying amount of recombinant human bone morphogenetic protein 2 (rhBMP-2) per level on fusion rates and complications in posterolateral spinal fusions. Overview of Literature rhBMP-2 has been utilized for lumbar posterolateral fusions for many years. Initial rhBMP-2 recommendations were 20 mg/level of fusion. Dose and concentration per level in current studies vary from 4.2 to 40 mg and 1.5 to 2.0 mg/mL, respectively. Variable fusion and complication rates have been reported. Methods Patients (n=1,610) undergoing instrumented lumbar spinal fusion (2003–2009) with utilization of rhBMP-2 were retrospectively evaluated. Patient demographics, body mass index (BMI), comorbidities, number of levels, associated interbody fusion, and types of bone void filler were analyzed. Fusions rates and nonunions were subdivided into number of levels and amount of rhBMP-2 used per level. Results Patients (n=559) were evaluated with 58.5% females having an average age of 63 years, BMI of 31 kg/m2. Number of levels fused ranged from 1 to 8. rhBMP-2 averaged 7.3 mg/level (range, 1.5–24 mg/level) based upon length of collagen sponge in relation to length of fusion levels. Patients with non-union formation had lower rhBMP-2 dose per level (p=0.016). A significant difference in non-union rate was found between patients undergoing fusion with <6 mg/level compared to those with >6 mg/level (9.1% vs. 2.4%, χ2=0.012). No significant differences were noted between 6–11.9 mg/level and ≥12 mg/level. No threshold was found for seroma formation or bone overgrowth. Conclusions Previous recommendation of 20 mg/level of rhBMP-2 is more than what is required for predictable fusion rates of 98%. No dose related increase of infection, seroma formation, and bone overgrowth has been found. In order to provide variable dosing and cost reduction, industry generated rhBMP-2 kit size should be

  16. The BAR Domain Proteins: Molding Membranes in Fission, Fusion, and Phagy

    PubMed Central

    Ren, Gang; Vajjhala, Parimala; Lee, Janet S.; Winsor, Barbara; Munn, Alan L.

    2006-01-01

    The Bin1/amphiphysin/Rvs167 (BAR) domain proteins are a ubiquitous protein family. Genes encoding members of this family have not yet been found in the genomes of prokaryotes, but within eukaryotes, BAR domain proteins are found universally from unicellular eukaryotes such as yeast through to plants, insects, and vertebrates. BAR domain proteins share an N-terminal BAR domain with a high propensity to adopt α-helical structure and engage in coiled-coil interactions with other proteins. BAR domain proteins are implicated in processes as fundamental and diverse as fission of synaptic vesicles, cell polarity, endocytosis, regulation of the actin cytoskeleton, transcriptional repression, cell-cell fusion, signal transduction, apoptosis, secretory vesicle fusion, excitation-contraction coupling, learning and memory, tissue differentiation, ion flux across membranes, and tumor suppression. What has been lacking is a molecular understanding of the role of the BAR domain protein in each process. The three-dimensional structure of the BAR domain has now been determined and valuable insight has been gained in understanding the interactions of BAR domains with membranes. The cellular roles of BAR domain proteins, characterized over the past decade in cells as distinct as yeasts, neurons, and myocytes, can now be understood in terms of a fundamental molecular function of all BAR domain proteins: to sense membrane curvature, to bind GTPases, and to mold a diversity of cellular membranes. PMID:16524918

  17. The BAR domain proteins: molding membranes in fission, fusion, and phagy.

    PubMed

    Ren, Gang; Vajjhala, Parimala; Lee, Janet S; Winsor, Barbara; Munn, Alan L

    2006-03-01

    The Bin1/amphiphysin/Rvs167 (BAR) domain proteins are a ubiquitous protein family. Genes encoding members of this family have not yet been found in the genomes of prokaryotes, but within eukaryotes, BAR domain proteins are found universally from unicellular eukaryotes such as yeast through to plants, insects, and vertebrates. BAR domain proteins share an N-terminal BAR domain with a high propensity to adopt alpha-helical structure and engage in coiled-coil interactions with other proteins. BAR domain proteins are implicated in processes as fundamental and diverse as fission of synaptic vesicles, cell polarity, endocytosis, regulation of the actin cytoskeleton, transcriptional repression, cell-cell fusion, signal transduction, apoptosis, secretory vesicle fusion, excitation-contraction coupling, learning and memory, tissue differentiation, ion flux across membranes, and tumor suppression. What has been lacking is a molecular understanding of the role of the BAR domain protein in each process. The three-dimensional structure of the BAR domain has now been determined and valuable insight has been gained in understanding the interactions of BAR domains with membranes. The cellular roles of BAR domain proteins, characterized over the past decade in cells as distinct as yeasts, neurons, and myocytes, can now be understood in terms of a fundamental molecular function of all BAR domain proteins: to sense membrane curvature, to bind GTPases, and to mold a diversity of cellular membranes. PMID:16524918

  18. Small-Molecule Hydrophobic Tagging Induced Degradation of HaloTag Fusion Proteins

    PubMed Central

    Neklesa, Taavi K.; Tae, Hyun Seop; Schneekloth, Ashley R.; Stulberg, Michael J.; Corson, Timothy W.; Sundberg, Thomas B.; Raina, Kanak; Holley, Scott A.; Crews, Craig M.

    2011-01-01

    The ability to regulate any protein of interest in living systems with small molecules remains a challenge. We hypothesized that appending a hydrophobic moiety to the surface of a protein would mimic the partially denatured state of the protein, thus engaging the cellular quality control machinery to induce its proteasomal degradation. We designed and synthesized bifunctional small molecules that bind a bacterial dehalogenase (HaloTag protein) and present a hydrophobic group on its surface. Remarkably, hydrophobic tagging of the HaloTag protein with an adamantyl moiety induced the degradation of cytosolic, isoprenylated, and transmembrane fusion proteins in cell culture. We demonstrated the in vivo utility of hydrophobic tagging by degrading proteins expressed in zebrafish embryos and by inhibiting RasG12V-driven tumor progression in mice. Therefore, hydrophobic tagging of HaloTag fusion proteins affords small molecule control over any protein of interest, making it an ideal system for validating potential drug targets in disease models. PMID:21725302

  19. Drug Delivery Vehicles Based on Albumin-Polymer Conjugates.

    PubMed

    Jiang, Yanyan; Stenzel, Martina

    2016-06-01

    Albumin has been a popular building block to create nanoparticles for drug delivery purposes. The performance of albumin as a drug carrier can be enhanced by combining protein with polymers, which allows the design of carriers to encompass a broader spectrum of drugs while features unique to synthetic polymers such as stimuli-responsiveness are introduced. Nanoparticles based on polymer-albumin hybrids can be divided into two classes: one that carries album as a bioactive surface coating and the other that uses albumin as biocompatible, although nonbioactive, building block. Nanoparticles with bioactive albumin surface coating can either be prepared by self-assembly of albumin-polymer conjugates or by postcoating of existing nanoparticles with albumin. Albumin has also been used as building block, either in its native or denatured form. Existing albumin nanoparticles are coated with polymers, which can influence the degradation of albumin or impact on the drug release. Finally, an alternative way of using albumin by denaturing the protein to generate a highly functional chain, which can be modified with polymer, has been presented. These albumin nanoparticles are designed to be extremely versatile so that they can deliver a wide variety of drugs, including traditional hydrophobic drugs, metal-based drugs and even therapeutic proteins and siRNA. PMID:26947019

  20. Mitochondrial fusion and fission proteins as novel therapeutic targets for treating cardiovascular disease

    PubMed Central

    Ong, Sang-Bing; Kalkhoran, Siavash Beikoghli; Cabrera-Fuentes, Hector A.; Hausenloy, Derek J.

    2015-01-01

    The past decade has witnessed a number of exciting developments in the field of mitochondrial dynamics – a phenomenon in which changes in mitochondrial shape and movement impact on cellular physiology and pathology. By undergoing fusion and fission, mitochondria are able to change their morphology between elongated interconnected networks and discrete fragmented structures, respectively. The cardiac mitochondria, in particular, have garnered much interest due to their unique spatial arrangement in the adult cardiomyocyte, and the multiple roles they play in cell death and survival. In this article, we review the role of the mitochondrial fusion and fission proteins as novel therapeutic targets for treating cardiovascular disease. PMID:25987420

  1. Full Conversion of the Hemagglutinin-Neuraminidase Specificity of the Parainfluenza Virus 5 Fusion Protein by Replacement of 21 Amino Acids in Its Head Region with Those of the Simian Virus 41 Fusion Protein

    PubMed Central

    Nakahashi, Mito; Matsushima, Yoshiaki; Ito, Morihiro; Nishio, Machiko; Kawano, Mitsuo; Komada, Hiroshi; Nosaka, Tetsuya

    2013-01-01

    For most parainfluenza viruses, a virus type-specific interaction between the hemagglutinin-neuraminidase (HN) and fusion (F) proteins is a prerequisite for mediating virus-cell fusion and cell-cell fusion. The molecular basis of this functional interaction is still obscure partly because it is unknown which region of the F protein is responsible for the physical interaction with the HN protein. Our previous cell-cell fusion assay using the chimeric F proteins of parainfluenza virus 5 (PIV5) and simian virus 41 (SV41) indicated that replacement of two domains in the head region of the PIV5 F protein with the SV41 F counterparts bestowed on the PIV5 F protein the ability to induce cell-cell fusion on coexpression with the SV41 HN protein while retaining its ability to induce fusion with the PIV5 HN protein. In the study presented here, we furthered the chimeric analysis of the F proteins of PIV5 and SV41, finding that the PIV5 F protein could be converted to an SV41 HN-specific chimeric F protein by replacing five domains in the head region with the SV41 F counterparts. The five SV41 F-protein-derived domains of this chimera were then divided into 16 segments; 9 out of 16 proved to be not involved in determining its specificity for the SV41 HN protein. Finally, mutational analyses of a chimeric F protein, which harbored seven SV41 F-protein-derived segments, revealed that replacement of at most 21 amino acids of the PIV5 F protein with the SV41 F-protein counterparts was enough to convert its HN protein specificity. PMID:23698295

  2. SUMO fusion technology for enhanced protein expression and purification in prokaryotes and eukaryotes.

    PubMed

    Peroutka Iii, Raymond J; Orcutt, Steven J; Strickler, James E; Butt, Tauseef R

    2011-01-01

    The preparation of sufficient amounts of high-quality protein samples is the major bottleneck for structural proteomics. The use of recombinant proteins has increased significantly during the past decades. The most commonly used host, Escherichia coli, presents many challenges including protein misfolding, protein degradation, and low solubility. A novel SUMO fusion technology appears to enhance protein expression and solubility ( http://www.lifesensors.com ). Efficient removal of the SUMO tag by SUMO protease in vitro facilitates the generation of target protein with a native N-terminus. In addition to its physiological relevance in eukaryotes, SUMO can be used as a powerful biotechnology tool for enhanced functional protein expression in prokaryotes and eukaryotes. PMID:21125378

  3. Protein fusions of beta-galactosidase to the ferrichrome-iron receptor of Escherichia coli K-12.

    PubMed Central

    Coulton, J W; Mason, P; Cameron, D R; Carmel, G; Jean, R; Rode, H N

    1986-01-01

    The fusion-generating phage lambda plac Mu1 was used to produce fusions of lacZ to fhuA, the gene encoding the ferrichrome-iron receptor (FhuA protein) in the outer membrane of Escherichia coli K-12. Fusions to the fhuA gene in a delta (lac) strain were selected by their resistance to bacteriophage phi 80 vir. Ten independent (fhuA'-'lacZ) fusions were all Lac+ and were resistant to the lethal agents which require the FhuA protein as receptor, i.e., phi 80 vir, T5, T1, UC-1, and colicin M; none could utilize ferrichrome as the sole iron source. Specialized transducing phages were obtained by illegitimate excision from the chromosome of each of the fusion-bearing strains, and EcoRI fragments which encoded the fusions were subcloned into the high-copy plasmid pMLB524. Physical mapping of the fusion-containing plasmids confirmed the presence of three restriction sites which were also located on the chromosomal DNA of sequences near the fhuA gene. The direction of transcription of the fhuA gene was deduced from the direction of transcription of the (fhuA'-'lacZ) gene fusion. Identification of the chimeric proteins was made by both radiolabeling cells and immunoprecipitating the LacZ-containing proteins with antibody to beta-galactosidase and by preparing whole cell extracts from Lac+ cells containing the cloned gene fusions. Two sizes of (FhuA'-'LacZ) proteins were detected, 121 kDa and 124 kDa. The DNA sequences at the unique fusion joints were determined. The sequence information allowed us to identify three distinct fusion joints which were grouped as follows, type I fusions, 5'-ACT GCT CAG CCA A-3'; type IIa fusions, 5'-GCG GTT GAA CCG A-3'; and type IIb fusions: 5'-ACC GCT GCA CCT G-3'. To orient these fhuA fusion joints, the complete nucleotide sequence of the fhuA gene was determined from a 2,902-base-pair fragment of DNA. A single open reading frame was found which translated into a 747-amino acid polypeptide. The signal sequence of 33 amino acids was followed

  4. The Pneumovirinae fusion (F) protein: A common target for vaccines and antivirals.

    PubMed

    Melero, José A; Mas, Vicente

    2015-11-01

    The Pneumovirinae fusion (F) protein mediates fusion of the virus and cell membrane, an essential step for entry of the viral genome in the cell cytoplasm and initiation of a new infectious cycle. Accordingly, potent inhibitors of virus infectivity have been found among antibodies and chemical compounds that target the Pneumovirinae F protein. Recent developments in structure-based vaccines have led to a deeper understanding of F protein antigenicity, unveiling new conformations and epitopes which should assist in development of efficacious vaccines. Similarly, structure-based studies of potent antiviral inhibitors have provided information about their mode of action and mechanisms of resistance. The advantages and disadvantages of the different options to battle against important pathogens, such as human respiratory syncytial virus (hRSV) and human metapneumovirus (hMPV) are summarized and critically discussed in this review. PMID:25738581

  5. Preparation of λN-GST fusion protein for affinity immobilization of RNA.

    PubMed

    Di Tomasso, Geneviève; Lampron, Philipe; Omichinski, James G; Legault, Pascale

    2012-01-01

    Affinity purification of in vitro transcribed RNA is becoming an attractive alternative to purification using standard denaturing gel electrophoresis. Affinity purification is particularly advantageous because it can be performed in a few hours under non-denaturing conditions. However, the performance of affinity purification methods can vary tremendously depending on the RNA immobilization matrix. It was previously shown that RNA immobilization via an optimized λN-GST fusion protein bound to glutathione-Sepharose resin allows affinity purification of RNA with very high purity and yield. This Chapter outlines the experimental procedure employed to prepare the λN-GST fusion protein used for RNA immobilization in successful affinity purifications of RNA. It describes the details of protein expression and purification as well as routine quality control analyses. PMID:23065558

  6. Sorting of growth hormone-erythropoietin fusion proteins in rat salivary glands

    SciTech Connect

    Samuni, Yuval Zheng Changyu; Cawley, Niamh X.; Cotrim, Ana P.; Loh, Y. Peng; Baum, Bruce J.

    2008-08-15

    Neuroendocrine and exocrine cells secrete proteins in either a constitutive manner or via the regulated secretory pathway (RSP), but the specific sorting mechanisms involved are not fully understood. After gene transfer to rat salivary glands, the transgenic model proteins human growth hormone (hGH) and erythropoietin (hEpo) are secreted primarily into saliva (RSP; exocrine) and serum (constitutive; endocrine), respectively. We hypothesized that fusion of hGH at either the C-terminus or the N-terminus of hEpo would re-direct hEpo from the bloodstream into saliva. We constructed and expressed two fusion proteins, hEpo-hGH and hGH-hEpo, using serotype 5-adenoviral vectors, and delivered them to rat submandibular glands in vivo via retroductal cannulation. Both the hEpo-hGH and hGH-hEpo fusion proteins, but not hEpo alone, were secreted primarily into saliva (p < 0.0001 and p = 0.0083, respectively). These in vivo studies demonstrate for the first time that hGH, in an N- as well as C-terminal position, influences the secretion of a constitutive pathway protein.

  7. Fusion protein based on Grb2-SH2 domain for cancer therapy

    SciTech Connect

    Saito, Yuriko; Furukawa, Takako; Arano, Yasushi; Fujibayashi, Yasuhisa; Saga, Tsuneo

    2010-08-20

    Research highlights: {yields} Grb2 mediates EGFR signaling through binding to phosphorylate EGFR with SH2 domain. {yields} We generated fusion proteins containing 1 or 2 SH2 domains of Grb2 added with TAT. {yields} The one with 2 SH2 domains (TSSF) interfered ERK phosphorylation. {yields} TSSF significantly delayed the growth of EGFR overexpressing tumor in a mouse model. -- Abstract: Epidermal growth factor receptor (EGFR) is one of the very attractive targets for cancer therapy. In this study, we generated fusion proteins containing one or two Src-homology 2 (SH2) domains of growth factor receptor bound protein 2 (Grb2), which bind to phosphorylated EGFR, added with HIV-1 transactivating transcription for cell membrane penetration (termed TSF and TSSF, respectively). We examined if they can interfere Grb2-mediated signaling pathway and suppress tumor growth as expected from the lack of SH3 domain, which is necessary to intermediate EGFR-Grb2 cell signaling, in the fusion proteins. The transduction efficiency of TSSF was similar to that of TSF, but the binding activity of TSSF to EGFR was higher than that of TSF. Treatment of EGFR-overexpressing cells showed that TSSF decreased p42-ERK phosphorylation, while TSF did not. Both the proteins delayed cell growth but did not induce cell death in culture. TSSF also significantly suppressed tumor growth in vivo under consecutive administration. In conclusion, TSSF showed an ability to inhibit EGFR-Grb2 signaling and could have a potential to treat EGFR-activated cancer.

  8. [Construction of cTnC-linker-TnI (P) Genes, Expression of Fusion Protein and Preparation of Lyophilized Protein].

    PubMed

    Song, Xiaoli; Liu, Xiaoyun; Cai, Lei; Wu, Jianwei; Wang, Jihua

    2015-12-01

    In order to construct and express human cardiac troponin C-linker-troponin I(P) [ cTnC-linker-TnI(P)] fusion protein, detect its activity and prepare lyophilized protein, we searched the CDs of human cTnC and cTnI from GenBank, synthesized cTnC and cTnI(30-110aa) into cloning vector by a short DNA sequence coding for 15 neutral amino acid residues. pCold I-cTnC-linker-TnI(P) was constructed and transformed into E. coli BL21(DE3). Then, cTnC-linker-TnI(P) fusion protein was induced by isopropyl-β-D-thiogalactopyranoside (IPTG). Soluable expression of cTnC-linker-TnI(P) in prokaryotic system was successfully obtained. The fusion protein was purified by Ni²⁺ Sepharose 6 Fast Flow affinity chromatography with over 95% purity and prepared into lyophilized protein. The activity of purified cTnC-linker-TnI(P) and its lyophilized protein were detected by Wondfo Finecare™ cTnI Test. Lyophilized protein of cTnC-linker-TnI(P) was stable for 10 or more days at 37 °C and 4 or more months at 25 °C and 4 °C. The expression system established in this research is feasible and efficient. Lyophilized protein is stable enough to be provided as biological raw materials for further research. PMID:27079099

  9. The conserved glycine residues in the transmembrane domain of the Semliki Forest virus fusion protein are not required for assembly and fusion

    SciTech Connect

    Liao Maofu; Kielian, Margaret . E-mail: kielian@aecom.yu.edu

    2005-02-05

    The alphavirus Semliki Forest virus (SFV) infects cells via a low pH-triggered fusion reaction mediated by the viral E1 protein. Both the E1 fusion peptide and transmembrane (TM) domain are essential for membrane fusion, but the functional requirements for the TM domain are poorly understood. Here we explored the role of the five TM domain glycine residues, including the highly conserved glycine pair at E1 residues 415/416. SFV mutants with alanine substitutions for individual or all five glycine residues (5G/A) showed growth kinetics and fusion pH dependence similar to those of wild-type SFV. Mutants with increasing substitution of glycine residues showed an increasingly more stringent requirement for cholesterol during fusion. The 5G/A mutant showed decreased fusion kinetics and extent in fluorescent lipid mixing assays. TM domain glycine residues thus are not required for efficient SFV fusion or assembly but can cause subtle effects on the properties of membrane fusion.

  10. Combining random gene fission and rational gene fusion to discover near-infrared fluorescent protein fragments that report on protein-protein interactions.

    PubMed

    Pandey, Naresh; Nobles, Christopher L; Zechiedrich, Lynn; Maresso, Anthony W; Silberg, Jonathan J

    2015-05-15

    Gene fission can convert monomeric proteins into two-piece catalysts, reporters, and transcription factors for systems and synthetic biology. However, some proteins can be challenging to fragment without disrupting function, such as near-infrared fluorescent protein (IFP). We describe a directed evolution strategy that can overcome this challenge by randomly fragmenting proteins and concomitantly fusing the protein fragments to pairs of proteins or peptides that associate. We used this method to create libraries that express fragmented IFP as fusions to a pair of associating peptides (IAAL-E3 and IAAL-K3) and proteins (CheA and CheY) and screened for fragmented IFP with detectable near-infrared fluorescence. Thirteen novel fragmented IFPs were identified, all of which arose from backbone fission proximal to the interdomain linker. Either the IAAL-E3 and IAAL-K3 peptides or CheA and CheY proteins could assist with IFP fragment complementation, although the IAAL-E3 and IAAL-K3 peptides consistently yielded higher fluorescence. These results demonstrate how random gene fission can be coupled to rational gene fusion to create libraries enriched in fragmented proteins with AND gate logic that is dependent upon a protein-protein interaction, and they suggest that these near-infrared fluorescent protein fragments will be suitable as reporters for pairs of promoters and protein-protein interactions within whole animals. PMID:25265085

  11. A new fusion protein platform for quantitatively measuring activity of multiple proteases

    PubMed Central

    2014-01-01

    Background Recombinant proteins fused with specific cleavage sequences are widely used as substrate for quantitatively analyzing the activity of proteases. Here we propose a new fusion platform for multiple proteases, by using diaminopropionate ammonia-lyase (DAL) as the fusion protein. It was based on the finding that a fused His6-tag could significantly decreases the activities of DAL from E. coli (eDAL) and Salmonella typhimurium (sDAL). Previously, we have shown that His6GST-tagged eDAL could be used to determine the activity of tobacco etch virus protease (TEVp) under different temperatures or in the denaturant at different concentrations. In this report, we will assay different tags and cleavage sequences on DAL for expressing yield in E. coli, stability of the fused proteins and performance of substrate of other common proteases. Results We tested seven different protease cleavage sequences (rhinovirus 3C, TEV protease, factor Xa, Ssp DnaB intein, Sce VMA1 intein, thrombin and enterokinase), three different tags (His6, GST, CBD and MBP) and two different DALs (eDAL and sDAL), for their performance as substrate to the seven corresponding proteases. Among them, we found four active DAL-fusion substrates suitable for TEVp, factor Xa, thrombin and DnaB intein. Enterokinase cleaved eDAL at undesired positions and did not process sDAL. Substitution of GST with MBP increase the expression level of the fused eDAL and this fusion protein was suitable as a substrate for analyzing activity of rhinovirus 3C. We demonstrated that SUMO protease Ulp1 with a N-terminal His6-tag or MBP tag displayed different activity using the designed His6SUMO-eDAL as substrate. Finally, owing to the high level of the DAL-fusion protein in E. coli, these protein substrates can also be detected directly from the crude extract. Conclusion The results show that our designed DAL-fusion proteins can be used to quantify the activities of both sequence- and conformational-specific proteases, with

  12. Affinity purification of recombinant proteins using a novel silica-binding peptide as a fusion tag.

    PubMed

    Abdelhamid, Mohamed A A; Motomura, Kei; Ikeda, Takeshi; Ishida, Takenori; Hirota, Ryuichi; Kuroda, Akio

    2014-06-01

    We recently reported that silica is deposited on the coat of Bacillus cereus spores as a layer of nanometer-sized particles (Hirota et al. 2010 J Bacteriol 192: 111-116). Gene disruption analysis revealed that the spore coat protein CotB1 mediates the accumulation of silica (our unpublished results). Here, we report that B. cereus CotB1 (171 amino acids [aa]) and its C-terminal 14-aa region (corresponding to residues 158-171, designated CotB1p) show strong affinity for silica particles, with dissociation constants at pH 8.0 of 2.09 and 1.24 nM, respectively. Using CotB1 and CotB1p as silica-binding tags, we developed a silica-based affinity purification method in which silica particles are used as an adsorbent for CotB1/CotB1p fusion proteins. Small ubiquitin-like modifier (SUMO) technology was employed to release the target proteins from the adsorbed fusion proteins. SUMO-protease-mediated site-specific cleavage at the C-terminus of the fused SUMO sequence released the tagless target proteins into the liquid phase while leaving the tag region still bound to the solid phase. Using the fluorescent protein mCherry as a model, our purification method achieved 85 % recovery, with a purity of 95 % and yields of 0.60 ± 0.06 and 1.13 ± 0.13 mg per 10-mL bacterial culture for the CotB1-SUMO-mCherry and CotB1p-SUMO-mCherry fusions, respectively. CotB1p, a short 14-aa peptide, which demonstrates high affinity for silica, could be a promising fusion tag for both affinity purification and enzyme immobilization on silica supports. PMID:24756322

  13. The Fusion Protein Specificity of the Parainfluenza Virus Hemagglutinin-Neuraminidase Protein Is Not Solely Defined by the Primary Structure of Its Stalk Domain

    PubMed Central

    Ito, Morihiro; Ohtsuka, Junpei; Hara, Kenichiro; Komada, Hiroshi; Nishio, Machiko; Nosaka, Tetsuya

    2015-01-01

    ABSTRACT Virus-specific interaction between the attachment protein (HN) and the fusion protein (F) is prerequisite for the induction of membrane fusion by parainfluenza viruses. This HN-F interaction presumably is mediated by particular amino acids in the HN stalk domain and those in the F head domain. We found in the present study, however, that a simian virus 41 (SV41) F-specific chimeric HPIV2 HN protein, SCA, whose cytoplasmic, transmembrane, and stalk domains were derived from the SV41 HN protein, could not induce cell-cell fusion of BHK-21 cells when coexpressed with an SV41 HN-specific chimeric PIV5 F protein, no. 36. Similarly, a headless form of the SV41 HN protein failed to induce fusion with chimera no. 36, whereas it was able to induce fusion with the SV41 F protein. Interestingly, replacement of 13 amino acids of the SCA head domain, which are located at or around the dimer interface of the head domain, with SV41 HN counterparts resulted in a chimeric HN protein, SCA-RII, which induced fusion with chimera no. 36 but not with the SV41 F protein. More interestingly, retroreplacement of 11 out of the 13 amino acids of SCA-RII with the SCA counterparts resulted in another chimeric HN protein, IM18, which induced fusion either with chimera no. 36 or with the SV41 F protein, similar to the SV41 HN protein. Thus, we conclude that the F protein specificity of the HN protein that is observed in the fusion event is not solely defined by the primary structure of the HN stalk domain. IMPORTANCE It is appreciated that the HN head domain initially conceals the HN stalk domain but exposes it after the head domain has bound to the receptors, which allows particular amino acids in the stalk domain to interact with the F protein and trigger it to induce fusion. However, other regulatory roles of the HN head domain in the fusion event have been ill defined. We have shown in the current study that removal of the head domain or amino acid substitutions in a particular

  14. Single Particle Dynamic Imaging and Fe3+ Sensing with Bright Carbon Dots Derived from Bovine Serum Albumin Proteins

    NASA Astrophysics Data System (ADS)

    Yang, Qingxiu; Wei, Lin; Zheng, Xuanfang; Xiao, Lehui

    2015-12-01

    In this work, we demonstrated a convenient and green strategy for the synthesis of highly luminescent and water-soluble carbon dots (Cdots) by carbonizing carbon precursors, i.e., Bovine serum albumin (BSA) nanoparticles, in water solution. Without post surface modification, the as-synthesized Cdots exhibit fluorescence quantum yield (Q.Y.) as high as 34.8% and display superior colloidal stability not only in concentrated salt solutions (e.g. 2 M KCl) but also in a wide range of pH solutions. According to the FT-IR measurements, the Cdots contain many carboxyl groups, providing a versatile route for further chemical and biological functionalization. Through conjugation of Cdots with the transacting activator of transcription (TAT) peptide (a kind of cell penetration peptide (CPP)) derived from human immunodeficiency virus (HIV), it is possible to directly monitor the dynamic interactions of CPP with living cell membrane at single particle level. Furthermore, these Cdots also exhibit a dosage-dependent selectivity toward Fe3+ among other metal ions, including K+, Na+, Mg2+, Hg2+, Co2+, Cu2+, Pb2+ and Al3+. We believed that the Cdots prepared by this strategy would display promising applications in various areas, including analytical chemistry, nanomedicine, biochemistry and so on.

  15. Single Particle Dynamic Imaging and Fe3+ Sensing with Bright Carbon Dots Derived from Bovine Serum Albumin Proteins

    PubMed Central

    Yang, Qingxiu; Wei, Lin; Zheng, Xuanfang; Xiao, Lehui

    2015-01-01

    In this work, we demonstrated a convenient and green strategy for the synthesis of highly luminescent and water-soluble carbon dots (Cdots) by carbonizing carbon precursors, i.e., Bovine serum albumin (BSA) nanoparticles, in water solution. Without post surface modification, the as-synthesized Cdots exhibit fluorescence quantum yield (Q.Y.) as high as 34.8% and display superior colloidal stability not only in concentrated salt solutions (e.g. 2 M KCl) but also in a wide range of pH solutions. According to the FT-IR measurements, the Cdots contain many carboxyl groups, providing a versatile route for further chemical and biological functionalization. Through conjugation of Cdots with the transacting activator of transcription (TAT) peptide (a kind of cell penetration peptide (CPP)) derived from human immunodeficiency virus (HIV), it is possible to directly monitor the dynamic interactions of CPP with living cell membrane at single particle level. Furthermore, these Cdots also exhibit a dosage-dependent selectivity toward Fe3+ among other metal ions, including K+, Na+, Mg2+, Hg2+, Co2+, Cu2+, Pb2+ and Al3+. We believed that the Cdots prepared by this strategy would display promising applications in various areas, including analytical chemistry, nanomedicine, biochemistry and so on. PMID:26634992

  16. Startling temperature effect on proteins when confined: single molecular level behaviour of human serum albumin in a reverse micelle.

    PubMed

    Sengupta, Bhaswati; Yadav, Rajeev; Sen, Pratik

    2016-06-01

    The present work reports the effect of confinement, and temperature therein, on the conformational fluctuation dynamics of domain-I of human serum albumin (HSA) by fluorescence correlation spectroscopy (FCS). The water-pool of a sodium bis(2-ethylhexyl)sulfosuccinate (AOT) reverse micelle has been used as the confined environment. It was observed that the conformational fluctuation time is about 6 times smaller compared to bulk medium when confined in a water-pool of 3.5 nm radius. On increasing the size of the water-pool the conformational fluctuation time was found to increase monotonically and approaches the bulk value. The effect of confinement is on par with the general belief about the restricted motion of a macromolecule upon confinement. However, the effect of temperature was found to be surprising. An increase in the temperature from 298 K to 313 K induces a larger change in the conformational fluctuation time in HSA, when confined. In the bulk medium, apparently there is no change in the conformational fluctuation time in the aforementioned temperature range, whereas, when HSA is present in an AOT water-pool of radius 3.5 nm, about an 88% increase in the fluctuation time was observed. The observed prominent thermal effect on the conformational dynamics of domain-I of HSA in the water-pool of an AOT reverse micelle as compared to in the bulk medium was concluded to arise from the confined solvent effect. PMID:27166785

  17. Statin-AE: a novel angiostatin-endostatin fusion protein with enhanced antiangiogenic and antitumor activity.

    PubMed

    Scappaticci, F A; Contreras, A; Smith, R; Bonhoure, L; Lum, B; Cao, Y; Engleman, E G; Nolan, G P

    2001-01-01

    The combination of angiostatin and endostatin has been shown to have synergistic antiangiogenic and antitumor effects when the genes for these proteins are delivered to tumor cells by retroviral gene transfer. Here we report the construction of a murine angiostatin-endostatin fusion gene (Statin-AE) which shows enhanced antiangiogenic activity on human umbilical vein endothelial cell (HUVEC) tube formation in vitro compared with angiostatin or endostatin alone. Similarly, the fusion gene demonstrates antiangiogenic effects in vivo and antitumor activity in a B16F10 melanoma model when co-delivered by retroviral packaging cell inoculation in mice. The fusion gene demonstrates significantly greater inhibition of tumor growth compared with angiostatin, endostatin or the combination of genes. PMID:12197471

  18. Choline and acetylcholine detection based on peroxidase-like activity and protein antifouling property of platinum nanoparticles in bovine serum albumin scaffold.

    PubMed

    He, Shao-Bin; Wu, Gang-Wei; Deng, Hao-Hua; Liu, Ai-Lin; Lin, Xin-Hua; Xia, Xing-Hua; Chen, Wei

    2014-12-15

    Platinum nanoparticles (PtNPs) in the scaffold of bovine serum albumin (BSA) through biomineralization are found to possess excellent peroxidase-like activity that can catalyze N-ethyl-N-(3-sulfopropyl)-3-methylaniline sodium salt (TOPS) coupled with 4-amino-antipyrine (4-AAP) by the action of hydrogen peroxide to give an obvious purple product. Based on this phenomenon, acetylcholinesterase (AChE) and choline oxidase (ChOx) are used to catalyze ACh and choline to form the active product H2O2 and the as-produced H2O2 is detected optically. Owning to the protection effect of the protein shell, BSA-PtNPs turn out to be very stable and preserve the catalytic activity in the presence of protein and even in the real plasma samples. This protein antifouling property makes the BSA-PtNPs suitable for a wide range of applications in sensors for biological samples. Choline in infant formula and ACh in plasma have been successfully detected. PMID:25038538

  19. Molecular Aspects of the Interaction of Iminium and Alkanolamine Forms of the Anticancer Alkaloid Chelerythrine with Plasma Protein Bovine Serum Albumin.

    PubMed

    Bhuiya, Sutanwi; Pradhan, Ankur Bikash; Haque, Lucy; Das, Suman

    2016-01-14

    The interaction between a quaternary benzophenanthridine alkaloid chelerythrine (herein after, CHL) and bovine serum albumin (herein after, BSA) was probed by employing various spectroscopic tools and isothermal titration calorimetry (ITC). Fluorescence studies revealed that the binding affinity of the alkanolamine form of the CHL is higher compared to the iminium counterpart. This was further established by fluorescence polarization anisotropy measurement and ITC. Fluorescence quenching study along with time-resolved fluorescence measurements establish that both forms of CHL quenched the fluorescence intensity of BSA through the mechanism of static quenching. Site selective binding and molecular modeling studies revealed that the alkaloid binds predominantly in the BSA subdomain IIA by electrostatic and hydrophobic forces. From Forster resonance energy transfer (FRET) studies, the average distances between the protein donor and the alkaloid acceptor were found to be 2.71 and 2.30 nm between tryptophan (Trp) 212 (donor) and iminium and alkanolamine forms (acceptor), respectively. Circular dichroism (CD) study demonstrated that the α-helical organization of the protein is reduced due to binding with CHL along with an increase in the coiled structure. This is indicative of a small but definitive partial unfolding of the protein. Thermodynamic parameters obtained from ITC experiments revealed that the interaction is favored by negative enthalpy change and positive entropy change. PMID:26653994

  20. Interaction between Human Serum Albumin and antidiabetic compounds and its influence on the O2((1)Δg)-mediated degradation of the protein.

    PubMed

    Challier, C; Beassoni, P; Boetsch, C; García, N A; Biasutti, M A; Criado, S

    2015-01-01

    The complexity depicted by disease scenarios as diabetes mellitus, constitutes a very interesting field of study when drugs and biologically relevant components may be affected by such environments. In this report, the interaction between the protein Human Serum Albumin (HSA) and two antidiabetics (Andb), Gliclazide (Gli) and Glipizide (Glip) was studied through fluorescence and docking assays, in order to characterize these systems. On the basis that HSA and Andb can be exposed in vivo at high Reactive Oxygen Species (ROS) concentrations in diabetic patients, the degradative process of the protein free and bound to Andb, in presence of the species singlet molecular oxygen (O2((1)Δg)), was evaluated. Fluorescence and docking assays indicated that Gli, as well as Glip bind to HSA on two sites, with binding constants values in the order of 10(4)-10(5)M(-1). Likewise, docking assays revealed that the location of Gli or Glip on the protein may be the HSA binding sites II and III. Thermodynamic parameters showed that the interaction between HSA and Glip is a favored, enthalpically-controlled process. Oxygen uptake experiments indicated that Glip is less photooxidizable than Gli through a O2((1)Δg)-mediated process. Besides, the protein-Andb binding produced a decrease in the overall rate constant for O2((1)Δg) quenching as compared to the value for the free protein. This fact could be interpreted in terms of a reduction in the availability of Tyrosine residues in the bonded protein, with a concomitant decrease in the physical quenching deactivation of the oxidative species. PMID:25490375

  1. The role of albumin receptors in regulation of albumin homeostasis: Implications for drug delivery.

    PubMed

    Bern, Malin; Sand, Kine Marita Knudsen; Nilsen, Jeannette; Sandlie, Inger; Andersen, Jan Terje

    2015-08-10

    Albumin is the most abundant protein in blood and acts as a molecular taxi for a plethora of small insoluble substances such as nutrients, hormones, metals and toxins. In addition, it binds a range of medical drugs. It has an unusually long serum half-life of almost 3weeks, and although the structure and function of albumin has been studied for decades, a biological explanation for the long half-life has been lacking. Now, recent research has unravelled that albumin-binding cellular receptors play key roles in the homeostatic regulation of albumin. Here, we review our current understanding of albumin homeostasis with a particular focus on the impact of the cellular receptors, namely the neonatal Fc receptor (FcRn) and the cubilin-megalin complex, and we discuss their importance on uses of albumin in drug delivery. PMID:26055641

  2. Construction and structural modeling of a single-chain Fv-asparaginase fusion protein resistant to proteolysis.

    PubMed

    Guo, L; Wang, J; Qian, S; Yan, X; Chen, R; Meng, G

    2000-11-20

    In this study, we construct a fusion protein composed of L-asparaginase (ASNase; from Escherichia coli AS 1.357) and a protective single-chain Fv (scFv), which was selected from a phage-display scFv library from our previous studies. The antibody moiety of the fusion protein was fused to the N-terminus of the enzyme moiety via a linker peptide, (Gly(4)Ser)(6). Recombinant plasmid pET-SLA was constructed to express scFv-ASNase fusion to high levels in E. coli and the expressed product was found to form inclusion bodies. We obtained a soluble fusion protein by refolding and purification. The soluble fusion protein exhibited about 82% of the enzymatic activity of the native ASNase at the same molar concentration, and had a K(m) value similar to that of the native enzyme for the substrate L-asparagine. Importantly, the fusion protein was more stable than native ASNase. In addition: (1) following treatment with trypsin, alpha-chymotrypsin, and rennet, at 37 degrees C for 30 min, scFv-ASNase fusion retained 94.0%, 88.8%, and 84.5% of its original activity, respectively, whereas native ASNase became inactive; and (2) ScFv-ASNase fusion had a much longer in vitro half-life (9 h) in serum than the native enzyme (2 h). The three-dimensional structure of the fusion protein was obtained by modeling with the Homology and Discover modules of the INSIGHT II software package. On the basis of the structural evidence and biochemical properties, we propose that the scFv moiety of the fusion protein may confer ASNase moiety resistance to proteolysis as a result of both steric hindrance and a change in the electrostatic surface of the enzyme. PMID:11005928

  3. Ion-specific modulation of protein interactions: Anion-induced, reversible oligomerization of a fusion protein

    PubMed Central

    Gokarn, Yatin R; Fesinmeyer, R Matthew; Saluja, Atul; Cao, Shawn; Dankberg, Jane; Goetze, Andrew; Remmele, Richard L; Narhi, Linda O; Brems, David N

    2009-01-01

    Ions can significantly modulate the solution interactions of proteins. We aim to demonstrate that the salt-dependent reversible heptamerization of a fusion protein called peptibody A or PbA is governed by anion-specific interactions with key arginyl and lysyl residues on its peptide arms. Peptibody A, an E. coli expressed, basic (pI = 8.8), homodimer (65.2 kDa), consisted of an IgG1-Fc with two, C-terminal peptide arms linked via penta-glycine linkers. Each peptide arm was composed of two, tandem, active sequences (SEYQGLPPQGWK) separated by a spacer (GSGSATGGSGGGASSGSGSATG). PbA was monomeric in 10 mM acetate, pH 5.0 but exhibited reversible self-association upon salt addition. The sedimentation coefficient (sw) and hydrodynamic diameter (DH) versus PbA concentration isotherms in the presence of 140 mM NaCl (A5N) displayed sharp increases in sw and DH, reaching plateau values of 9 s and 16 nm by 10 mg/mL PbA. The DH and sedimentation equilibrium data in the plateau region (>12 mg/mL) indicated the oligomeric ensemble to be monodisperse (PdI = 0.05) with a z-average molecular weight (Mz) of 433 kDa (stoichiometry = 7). There was no evidence of reversible self-association for an IgG1-Fc molecule in A5N by itself or in a mixture containing fluorescently labeled IgG1-Fc and PbA, indicative of PbA self-assembly being mediated through its peptide arms. Self-association increased with pH, NaCl concentration, and anion size (I− > Br− > Cl− > F−) but could be inhibited using soluble Trp-, Phe-, and Leu-amide salts (Trp > Phe > Leu). We propose that in the presence of salt (i) anion binding renders PbA self-association competent by neutralizing the peptidyl arginyl and lysyl amines, (ii) self-association occurs via aromatic and hydrophobic interactions between the..xx..xxx..xx.. motifs, and (iii) at >10 mg/mL, PbA predominantly exists as heptameric clusters. PMID:19177361

  4. Effect of COR proteins on the freeze-induced fusion of liposomes

    SciTech Connect

    Uemura, M.; Steponkus, P.L. ); Gilmour, S.J.; Thomashow, M.F. )

    1993-05-01

    Although substantial progress has been made in identifying genes that are regulated during cold acclimation, with few exceptions, the function of the COR proteins encoded by these genes is not known. The objectives of this study were to determine if COR6.6 and COR15am, which are synthesized in Arabidopsis thaliana, influence the freeze-induced fusion of lipid bilayers. When small unilamellar vesicles (SUVs) composed of either POPC (16:0/18:1) or DOPC (18:1/18:1) were frozen to temperatures over the range of [minus]2[degrees]C to [minus]30[degrees]C liposome fusion occurred at temperatures below [minus]2[degrees]C, with a substantial increase occurring at temperatures below the T[sub m] of the lipids ([minus]3[degrees]C for POPC and [minus]18[degrees]C for DOPC). Freeze-induced fusion was not observed SUVs composed of BL[sub 2]PC (18:2/18:2), which has a t[sub m] of [minus]55[degrees]C. Addition of either COR6.6 or COR15am significantly decreased the incidence of fusion of large unilamellar vesicles of DOPC if proteins were added to the liposome suspension before freezing; there was no effect if the COR proteins were only encapsulated within the liposomes. With SUVs formed from the total lipid extract of the plasma membrane of either non-acclimated (NA) or cold-acclimated (ACC) rye leave, there was differential response to freezing. In NA-SUVs, the incidence of fusion increased after freezing to [minus]5[degrees]C and reached a maximum at [minus]10[degrees]C; in ACC-SUVs, the maximum incidence of fusion occurred at [minus]20[degrees]C. The difference in cryostability reflects differences in the lipid composition of the plasma membrane after cold acclimation. But there was a significant decrease in the incidence of fusion in both NA- and ACC-SUVs when frozen in the presence of either COR6.6 or COR15am. Thus, although the cryostability of lipid bilayers is primarily influenced by the lipid composition of the bilayers, there is an additive effect of the COR proteins.

  5. Proteolytic Activation of the Porcine Epidemic Diarrhea Coronavirus Spike Fusion Protein by Trypsin in Cell Culture

    PubMed Central

    Wicht, Oliver; Li, Wentao; Willems, Lione; Meuleman, Tom J.; Wubbolts, Richard W.; van Kuppeveld, Frank J. M.; Rottier, Peter J. M.

    2014-01-01

    ABSTRACT Isolation of porcine epidemic diarrhea coronavirus (PEDV) from clinical material in cell culture requires supplementation of trypsin. This may relate to the confinement of PEDV natural infection to the protease-rich small intestine of pigs. Our study focused on the role of protease activity on infection by investigating the spike protein of a PEDV isolate (wtPEDV) using a reverse genetics system based on the trypsin-independent cell culture-adapted strain DR13 (caPEDV). We demonstrate that trypsin acts on the wtPEDV spike protein after receptor binding. We mapped the genetic determinant for trypsin-dependent cell entry to the N-terminal region of the fusion subunit of this class I fusion protein, revealing a conserved arginine just upstream of the putative fusion peptide as the potential cleavage site. Whereas coronaviruses are typically processed by endogenous proteases of the producer or target cell, PEDV S protein activation strictly required supplementation of a protease, enabling us to study mechanistic details of proteolytic processing. IMPORTANCE Recurring PEDV epidemics constitute a serious animal health threat and an economic burden, particularly in Asia but, as of recently, also on the North-American subcontinent. Understanding the biology of PEDV is critical for combatting the infection. Here, we provide new insight into the protease-dependent cell entry of PEDV. PMID:24807723

  6. Interleukin 2-diphtheria toxin fusion protein can abolish cell-mediated immunity in vivo.

    PubMed Central

    Kelley, V E; Bacha, P; Pankewycz, O; Nichols, J C; Murphy, J R; Strom, T B

    1988-01-01

    De novo expression of the interleukin 2 receptor (IL-2R) is a critical and pivotal event in initiation of an immune response. Targeting the low-affinity IL-2-binding p55 subunit of the high-affinity IL-2R with the rat anti-mouse IgM monoclonal antibody M7/20 suppresses a variety of T-cell-mediated reactions, including transplant rejection, autoimmunity, and delayed-type hypersensitivity (DTH). A hybrid IL-2-toxin gene was constructed from the diphtheria toxin gene by replacing the DNA encoding the diphtheria toxin receptor-binding domain with the DNA encoding the receptor-binding domain of IL-2, and the fusion protein encoded by the hybrid gene was expressed in Escherichia coli [Williams, D.P., Parker, K., Bacha, P., Bishai, W., Borowski, M., Genbauffe, F., Strom, T.B. & Murphy, J.R. (1987) Protein Eng. 1, 493-498]. We examined the action of the chimeric IL-2-toxin fusion protein on an in vivo T-cell mediated response, DTH. The IL-2-toxin fusion protein was found to be a potent immunosuppressive agent. Treatment of mice with the IL-2-toxin blocks DTH and prevents expansion of IL-2R+ T cells. Indeed, IL-2-toxin treatment targets IL-2R+ T cells in vivo and is shown to selectively eliminate their appearance in draining lymph nodes. DTH suppression was observed even in mice possessing high titers of antibodies to diphtheria toxoid. PMID:3131768

  7. Proteolytic activation of the porcine epidemic diarrhea coronavirus spike fusion protein by trypsin in cell culture.

    PubMed

    Wicht, Oliver; Li, Wentao; Willems, Lione; Meuleman, Tom J; Wubbolts, Richard W; van Kuppeveld, Frank J M; Rottier, Peter J M; Bosch, Berend Jan

    2014-07-01

    Isolation of porcine epidemic diarrhea coronavirus (PEDV) from clinical material in cell culture requires supplementation of trypsin. This may relate to the confinement of PEDV natural infection to the protease-rich small intestine of pigs. Our study focused on the role of protease activity on infection by investigating the spike protein of a PEDV isolate (wtPEDV) using a reverse genetics system based on the trypsin-independent cell culture-adapted strain DR13 (caPEDV). We demonstrate that trypsin acts on the wtPEDV spike protein after receptor binding. We mapped the genetic determinant for trypsin-dependent cell entry to the N-terminal region of the fusion subunit of this class I fusion protein, revealing a conserved arginine just upstream of the putative fusion peptide as the potential cleavage site. Whereas coronaviruses are typically processed by endogenous proteases of the producer or target cell, PEDV S protein activation strictly required supplementation of a protease, enabling us to study mechanistic details of proteolytic processing. Importance: Recurring PEDV epidemics constitute a serious animal health threat and an economic burden, particularly in Asia but, as of recently, also on the North-American subcontinent. Understanding the biology of PEDV is critical for combatting the infection. Here, we provide new insight into the protease-dependent cell entry of PEDV. PMID:24807723

  8. Transient fusion and selective secretion of vesicle proteins in Phytophthora nicotianae zoospores.

    PubMed

    Zhang, Weiwei; Blackman, Leila M; Hardham, Adrienne R

    2013-01-01

    Secretion of pathogen proteins is crucial for the establishment of disease in animals and plants. Typically, early interactions between host and pathogen trigger regulated secretion of pathogenicity factors that function in pathogen adhesion and host penetration. During the onset of plant infection by spores of the Oomycete, Phytophthora nicotianae, proteins are secreted from three types of cortical vesicles. Following induction of spore encystment, two vesicle types undergo full fusion, releasing their entire contents onto the cell surface. However, the third vesicle type, so-called large peripheral vesicles, selectively secretes a small Sushi domain-containing protein, PnCcp, while retaining a large glycoprotein, PnLpv, before moving away from the plasma membrane. Selective secretion of PnCcp is associated with its compartmentalization within the vesicle periphery. Pharmacological inhibition of dynamin function, purportedly in vesicle fission, by dynasore treatment provides evidence that selective secretion of PnCcp requires transient fusion of the large peripheral vesicles. This is the first report of selective protein secretion via transient fusion outside mammalian cells. Selective secretion is likely to be an important aspect of plant infection by this destructive pathogen. PMID:24392285

  9. An optimized antibody-single-chain TRAIL fusion protein for cancer therapy.

    PubMed

    Siegemund, Martin; Seifert, Oliver; Zarani, Maria; Džinić, Tamara; De Leo, Valentino; Göttsch, Doris; Münkel, Sabine; Hutt, Meike; Pfizenmaier, Klaus; Kontermann, Roland E

    2016-07-01

    Fusion proteins combining oligomeric assemblies of a genetically obtained single-chain (sc) variant of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) with antibodies directed against tumor-associated antigens represent a promising strategy to overcome the limited therapeutic activity of conventional soluble TRAIL. To further improve the scTRAIL module in order to obtain a robust, thermostable molecule of high activity, we performed a comprehensive analysis of the minimal TNF homology domain (THD) and optimized linkers between the 3 TRAIL subunits constituting a scTRAIL. Through a stepwise mutagenesis of the N- and C-terminal region and the joining linker sequences, we generated bioactive scTRAIL molecules comprising a covalent linkage of the C-terminal Val280 and the N-terminal position 122 by only 2 amino acid residues in combination with conservative exchanges at positions 122 and 279. The increased thermal stability and solubility of such optimized scTRAIL molecules translated into increased bioactivity in the diabody-scTRAIL (Db-scTRAIL) format, exemplified here for an epidermal growth factor receptor-specific Db-scTRAIL. Additional modifications within the diabody linkers resulted in a fusion protein exerting high, target-dependent apoptosis induction in tumor cell lines in vitro and potent antitumor activity in vivo. Our results illustrate that protein engineering of scTRAIL and associated peptide linkers provides a promising strategy to develop antibody-scTRAIL fusion proteins as effective antitumor therapeutics. PMID:27064440

  10. Type II integral membrane protein, TM of J paramyxovirus promotes cell-to-cell fusion

    PubMed Central

    Li, Zhuo; Hung, Cher; Paterson, Reay G.; Michel, Frank; Fuentes, Sandra; Place, Ryan; Lin, Yuan; Hogan, Robert J.; Lamb, Robert A.; He, Biao

    2015-01-01

    Paramyxoviruses include many important animal and human pathogens. Most paramyxoviruses have two integral membrane proteins: fusion protein (F) and attachment proteins hemagglutinin, hemagglutinin–neuraminidase, or glycoprotein (G), which are critical for viral entry into cells. J paramyxovirus (JPV) encodes four integral membrane proteins: F, G, SH, and transmembrane (TM). The function of TM is not known. In this work, we have generated a viable JPV lacking TM (JPV∆TM). JPV∆TM formed opaque plaques compared with JPV. Quantitative syncytia assays showed that JPV∆TM was defective in promoting cell-to-cell fusion (i.e., syncytia formation) compared with JPV. Furthermore, cells separately expressing F, G, TM, or F plus G did not form syncytia whereas cells expressing F plus TM formed some syncytia. However, syncytia formation was much greater with coexpression of F, G, and TM. Biochemical analysis indicates that F, G, and TM interact with each other. A small hydrophobic region in the TM ectodomain from amino acid residues 118 to 132, the hydrophobic loop (HL), was important for syncytial promotion, suggesting that the TM HL region plays a critical role in cell-to-cell fusion. PMID:26392524

  11. Purification method for recombinant proteins based on a fusion between the target protein and the C-terminus of calmodulin

    NASA Technical Reports Server (NTRS)

    Schauer-Vukasinovic, Vesna; Deo, Sapna K.; Daunert, Sylvia

    2002-01-01

    Calmodulin (CaM) was used as an affinity tail to facilitate the purification of the green fluorescent protein (GFP), which was used as a model target protein. The protein GFP was fused to the C-terminus of CaM, and a factor Xa cleavage site was introduced between the two proteins. A CaM-GFP fusion protein was expressed in E. coli and purified on a phenothiazine-derivatized silica column. CaM binds to the phenothiazine on the column in a Ca(2+)-dependent fashion and it was, therefore, used as an affinity tail for the purification of GFP. The fusion protein bound to the affinity column was then subjected to a proteolytic digestion with factor Xa. Pure GFP was eluted with a Ca(2+)-containing buffer, while CaM was eluted later with a buffer containing the Ca(2+)-chelating agent EGTA. The purity of the isolated GFP was verified by SDS-PAGE, and the fluorescence properties of the purified GFP were characterized.

  12. A cell penetrating peptide-integrated and enediyne-energized fusion protein shows potent antitumor activity.

    PubMed

    Ru, Qin; Shang, Bo-Yang; Miao, Qing-Fang; Li, Liang; Wu, Shu-Ying; Gao, Rui-Juan; Zhen, Yong-Su

    2012-11-20

    Arginine-rich peptides belong to a subclass of cell penetrating peptides that are taken up by living cells and can be detected freely diffusing inside the cytoplasm and nucleoplasm. This phenomenon has been attributed to either an endocytotic mode of uptake and a subsequent release from vesicles or a direct membrane penetration. Lidamycin is an antitumor antibiotic, which consists of an active enediyne chromophore (AE) and a noncovalently bound apoprotein (LDP). In the present study, a fusion protein (Arg)(9)-LDP composed of cell penetrating peptide (Arg)(9) and LDP was prepared by DNA recombination, and the enediyne-energized fusion protein (Arg)(9)-LDP-AE was prepared by molecular reconstitution. The data in fixed cells demonstrated that (Arg)(9)-LDP could rapidly enter cells, and the results based on fluorescence activated cell sorting indicated that the major route for (Arg)(9)-mediated cellular uptake of protein molecules was endocytosis. (Arg)(9)-LDP-AE demonstrated more potent cytotoxicity against different carcinoma cell lines than lidamycin in vitro. In the mouse hepatoma 22 model, (Arg)(9)-LDP-AE (0.3mg/kg) suppressed the tumor growth by 89.2%, whereas lidamycin (0.05 mg/kg) by 74.6%. Furthermore, in the glioma U87 xenograft model in nude mice, (Arg)(9)-LDP-AE at 0.2mg/kg suppressed tumor growth by 88.8%, compared with that of lidamycin by 62.9% at 0.05 mg/kg. No obvious toxic effects were observed in all groups during treatments. The results showed that energized fusion protein (Arg)(9)-LDP-AE was more effective than lidamycin and would be a promising candidate for glioma therapy. In addition, this approach to manufacturing fusion proteins might serve as a technology platform for the development of new cell penetrating peptides-based drugs. PMID:22982402

  13. Flagellar membrane fusion and protein exchange in trypanosomes; a new form of cell-cell communication?

    PubMed Central

    Imhof, Simon; Fragoso, Cristina; Hemphill, Andrew; von Schubert, Conrad; Li, Dong; Legant, Wesley; Betzig, Eric; Roditi, Isabel

    2016-01-01

    Diverse structures facilitate direct exchange of proteins between cells, including plasmadesmata in plants and tunnelling nanotubes in bacteria and higher eukaryotes.  Here we describe a new mechanism of protein transfer, flagellar membrane fusion, in the unicellular parasite Trypanosoma brucei. When fluorescently tagged trypanosomes were co-cultured, a small proportion of double-positive cells were observed. The formation of double-positive cells was dependent on the presence of extracellular calcium and was enhanced by placing cells in medium supplemented with fresh bovine serum. Time-lapse microscopy revealed that double-positive cells arose by bidirectional protein exchange in the absence of nuclear transfer.  Furthermore, super-resolution microscopy showed that this process occurred in ≤1 minute, the limit of temporal resolution in these experiments. Both cytoplasmic and membrane proteins could be transferred provided they gained access to the flagellum. Intriguingly, a component of the RNAi machinery (Argonaute) was able to move between cells, raising the possibility that small interfering RNAs are transported as cargo. Transmission electron microscopy showed that shared flagella contained two axonemes and two paraflagellar rods bounded by a single membrane. In some cases flagellar fusion was partial and interactions between cells were transient. In other cases fusion occurred along the entire length of the flagellum, was stable for several hours and might be irreversible. Fusion did not appear to be deleterious for cell function: paired cells were motile and could give rise to progeny while fused. The motile flagella of unicellular organisms are related to the sensory cilia of higher eukaryotes, raising the possibility that protein transfer between cells via cilia or flagella occurs more widely in nature. PMID:27239276

  14. Development of a fusion protein SNVP as substrate for assaying multi-serotype botulinum neurotoxins.

    PubMed

    Luo, Sen; Li, Tao; Wang, Qin; Tian, Renmao; Liu, Hao; Fang, Huali; Chen, Fanghong; Wang, Hui

    2014-10-15

    The SNARE super family has three core members, namely SNAP-25, VAMP-2, and syntaxin. SNAP-25 is cleaved by botulinum toxins (BoNTs)/A, /C, and /E, whereas VAMP-2 is the substrate for proteolytic BoNTs/B, /D, /F, and /G. In this study, we constructed a hybrid gene encoding the fusion protein SNVP that encompasses SNAP-25 residues Met1 to Gly206 and VAMP-2 residues Met1 to Lys94. The hybrid gene was cloned in a prokaryotic vector carrying an N-terminal pelB signal sequence and overexpressed in Escherichia coli BL21(DE3) Rosetta. To easily purify the protein, 6× His double-affinity tags were designed as the linker and C terminus of the fusion protein. SNVP was purified to homogeneity by affinity chromatography on a HisTrap FF column and determined to be more than 97% pure by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. N-terminal sequencing of the purified protein showed that signal peptide was successfully removed. The fusion protein SNVP contained the protease cleavage sites of all seven serotypes of BoNTs. SNVP was also proved to be recognized and cleaved by the endopeptidase of BoNTs (BoNT/A-LC, BoNT/B-LC, BoNT/E-LC, and BoNT/G-LC). The novel fusion substrate SNVP exhibited high biological activity under the optimal conditions, suggesting its potential use as a reagent for BoNT assay. PMID:23851341

  15. Construction of human LRIG1-TAT fusions and TAT-mediated LRIG1 protein delivery.

    PubMed

    Wang, Yuchun; Fu, Liqi; Liu, Bo; Wang, Xiaomin; Wang, Kai; Ye, Ming

    2015-02-01

    Human leucine-rich repeats and immunoglobulin-like domains (LRIG1) is a tumor suppressor in animals and also functions as an endogenous suppressor in human tumor. The level of LRIG1 expression is highly associated with patient survival in clinic. The exploration of LRIG1 as a protein drug is an important task. HIV-1 transactivator of transcription peptide (TAT) is an excellent candidate for protein transduction. In this study, human LRIG1 was cloned and LRIG1-TAT fusion gene was constructed. The fusion proteins were produced by an Escherichia coli strain and purified by Ni(2+)-resin. Western blot assay and immunofluorescence microscopy were employed for monitoring LRIG1-TAT protein transduction into human neuroblastoma cells. Cell proliferation and invasion were measured for evaluating the effect of LRIG1-TAT on neuroblastoma cell. Our data showed that LRIG1 protein can be delivered into cells or organs in living animals by TAT. One-time transduction of LRIG1 proteins into human neuroblastoma cells enhanced cell proliferation and increased cell invasion. In vivo transduction showed that LRIG1-TAT protein can be presented in living animal organs. Our experiments provide a new vision on LRIG1 applications and also offer a therapy window for revealing the intrinsic function of LRIG1 on cells. PMID:25661388

  16. The Plasma Membrane Proteins Prm1 and Fig1 Ascertain Fidelity of Membrane Fusion during Yeast Mating

    PubMed Central

    Engel, Alex; Walter, Peter

    2007-01-01

    As for most cell–cell fusion events, the molecular details of membrane fusion during yeast mating are poorly understood. The multipass membrane protein Prm1 is the only known component that acts at the step of bilayer fusion. In its absence, mutant mating pairs lyse or arrest in the mating reaction with tightly apposed plasma membranes. We show that deletion of FIG 1, which controls pheromone-induced Ca2+ influx, yields similar cell fusion defects. Although extracellular Ca2+ is not required for efficient cell fusion of wild-type cells, cell fusion in prm1 mutant mating pairs is dramatically reduced when Ca2+ is removed. This enhanced fusion defect is due to lysis. Time-lapse microscopy reveals that fusion and lysis events initiate with identical kinetics, suggesting that both outcomes result from engagement of the fusion machinery. The yeast synaptotagmin orthologue and Ca2+ binding protein Tcb3 has a role in reducing lysis of prm1 mutants, which opens the possibility that the observed role of Ca2+ is to engage a wound repair mechanism. Thus, our results suggest that Prm1 and Fig1 have a role in enhancing membrane fusion and maintaining its fidelity. Their absence results in frequent mating pair lysis, which is counteracted by Ca2+-dependent membrane repair. PMID:17151357

  17. Composites containing albumin protein or cyanoacrylate adhesives and biodegradable scaffolds: I. Acute wound closure study in a rat model

    NASA Astrophysics Data System (ADS)

    Hoffman, Grant T.; Soller, Eric C.; Heintzelman, Douglas L.; Duffy, Mark T.; Bloom, Jeffrey N.; Gilmour, Travis M.; Gonnerman, Krista N.; McNally-Heintzelman, Karen M.

    2004-07-01

    Composite adhesives composed of biodegradable scaffolds impregnated with a biological or synthetic adhesive were investigated for use in wound closure as an alternative to using either one of the adhesives alone. Two different scaffold materials were investigated: (i) a synthetic biodegradable material fabricated from poly(L-lactic-co-glycolic acid); and (ii) a biological material, small intestinal sub mucosa, manufactured by Cook BioTech. The biological adhesive was composed of 50%(w/v) bovine serum albumin solder and 0.5mg/ml indocyanine green dye mixed in deionized water, and activated with an 808-nm diode laser. The synthetic adhesive was Ethicon's Dermabond, a 2-octyl-cyanoacrylate. The tensile strength of skin incisions repaired ex vivo in a rat model, by adhesive alone or in combination with a scaffold, as well as the time-to-failure, were measured and compared. The tensile strength of repairs formed using the scaffold-enhanced biological adhesives were on average, 80% stronger than their non-enhanced counterparts, with an accompanying increase in the time-to-failure of the repairs. These results support the theory that a scaffold material with an irregular surface that bridges the wound provides a stronger, more durable and consistent adhesion, due to the distribution of the tensile stress forces over the many micro-adhesions provided by the irregular surface, rather than the one large continuous adhesive contact. This theory is also supported by several previous ex vivo experiments demonstrating enhanced tensile strength of irregular versus smooth scaffold surfaces in identical tissue repairs performed on bovine thoracic aorta, liver, spleen, small intestine and lung tissue.

  18. Delivery of membrane proteins into small and giant unilamellar vesicles by charge-mediated fusion.

    PubMed

    Biner, Olivier; Schick, Thomas; Müller, Yannic; von Ballmoos, Christoph

    2016-07-01

    One of the current challenges in synthetic biology is the production of stable membrane mimetic systems and the insertion of components in these systems. Here, we employ fusion of oppositely charged liposomes to deliver separately reconstituted membrane proteins into a common lipid bilayer. After a systematic evaluation of different lipid compositions by lipid mixing and size distribution analysis, suitable conditions were further investigated for proteoliposome fusion. With this technique, we functionally coreconstituted bo3 oxidase and ATP synthase from Escherichia coli into unilamellar liposomes ranging from 100 nm to 50 μm in size. The presented method is a simple and versatile tool for oriented membrane protein reconstitution to produce biomimetic systems with increased complexity. PMID:27264202

  19. Studies of the Interaction between Isoimperatorin and Human Serum Albumin by Multispectroscopic Method: Identification of Possible Binding Site of the Compound Using Esterase Activity of the Protein

    PubMed Central

    Ranjbar, Samira; Shokoohinia, Yalda; Ghobadi, Sirous; Gholamzadeh, Saeed; Moradi, Nastaran; Ashrafi-Kooshk, Mohammad Reza; Aghaei, Abbas

    2013-01-01

    Isoimperatorin is one of the main components of Prangos ferulacea as a linear furanocoumarin and used as anti-inflammatory, analgesic, antispasmodic, and anticancer drug. Human serum albumin (HSA) is a principal extracellular protein with a high concentration in blood plasma and carrier for many drugs to different molecular targets. Since the carrying of drug by HSA may affect on its structure and action, we decided to investigate the interaction between HSA and isoimperatorin using fluorescence and UV spectroscopy. Fluorescence data indicated that isoimperatorin quenches the intrinsic fluorescence of the HSA via a static mechanism and hydrophobic interaction play the major role in the drug binding. The binding average distance between isoimperatorin and Trp 214 of HSA was estimated on the basis of the theory of Förster energy transfer. Decrease of protein surface hydrophobicity (PSH) was also documented upon isoimperatorin binding. Furthermore, the synchronous fluorescence spectra show that the microenvironment of the tryptophan residues does not have obvious changes. Site marker compettive and fluorescence experiments revealed that the binding of isoimperatorin to HSA occurred at or near site I. Finally, the binding details between isoimperatorin and HSA were further confirmed by molecular docking and esterase activity inhibition studies which revealed that drug was bound at subdomain IIA. PMID:24319355

  20. Subchronic toxicity study in vivo and allergenicity study in vitro for genetically modified rice that expresses pharmaceutical protein (human serum albumin).

    PubMed

    Sheng, Yao; Qi, Xiaozhe; Liu, Yifei; Guo, Mingzhang; Chen, Siyuan; He, Xiaoyun; Huang, Kunlun; Xu, Wentao

    2014-10-01

    Genetically modified (GM) crops that express pharmaceutical proteins have become an important focus of recent genetic engineering research. Food safety assessment is necessary for the commercial development of these crops. Subchronic toxicity study in vivo and allergenicity study in vitro were designed to evaluate the food safety of the rice variety expressing human serum albumin (HSA). Animals were fed rodent diets containing 12.5%, 25.0% and 50.0% GM or non-GM rice for 90 days. The composition analysis of the GM rice demonstrated several significant differences. However, most of the differences remained within the ranges reported in the literature. In the animal study, a range of indexes including clinical observation, feed efficiency, hematology, serum chemistry, organ weights and histopathology were examined. Random changes unrelated to the GM rice exposure, within the range of historical control values and not associated with any signs of illness were observed. The results of heat stability and in vitro digestion of HSA indicated no evidence of potential allergenicity of the protein. Overall, the results of these studies suggest that the GM rice appears to be safe as a dietary ingredient when it is used at up to 50% in the diet on a subchronic basis. PMID:25086369

  1. Facile synthesis of nano-sized agarose based amino acid-Its pH-dependent protein-like behavior and interactions with bovine serum albumin.

    PubMed

    Chudasama, Nishith A; Siddhanta, A K

    2015-11-19

    In a facile synthesis agarose was amphoterically functionalized to afford nano-sized agarose amino acids, aminoagarose succinate half-esters (AAE) containing one pendant carboxyl group. Nano-sized AAEs (<10 nm; DLS) were characterized and they had three various degrees of substitution [overall DSs 0.88, 0.89 and 0.96], both the amino and half-ester groups were placed on C-6 positions of the 1,3 beta-d-galactopyranose moieties of agarose backbone ((13)C NMR). AAEs performed like large protein molecules exhibiting pH-responsive structural variations (optical rotatory dispersion), presenting a mixed solubility pattern like random coil (soluble) and aggregate (precipitation) formations. Circular dichroism studies showed their pH-dependent associative interactions with bovine serum albumin, which indicated complexation at acidic and basic pHs, and decomplexation at pH 6.8 with AAE (DS 0.96). Thus, these nano-sized AAE based systems may be of potential utility in the domains demanding the merits of preferential protein bindings e.g. pH-responsive cationic/anionic drug carrier, separations or chiral sensing applications. PMID:26413976

  2. Functional NifD-K fusion protein in Azotobacter vinelandii is a homodimeric complex equivalent to the native heterotetrameric MoFe protein

    SciTech Connect

    Lahiri, Surobhi; Pulakat, Lakshmi; Gavini, Nara . E-mail: gavini@biology.msstate.edu

    2005-11-18

    The MoFe protein of the complex metalloenzyme nitrogenase folds as a heterotetramer containing two copies each of the homologous {alpha} and {beta} subunits, encoded by the nifD and the nifK genes respectively. Recently, the functional expression of a fusion NifD-K protein of nitrogenase was demonstrated in Azotobacter vinelandii, strongly implying that the MoFe protein is flexible as it could accommodate major structural changes, yet remain functional [M.H. Suh, L. Pulakat, N. Gavini, J. Biol. Chem. 278 (2003) 5353-5360]. This finding led us to further explore the type of interaction between the fused MoFe protein units. We aimed to determine whether an interaction exists between the two fusion MoFe proteins to form a homodimer that is equivalent to native heterotetrameric MoFe protein. Using the Bacteriomatch Two-Hybrid System, translationally fused constructs of NifD-K (fusion) with the full-length {lambda}CI of the pBT bait vector and also NifD-K (fusion) with the N-terminal {alpha}-RNAP of the pTRG target vector were made. To compare the extent of interaction between the fused NifD-K proteins to that of the {beta}-{beta} interactions in the native MoFe protein, we proceeded to generate translationally fused constructs of NifK with the {alpha}-RNAP of the pTRG vector and {lambda}CI protein of the pBT vector. The strength of the interaction between the proteins in study was determined by measuring the {beta}-galactosidase activity and extent of ampicillin resistance of the colonies expressing these proteins. This analysis demonstrated that direct protein-protein interaction exists between NifD-K fusion proteins, suggesting that they exist as homodimers. As the interaction takes place at the {beta}-interfaces of the NifD-K fusion proteins, we propose that these homodimers of NifD-K fusion protein may function in a similar manner as that of the heterotetrameric native MoFe protein. The observation that the extent of protein-protein interaction between the {beta

  3. IQCJ-SCHIP1, a novel fusion transcript encoding a calmodulin-binding IQ motif protein

    SciTech Connect

    Kwasnicka-Crawford, Dorota A. . E-mail: dakc@yorku.ca; Carson, Andrew R.; Scherer, Stephen W.

    2006-12-01

    The existence of transcripts that span two adjacent, independent genes is considered rare in the human genome. This study characterizes a novel human fusion gene named IQCJ-SCHIP1. IQCJ-SCHIP1 is the longest isoform of a complex transcriptional unit that bridges two separate genes that encode distinct proteins, IQCJ, a novel IQ motif containing protein and SCHIP1, a schwannomin interacting protein that has been previously shown to interact with the Neurofibromatosis type 2 (NF2) protein. IQCJ-SCHIP1 is located on the chromosome 3q25 and comprises a 1692-bp transcript encompassing 11 exons spanning 828 kb of the genomic DNA. We show that IQCJ-SCHIP1 mRNA is highly expressed in the brain. Protein encoded by the IQCJ-SCHIP1 gene was localized to cytoplasm and actin-rich regions and in differentiated PC12 cells was also seen in neurite extensions.

  4. Efficient refolding of the bifunctional therapeutic fusion protein VAS-TRAIL by a triple agent solution.

    PubMed

    Fan, Jiying; Wang, Zhanqing; Huang, Liying; Shen, Yaling

    2016-09-01

    VAS-TRAIL is a bifunctional fusion protein that combines anti-angiogenic activity with tumor-selective apoptotic activity for enhanced anti-tumor efficacy. VAS-TRAIL is expressed as inclusion body in Escherichia coli, but protein refolding is difficult to achieve and results in low yields of bioactive protein. In this study, we describe an efficient method for VAS-TRAIL refolding. The solubilization of aggregated VAS-TRAIL was achieved by a triple agent solution, which consists of an alkaline solution (pH 11.5) containing 0.4M l-arginine and 2M urea. The solubilized protein showed high purity and preserved secondary structure according to fluorescence properties. VAS-TRAIL refolding was performed through stepwise dialysis and resulted in more than 50% recovery of the soluble protein. The function of l-arginine was additive with alkaline pH, as shown by the significant improvement in refolding yield (≈30%) by l-arginine-containing solubilization solutions compared with alkaline solubilization solutions without l-arginine. The refolded VAS-TRAIL also showed β-sheet structures and the propensity for oligomerization. Bioassays showed that the refolded fusion protein exhibited the expected activities, including its apoptotic activities toward tumor and endothelial cells, which proposed its promising therapeutic potential. PMID:26358405

  5. Identification and quantification of major maillard cross-links in human serum albumin and lens protein. Evidence for glucosepane as the dominant compound.

    PubMed

    Biemel, Klaus M; Friedl, D Alexander; Lederer, Markus O

    2002-07-12

    Glycation reactions leading to protein modifications (advanced glycation end products) contribute to various pathologies associated with the general aging process and long term complications of diabetes. However, only few relevant compounds have so far been detected in vivo. We now report on the first unequivocal identification of the lysine-arginine cross-links glucosepane 5, DOGDIC 6, MODIC 7, and GODIC 8 in human material. For their accurate quantification by coupled liquid chromatography-electrospray ionization mass spectrometry, (13)C-labeled reference compounds were synthesized independently. Compounds 5-8 are formed via the alpha-dicarbonyl compounds N(6)-(2,3-dihydroxy-5,6-dioxohexyl)-l-lysinate (1a,b), 3-deoxyglucosone (), methylglyoxal (), and glyoxal (), respectively. The protein-bound dideoxyosone 1a,b seems to be of prime significance for cross-linking because it presumably is not detoxified by mammalian enzymes as readily as 2-4. Hence, the follow-up product glucosepane 5 was found to be the dominant compound. Up to 42.3 pmol of 5/mg of protein was identified in human serum albumin of diabetics; the level of 5 correlates markedly with the glycated hemoglobin HbA(1c). In the water-insoluble fraction of lens proteins from normoglycemics, concentration of 5 ranges between 132.3 and 241.7 pmol/mg. The advanced glycoxidation end product GODIC 8 is elevated significantly in brunescent lenses, indicating enhanced oxidative stress in this material. Compounds 5-8 thus appear predestined as markers for pathophysiological processes. PMID:11978796

  6. Analysis of multi-site drug-protein interactions by high-performance affinity chromatography: Binding by glimepiride to normal or glycated human serum albumin.

    PubMed

    Matsuda, Ryan; Li, Zhao; Zheng, Xiwei; Hage, David S

    2015-08-21

    High-performance affinity chromatography (HPAC) was used in a variety of formats to examine multi-site interactions between glimepiride, a third-generation sulfonylurea drug, and normal or in vitro glycated forms of the transport protein human serum albumin (HSA). Frontal analysis revealed that glimepiride interacts with normal HSA and glycated HSA at a group of high affinity sites (association equilibrium constant, or Ka, 9.2-11.8×10(5)M(-1) at pH 7.4 and 37°C) and a group of lower affinity regions (Ka, 5.9-16×10(3)M(-1)). Zonal elution competition studies were designed and carried out in both normal- and reversed-role formats to investigate the binding by this drug at specific sites. These experiments indicated that glimepiride was interacting at both Sudlow sites I and II. Allosteric effects were also noted with R-warfarin at Sudlow site I and with tamoxifen at the tamoxifen site on HSA. The binding at Sudlow site I had a 2.1- to 2.3-fold increase in affinity in going from normal HSA to the glycated samples of HSA. There was no significant change in the affinity for glimepiride at Sudlow site II in going from normal HSA to a moderately glycated sample of HSA, but a slight decrease in affinity was seen in going to a more highly glycated HSA sample. These results demonstrated how various HPAC-based methods can be used to profile and characterize multi-site binding by a drug such as glimepiride to a protein and its modified forms. The information obtained from this study should be useful in providing a better understanding of how drug-protein binding may be affected by glycation and of how separation and analysis methods based on HPAC can be employed to study systems with complex interactions or that involve modified proteins. PMID:26189669

  7. Analysis of Multi-Site Drug-Protein Interactions by High-Performance Affinity Chromatography: Binding by Glimepiride to Normal or Glycated Human Serum Albumin

    PubMed Central

    Matsuda, Ryan; Li, Zhao; Zheng, Xiwei; Hage, David S.

    2015-01-01

    High-performance affinity chromatography (HPAC) was used in a variety of formats to examine multi-site interactions between glimepiride, a third-generation sulfonylurea drug, and normal or in vitro glycated forms of the transport protein human serum albumin (HSA). Frontal analysis revealed that glimepiride interacts with normal HSA and glycated HSA at a group of high affinity sites (association equilibrium constant, or Ka, 9.2–11.8 × 105 M−1 at pH 7.4 and 37°C) and a group of lower affinity regions (Ka, 5.9–16.2 × 103 M−1). Zonal elution competition studies were designed and carried out in both normal- and reversed-role formats to investigate the binding by this drug at specific sites. These experiments indicated that glimepiride was interacting at both Sudlow sites I and II. Allosteric effects were also noted with R-warfarin at Sudlow site I and with tamoxifen at the tamoxifen site on HSA. The binding at Sudlow site I had a 2.1- to 2.3-fold increase in affinity in going from normal HSA to the glycated samples of HSA. There was no significant change in the affinity for glimepiride at Sudlow site II in going from normal HSA to a moderately glycated sample of HSA, but a slight decrease in affinity was seen in going to a more highly glycated HSA sample. These results demonstrated how various HPAC-based methods can be used to profile and characterize multi-site binding by a drug such as glimepiride to a protein and its modified forms. The information obtained from this study should be useful in providing a better understanding of how drug-protein binding may be affected by glycation and of how separation and analysis methods based on HPAC can be employed to study systems with complex interactions or that involve modified proteins. PMID:26189669

  8. Sequence motifs and prokaryotic expression of the reptilian paramyxovirus fusion protein

    USGS Publications Warehouse

    Franke, J.; Batts, W.N.; Ahne, W.; Kurath, G.; Winton, J.R.

    2006-01-01

    Fourteen reptilian paramyxovirus isolates were chosen to represent the known extent of genetic diversity among this novel group of viruses. Selected regions of the fusion (F) gene were sequenced, analyzed and compared. The F gene of all isolates contained conserved motifs homologous to those described for other members of the family Paramyxoviridae including: signal peptide, transmembrane domain, furin cleavage site, fusion peptide, N-linked glycosylation sites, and two heptad repeats, the second of which (HRB-LZ) had the characteristics of a leucine zipper. Selected regions of the fusion gene of isolate Gono-GER85 were inserted into a prokaryotic expression system to generate three recombinant protein fragments of various sizes. The longest recombinant protein was cleaved by furin into two fragments of predicted length. Western blot analysis with virus-neutralizing rabbit-antiserum against this isolate demonstrated that only the longest construct reacted with the antiserum. This construct was unique in containing 30 additional C-terminal amino acids that included most of the HRB-LZ. These results indicate that the F genes of reptilian paramyxoviruses contain highly conserved motifs typical of other members of the family and suggest that the HRB-LZ domain of the reptilian paramyxovirus F protein contains a linear antigenic epitope. ?? Springer-Verlag 2005.

  9. The recombinant expression and activity detection of MAF-1 fusion protein

    PubMed Central

    Fu, Ping; Wu, Jianwei; Gao, Song; Guo, Guo; Zhang, Yong; Liu, Jian

    2015-01-01

    This study establishes the recombinant expression system of MAF-1 (Musca domestica antifungal peptide-1) and demonstrates the antifungal activity of the expression product and shows the relationship between biological activity and structure. The gene segments on mature peptide part of MAF-1 were cloned, based on the primers designed according to the cDNA sequence of MAF-1. We constructed the recombinant prokaryotic expression plasmid using prokaryotic expression vector (pET-28a(+)) and converted it to the competent cell of BL21(DE3) to gain recombinant MAF-1 fusion protein with His tag sequence through purifying affinity chromatographic column of Ni-NTA. To conduct the Western Blotting test, recombinant MAF-1 fusion protein was used to produce the polyclonal antibody of rat. The antifungal activity of the expression product was detected using Candida albicans (ATCC10231) as the indicator. The MAF-1 recombinant fusion protein was purified to exhibit obvious antifungal activity, which lays the foundation for the further study of MAF-1 biological activity, the relationship between structure and function, as well as control of gene expression. PMID:26423137

  10. Ebola virus glycoprotein Fc fusion protein confers protection against lethal challenge in vaccinated mice

    PubMed Central

    Konduru, Krishnamurthy; Bradfute, Steven B.; Jacques, Jerome; Manangeeswaran, Mohanraj; Nakamura, Siham; Morshed, Sufi; Wood, Steven C.; Bavari, Sina

    2011-01-01

    Ebola virus is a Filoviridae that causes hemorrhagic fever in humans and induces high morbidity and mortality rates. Filoviruses are classified as "Category A bioterrorism agents", and currently there are no licensed therapeutics or vaccines to treat and prevent infection. The Filovirus glycoprotein (GP) is sufficient to protect individuals against infection, and several vaccines based on GP are under development including recombinant adenovirus, parainfluenza virus, Venezuelan equine encephalitis virus, vesicular stomatitis virus (VSV) and virus-like particles. Here we describe the development of a GP Fc fusion protein as a vaccine candidate. We expressed the extracellular domain of the Zaire Ebola virus (ZEBOV) GP fused to the Fc fragment of human IgG1 (ZEBOVGP-Fc) in mammalian cells and showed that GP undergoes the complex furin cleavage and processing observed in the native membrane-bound GP. Mice immunized with ZEBOVGP-Fc developed T-cell immunity against ZEBOV GP and neutralizing antibodies against replication-competent VSV-G deleted recombinant VSV containing ZEBOV GP. The ZEBOVGP-Fc vaccinated mice were protected against challenge with a lethal dose of ZEBOV. These results show that vaccination with the ZEBOVGP-Fc fusion protein alone without the need of a viral vector or assembly into virus-like particles is sufficient to induce protective immunity against ZEBOV in mice. Our data suggested that Filovirus GP Fc fusion proteins could be developed as a simple, safe, efficacious, and cost effective vaccine against Filovirus infection for human use. PMID:21329775

  11. ANTIBODY-CYTOKINE FUSION PROTEINS FOR TREATMENT OF CANCER: ENGINEERING CYTOKINES FOR IMPROVED EFFICACY AND SAFETY

    PubMed Central

    Young, Patricia A.; Morrison, Sherie L.; Timmerman, John M.

    2014-01-01

    The true potential of cytokine therapies in cancer treatment is limited by the inability to deliver optimal concentrations into tumor sites due to dose-limiting systemic toxicities. To maximize the efficacy of cytokine therapy, recombinant antibody-cytokine fusion proteins have been constructed by a number of groups to harness the tumor-targeting ability of monoclonal antibodies. The aim is to guide cytokines specifically to tumor sites where they might stimulate more optimal anti-tumor immune responses while avoiding the systemic toxicities of free cytokine therapy. Antibody-cytokine fusion proteins containing IL-2, IL-12, IL-21, TNFα, and interferons α, β and γ have been constructed and have shown anti-tumor activity in pre-clinical and early phase clinical studies. Future priorities for development of this technology include optimization of tumor targeting, bioactivity of the fused cytokine, and choice of appropriate agents for combination therapies. This review is intended to serve as a framework for engineering an ideal antibody-cytokine fusion protein, focusing on previously developed constructs and their clinical trial results. PMID:25440607

  12. Production, purification, and characterization of scFv TNF ligand fusion proteins.

    PubMed

    Fick, Andrea; Wyzgol, Agnes; Wajant, Harald

    2012-01-01

    Single-chain variable fragments (scFvs) specific for tumor-associated cell surface antigens are the most broadly used reagents to direct therapeutic or diagnostic effector molecules, such as toxins, radioisotopes, and CD3-stimulating scFvs, to tumors. One novel class of effector molecules that can be targeted to tumors by scFvs are ligands of the tumor necrosis factor (TNF) family. Typically, these molecules have apoptosis inducing and/or immune stimulating properties and are therefore highly attractive for cancer treatment. N-terminal fusion of scFvs does not interfere with the receptor binding capabilities of TNF ligands and thus allows the straightforward generation of scFv TNF ligand fusion proteins. We report here a protocol for the purification of eukaryotically produced scFv TNF ligand fusion proteins based on affinity chromatography on anti-Flag agarose and further describe assays for the determination of the targeting index of this type of scFv-targeted proteins. PMID:22907375

  13. Immobilization of the N-terminal helix stabilizes prefusion paramyxovirus fusion proteins.

    PubMed

    Song, Albert S; Poor, Taylor A; Abriata, Luciano A; Jardetzky, Theodore S; Dal Peraro, Matteo; Lamb, Robert A

    2016-07-01

    Parainfluenza virus 5 (PIV5) is an enveloped, single-stranded, negative-sense RNA virus of the Paramyxoviridae family. PIV5 fusion and entry are mediated by the coordinated action of the receptor-binding protein, hemagglutinin-neuraminidase (HN), and the fusion protein (F). Upon triggering by HN, F undergoes an irreversible ATP- and pH-independent conformational change, going down an energy gradient from a metastable prefusion state to a highly stable postfusion state. Previous studies have highlighted key conformational changes in the F-protein refolding pathway, but a detailed understanding of prefusion F-protein metastability remains elusive. Here, using two previously described F-protein mutations (S443D or P22L), we examine the capacity to modulate PIV5 F stability and the mechanisms by which these point mutants act. The S443D mutation destabilizes prefusion F proteins by disrupting a hydrogen bond network at the base of the F-protein globular head. The introduction of a P22L mutation robustly rescues destabilized F proteins through a local hydrophobic interaction between the N-terminal helix and a hydrophobic pocket. Prefusion stabilization conferred by a P22L-homologous mutation is demonstrated in the F protein of Newcastle disease virus, a paramyxovirus of a different genus, suggesting a conserved stabilizing structural element within the paramyxovirus family. Taken together, the available data suggest that movement of the N-terminal helix is a necessary early step for paramyxovirus F-protein refolding and presents a novel target for structure-based drug design. PMID:27335462

  14. Expression and activity analysis of a new fusion protein targeting ovarian cancer cells.

    PubMed

    Su, Manman; Chang, Weiqin; Wang, Dingding; Cui, Manhua; Lin, Yang; Wu, Shuying; Xu, Tianmin

    2015-09-01

    The aim of the present study was to develop a new therapeutic drug to improve the prognosis of ovarian cancer patients. Human urokinase-type plasminogen activator (uPA)17-34-kunitz-type protease inhibitor (KPI) eukaryotic expression vector was constructed and recombinant human uPA17-34-KPI (rhuPA17-34-KPI) in P. pastoris was expressed. In the present study, the DNA sequences that encode uPA 17-34 amino acids were created according to the native amino acids sequence and inserted into the KPI-pPICZαC vector, which was constructed. Then, uPA17‑34-KPI-pPICZαC was transformed into P. pastoris X-33, and rhuPA17-34-KPI was expressed by induction of methanol. The bioactivities of a recombinant fusion protein were detected with trypsin inhibition analysis, and the inhibitory effects on the growth of ovarian cancer cells were identified using the TUNEL assay, in vitro wound‑healing assay and Matrigel model analysis. The results of the DNA sequence analysis of the recombinant vector uPA17-34-KPI‑pPICZα demonstrated that the DNA‑encoding human uPA 17-34 amino acids, 285-288 amino acids of amyloid precursor protein (APP) and 1-57 amino acids of KPI were correctly inserted into the pPICZαC vector. Following induction by methonal, the fusion protein with a molecular weight of 8.8 kDa was observed using SDS-PAGE and western blot analysis. RhuPA17-34-KPI was expressed in P. pastoris with a yield of 50 mg/l in a 50-ml tube. The recombinant fusion protein was able to inhibit the activity of trypsin, inhibit growth and induce apoptosis of SKOV3 cells, and inhibit the invasion and metastasis of ovarian cancer cells. By considering uPA17-34 amino acid specific binding uPAR as the targeted part of fusion protein and utilizing the serine protease inhibitor activity of KPI, it was found that the recombinant fusion protein uPA17-34-KPI inhibited the invasion and metastasis of ovarian tumors, and may therefore be regarded as effective in targeted treatment. PMID

  15. Sequential Conformational Changes in the Morbillivirus Attachment Protein Initiate the Membrane Fusion Process

    PubMed Central

    Ader-Ebert, Nadine; Khosravi, Mojtaba; Herren, Michael; Avila, Mislay; Alves, Lisa; Bringolf, Fanny; Örvell, Claes; Langedijk, Johannes P.; Zurbriggen, Andreas; Plemper, Richard K.; Plattet, Philippe

    2015-01-01

    Despite large vaccination campaigns, measles virus (MeV) and canine distemper virus (CDV) cause major morbidity and mortality in humans and animals, respectively. The MeV and CDV cell entry system relies on two interacting envelope glycoproteins: the attachment protein (H), consisting of stalk and head domains, co-operates with the fusion protein (F) to mediate membrane fusion. However, how receptor-binding by the H-protein leads to F-triggering is not fully understood. Here, we report that an anti-CDV-H monoclonal antibody (mAb-1347), which targets the linear H-stalk segment 126-133, potently inhibits membrane fusion without interfering with H receptor-binding or F-interaction. Rather, mAb-1347 blocked the F-triggering function of H-proteins regardless of the presence or absence of the head domains. Remarkably, mAb-1347 binding to headless CDV H, as well as standard and engineered bioactive stalk-elongated CDV H-constructs treated with cells expressing the SLAM receptor, was enhanced. Despite proper cell surface expression, fusion promotion by most H-stalk mutants harboring alanine substitutions in the 126-138 “spacer” section was substantially impaired, consistent with deficient receptor-induced mAb-1347 binding enhancement. However, a previously reported F-triggering defective H-I98A variant still exhibited the receptor-induced “head-stalk” rearrangement. Collectively, our data spotlight a distinct mechanism for morbillivirus membrane fusion activation: prior to receptor contact, at least one of the morbillivirus H-head domains interacts with the membrane-distal “spacer” domain in the H-stalk, leaving the F-binding site located further membrane-proximal in the stalk fully accessible. This “head-to-spacer” interaction conformationally stabilizes H in an auto-repressed state, which enables intracellular H-stalk/F engagement while preventing the inherent H-stalk’s bioactivity that may prematurely activate F. Receptor-contact disrupts the

  16. Sequential conformational changes in the morbillivirus attachment protein initiate the membrane fusion process.

    PubMed

    Ader-Ebert, Nadine; Khosravi, Mojtaba; Herren, Michael; Avila, Mislay; Alves, Lisa; Bringolf, Fanny; Örvell, Claes; Langedijk, Johannes P; Zurbriggen, Andreas; Plemper, Richard K; Plattet, Philippe

    2015-05-01

    Despite large vaccination campaigns, measles virus (MeV) and canine distemper virus (CDV) cause major morbidity and mortality in humans and animals, respectively. The MeV and CDV cell entry system relies on two interacting envelope glycoproteins: the attachment protein (H), consisting of stalk and head domains, co-operates with the fusion protein (F) to mediate membrane fusion. However, how receptor-binding by the H-protein leads to F-triggering is not fully understood. Here, we report that an anti-CDV-H monoclonal antibody (mAb-1347), which targets the linear H-stalk segment 126-133, potently inhibits membrane fusion without interfering with H receptor-binding or F-interaction. Rather, mAb-1347 blocked the F-triggering function of H-proteins regardless of the presence or absence of the head domains. Remarkably, mAb-1347 binding to headless CDV H, as well as standard and engineered bioactive stalk-elongated CDV H-constructs treated with cells expressing the SLAM receptor, was enhanced. Despite proper cell surface expression, fusion promotion by most H-stalk mutants harboring alanine substitutions in the 126-138 "spacer" section was substantially impaired, consistent with deficient receptor-induced mAb-1347 binding enhancement. However, a previously reported F-triggering defective H-I98A variant still exhibited the receptor-induced "head-stalk" rearrangement. Collectively, our data spotlight a distinct mechanism for morbillivirus membrane fusion activation: prior to receptor contact, at least one of the morbillivirus H-head domains interacts with the membrane-distal "spacer" domain in the H-stalk, leaving the F-binding site located further membrane-proximal in the stalk fully accessible. This "head-to-spacer" interaction conformationally stabilizes H in an auto-repressed state, which enables intracellular H-stalk/F engagement while preventing the inherent H-stalk's bioactivity that may prematurely activate F. Receptor-contact disrupts the "head

  17. Physical Instability of a Therapeutic Fc Fusion Protein: Domain Contributions to Conformational and Colloidal Stability†

    PubMed Central

    Fast, Jonas L; Cordes, Amanda A; Carpenter, John F; Randolph, Theodore W

    2009-01-01

    Protein therapeutics made up of artificially combined proteins or protein domains, so called fusion proteins, are a novel and growing class of biopharmaceuticals. We have studied abatacept (Orencia®), a fusion protein that is constructed of a modified IgG Fc domain and the soluble part of the T-cell receptor CTLA-4. In accelerated degradation studies conducted at at 40 °C, a pH shift from 7.5 to 6.0 yields significantly faster aggregation kinetics, as measured by size-exclusion chromatography. To understand how the fusion domains and their interactions contribute to this result, we considered aggregation in light of the modified Lumry-Eyring reaction pathway. Protein conformational stabilities against chaotropes and temperature were measured. The structural consequences of these perturbations were observed by a variety of experimental techniques, including differential scanning calorimetry, circular dichroism, and intrinsic fluorescence. Abatacept’s colloidal stability was studied by measuring zeta potentials and osmotic second virial coefficients, as well as by modeling electrostatic potentials on the protein’s surface. The domains of abatacept exhibit different conformational stabilities that are highly pH dependent, whereas abatacept was weakly colloidally unstable at pH 6 or pH 7.5. These results are ascribed to conformational instability of the CTLA-4 and CH2 domains, which unfold to form a molten globule-like structure that is aggregation-prone. We suggest the instability against aggregation is determined by the least stable domains. PMID:19899812

  18. Luteinizing hormone-releasing hormone fusion protein vaccines block estrous cycle activity in beef heifers.

    PubMed

    Stevens, J D; Sosa, J M; deAvila, D M; Oatley, J M; Bertrand, K P; Gaskins, C T; Reeves, J J

    2005-01-01

    Two LHRH fusion proteins, thioredoxin and ovalbumin, each containing seven LHRH inserts were tested for their ability to inhibit estrous cycle activity. The objective was to evaluate immune and biological responses from alternating the two fusion proteins in an immunization schedule. One hundred ten heifers were divided equally into 11 groups. Two control groups consisted of either spayed or intact, untreated heifers. Heifers in the other nine groups were immunized on wk 0, 4, and 9. Treatments were immunizations of the same protein throughout or alternating the proteins in different booster sequences. Blood was collected weekly for 22 wk, and serum was assayed for concentrations of progesterone and titers of anti-LHRH. At slaughter, reproductive tracts were removed from each heifer and weighed. Heifers with >or=1 ng/mL of progesterone were considered to have a functional corpus luteum and thus to have estrous cycle activity. All LHRH-immunized groups of heifers had a smaller (P < 0.05) proportion of heifers showing estrous cycle activity after 6 wk than the intact, untreated control group. There was no difference in number of heifers cycling between the immunized groups and the spayed heifers during wk 9 to 22. Anti-LHRH did not differ among immunized groups during wk 1 to 9. Starting at wk 10 and continuing through the conclusion of the study, there was an overall difference among treatment groups for anti-LHRH (P < 0.05). Uterine weights differed among treatments (P < 0.05), with intact control animals having heavier uteri than all other groups (P < 0.05). Uterine weights were negatively correlated with maximum LHRH antibody binding (r = -0.44). In summary, the LHRH fusion proteins were as effective as surgical spaying in suppression of estrous cycle activity, but alternating the two proteins in an immunization schedule did not enhance the immunological or biological effectiveness of the vaccine. PMID:15583055

  19. Detection of an unknown fusion protein in confiscated black market products.

    PubMed

    Walpurgis, Katja; Krug, Oliver; Thomas, Andreas; Laussmann, Tim; Schänzer, Wilhelm; Thevis, Mario

    2014-01-01

    Even without clinical approval, many performance-enhancing drugs are available on the black market and can therefore be easily obtained by cheating athletes. The misuse of these preparations can be associated with unforeseeable health risks - either due to a poor quality of the drugs or as a result of an insufficient clinical assessment. Moreover, confiscated black market products have frequently been shown to contain ingredients other than those declared on the label as well as additional by-products or compounds with a modified molecular structure. This communication describes the identification of an unknown fusion protein observed in several unlabelled black market products obtained from independent sources. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis of the confiscated preparations indicated the presence of an 18-kDa fusion protein consisting of the bacterial redox protein thioredoxin-1 (Trx, 12 kDa) and a 6-kDa peptide of unassigned composition. Trx has no relevance as performance enhancing agent but is routinely used as solubility tag for recombinant protein production. Further evaluation of the acquired MS/MS data revealed both an additional His tag and a thrombin cleavage site between the tags and the presumed bioactive peptide. However, thrombin cleavage of the fusion protein and LC-MS/MS analysis of the resulting peptide fragment finally suggested that the unknown protein is only the product of an empty expression vector without the DNA insert of interest. These findings are a further alarming example for the high level of risk that athletes take when misusing drugs obtained from the black market. PMID:25219942

  20. Physico-chemical characterization of protein-pigment interactions in tempera paint reconstructions: casein/cinnabar and albumin/cinnabar.

    PubMed

    Duce, Celia; Ghezzi, Lisa; Onor, Massimo; Bonaduce, Ilaria; Colombini, Maria Perla; Tine', Maria Rosaria; Bramanti, Emilia

    2012-02-01

    In this work, we characterized paint reconstructions using ovalbumin and casein as binders, and cinnabar (HgS) as a pigment, before and after artificial ageing. Egg and casein are common paint binders that were used historically in the technique of tempera painting. Despite extensive research on the identification of proteinaceous binders in paintings, there is a substantial lack of knowledge regarding the ageing pathway of their protein content, and their chemical interaction with inorganic pigments. Thermogravimetric analysis, infrared spectroscopy and size-exclusion chromatography (SEC) were used to reveal the physico-chemical processes involved in the ageing of proteins in paintings. Taken together, the three techniques highlighted that proteins are subject to both cross-linking and hydrolysis upon ageing, and to a lesser extent, to oxidation of the side chains. Mercury-protein interactions were also revealed using a cold vapour generation atomic fluorescence spectrometer mercury-specific detector coupled to SEC. The study clearly showed that HgS forms stable complexes with proteins and acts as a sensitizer in cross-linking, hydrolysis and oxidation. PMID:22231511

  1. Efficient replication of a paramyxovirus independent of full zippering of the fusion protein six-helix bundle domain

    PubMed Central

    Brindley, Melinda A.; Plattet, Philippe; Plemper, Richard Karl

    2014-01-01

    Enveloped viruses such as HIV and members of the paramyxovirus family use metastable, proteinaceous fusion machineries to merge the viral envelope with cellular membranes for infection. A hallmark of the fusogenic glycoproteins of these pathogens is refolding into a thermodynamically highly stable fusion core structure composed of six antiparallel α-helices, and this structure is considered instrumental for pore opening and/or enlargement. Using a paramyxovirus fusion (F) protein, we tested this paradigm by engineering covalently restricted F proteins that are predicted to be unable to close the six-helix bundle core structure fully. Several candidate bonds formed efficiently, resulting in F trimers and higher-order complexes containing covalently linked dimers. The engineered F complexes were incorporated into recombinant virions efficiently and were capable of refolding into a postfusion conformation without temporary or permanent disruption of the disulfide bonds. They efficiently formed fusion pores based on virus replication and quantitative cell-to-cell and virus-to-cell fusion assays. Complementation of these F mutants with a monomeric, fusion-inactive F variant enriched the F oligomers for heterotrimers containing a single disulfide bond, without affecting fusion complementation profiles compared with standard F protein. Our demonstration that complete closure of the fusion core does not drive paramyxovirus entry may aid the design of strategies for inhibiting virus entry. PMID:25157143

  2. A role for sperm surface protein disulfide isomerase activity in gamete fusion: evidence for the participation of ERp57.

    PubMed

    Ellerman, Diego A; Myles, Diana G; Primakoff, Paul

    2006-06-01

    In mammals, sperm-egg interaction is based on molecular events either unique to gametes or also present in somatic cells. In gamete fusion, it is unknown which features are gamete specific and which are shared with other systems. Conformational changes mediated by thiol-disulfide exchange are involved in the activation of some virus membrane fusion proteins. Here we asked whether that mechanism is also operative in sperm-egg fusion. Different inhibitors of protein disulfide isomerase (PDI) activity were able to inhibit sperm-egg fusion in vitro. While pretreatment of oocytes had no effect, pretreatment of sperm reduced their fusion ability. Some members of the PDI family were detected on the sperm head, and use of specific antibodies and substrates suggested that the oxidoreductase ERp57 has a role in gamete fusion. The results support the idea that thiol-disulfide exchange is a mechanism that may act in gamete fusion to produce conformational changes in fusion-active proteins. PMID:16740484

  3. Effects of retroviral envelope-protein cleavage upon trafficking, incorporation, and membrane fusion

    SciTech Connect

    Apte, Swapna; Sanders, David Avram

    2010-09-15

    Retroviral envelope glycoproteins undergo proteolytic processing by cellular subtilisin-like proprotein convertases at a polybasic amino-acid site in order to produce the two functional subunits, SU and TM. Most previous studies have indicated that envelope-protein cleavage is required for rendering the protein competent for promoting membrane fusion and for virus infectivity. We have investigated the role of proteolytic processing of the Moloney murine leukemia virus envelope-protein through site-directed mutagenesis of the residues near the SU-TM cleavage site and have established that uncleaved glycoprotein is unable either to be incorporated into virus particles efficiently or to induce membrane fusion. Additionally, the results suggest that cleavage of the envelope protein plays an important role in intracellular trafficking of protein via the cellular secretory pathway. Based on our results it was concluded that a positively charged residue located at either P2 or P4 along with the arginine at P1 is essential for cleavage.

  4. S-layer-streptavidin fusion proteins as template for nanopatterned molecular arrays

    NASA Astrophysics Data System (ADS)

    Moll, Dieter; Huber, Carina; Schlegel, Birgit; Pum, Dietmar; Sleytr, Uwe B.; Sára, Margit

    2002-11-01

    Biomolecular self-assembly can be used as a powerful tool for nanoscale engineering. In this paper, we describe the development of building blocks for nanobiotechnology, which are based on the fusion of streptavidin to a crystalline bacterial cell surface layer (S-layer) protein with the inherent ability to self-assemble into a monomolecular protein lattice. The fusion proteins and streptavidin were produced independently in Escherichia coli, isolated, and mixed to refold and purify heterotetramers of 1:3 stoichiometry. Self-assembled chimeric S-layers could be formed in suspension, on liposomes, on silicon wafers, and on accessory cell wall polymer containing cell wall fragments. The two-dimensional protein crystals displayed streptavidin in defined repetitive spacing, and they were capable of binding D-biotin and biotinylated proteins. Therefore, the chimeric S-layer can be used as a self-assembling nanopatterned molecular affinity matrix to arrange biotinylated compounds on a surface. In addition, it has application potential as a functional coat of liposomes.

  5. The destructive effect of botulinum neurotoxins on the SNARE protein: SNAP-25 and synaptic membrane fusion

    PubMed Central

    2015-01-01

    Synaptic exocytosis requires the assembly of syntaxin 1A and SNAP-25 on the plasma membrane and synaptobrevin 2 (VAMP2) on the vesicular membrane to bridge the two opposite membranes. It is believed that the three SNARE proteins assemble in steps along the dynamic assembly pathway. The C-terminus of SNAP-25 is known to be the target of botulinum neurotoxins (BoNT/A and BoNT/E) that block neurotransmitters release in vivo. In this study, we employed electron paramagnetic resonance (EPR) spectroscopy to investigate the conformation of the SNAP-25 C-terminus in binary and ternary SNARE complexes. The fluorescence lipid mixing assay shows that the C-terminal of SNAP-25 is essential for membrane fusion, and that the truncated SNAP-25 mutants cleaved by BoNT/A and BoNT/E display different inhibition effects on membrane fusion: SNAP-25E (Δ26) abolishes the fusion activity of the SNARE complex, while SNAP-25A (Δ9) loses most of its function, although it can still form a SDS-resistant SNARE complex as the wild-type SNAP-25. CW-EPR spectra validate the unstable structures of the SNARE complex formed by SNAP-25 mutants. We propose that the truncated SNAP-25 mutants will disrupt the assembly of the SNARE core complex, and then inhibit the synaptic membrane fusion accordingly. PMID:26157630

  6. Purification, crystallization and X-ray diffraction analysis of human dynamin-related protein 1 GTPase-GED fusion protein

    PubMed Central

    Klinglmayr, Eva; Wenger, Julia; Mayr, Sandra; Bossy-Wetzel, Ella; Puehringer, Sandra

    2012-01-01

    The mechano-enzyme dynamin-related protein 1 plays an important role in mitochondrial fission and is implicated in cell physiology. Dysregulation of Drp1 is associated with abnormal mitochondrial dynamics and neuronal damage. Drp1 shares structural and functional similarities with dynamin 1 with respect to domain organization, ability to self-assemble into spiral-like oligomers and GTP-cycle-dependent membrane scission. Structural studies of human dynamin-1 have greatly improved the understanding of this prototypical member of the dynamin superfamily. However, high-resolution structural information for full-length human Drp1 covering the GTPase domain, the middle domain and the GTPase effector domain (GED) is still lacking. In order to obtain mechanistic insights into the catalytic activity, a nucleotide-free GTPase-GED fusion protein of human Drp1 was expressed, purified and crystallized. Initial X-ray diffraction experiments yielded data to 2.67 Å resolution. The hexagonal-shaped crystals belonged to space group P21212, with unit-cell parameters a = 53.59, b = 151.65, c = 43.53 Å, one molecule per asymmetric unit and a solvent content of 42%. Expression of selenomethionine-labelled protein is currently in progress. Here, the expression, purification, crystallization and X-ray diffraction analysis of the Drp1 GTPase-GED fusion protein are presented, which form a basis for more detailed structural and biophysical analysis. PMID:23027751

  7. TRAIL-CM4 fusion protein shows in vitro antibacterial activity and a stronger antitumor activity than solo TRAIL protein.

    PubMed

    Sang, Ming; Zhang, Jiaxin; Li, Bin; Chen, Yuqing

    2016-06-01

    A TRAIL-CM4 fusion protein in soluble form with tumor selective apoptosis and antibacterial functions was expressed in the Escherichia coli expression system and isolated through dialysis refolding and histidine-tag Nickel-affinity purification. Fresh Jurkat cells were treated with the TRAIL-CM4 fusion protein. Trypan blue staining and MTT analyses showed that, similar to a TRAIL positive control, Jurkat cell proliferation was significantly inhibited. Flow cytometry analyses using Annexin V-fluorescein revealed that Jurkat cells treated with the TRAIL-CM4 fusion protein exhibited increased apoptosis. Laser confocal microscopy showed that APB-CM4 and the fusion protein TRAIL-CM4 can bind to Jurkat cell membranes and initiate their destruction. ABP-CM4 enhances the antitumor activity of TRAIL by targeting and damaging the tumor cell membrane. In antibacterial experiments, agar well diffusion and bacterial growth inhibition curve assays revealed concentration-dependent TRAIL-CM4 antibacterial activity against Escherichia coli K12D31. The expressed TRAIL-CM4 fusion protein exhibited enhanced antitumor and antibacterial activities. Fusion protein expression allowed the two different proteins to function in combination. PMID:26926590

  8. HIGH-PERFORMANCE AFFINITY CHROMATOGRAPHY AND THE ANALYSIS OF DRUG INTERACTIONS WITH MODIFIED PROTEINS: BINDING OF GLICLAZIDE WITH GLYCATED HUMAN SERUM ALBUMIN

    PubMed Central

    Matsuda, Ryan; Anguizola, Jeanethe; Joseph, K.S.; Hage, David S.

    2011-01-01

    This study used high-performance affinity chromatography (HPAC) to examine the binding of gliclazide (i.e., a sulfonylurea drug used to treat diabetes) with the protein human serum albumin (HSA) at various stages of modification due to glycation. Frontal analysis conducted with small HPAC columns was first used to estimate the number of binding sites and association equilibrium constants (Ka) for gliclazide with normal HSA and glycated HSA. Both normal and glycated HSA interacted with gliclazide according to a two-site model, with a class of high affinity sites (average Ka, 7.1-10 × 104 M−1) and a group of lower affinity sites (average Ka, 5.7-8.9 × 103 M−1) at pH 7.4 and 37°C. Competition experiments indicated that Sudlow sites I and II of HSA were both involved in these interactions, with the Ka values for gliclazide at these sites being 1.9 × 104 M−1 and 6.0 × 104 M−1, respectively, for normal HSA. Two samples of glycated HSA had similar affinities to normal HSA for gliclazide at Sudlow site I, but one sample had a 1.9-fold increase in affinity at this site. All three glycated HSA samples differed from normal HSA in their affinity for gliclazide at Sudlow site II. This work illustrated how HPAC can be used to examine both the overall binding of a drug with normal or modified proteins and the site-specific changes that can occur in these interactions as a result of protein modification. PMID:21922305

  9. Roles of the Putative Integrin-Binding Motif of the Human Metapneumovirus Fusion (F) Protein in Cell-Cell Fusion, Viral Infectivity, and Pathogenesis

    PubMed Central

    Wei, Yongwei; Zhang, Yu; Cai, Hui; Mirza, Anne M.; Iorio, Ronald M.; Peeples, Mark E.; Niewiesk, Stefan

    2014-01-01

    ABSTRACT Human metapneumovirus (hMPV) is a relatively recently identified paramyxovirus that causes acute upper and lower respiratory tract infection. Entry of hMPV is unusual among the paramyxoviruses, in that fusion is accomplished by the fusion (F) protein without the attachment glycoprotein (G protein). It has been suggested that hMPV F protein utilizes integrin αvβ1 as a cellular receptor. Consistent with this, the F proteins of all known hMPV strains possess an integrin-binding motif (329RGD331). The role of this motif in viral entry, infectivity, and pathogenesis is poorly understood. Here, we show that α5β1 and αv integrins are essential for cell-cell fusion and hMPV infection. Mutational analysis found that residues R329 and G330 in the 329RGD331 motif are essential for cell-cell fusion, whereas mutations at D331 did not significantly impact fusion activity. Furthermore, fusion-defective RGD mutations were either lethal to the virus or resulted in recombinant hMPVs that had defects in viral replication in cell culture. In cotton rats, recombinant hMPV with the R329K mutation in the F protein (rhMPV-R329K) and rhMPV-D331A exhibited significant defects in viral replication in nasal turbinates and lungs. Importantly, inoculation of cotton rats with these mutants triggered a high level of neutralizing antibodies and protected against hMPV challenge. Taken together, our data indicate that (i) α5β1 and αv integrins are essential for cell-cell fusion and viral replication, (ii) the first two residues in the RGD motif are essential for fusion activity, and (iii) inhibition of the interaction of the integrin-RGD motif may serve as a new target to rationally attenuate hMPV for the development of live attenuated vaccines. IMPORTANCE Human metapneumovirus (hMPV) is one of the major causative agents of acute respiratory disease in humans. Currently, there is no vaccine or antiviral drug for hMPV. hMPV enters host cells via a unique mechanism, in that viral

  10. Eradication of Human Hepatic and Pulmonary Melanoma Metastases in SCID Mice by Antibody--Interleukin 2 Fusion Proteins

    NASA Astrophysics Data System (ADS)

    Becker, Jurgen C.; Pancook, James D.; Gillies, Stephen D.; Mendelsohn, John; Reisfeld, Ralph A.

    1996-04-01

    Antibody--cytokine fusion proteins combine the unique targeting ability of antibodies with the multifunctional activity of cytokines. Here, we demonstrate the therapeutic efficacy of such constructs for the treatment of hepatic and pulmonary metastases of different melanoma cell lines. Two antibody--interleukin 2 (IL-2) fusion proteins, ch225-IL2 and ch14.18-IL2, constructed by fusion of a synthetic sequence coding for human IL-2 to the carboxyl end of the Cγ 1 gene of the corresponding antibodies, were tested for their therapeutic efficacy against xenografted human melanoma in vivo. Tumorspecific fusion proteins completely inhibited the growth of hepatic and pulmonary metastases in C.B-17 scid/scid mice previously reconstituted with human lymphokine-activated killer cells, whereas treatment with combinations of the corresponding antibodies plus recombinant IL-2 only reduced the tumor load. Even when treatment with fusion proteins was delayed up to 8 days after inoculation of tumor cells, it still resulted in complete eradication of micrometastases that were established at that time point. Selection of tumor cell lines expressing or lacking the targeted antigen of the administered fusion protein proved the specificity of the observed antitumor effect. Biodistribution analysis demonstrated that the tumorspecific fusion protein accumulated not only in subcutaneous tumors but also in lungs and livers affected with micrometastases. Survival times of animals treated with the fusion protein were more than doubled as compared to those treated with the combination of the corresponding antibody plus IL-2. Our data demonstrate that an immunotherapeutic approach using cytokines targeted by antibodies to tumor sites has potent effects against disseminated human melanoma.

  11. Time Resolved EPR Study on the Photoinduced Long-Range Charge-Separated State in Protein: Electron Tunneling Mediated by Arginine Residue in Human Serum Albumin.

    PubMed

    Fuki, Masaaki; Murai, Hisao; Tachikawa, Takashi; Kobori, Yasuhiro

    2016-05-19

    To elucidate how local molecular conformations play a role on electronic couplings for the long-range photoinduced charge-separated (CS) states in protein systems, we have analyzed time-resolved electron paramagnetic resonance (TREPR) spectra by polarized laser irradiations of 9,10-anthraquinone-1-sulfonate (AQ1S(-)) bound to human serum albumin (HSA). Analyses of the magnetophotoselection effects on the EPR spectra and a docking simulation clarified the molecular geometry and the electronic coupling of the long-range CS states of AQ1S(•2-)-tryptophan214 radical cation (W214(•+)) separated by 1.2 nm. The ligand of AQ1S(-) has been demonstrated to be bound to the drug site I in HSA. Molecular conformations of the binding region were estimated by the docking simulations, indicating that an arginine218 (R218(+)) residue bound to AQ1S(•2-) mediates the long-range electron-transfer. The energetics of triad states of AQ1S(•2-)-R218(+)-W214(•+) and AQ1S(-)-R218(•)-W214(•+) have been computed on the basis of the density functional molecular orbital calculations, providing the clear evidence for the long-range electronic couplings of the CS states in terms of the superexchange tunneling model through the arginine residue. PMID:27116363

  12. Penetrable silica microspheres for immobilization of bovine serum albumin and their application to the study of the interaction between imatinib mesylate and protein by frontal affinity chromatography.

    PubMed

    Ma, Liyun; Li, Jing; Zhao, Juan; Liao, Han; Xu, Li; Shi, Zhi-guo

    2016-01-01

    In the current study, novel featured silica, named penetrable silica, simultaneously containing macropores and mesopores, was immobilized with bovine serum albumin (BSA) via Schiff base method. The obtained BSA-SiO2 was employed as the high-performance liquid chromatographic (HPLC) stationary phase. Firstly, D- and L-tryptophan were used as probes to investigate the chiral separation ability of the BSA-SiO2 stationary phase. An excellent enantioseparation factor was obtained up to 4.3 with acceptable stability within at least 1 month. Next, the BSA-SiO2 stationary phase was applied to study the interaction between imatinib mesylate (IM) and BSA by frontal affinity chromatography. A single type of binding site was found for IM with the immobilized BSA, and the hydrogen-bonding and van der Waals interactions were expected to be contributing interactions based on the thermodynamic studies, and this was a spontaneous process. Compared to the traditional silica for HPLC stationary phase, the proposed penetrable silica microsphere possessed a larger capacity to bond more BSA, minimizing column overloading effects and enhancing enantioseparation ability. In addition, the lower running column back pressure and fast mass transfer were meaningful for the column stability and lifetime. It was a good substrate to immobilize biomolecules for fast chiral resolution and screening drug-protein interactions. PMID:26573171

  13. ANALYSIS OF DRUG INTERACTIONS WITH MODIFIED PROTEINS BY HIGH-PERFORMANCE AFFINITY CHROMATOGRAPHY: BINDING OF GLIBENCLAMIDE TO NORMAL AND GLYCATED HUMAN SERUM ALBUMIN

    PubMed Central

    Matsuda, Ryan; Anguizola, Jeanethe; Joseph, K.S.; Hage, David S.

    2012-01-01

    High-performance affinity chromatography (HPAC) was used to examine the changes in binding that occur for the sulfonylurea drug glibenclamide with human serum albumin (HSA) at various stages of glycation for HSA. Frontal analysis on columns containing normal HSA or glycated HSA indicated glibenclamide was interacting through both high affinity sites (association equilibrium constant, Ka, 1.4–1.9 × 106 M−1 at pH 7.4 and 37°C) and lower affinity sites (Ka, 4.4–7.2 × 104 M−1). Competition studies were used to examine the effect of glycation at specific binding sites of HSA. An increase in affinity of 1.7- to 1.9-fold was seen at Sudlow site I with moderate to high levels of glycation. An even larger increase of 4.3- to 6.0-fold in affinity was noted at Sudlow site II for all of the tested samples of glycated HSA. A slight decrease in affinity may have occurred at the digitoxin site, but this change was not significant for any individual glycated HSA sample. These results illustrate how HPAC can be used as tool for examining the interactions of relatively non-polar drugs like glibenclamide with modified proteins and should lead to a more complete understanding of how glycation can alter the binding of drugs in blood. PMID:23092871

  14. Trans-splicing as a novel method to rapidly produce antibody fusion proteins

    SciTech Connect

    Iwasaki, Ryohei; Kiuchi, Hiroki; Ihara, Masaki; Mori, Toshihiro; Kawakami, Masayuki; Ueda, Hiroshi

    2009-07-03

    To cultivate the use of trans-splicing as a novel means to rapidly express various antibody fusion proteins, we tried to express antibody-reporter enzyme fusions in a COS-1 co-transfection model. When a vector designed to induce trans-splicing with IgH pre-mRNA was co-transfected with a vector encoding the mouse IgM locus, the expression of V{sub H}-secreted human placental alkaline phosphatase (SEAP) as well as Fab-SEAP were successfully expressed both in mRNA and protein levels. Especially, the vectors encoding complementary sequence to S{mu} as a binding domain was accurate and efficient, producing trans-spliced mRNA of up to 2% of cis-spliced one. Since S{mu} sequence should exist in every IgH pre-mRNA, our finding will lead to the rapid production and analysis of various antibody-enzyme fusions suitable for enzyme-linked immunosorbent assay (ELISA) or antibody-dependent enzyme prodrug therapy (ADEPT).

  15. Equatorial Segment Protein (ESP) Is a Human Alloantigen Involved in Sperm-Egg Binding and Fusion

    PubMed Central

    Wolkowicz, M. J.; Digilio, L.; Klotz, K.; Shetty, J.; Flickinger, C. J.; Herr, J. C.

    2010-01-01

    The equatorial segment of the sperm head is known to play a role in fertilization; however, the specific sperm molecules contributing to the integrity of the equatorial segment and in binding and fusion at the oolemma remain incomplete. Moreover, identification of molecular mediators of fertilization that are also immunogenic in humans is predicted to advance both the diagnosis and treatment of immune infertility. We previously reported the cloning of Equatorial Segment Protein (ESP), a protein localized to the equatorial segment of ejaculated human sperm. ESP is a biomarker for a subcompartment of the acrosomal matrix that can be traced through all stages of acrosome biogenesis (Wolkowicz et al, 2003). In the present study, ESP immunoreacted on Western blots with 4 (27%) of 15 antisperm antibody (ASA)–positive serum samples from infertile male patients and 2 (40%) of 5 ASA-positive female sera. Immunofluorescent studies revealed ESP in the equatorial segment of 89% of acrosome-reacted sperm. ESP persisted as a defined equatorial segment band on 100% of sperm tightly bound to the oolemma of hamster eggs. Antisera to recombinant human ESP inhibited both oolemmal binding and fusion of human sperm in the hamster egg penetration assay. The results indicate that ESP is a human alloantigen involved in sperm-egg binding and fusion. Defined recombinant sperm immunogens, such as ESP, may offer opportunities for differential diagnosis of immune infertility. PMID:17978344

  16. A Compact, Multifunctional Fusion Module Directs Cholesterol-Dependent Homomultimerization and Syncytiogenic Efficiency of Reovirus p10 FAST Proteins

    PubMed Central

    Key, Tim; Duncan, Roy

    2014-01-01

    The homologous p10 fusion-associated small transmembrane (FAST) proteins of the avian (ARV) and Nelson Bay (NBV) reoviruses are the smallest known viral membrane fusion proteins, and are virulence determinants of the fusogenic reoviruses. The small size of FAST proteins is incompatible with the paradigmatic membrane fusion pathway proposed for enveloped viral fusion proteins. Understanding how these diminutive viral fusogens mediate the complex process of membrane fusion is therefore of considerable interest, from both the pathogenesis and mechanism-of-action perspectives. Using chimeric ARV/NBV p10 constructs, the 36–40-residue ectodomain was identified as the major determinant of the differing fusion efficiencies of these homologous p10 proteins. Extensive mutagenic analysis determined the ectodomain comprises two distinct, essential functional motifs. Syncytiogenesis assays, thiol-specific surface biotinylation, and liposome lipid mixing assays identified an ∼25-residue, N-terminal motif that dictates formation of a cystine loop fusion peptide in both ARV and NBV p10. Surface immunofluorescence staining, FRET analysis and cholesterol depletion/repletion studies determined the cystine loop motif is connected through a two-residue linker to a 13-residue membrane-proximal ectodomain region (MPER). The MPER constitutes a second, independent motif governing reversible, cholesterol-dependent assembly of p10 multimers in the plasma membrane. Results further indicate that: (1) ARV and NBV homomultimers segregate to distinct, cholesterol-dependent microdomains in the plasma membrane; (2) p10 homomultimerization and cholesterol-dependent microdomain localization are co-dependent; and (3) the four juxtamembrane MPER residues present in the multimerization motif dictate species-specific microdomain association and homomultimerization. The p10 ectodomain therefore constitutes a remarkably compact, multifunctional fusion module that directs syncytiogenic efficiency and

  17. Chemotropism and Cell Fusion in Neurospora crassa Relies on the Formation of Distinct Protein Complexes by HAM-5 and a Novel Protein HAM-14.

    PubMed

    Jonkers, Wilfried; Fischer, Monika S; Do, Hung P; Starr, Trevor L; Glass, N Louise

    2016-05-01

    In filamentous fungi, communication is essential for the formation of an interconnected, multinucleate, syncytial network, which is constructed via hyphal fusion or fusion of germinated asexual spores (germlings). Anastomosis in filamentous fungi is comparable to other somatic cell fusion events resulting in syncytia, including myoblast fusion during muscle differentiation, macrophage fusion, and fusion of trophoblasts during placental development. In Neurospora crassa, fusion of genetically identical germlings is a highly dynamic and regulated process that requires components of a MAP kinase signal transduction pathway. The kinase pathway components (NRC-1, MEK-2 and MAK-2) and the scaffold protein HAM-5 are recruited to hyphae and germling tips undergoing chemotropic interactions. The MAK-2/HAM-5 protein complex shows dynamic oscillation to hyphae/germling tips during chemotropic interactions, and which is out-of-phase to the dynamic localization of SOFT, which is a scaffold protein for components of the cell wall integrity MAP kinase pathway. In this study, we functionally characterize HAM-5 by generating ham-5 truncation constructs and show that the N-terminal half of HAM-5 was essential for function. This region is required for MAK-2 and MEK-2 interaction and for correct cellular localization of HAM-5 to "fusion puncta." The localization of HAM-5 to puncta was not perturbed in 21 different fusion mutants, nor did these puncta colocalize with components of the secretory pathway. We also identified HAM-14 as a novel member of the HAM-5/MAK-2 pathway by mining MAK-2 phosphoproteomics data. HAM-14 was essential for germling fusion, but not for hyphal fusion. Colocalization and coimmunoprecipitation data indicate that HAM-14 interacts with MAK-2 and MEK-2 and may be involved in recruiting MAK-2 (and MEK-2) to complexes containing HAM-5. PMID:27029735

  18. Transposon assisted gene insertion technology (TAGIT): a tool for generating fluorescent fusion proteins.

    PubMed

    Gregory, James A; Becker, Eric C; Jung, James; Tuwatananurak, Ida; Pogliano, Kit

    2010-01-01

    We constructed a transposon (transposon assisted gene insertion technology, or TAGIT) that allows the random insertion of gfp (or other genes) into chromosomal loci without disrupting operon structure or regulation. TAGIT is a modified Tn5 transposon that uses Kan(R) to select for insertions on the chromosome or plasmid, beta-galactosidase to identify in-frame gene fusions, and Cre recombinase to excise the kan and lacZ genes in vivo. The resulting gfp insertions maintain target gene reading frame (to the 5' and 3' of gfp) and are integrated at the native chromosomal locus, thereby maintaining native expression signals. Libraries can be screened to identify GFP insertions that maintain target protein function at native expression levels, allowing more trustworthy localization studies. We here use TAGIT to generate a library of GFP insertions in the Escherichia coli lactose repressor (LacI). We identified fully functional GFP insertions and partially functional insertions that bind DNA but fail to repress the lacZ operon. Several of these latter GFP insertions localize to lacO arrays integrated in the E. coli chromosome without producing the elongated cells frequently observed when functional LacI-GFP fusions are used in chromosome tagging experiments. TAGIT thereby faciliates the isolation of fully functional insertions of fluorescent proteins into target proteins expressed from the native chromosomal locus as well as potentially useful partially functional proteins. PMID:20090956

  19. Adhesion and fusion efficiencies of human immunodeficiency virus type 1 (HIV-1) surface proteins

    NASA Astrophysics Data System (ADS)

    Dobrowsky, Terrence M.; Rabi, S. Alireza; Nedellec, Rebecca; Daniels, Brian R.; Mullins, James I.; Mosier, Donald E.; Siliciano, Robert F.; Wirtz, Denis

    2013-10-01

    In about half of patients infected with HIV-1 subtype B, viral populations shift from utilizing the transmembrane protein CCR5 to CXCR4, as well as or instead of CCR5, during late stage progression of the disease. How the relative adhesion efficiency and fusion competency of the viral Env proteins relate to infection during this transition is not well understood. Using a virus-cell fusion assay and live-cell single-molecule force spectroscopy, we compare the entry competency of viral clones to tensile strengths of the individual Env-receptor bonds of Env proteins obtained from a HIV-1 infected patient prior to and during coreceptor switching. The results suggest that the genetic determinants of viral entry were predominantly enriched in the C3, HR1 and CD regions rather than V3. Env proteins can better mediate entry into cells after coreceptor switch; this effective entry capacity does not correlate with the bond strengths between viral Env and cellular receptors.

  20. Protein Sub-Nuclear Localization Based on Effective Fusion Representations and Dimension Reduction Algorithm LDA

    PubMed Central

    Wang, Shunfang; Liu, Shuhui

    2015-01-01

    An effective representation of a protein sequence plays a crucial role in protein sub-nuclear localization. The existing representations, such as dipeptide composition (DipC), pseudo-amino acid composition (PseAAC) and position specific scoring matrix (PSSM), are insufficient to represent protein sequence due to their single perspectives. Thus, this paper proposes two fusion feature representations of DipPSSM and PseAAPSSM to integrate PSSM with DipC and PseAAC, respectively. When constructing each fusion representation, we introduce the balance factors to value the importance of its components. The optimal values of the balance factors are sought by genetic algorithm. Due to the high dimensionality of the proposed representations, linear discriminant analysis (LDA) is used to find its important low dimensional structure, which is essential for classification and location prediction. The numerical experiments on two public datasets with KNN classifier and cross-validation tests showed that in terms of the common indexes of sensitivity, specificity, accuracy and MCC, the proposed fusing representations outperform the traditional representations in protein sub-nuclear localization, and the representation treated by LDA outperforms the untreated one. PMID:26703574

  1. Prolonged activity of factor IX as a monomeric Fc fusion protein.

    PubMed

    Peters, Robert T; Low, Susan C; Kamphaus, George D; Dumont, Jennifer A; Amari, John V; Lu, Qi; Zarbis-Papastoitsis, Greg; Reidy, Thomas J; Merricks, Elizabeth P; Nichols, Timothy C; Bitonti, Alan J

    2010-03-11

    Treatment of hemophilia B requires frequent infusions of factor IX (FIX) to prophylax against bleeding episodes. Hemophilia B management would benefit from a FIX protein with an extended half-life. A recombinant fusion protein (rFIXFc) containing a single FIX molecule attached to the Fc region of immunoglobulin G was administered intravenously and found to have an extended half-life, compared with recombinant FIX (rFIX) in normal mice, rats, monkeys, and FIX-deficient mice and dogs. Recombinant FIXFc protein concentration was determined in all species, and rFIXFc activity was measured in FIX-deficient animals. The half-life of rFIXFc was approximately 3- to 4-fold longer than that of rFIX in all species. In contrast, in mice in which the neonatal Fc receptor (FcRn) was deleted, the half-life of rFIXFc was similar to rFIX, confirming the increased circulatory time was due to protection of the rFIXFc via the Fc/FcRn interaction. Whole blood clotting time in FIX-deficient mice was corrected through 144 hours for rFIXFc, compared with 72 hours for rFIX; similar results were observed in FIX-deficient dogs. Taken together, these studies show the enhanced pharmacodynamic and pharmacokinetic properties of the rFIXFc fusion protein and provide the basis for evaluating rFIXFc in patients with hemophilia B. PMID:20056791

  2. Structure and expression of the Drosophila ubiquitin-80-amino-acid fusion-protein gene.

    PubMed Central

    Barrio, R; del Arco, A; Cabrera, H L; Arribas, C

    1994-01-01

    In the fruitfly Drosophila, as in all eukaryotes examined so far, some ubiquitin-coding sequences appear fused to unrelated open reading frames. Two of these fusion genes have been previously described (the homologues of UBI1-UBI2 and UBI4 in yeast), and we report here the organization and expression of a third one, the DUb80 gene (the homologue of UBI3 in yeast). This gene encodes a ubiquitin monomer fused to an 80-amino-acid extension which is homologous with the ribosomal protein encoded by the UB13 gene. The 5' regulatory region of DUb80 shares common features with another ubiquitin fusion gene, DUb52, and with the ribosomal protein genes of Drosophila, Xenopus and mouse. We also find helix-loop-helix protein-binding sequences (E-boxes). The DUb80 gene is transcribed to a 0.9 kb mRNA which is particularly abundant under conditions of high protein synthesis, such as in ovaries and exponentially growing cells. Images Figure 3 Figure 4 PMID:8068011

  3. Resolution of Disulfide Heterogeneity in Nogo Receptor 1 Fusion Proteins by Molecular Engineering

    SciTech Connect

    P Weinreb; D Wen; F Qian; C Wildes; E Garber; L Walus; M Jung; J Wang; J Relton; et al.

    2011-12-31

    NgRI (Nogo-66 receptor) is part of a signalling complex that inhibits axon regeneration in the central nervous system. Truncated soluble versions of NgRI have been used successfully to promote axon regeneration in animal models of spinal-cord injury, raising interest in this protein as a potential therapeutic target. The LRR (leucine-rich repeat) regions in NgRI are flanked by N- and C-terminal disulfide-containing 'cap' domains (LRRNT and LRRCT respectively). In the present work we show that, although functionally active, the NgRI(310)-Fc fusion protein contains mislinked and heterogeneous disulfide patterns in the LRRCT domain, and we report the generation of a series of variant molecules specifically designed to prevent this heterogeneity. Using these variants we explored the effects of modifying the NgRI truncation site or the spacing between the NgRI and Fc domains, or replacing cysteines within the NgRI or IgG hinge regions. One variant, which incorporates replacements of Cys{sup 266} and Cys{sup 309} with alanine residues, completely eliminated disulfide scrambling while maintaining functional in vitro and in vivo efficacy. This modified NgRI-Fc molecule represents a significantly improved candidate for further pharmaceutical development, and may serve as a useful model for the optimization of other IgG fusion proteins made from LRR proteins.

  4. DELIVERY OF siRNA INTO BREAST CANCER CELLS VIA PHAGE FUSION PROTEIN-TARGETED LIPOSOMES

    PubMed Central

    Bedi, Deepa; Musacchio, Tiziana; Fagbohun, Olusegun A.; Gillespie, James W.; Deinnocentes, Patricia; Bird, R. Curtis; Bookbinder, Lonnie; Torchilin, Vladimir P.; Petrenko, Valery A.

    2011-01-01

    Efficacy of siRNAs as potential anticancer therapeutics can be increased by their targeted delivery into cancer cells via tumor-specific ligands. Phage display offers an unique approach to identify highly specific and selective ligands that can deliver nanocarriers to the site of disease. In this study, we proved a novel approach for intracellular delivery of siRNAs into breast cancer cells through their encapsulation into liposomes targeted to the tumor cells with preselected intact phage proteins. The targeted siRNA liposomes were obtained by a fusion of two parental liposomes containing spontaneously inserted siRNA and fusion phage proteins. The presence of pVIII coat protein fused to a MCF-7 cell-targeting peptide DMPGTVLP in the liposomes was confirmed by Western blotting. The novel phage-targeted siRNA-nanopharmaceuticals demonstrate significant down-regulation of PRDM14 gene expression and PRDM14 protein synthesis in the target MCF- 7 cells. This approach offers the potential for development of new anticancer siRNA-based targeted nanomedicines. PMID:21050894

  5. Antigen Binding and Site-Directed Labeling of Biosilica-Immobilized Fusion Proteins Expressed in Diatoms.

    PubMed

    Ford, Nicole R; Hecht, Karen A; Hu, DeHong; Orr, Galya; Xiong, Yijia; Squier, Thomas C; Rorrer, Gregory L; Roesijadi, Guritno

    2016-03-18

    The diatom Thalassiosira pseudonana was genetically modified to express biosilica-targeted fusion proteins comprising either enhanced green fluorescent protein (EGFP) or single chain antibodies engineered with a tetracysteine tagging sequence. Of interest were the site-specific binding of (1) the fluorescent biarsenical probe AsCy3 and AsCy3e to the tetracysteine tagged fusion proteins and (2) high and low molecular mass antigens, the Bacillus anthracis surface layer protein EA1 or small molecule explosive trinitrotoluene (TNT), to biosilica-immobilized single chain antibodies. Analysis of biarsenical probe binding using fluorescence and structured illumination microscopy indicated differential colocalization with EGFP in nascent and mature biosilica, supporting the use of either EGFP or bound AsCy3 and AsCy3e in studying biosilica maturation. Large increases in the lifetime of a fluorescent analogue of TNT upon binding single chain antibodies provided a robust signal capable of discriminating binding to immobilized antibodies in the transformed frustule from nonspecific binding to the biosilica matrix. In conclusion, our results demonstrate an ability to engineer diatoms to create antibody-functionalized mesoporous silica able to selectively bind chemical and biological agents for the development of sensing platforms. PMID:26746113

  6. Putative sperm fusion protein IZUMO and the role of N-glycosylation

    SciTech Connect

    Inoue, Naokazu; Ikawa, Masahito; Okabe, Masaru

    2008-12-19

    IZUMO is the mouse sperm protein proven to be essential for fusion with eggs. It contains one immunoglobulin-like domain with a conserved glycosylation site within. In the present paper, we produced transgenic mouse lines expressing unglycosylated IZUMO (N204Q-IZUMO) in Izumo1 -/- background. The expression of N204Q-IZUMO rescued the infertile phenotype of IZUMO disrupted mice, indicating glycosylation is not essential for fusion-facilitating activity of IZUMO. The N204Q-IZUMO was produced in testis in comparable amounts to wild-type IZUMO, but the amount of N204Q-IZUMO on sperm was significantly decreased by the time sperm reached the cauda epididymis. These data suggest that glycosylation is not essential for the function of IZUMO, but has a role in protecting it from fragmentation in cauda epididymis.

  7. Investigation of bovine serum albumin glycation by THz spectroscopy

    NASA Astrophysics Data System (ADS)

    Cherkasova, Olga P.; Nazarov, Maxim M.; Shkurinov, Alexander P.

    2016-04-01

    Protein glycation is accelerated under hyperglycemic conditions resulting to loss in the structure and biological functions of proteins. The transmission THz spectroscopy has been used for measuring of bovine serum albumin glycation dynamics. It was found that amplitude of albumin THz absorption depends on type of sugars and incubation time.

  8. Probing the paramyxovirus fusion (F) protein-refolding event from pre- to postfusion by oxidative footprinting

    PubMed Central

    Poor, Taylor A.; Jones, Lisa M.; Sood, Amika; Leser, George P.; Plasencia, Manolo D.; Rempel, Don L.; Jardetzky, Theodore S.; Woods, Robert J.; Gross, Michael L.; Lamb, Robert A.

    2014-01-01

    To infect a cell, the Paramyxoviridae family of enveloped viruses relies on the coordinated action of a receptor-binding protein (variably HN, H, or G) and a more conserved metastable fusion protein (F) to effect membrane fusion and allow genomic transfer. Upon receptor binding, HN (H or G) triggers F to undergo an extensive refolding event to form a stable postfusion state. Little is known about the intermediate states of the F refolding process. Here, a soluble form of parainfluenza virus 5 F was triggered to refold using temperature and was footprinted along the refolding pathway using fast photochemical oxidation of proteins (FPOP). Localization of the oxidative label to solvent-exposed side chains was determined by high-resolution MS/MS. Globally, metastable prefusion F is oxidized more extensively than postfusion F, indicating that the prefusion state is more exposed to solvent and is more flexible. Among the first peptides to be oxidatively labeled after temperature-induced triggering is the hydrophobic fusion peptide. A comparison of peptide oxidation levels with the values of solvent-accessible surface area calculated from molecular dynamics simulations of available structural data reveals regions of the F protein that lie at the heart of its prefusion metastability. The strong correlation between the regions of F that experience greater-than-expected oxidative labeling and epitopes for neutralizing antibodies suggests that FPOP has a role in guiding the development of targeted therapeutics. Analysis of the residue levels of labeled F intermediates provides detailed insights into the mechanics of this critical refolding event. PMID:24927585

  9. Effect of protein aggregates on characterization of FcRn binding of Fc-fusion therapeutics.

    PubMed

    Bajardi-Taccioli, Adriana; Blum, Andrew; Xu, Chongfeng; Sosic, Zoran; Bergelson, Svetlana; Feschenko, Marina

    2015-10-01

    Recycling of antibodies and Fc containing therapeutic proteins by the neonatal Fc receptor (FcRn) is known to prolong their persistence in the bloodstream. Fusion of Fc fragment of IgG1 to other proteins is one of the strategies to improve their pharmacokinetic properties. Accurate measurement of Fc-FcRn binding provides information about the strength of this interaction, which in most cases correlates with serum half-life of the protein. It can also offer insight into functional integrity of Fc region. We investigated FcRn binding activity of a large set of Fc-fusion samples after thermal stress by the method based on AlphaScreen technology. An unexpected significant increase in FcR binding was found to correlate with formation of aggregates in these samples. Monomer purified from a thermally-stressed sample had normal FcRn binding, confirming that its Fc portion was intact. Experiments with aggregates spiked into a sample with low initial aggregation level, demonstrated strong correlation between the level of aggregates and FcRn binding. This correlation varied significantly in different methods. By introducing modifications to the assay format we were able to minimize the effects of aggregated species on FcRn binding, which should prevent masking functional changes of Fc-fusion protein. Biolayer interferometry (BLI) was used as an alternative method to measure FcRn binding. Both optimized AlphaScreen- and BLI-based assays were sensitive to structural changes in Fc portion of the molecule, such as oxidation of methionines 252 and 428, and therefore suitable for characterization of FcRn binding. PMID:26254986

  10. Oscillatory recruitment of signaling proteins to cell tips promotes coordinated behavior during cell fusion.

    PubMed

    Fleissner, André; Leeder, Abigail C; Roca, M Gabriela; Read, Nick D; Glass, N Louise

    2009-11-17

    Cell-cell communication is essential for coordinating physiological responses in multicellular organisms and is required for various developmental processes, including cell migration, differentiation, and fusion. To facilitate communication, functional differences are usually required between interacting cells, which can be established either genetically or developmentally. However, genetically identical cells in the same developmental state are also capable of communicating, but must avoid self-stimulation. We hypothesized that such cells must alternate their physiological state between signal sending and receiving to allow recognition and behavioral changes. To test this hypothesis, we studied cell communication in the filamentous fungus Neurospora crassa, a simple and experimentally amenable model system. In N. crassa, germinating asexual spores (germlings) of identical genotype chemotropically sense others in close proximity, show attraction-mediated directed growth, and ultimately undergo cell fusion. Here, we report that two proteins required for cell fusion, a MAP kinase (MAK-2) and a protein of unknown molecular function (SO), exhibit rapid oscillatory recruitment to the plasma membranes of interacting germlings undergoing chemotropic interactions via directed growth. Using an inhibitable MAK-2 variant, we show that MAK-2 kinase activity is required both for chemotropic interactions and for oscillation of MAK-2 and SO to opposing cell tips. Thus, N. crassa germlings undergoing chemotropic interactions rapidly alternate between two different physiological states, associated with signal delivery and response. Such spatiotemporal coordination of signaling allows genetically identical and developmentally equivalent cells to avoid self-stimulation and to coordinate their behavior to achieve the beneficial physiological outcome of cell fusion. PMID:19884508

  11. Contraceptive efficacy of recombinant fusion protein comprising zona pellucida glycoprotein-3 fragment and gonadotropin releasing hormone.

    PubMed

    Arukha, Ananta Prasad; Minhas, Vidisha; Shrestha, Abhinav; Gupta, Satish Kumar

    2016-04-01

    Contraceptive vaccines have been used for the management of wildlife population. In the present study, we have examined the contraceptive potential of Escherichia coli-expressed recombinant fusion protein comprising of 'promiscuous' T cell epitope of tetanus toxoid [TT; amino acid (aa) residues 830-844] followed by dilysine linker (KK), dog ZP3 fragment (aa residues 307-346), triglycine spacer (GGG), T cell epitope of bovine RNase (bRNase; aa residues 94-104), GnRH, T cell epitope of circumsporozoite protein of Plasmodium falciparum (CSP; aa residues 362-383), and GnRH. SDS-PAGE analysis of the purified refolded protein revealed a dominant ∼12 kDa band, which in Western blot reacted with mouse polyclonal antibodies against dog ZP3 fragment and mouse monoclonal antibodies against GnRH. Immunization of female FvB/J mice following two booster schedule with the above recombinant protein supplemented with alum led to high antibody titres against the immunogen as well as ZP3 and GnRH as determined by ELISA. The immune sera reacted with zona pellucida of mouse oocyte and also inhibited in-vitro fertilization. The qRT-PCR studies showed decrease in the ovarian GnRH receptor in mice immunized with the recombinant fusion protein. Mating studies revealed high contraceptive efficacy of the recombinant protein as in two independent experiments, 90% of the immunized female mice failed to conceive. Following one booster immunization schedule, 50% of the immunized female mice failed to conceive. However, in adjuvanted controls, all the female mice became pregnant. To conclude, the recombinant protein described herein has a good potential to be developed as candidate contraceptive vaccine. PMID:26859695

  12. Influence of silk-silica fusion protein design on silica condensation in vitro and cellular calcification

    PubMed Central

    Plowright, Robyn; Dinjaski, Nina; Zhou, Shun; Belton, David J.; Kaplan, David L.; Perry, Carole C.

    2016-01-01

    Biomaterial design via genetic engineering can be utilized for the rational functionalization of proteins to promote biomaterial integration and tissue regeneration. Spider silk has been extensively studied for its biocompatibility, biodegradability and extraordinary material properties. As a protein-based biomaterial, recombinant DNA derived derivatives of spider silks have been modified with biomineralization domains which lead to silica deposition and potentially accelerated bone regeneration. However, the influence of the location of the R5 (SSKKSGSYSGSKGSKRRIL) silicifying domain fused with the spider silk protein sequence on the biosilicification process remains to be determined. Here we designed two silk-R5 fusion proteins that differed in the location of the R5 peptide, C- vs. N-terminus, where the spider silk domain consisted of a 15mer repeat of a 33 amino acid consensus sequence of the major ampullate dragline Spidroin 1 from Nephila clavipes (SGRGGLGGQG AGAAAAAGGA GQGGYGGLGSQGT). The chemical, physical and silica deposition properties of these recombinant proteins were assessed and compared to a silk 15mer control without the R5 present. The location of the R5 peptide did not have a significant effect on wettability and surface energies, while the C-terminal location of the R5 promoted more controlled silica precipitation, suggesting differences in protein folding and possibly different access to charged amino acids that drive the silicification process. Further, cell compatibility in vitro, as well as the ability to promote human bone marrow derived mesenchymal stem cell (hMSC) differentiation were demonstrated for both variants of the fusion proteins. PMID:26989487

  13. Mitochondrial remodeling following fission inhibition by 15d-PGJ2 involves molecular changes in mitochondrial fusion protein OPA1

    SciTech Connect

    Kar, Rekha; Mishra, Nandita; Singha, Prajjal K.; Venkatachalam, Manjeri A.; Saikumar, Pothana

    2010-09-03

    Research highlights: {yields} Chemical inhibition of fission protein Drp1 leads to mitochondrial fusion. {yields} Increased fusion stimulates molecular changes in mitochondrial fusion protein OPA1. {yields} Proteolysis of larger isoforms, new synthesis and ubiquitination of OPA1 occur. {yields} Loss of mitochondrial tubular rigidity and disorganization of cristae. {yields} Generation of large swollen dysfunctional mitochondria. -- Abstract: We showed earlier that 15 deoxy {Delta}{sup 12,14} prostaglandin J2 (15d-PGJ2) inactivates Drp1 and induces mitochondrial fusion . However, prolonged incubation of cells with 15d-PGJ2 resulted in remodeling of fused mitochondria into large swollen mitochondria with irregular cristae structure. While initial fusion of mitochondria by 15d-PGJ2 required the presence of both outer (Mfn1 and Mfn2) and inner (OPA1) mitochondrial membrane fusion proteins, later mitochondrial changes involved increased degradation of the fusion protein OPA1 and ubiquitination of newly synthesized OPA1 along with decreased expression of Mfn1 and Mfn2, which likely contributed to the loss of tubular rigidity, disorganization of cristae, and formation of large swollen degenerated dysfunctional mitochondria. Similar to inhibition of Drp1 by 15d-PGJ2, decreased expression of fission protein Drp1 by siRNA also resulted in the loss of fusion proteins. Prevention of 15d-PGJ2 induced mitochondrial elongation by thiol antioxidants prevented not only loss of OPA1 isoforms but also its ubiquitination. These findings provide novel insights into unforeseen complexity of molecular events that modulate mitochondrial plasticity.

  14. Bovine Serum Albumin-Catalyzed Deprotonation of [1-13C]-Glycolaldehyde: Protein Reactivity Toward Deprotonation of α–Hydroxy α–Carbonyl Carbon

    PubMed Central

    Go, Maybelle K.; Malabanan, M. Merced; Amyes, Tina L.; Richard, John P.

    2010-01-01

    Bovine serum albumin (BSA) in D2O at 25 °C and pD 7.0 was found to catalyze the deuterium exchange reactions of [1-13C]-glycolaldehyde ([1-13C]-GA) to form [1-13C, 2-2H]-GA and [1-13C, 2,2-di-2H]-GA. The formation of [1-13C, 2-2H]-GA and [1-13C, 2,2-di-2H]-GA in a total yield of 51 ± 3% was observed at early reaction times, and at latter times [1-13C, 2-2H]-GA was observed to undergo BSA-catalyzed conversion to [1-13C, 2,2-di-2H]-GA. The overall second-order rate constant for these deuterium exchange reactions is (kE)P = 0.25 M−1 s−1. By comparison, values of (kE)P = 0.04 M−1 s−1 (Go, M. K., Amyes, T. L., and Richard, J. P. (2009), Biochemistry 48, 5769–5778) and 0.06 M−1 s−1 (Go, M. K., Koudelka, A., Amyes, T. L., and Richard, J. P. (2010), Biochemistry 49, 5377–5389) have been determined, respectively, for the wildtype- and K12G mutant TIM-catalyzed deuterium exchange reactions of [1-13C]-GA to form [1-13C, 2,2-di-2H]-GA. These data show that TIM and BSA exhibit a modest catalytic activity towards deprotonation of α-hydroxy α-carbonyl carbon. It is suggested that this activity is intrinsic to many globular proteins, and that it must be enhanced to demonstrate successful de novo design of protein catalysts of reactions through enamine intermediates. PMID:20687575

  15. Construction and Production of Foxp3-Fc (IgG) DNA Vaccine/Fusion Protein

    PubMed Central

    Mousavi Niri, Neda; Memarnejadian, Arash; Hadjati, Jamshid; Aghasadeghi, Mohammad Reza; Shokri, Mehdi; Pilehvar-soltanahmadi, Yones; Akbarzadeh, Abolfazl; Zarghami, Nosratollah

    2016-01-01

    Background: It seems that the success of vaccination for cancer immunotherapy such as Dendritic Cell (DC) based cancer vaccine is hindered through a powerful network of immune system suppressive elements in which regulatory T cell is the common factor. Foxp3 transcription factor is the most specific marker of regulatory T cells. In different studies, targeting an immune response against regulatory cells expressing Foxp3 and their removal have been assessed. As these previous studies could not efficiently conquer the suppressive effect of regulatory cells by their partial elimination, an attempt was made to search for constructing more effective vaccines against regulatory T cells by which to improve the effect of combined means of immunotherapy in cancer. In this study, a DNA vaccine and its respective protein were constructed in which Foxp3 fused to Fc(IgG) can be efficiently captured and processed by DC via receptor mediated endocytosis and presented to MHCII and I (cross priming). Methods: DNA construct containing fragment C (Fc) portion of IgG fused to Foxp3 was designed. DNA construct was transfected into HEK cells to investigate its expression through fluorescent microscopy and flow cytometry. Its specific expression was also assessed by western blot. For producing recombinant protein, FOXP3-Fc fusion construct was inserted into pET21a vector and consequently, Escherichia coli (E. coli) strain BL21 was selected as host cells. The expression of recombinant fusion protein was assayed by western blot analysis. Afterward, fusion protein was purified by SDS PAGE reverse staining. Results: The expression analysis of DNA construct by flow cytometry and fluorescent microscopy showed that this construct was successfully expressed in eukaryotic cells. Moreover, the Foxp3-Fc expression was confirmed by SDS-PAGE followed by western blot analysis. Additionally, the presence of fusion protein was shown by specific antibody after purification. Conclusion: Due to successful

  16. Evaluation of a Novel Methacrylate-Based Protein A Resin for the Purification of Immunoglobulins and Fc-Fusion Proteins

    PubMed Central

    McCaw, Tyler R; Koepf, Edward K; Conley, Lynn

    2014-01-01

    Protein A affinity chromatography is a central part of most commercial monoclonal antibody and Fc-fusion protein purification processes. In the last couple years an increasing number of new Protein A technologies have emerged. One of these new Protein A technologies consists of a novel, alkaline-tolerant, Protein A ligand coupled to a macroporous polymethacrylate base matrix that has been optimized for immunoglobulin (Ig) G capture. The resin is interesting from a technology perspective because the particle size and pore distribution of the base beads are reported to have been optimized for high IgG binding and fast mass transfer, while the Protein A ligand has been engineered for enhanced alkaline tolerance. This resin was subjected to a number of technical studies including evaluating dynamic and static binding capacities, alkaline stability, Protein A leachate propensity, impurity clearance, and pressure–flow behavior. The results demonstrated similar static binding capacities as those achieved with industry standard agarose Protein A resins, but marginally lower dynamic binding capacities. Removal of impurities from the process stream, particularly host cell proteins, was molecule dependent, but in most instances matched the performance of the agarose resins. This resin was stable in 0.1 M NaOH for at least 100 h with little loss in binding capacity, with Protein A ligand leakage levels comparable to values for the agarose resins. Pressure–flow experiments in lab-scale chromatography columns demonstrated minimal resin compression at typical manufacturing flow rates. Prediction of resin compression in manufacturing scale columns did not suggest any pressure limitations upon scale up. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:1125–1136, 2014 PMID:25045034

  17. RESOLFT Nanoscopy of Fixed Cells Using a Z-Domain Based Fusion Protein for Labelling

    PubMed Central

    Kilisch, Markus; Hell, Stefan W.; Jakobs, Stefan

    2015-01-01

    RESOLFT super-resolution microscopy allows subdiffraction resolution imaging of living cells using low intensities of light. It relies on the light-driven switching of reversible switchable fluorescent proteins (RSFPs). So far, RESOLFT imaging was restricted to living cells, because chemical fixation typically affects the switching characteristics of RSFPs. In this study we created a fusion construct (FLASR) consisting of the RSFP rsEGFP2 and the divalent form of the antibody binding Z domain from protein A. FLASR can be used analogous to secondary antibodies in conventional immunochemistry, facilitating simple and robust sample preparation. We demonstrate RESOLFT super-resolution microscopy on chemically fixed mammalian cells. The approach may be extended to other super-resolution approaches requiring fluorescent proteins in an aqueous environment. PMID:26375606

  18. Direct CRISPR spacer acquisition from RNA by a natural reverse transcriptase-Cas1 fusion protein.

    PubMed

    Silas, Sukrit; Mohr, Georg; Sidote, David J; Markham, Laura M; Sanchez-Amat, Antonio; Bhaya, Devaki; Lambowitz, Alan M; Fire, Andrew Z

    2016-02-26

    CRISPR systems mediate adaptive immunity in diverse prokaryotes. CRISPR-associated Cas1 and Cas2 proteins have been shown to enable adaptation to new threats in type I and II CRISPR systems by the acquisition of short segments of DNA (spacers) from invasive elements. In several type III CRISPR systems, Cas1 is naturally fused to a reverse transcriptase (RT). In the marine bacterium Marinomonas mediterranea (MMB-1), we showed that a RT-Cas1 fusion protein enables the acquisition of RNA spacers in vivo in a RT-dependent manner. In vitro, the MMB-1 RT-Cas1 and Cas2 proteins catalyze the ligation of RNA segments into the CRISPR array, which is followed by reverse transcription. These observations outline a host-mediated mechanism for reverse information flow from RNA to DNA. PMID:26917774

  19. Antitransferrin receptor antibody-RNase fusion protein expressed in the mammary gland of transgenic mice.

    PubMed

    Newton, D L; Pollock, D; DiTullio, P; Echelard, Y; Harvey, M; Wilburn, B; Williams, J; Hoogenboom, H R; Raus, J C; Meade, H M; Rybak, S M

    1999-12-10

    Antibodies fused to human enzymes offer an alternative to specifically targeting tumors with antibodies linked to plant or bacterial toxins. Since large amounts of these reagents can be administered without eliciting non-specific toxicities, efficient methods of production are needed. The goal of this work was to express a complex immunoenzyme fusion protein (immunotoxin) in the mammary gland of transgenic mice. A chimeric mouse/human antibody directed against the human transferrin receptor (E6) was fused at its CH2 domain to the gene for a human angiogenic ribonuclease, angiogenin (Ang). It was expressed in the mammary gland of mice and secreted into mouse milk. Expression levels in milk were approximately 0.8 g/l. The chimeric protein retained antibody binding activity and protein synthesis inhibitory activity equivalent to that of free Ang. It was specifically cytotoxic to human tumor cells in vitro. PMID:10648935

  20. RESOLFT Nanoscopy of Fixed Cells Using a Z-Domain Based Fusion Protein for Labelling.

    PubMed

    Ilgen, Peter; Grotjohann, Tim; Jans, Daniel C; Kilisch, Markus; Hell, Stefan W; Jakobs, Stefan

    2015-01-01

    RESOLFT super-resolution microscopy allows subdiffraction resolution imaging of living cells using low intensities of light. It relies on the light-driven switching of reversible switchable fluorescent proteins (RSFPs). So far, RESOLFT imaging was restricted to living cells, because chemical fixation typically affects the switching characteristics of RSFPs. In this study we created a fusion construct (FLASR) consisting of the RSFP rsEGFP2 and the divalent form of the antibody binding Z domain from protein A. FLASR can be used analogous to secondary antibodies in conventional immunochemistry, facilitating simple and robust sample preparation. We demonstrate RESOLFT super-resolution microscopy on chemically fixed mammalian cells. The approach may be extended to other super-resolution approaches requiring fluorescent proteins in an aqueous environment. PMID:26375606

  1. A faster way to make GFP-based biosensors: Two new transposons for creating multicolored libraries of fluorescent fusion proteins

    PubMed Central

    Sheridan, Douglas L; Hughes, Thomas E

    2004-01-01

    Background There are now several ways to generate fluorescent fusion proteins by randomly inserting DNA encoding the Green Fluorescent Protein (GFP) into another protein's coding sequence. These approaches can be used to map regions in a protein that are permissive for GFP insertion or to create novel biosensors. While remarkably useful, the current insertional strategies have two major limitations: (1) they only produce one kind, or color, of fluorescent fusion protein and (2) one half of all GFP insertions within the target coding sequence are in the wrong orientation. Results We have overcome these limitations by incorporating two different fluorescent proteins coding sequences in a single transposon, either in tandem or antiparallel. Our initial tests targeted two mammalian integral membrane proteins: the voltage sensitive motor, Prestin, and an ER ligand gated Ca2+ channel (IP3R). Conclusions These new designs increase the efficiency of random fusion protein generation in one of two ways: (1) by creating two different fusion proteins from each insertion or (2) by being independent of orientation. PMID:15317651

  2. A Bidirectional System for the Dynamic Small Molecule Control of Intracellular Fusion Proteins

    PubMed Central

    Kuzin, Alexander P.; Lew, Scott; Seetharaman, Jayaraman; Acton, Thomas B.; Kornhaber, Gregory J.; Xiao, Rong; Montelione, Gaetano Thomas; Tong, Liang; Crews, Craig M.

    2014-01-01

    Small molecule control of intracellular protein levels allows temporal and dose-dependent regulation of protein function. Recently, we developed a method to degrade proteins fused to a mutant dehalogenase (HaloTag2) using small molecule hydrophobic tags (HyTs). Here, we introduce a complementary method to stabilize the same HaloTag2 fusion proteins, resulting in a unified system allowing bidirectional control of cellular protein levels in a temporal and dose-dependent manner. From a small molecule screen, we identified N-(3,5-dichloro-2-ethoxybenzyl)-2H-tetrazol-5-amine as a nanomolar HALoTag2 Stabilizer (HALTS1) that reduces the Hsp70:HaloTag2 interaction, thereby preventing HaloTag2 ubiquitination. Finally, we demonstrate the utility of the HyT/HALTS system in probing the physiological role of therapeutic targets by modulating HaloTag2-fused oncogenic H-Ras, which resulted in either the cessation (HyT) or acceleration (HALTS) of cellular transformation. In sum, we present a general platform to study protein function, whereby any protein of interest fused to HaloTag2 can be either degraded 10-fold or stabilized 5-fold using two corresponding compounds. PMID:23978068

  3. Effects of antibodies against cell surface protein antigen PAc-glucosyltransferase fusion proteins on glucan synthesis and cell adhesion of Streptococcus mutans.

    PubMed Central

    Yu, H; Nakano, Y; Yamashita, Y; Oho, T; Koga, T

    1997-01-01

    Cell surface protein antigen (PAc) and glucosyltransferases (GTFs) produced by Streptococcus mutans are considered to be major colonization factors of the organism, and the inhibition of these two factors is predicted to provide protection against dental caries. In this study, we have constructed fusion protein PAcA-GB, a fusion of the saliva-binding alanine-rich region (PAcA) of PAc with the glucan binding (GB) domain of GTF-I, an enzyme catalyzing the synthesis of water-insoluble glucan from sucrose, and fusion protein PAcA-SB, a fusion of PAcA with the sucrose binding (SB) domain of GTF-I. The recombinant fusion proteins were purified from cell extracts of Escherichia coli harboring the fusion genes, and rabbit antibodies against these fusion proteins were prepared. Water-insoluble glucan synthesis by cell-associated and cell-free GTF preparations from S. mutans as well as total glucan synthesis by GTF-I was markedly inhibited by anti-PAcA-GB immunoglobulin G (IgG) antibodies but not by anti-PAcA-SB IgG antibodies. Significant inhibition of the sucrose-independent and sucrose-dependent adhesion of S. mutans to saliva-coated hydroxyapatite beads was observed when anti-PAcA-GB antibodies were added to the reaction mixture. Anti-PAcA-SB antibodies inhibited the adhesion of S. mutans to the beads in the absence of sucrose but not in the presence of sucrose. Immunization with the fusion protein PAcA-GB may be useful for controlling the colonization of teeth by S. mutans. PMID:9169766

  4. Adenoviral-Mediated Imaging of Gene Transfer Using a Somatostatin Receptor-Cytosine Deaminase Fusion Protein

    PubMed Central

    Lears, Kimberly A.; Parry, Jesse J.; Andrews, Rebecca; Nguyen, Kim; Wadas, Thaddeus J.; Rogers, Buck E.

    2015-01-01

    Suicide gene therapy is a process by which cells are administered a gene that encodes a protein capable of converting a nontoxic prodrug into an active toxin. Cytosine deaminase (CD) has been widely investigated as a means of suicide gene therapy due to the enzyme’s ability to convert the prodrug 5-fluorocytosine (5-FC) into the toxic compound 5-fluorouracil (5-FU). However, the extent of gene transfer is a limiting factor in predicting therapeutic outcome. The ability to monitor gene transfer, non-invasively, would strengthen the efficiency of therapy. In this regard, we have constructed and evaluated a replication-deficient adenovirus (Ad) containing the human somatostatin receptor subtype 2 (SSTR2) fused with a C-terminal yeast CD gene for the non-invasive monitoring of gene transfer and therapy. The resulting Ad (AdSSTR2-yCD) was evaluated in vitro in breast cancer cells to determine the function of the fusion protein. These studies demonstrated that the both the SSTR2 and yCD were functional in binding assays, conversion assays, and cytotoxicity assays. In vivo studies similarly demonstrated the functionality using conversion assays, biodistribution studies, and small animal positron-emission tomography (PET) imaging studies. In conclusion, the fusion protein has been validated as useful for the non-invasive imaging of yCD expression and will be evaluated in the future for monitoring yCD-based therapy. PMID:25837665

  5. Efficient killing of CD22{sup +} tumor cells by a humanized diabody-RNase fusion protein

    SciTech Connect

    Krauss, Juergen . E-mail: juergen.krauss@uni-essen.de; Arndt, Michaela A.E.; Vu, Bang K.; Newton, Dianne L.; Seeber, Siegfried; Rybak, Susanna M.

    2005-06-03

    We report on the generation of a dimeric immunoenzyme capable of simultaneously delivering two ribonuclease (RNase) effector domains on one molecule to CD22{sup +} tumor cells. As targeting moiety a diabody derived from the previously humanized scFv SGIII with grafted specificity of the murine anti-CD22 mAb RFB4 was constructed. Further engineering the interface of this construct (V{sub L}36{sub Leu{yields}}{sub Tyr}) resulted in a highly robust bivalent molecule that retained the same high affinity as the murine mAb RFB4 (K{sub D} 0.2 nM). A dimeric immunoenzyme comprising this diabody and Rana pipiens liver ribonuclease I (rapLRI) was generated, expressed as soluble protein in bacteria, and purified to homogeneity. The dimeric fusion protein killed several CD22{sup +} tumor cell lines with high efficacy (IC{sub 50} = 3-20 nM) and exhibited 9- to 48-fold stronger cytotoxicity than a monovalent rapLRI-scFv counterpart. Our results demonstrate that engineering of dimeric antibody-ribonuclease fusion proteins can markedly enhance their biological efficacy.

  6. [Expression and purification of GST-CML28 fusion protein and preparation of its polyclonal antibody].

    PubMed

    Mao, Xia; Zhang, Bing; Bai, Xue-Ling; Liu, Long-Long; Zhang, Dong-Hua

    2012-12-01

    This study was aimed to investigate the expression of GST-CML28 in Escherichia Coli and to prepare its antibody. The constructed recombinant expression vectors CML28-pGEX-3X were transformed into Escherichia Coli BL21 under IPTG induction. The protein was abstracted from the transformers, and purified by a GSTrap FF column. The rabbits were immunized by the purified fusion protein to produce serum with anti-CML28 antibody. The serum was purified by chromatographic column stuffed with glutathione Sephamse 4B to get the antibody. The specific antibody against CML28 was further identified by ELISA, Western blot, immunohistochemistry and quantum dot luminescence. The results indicated that GST-CML28 fusion protein was expressed in Escherichia coli and its specific polyclonal antibody was obtained. It is concluded that the anti-CML28 polyclonal antibodies with high titer and specificity are successfully prepared. These antibodies provide an useful experimental tool to profoundly research the physiological significance and biological function of the CML28 gene. PMID:23257421

  7. Dendritic-Tumor Fusion Cells Derived Heat Shock Protein70-Peptide Complex Has Enhanced Immunogenicity

    PubMed Central

    Chen, Jun; Liu, Yunyan; Luo, Wen

    2015-01-01

    Tumor-derived heat shock protein70-peptide complexes (HSP70.PC-Tu) have shown great promise in tumor immunotherapy due to numerous advantages. However, large-scale phase III clinical trials showed that the limited immunogenicity remained to be enhanced. In previous research, we demonstrated that heat shock protein 70-peptide complexes (HSP70.PC-Fc) derived from dendritic cell (DC)-tumor fusions exhibit enhanced immunogenicity compared with HSP70.PCs from tumor cells. However, the DCs used in our previous research were obtained from healthy donors and not from the patient population. In order to promote the clinical application of these complexes, HSP70.PC-Fc was prepared from patient-derived DC fused directly with patient-derived tumor cells in the current study. Our results showed that compared with HSP70.PC-Tu, HSP70.PC-Fc elicited much more powerful immune responses against the tumor from which the HSP70 was derived, including enhanced T cell activation, and CTL responses that were shown to be antigen specific and HLA restricted. Our results further indicated that the enhanced immunogenicity is related to the activation of CD4+ T cells and increased association with other heat shock proteins, such as HSP90. Therefore, the current study confirms the enhanced immunogenicity of HSP70.PC derived from DC-tumor fusions and may provide direct evidence promoting their future clinical use. PMID:25961716

  8. MLL1 and MLL1 fusion proteins have distinct functions in regulating leukemic transcription program

    PubMed Central

    Xu, Jing; Li, Li; Xiong, Jie; denDekker, Aaron; Ye, Andrew; Karatas, Hacer; Liu, Liu; Wang, He; Qin, Zhaohui S; Wang, Shaomeng; Dou, Yali

    2016-01-01

    Mixed lineage leukemia protein-1 (MLL1) has a critical role in human MLL1 rearranged leukemia (MLLr) and is a validated therapeutic target. However, its role in regulating global gene expression in MLLr cells, as well as its interplay with MLL1 fusion proteins remains unclear. Here we show that despite shared DNA-binding and cofactor interacting domains at the N terminus, MLL1 and MLL-AF9 are recruited to distinct chromatin regions and have divergent functions in regulating the leukemic transcription program. We demonstrate that MLL1, probably through C-terminal interaction with WDR5, is recruited to regulatory enhancers that are enriched for binding sites of E-twenty-six (ETS) family transcription factors, whereas MLL-AF9 binds to chromatin regions that have no H3K4me1 enrichment. Transcriptome-wide changes induced by different small molecule inhibitors also highlight the distinct functions of MLL1 and MLL-AF9. Taken together, our studies provide novel insights on how MLL1 and MLL fusion proteins contribute to leukemic gene expression, which have implications for developing effective therapies in the future.

  9. Production of Hev b5 as a fluorescent biotin-binding tripartite fusion protein in insect cells

    SciTech Connect

    Nordlund, Henri R. . E-mail: henri.nordlund@uta.fi; Laitinen, Olli H.; Uotila, Sanna T.H.; Kulmala, Minna; Kalkkinen, Nisse; Kulomaa, Markku S.

    2005-10-14

    The presented green fluorescent protein and streptavidin core-based tripartite fusion system provides a simple and efficient way for the production of proteins fused to it in insect cells. This fusion protein forms a unique tag, which serves as a multipurpose device enabling easy optimization of production, one-step purification via streptavidin-biotin interaction, and visualization of the fusion protein during downstream processing and in applications. In the present study, we demonstrate the successful production, purification, and detection of a natural rubber latex allergen Hev b5 with this system. We also describe the production of another NRL allergen with the system, Hev b1, which formed large aggregates and gave small yields in purification. The aggregates were detected at early steps by microscopical inspection of the infected insect cells producing this protein. Therefore, this fusion system can also be utilized as a fast indicator of the solubility of the expressed fusion proteins and may therefore be extremely useful in high-throughput expression approaches.

  10. F-18 Labeled Diabody-Luciferase Fusion Proteins for Optical-ImmunoPET

    SciTech Connect

    Wu, Anna M

    2013-01-18

    The goal of the proposed work is to develop novel dual-labeled molecular imaging probes for multimodality imaging. Based on small, engineered antibodies called diabodies, these probes will be radioactively tagged with Fluorine-18 for PET imaging, and fused to luciferases for optical (bioluminescence) detection. Performance will be evaluated and validated using a prototype integrated optical-PET imaging system, OPET. Multimodality probes for optical-PET imaging will be based on diabodies that are dually labeled with 18F for PET detection and fused to luciferases for optical imaging. 1) Two sets of fusion proteins will be built, targeting the cell surface markers CEA or HER2. Coelenterazine-based luciferases and variant forms will be evaluated in combination with native substrate and analogs, in order to obtain two distinct probes recognizing different targets with different spectral signatures. 2) Diabody-luciferase fusion proteins will be labeled with 18F using amine reactive [18F]-SFB produced using a novel microwave-assisted, one-pot method. 3) Sitespecific, chemoselective radiolabeling methods will be devised, to reduce the chance that radiolabeling will inactivate either the target-binding properties or the bioluminescence properties of the diabody-luciferase fusion proteins. 4) Combined optical and PET imaging of these dual modality probes will be evaluated and validated in vitro and in vivo using a prototype integrated optical-PET imaging system, OPET. Each imaging modality has its strengths and weaknesses. Development and use of dual modality probes allows optical imaging to benefit from the localization and quantitation offered by the PET mode, and enhances the PET imaging by enabling simultaneous detection of more than one probe.

  11. The human ubiquitin-52 amino acid fusion protein gene shares several structural features with mammalian ribosomal protein genes.

    PubMed Central

    Baker, R T; Board, P G

    1991-01-01

    Complementary DNA clones encoding ubiquitin fused to a 52 amino acid tail protein were isolated from human placental and adrenal gland cDNA libraries. The deduced human 52 amino acid tail protein is very similar to the homologous protein from other species, including the conservation of the putative metal-binding, nucleic acid-binding domain observed in these proteins. Northern blot analysis with a tail-specific probe indicated that the previously identified UbA mRNA species most likely represents comigrating transcripts of the 52 amino acid tail (UbA52) and 80 amino acid tail (UbA80) ubiquitin fusion genes. The UbA52 gene was isolated from a human genomic library and consists of five exons distributed over 3400 base pairs. One intron is in the 5' non-coding region, two interrupt the single ubiquitin coding unit, and the fourth intron is within the tail coding region. Several members of the Alu family of repetitive DNA are associated with the gene. The UbA52 promoter has several features in common with mammalian ribosomal protein genes, including its location in a CpG-rich island, initiation of transcription within a polypyrimidine tract, the lack of a consensus TATA motif, and the presence of Sp1 binding sites, observations that are consistent with the recent identification of the ubiquitin-free tail proteins as ribosomal proteins. Thus, in spite of its unusual feature of being translationally fused to ubiquitin, the 52 amino acid tail ribosomal protein is expressed from a structurally typical ribosomal protein gene. Images PMID:1850507

  12. Generation of a functional monomolecular protein lattice consisting of an s-layer fusion protein comprising the variable domain of a camel heavy chain antibody.

    PubMed

    Pleschberger, Magdalena; Neubauer, Angela; Egelseer, Eva M; Weigert, Stefan; Lindner, Brigitte; Sleytr, Uwe B; Muyldermans, Serge; Sára, Margit

    2003-01-01

    Crystalline bacterial cell surface layer (S-layer) proteins are composed of a single protein or glycoprotein species. Isolated S-layer subunits frequently recrystallize into monomolecular protein lattices on various types of solid supports. For generating a functional protein lattice, a chimeric protein was constructed, which comprised the secondary cell wall polymer-binding region and the self-assembly domain of the S-layer protein SbpA from Bacillus sphaericus CCM 2177, and a single variable region of a heavy chain camel antibody (cAb-Lys3) recognizing lysozyme as antigen. For construction of the S-layer fusion protein, the 3'-end of the sequence encoding the C-terminally truncated form rSbpA(31)(-)(1068) was fused via a short linker to the 5'-end of the sequence encoding cAb-Lys3. The functionality of the fused cAb-Lys3 in the S-layer fusion protein was proved by surface plasmon resonance measurements. Dot blot assays revealed that the accessibility of the fused functional sequence for the antigen was independent of the use of soluble or assembled S-layer fusion protein. Recrystallization of the S-layer fusion protein into the square lattice structure was observed on peptidoglycan-containing sacculi of B. sphaericus CCM 2177, on polystyrene or on gold chips precoated with thiolated secondary cell wall polymer, which is the natural anchoring molecule for the S-layer protein in the bacterial cell wall. Thereby, the fused cAb-Lys3 remained located on the outer S-layer surface and accessible for lysozyme binding. Together with solid supports precoated with secondary cell wall polymers, S-layer fusion proteins comprising rSbpA(31)(-)(1068) and cAbs directed against various antigens shall be exploited for building up monomolecular functional protein lattices as required for applications in nanobiotechnology. PMID:12643755

  13. A Maltose-Binding Protein Fusion Construct Yields a Robust Crystallography Platform for MCL1

    PubMed Central

    Clifton, Matthew C.; Dranow, David M.; Leed, Alison; Fulroth, Ben; Fairman, James W.; Abendroth, Jan; Atkins, Kateri A.; Wallace, Ellen; Fan, Dazhong; Xu, Guoping; Ni, Z. J.; Daniels, Doug; Van Drie, John; Wei, Guo; Burgin, Alex B.; Golub, Todd R.; Hubbard, Brian K.; Serrano-Wu, Michael H.

    2015-01-01

    Crystallization of a maltose-binding protein MCL1 fusion has yielded a robust crystallography platform that generated the first apo MCL1 crystal structure, as well as five ligand-bound structures. The ability to obtain fragment-bound structures advances structure-based drug design efforts that, despite considerable effort, had previously been intractable by crystallography. In the ligand-independent crystal form we identify inhibitor binding modes not observed in earlier crystallographic systems. This MBP-MCL1 construct dramatically improves the structural understanding of well-validated MCL1 ligands, and will likely catalyze the structure-based optimization of high affinity MCL1 inhibitors. PMID:25909780

  14. Designing Two-Dimensional Protein Arrays through Fusion of Multimers and Interface Mutations.

    PubMed

    Matthaei, James F; DiMaio, Frank; Richards, Jeffrey J; Pozzo, Lilo D; Baker, David; Baneyx, François

    2015-08-12

    We have combined fusion of oligomers with cyclic symmetry and alanine substitutions to eliminate clashes and produce proteins that self-assemble into 2-D arrays upon addition of calcium ions. Using TEM, AFM, small-angle X-ray scattering, and fluorescence microscopy, we show that the designed lattices which are 5 nm high with p3 space group symmetry and 7.25 nm periodicity self-assemble into structures that can exceed 100 μm in characteristic length. The versatile strategy, experimental approach, and hexagonal arrays described herein should prove valuable for the engineering of functional nanostructured materials in 2-D. PMID:25986921

  15. pH-Dependent Vesicle Fusion Induced by the Ectodomain of the Human Immunodeficiency Virus Membrane Fusion Protein gp41: Two Kinetically Distinct Processes and Fully-Membrane-Associated gp41 with Predominant β Sheet Fusion Peptide Conformation

    PubMed Central

    Ratnayake, Punsisi U.; Sackett, Kelly; Nethercott, Matthew J.; Weliky, David P.

    2014-01-01

    The gp41 protein of the Human Immunodeficiency Virus (HIV) catalyzes fusion between HIV and host cell membranes. The ~180-residue ectodomain of gp41 is outside the virion and is the most important gp41 region for membrane fusion. The ectodomain consists of an apolar fusion peptide (FP) region followed by N-heptad repeat (NHR), loop, and C-heptad repeat (CHR) regions. The FP is critical for fusion and is hypothesized to bind to the host cell membrane. Large ectodomain constructs either with or without the FP are highly aggregated at physiologic pH but soluble in the pH 3–4 range with hyperthermostable hairpin structure with antiparallel NHR and CHR helices. The present study focuses on the large gp41 ectodomain constructs “Hairpin” (HP) containing NHR+loop+CHR and “FP-Hairpin” (FP-HP) containing FP+NHR+loop+CHR. Both proteins induce rapid and extensive fusion of anionic vesicles at pH 4 where the protein is positively-charged but do not induce fusion at pH 7 where the protein is negatively charged. This observation, along with lack of fusion of neutral vesicles at either pH supports the significance of attractive protein/membrane electrostatics in fusion. The functional role of the hydrophobic FP is supported by increases in the rate and extent of fusion for FP-HP relative to HP. There are two kinetically distinct fusion processes at pH 4: (1) a faster ~100 ms−1 process with rate strongly positively correlated with vesicle charge; and (2) a slower ~5 ms−1 process with extent strongly inversely correlated with this charge. The faster charge-dependent process is likely related to the electrostatic energy released upon initial monomer protein binding to the vesicle. After dissipation of this energy, the subsequent slower process is likely due to the equilibrium membrane-associated structure of the protein. The slower process may be more physiologically relevant because HIV/host cell fusion occurs at physiologic pH with gp41 restricted to the narrow region

  16. Enhancing the production of Fc fusion protein in fed-batch fermentation of Pichia pastoris by design of experiments.

    PubMed

    Lin, Henry; Kim, Tina; Xiong, Fei; Yang, Xiaoming

    2007-01-01

    This study focuses on the feasibility of producing a therapeutic Fc fusion protein in Pichia pastoris (P. pastoris) and presents an optimization design of experiment (DOE) strategy in a well-defined experimental space. The parameters examined in this study include pH, temperature, salt supplementation, and batch glycerol concentration. The effects of these process conditions were captured by statistical analysis focusing on growth rate and titer responses. Batch medium and fermentation conditions were also investigated prior to the DOE study in order to provide a favorable condition to enable the production of this Fc fusion protein. The results showed that approximately 373 mg/L of the Fc fusion protein could be produced. The pH was found to be particularly critical for the production of this Fc fusion protein. It was significantly higher than the conventional, recommended pH for P. pastoris fermentation. The development of this process shows that protein production in P. pastoris is protein specific, and there is not a set of pre-defined conditions that can work well for all types of proteins. Thorough process development would need to be performed for every type of protein in order for large-scale production in P. pastoris to be feasible. PMID:17461547

  17. Use of phoA fusions to study the topology of the Escherichia coli inner membrane protein leader peptidase.

    PubMed Central

    San Millan, J L; Boyd, D; Dalbey, R; Wickner, W; Beckwith, J

    1989-01-01

    A topology of the Escherichia coli leader peptidase has been previously proposed on the basis of proteolytic studies. Here, a collection of alkaline phosphatase fusions to leader peptidase is described. Fusions to the periplasmic domain of this protein exhibit high alkaline phosphatase activity, while fusions to the cytoplasmic domain exhibit low activity. Elements within the cytoplasmic domain are necessary to stably anchor alkaline phosphatase in the cytoplasm. The amino-terminal hydrophobic segment of leader peptidase acts as a weak export signal for alkaline phosphatase. However, when this segment is preceded by four lysines, it acts as a highly efficient export signal. The coherence of in vitro studies with alkaline phosphatase fusion analysis of the topology of leader peptidase further indicates the utility of this genetic approach to membrane protein structure and insertion. Images PMID:2551889

  18. Regulated expression of the Leishmania major surface virulence factor lipophosphoglycan using conditionally destabilized fusion proteins

    PubMed Central

    Madeira da Silva, Luciana; Owens, Katherine L.; Murta, Silvane M. F.; Beverley, Stephen M.

    2009-01-01

    Surface glycoconjugates play important roles in the infectious cycle of Leishmania major, including the abundant lipophosphoglycan (LPG) implicated in parasite survival in the sand fly vector and the initial stages of establishment in the mammalian host macrophage. We describe a system for inducible expression of LPG, applying a novel protein-based system that allows controlled degradation of a key LPG biosynthetic enzyme, UDP-galactopyranose mutase (UGM). This methodology relies on a mutated FK506-binding protein (FKBP) destabilizing domain (dd) fused to the protein of interest; in the absence of rapamycin analogs, such as Shld1, the dd domain is destabilized, leading to proteasomal degradation, whereas drug treatment confers stabilization. Tests in L. major using dd fusions to a panel of reporters and cellular proteins confirmed its functionality, with a high degree of regulation and low background, and we established the kinetics of protein activation and/or loss. Two inexpensive and widely available ligands, FK506 and rapamycin, functioned similarly to Shld1, without effect on Leishmania growth or differentiation. We generated parasites lacking UGM through deletion of the GLF gene and substitution with a ddGLF fusion construct, either as chromosomal knockins or through episomal complementation; these showed little or no LPG expression in the absence of inducer, whereas in its presence, high levels of LPG were attained rapidly. Complement lysis tests confirmed the correct integrity of the Leishmania LPG coat. These data suggest that the dd approach has great promise in the study of LPG and other pathways relevant to parasite survival and virulence. PMID:19383793

  19. Secretion of the respiratory syncytial virus fusion protein from insect cells using the baculovirus expression system.

    PubMed

    Tan, Boon-Huan; Brown, Gaie; Sugrue, Richard J

    2007-01-01

    Sequences derived from the respiratory syncytial virus (RSV) fusion (F) protein were expressed in insect cells as recombinant glutathione-S-transferase (GST)-tagged proteins. The sequence covering the F2 subunit (GST-F2), and a truncated form of the F protein in which the transmembrane domain was removed (GST-F2/F1), were cloned into the baculovirus pAcSecG2T secretory vector. These virus sequences also had the endogenous virus signal sequence removed and replaced with a signal sequence derived from the baculovirus gp67 glycoprotein, which was present in pAcSecG2T. The recombinant RSV glycoproteins were successfully detected in expressing cells by immunofluorescence assay and in the tissue culture medium by western blot analysis. The secreted recombinant GST-F2/F1 protein was further analysed using glycosidases. Our results showed that the GST-F2/F1 protein were sensitive to peptide:N-glycosidase F (PNGase F) treatment, but not to Endoglycosidase H (EndoH) treatment. This indicates that the secreted recombinant proteins were modified by the addition of mature N-linked glycan chains. PMID:17502677

  20. Identification and characterization of LFD1, a novel protein involved in membrane merger during cell fusion in Neurospora crassa.

    PubMed

    Palma-Guerrero, Javier; Leeder, Abigail C; Welch, Juliet; Glass, N Louise

    2014-04-01

    Despite its essential role in development, molecular mechanisms of membrane merger during cell-cell fusion in most eukaryotic organisms remain elusive. In the filamentous fungus Neurospora crassa, cell fusion occurs during asexual spore germination, where genetically identical germlings show chemotropic interactions and cell-cell fusion. Fusion of germlings and hyphae is required for the formation of the interconnected mycelial network characteristic of filamentous fungi. Previously, a multipass membrane protein, PRM1, was characterized and acts at the step of bilayer fusion in N. crassa. Here we describe the identification and characterization of lfd-1, encoding a single pass transmembrane protein, which is also involved in membrane merger. lfd-1 was identified by a targeted analysis of a transcriptional profile of a transcription factor mutant (Δpp-1) defective in germling fusion. The Δlfd-1 mutant shows a similar, but less severe, membrane merger defect as a ΔPrm1 mutant. By genetic analyses, we show that LFD1 and PRM1 act independently, but share a redundant function. The cell fusion frequency of both Δlfd-1 and ΔPrm1 mutants was sensitive to extracellular calcium concentration and was associated with an increase in cell lysis, which was suppressed by a calcium-dependent mechanism involving a homologue to synaptotagmin. PMID:24673848

  1. Protein stability, conformational change and binding mechanism of human serum albumin upon binding of embelin and its role in disease control.

    PubMed

    Yeggoni, Daniel Pushparaju; Rachamallu, Aparna; Subramanyam, Rajagopal

    2016-07-01

    Here, we present the inclusive binding mode of phytochemical embelin, an anticancer drug with human serum albumin (HSA) established under physiological condition. Also, to understand the pharmacological role of embelin molecule, here, we have studied the anti-cancer activity of embelin on human cervical cancer cell line (HeLa cell line), which revealed that embelin showed dose dependent inhibition in the growth of cancer cells and also induces 26.3% of apoptosis at an IC50 value of 29μM. Further, embelin was titrated with HSA and the fluorescence emission quenching of HSA due to the formation of the HSA-embelin complex was observed. The binding constant of this complex is 5.9±.01×10(4)M(-1) and the number of bound embelin molecules is approximately 1.0. Consequently, molecular displacement and computational docking experiments show that the embelin is binding to subdomain IB to HSA. Further evidence from microTOF-Q mass spectrometry showed an increase in mass from 66,563Da to 66,857Da observed for free HSA and HSA+embelin complex, signifying that there is robust binding of embelin with HSA. In addition, the variations of HSA secondary structural elements in presence of embelin were confirmed by circular dichroism which indicates partial unfolding of protein. Furthermore, the transmission electron micrographs established that complex formation leads to aggregation of HSA plus embelin. Molecular dynamics simulations revealed that the stability of the HSA-embelin complexes and results suggests that at around 3500ps the complex reaches equilibration state which clearly contributes to the understanding of the stability of the HSA-embelin complexes. PMID:27130964

  2. Exploration and Validation of C-Reactive Protein/Albumin Ratio as a Novel Inflammation-Based Prognostic Marker in Nasopharyngeal Carcinoma

    PubMed Central

    Zhang, Yuan; Zhou, Guan-Qun; Liu, Xu; Chen, Lei; Li, Wen-Fei; Tang, Ling-Long; Liu, Qing; Sun, Ying; Ma, Jun

    2016-01-01

    Background: The prognostic value of C-reactive protein/albumin ratio (CRP/Alb), a novel inflammation-based marker, remains unknown in nasopharyngeal carcinoma (NPC). Methods: We conducted a retrospective review of 1572 consecutive patients with non-metastatic NPC. Patients were randomly divided into a training set (n = 514) and validation set (n = 1058). The prognostic value of the CRP/Alb ratio and the modified Glasgow prognostic score (mGPS; a well-recognized inflammation-based score) was assessed. Results: Receiver-operating characteristic analysis identified 0.05 as the optimal CRP/Alb cut-off value for disease failure in the training set. Patients with a CRP/Alb > 0.05 had poorer overall survival (OS), distant metastasis-free survival (DMFS) and disease-free survival (DFS) in the training set (all P < 0.05). These results were confirmed in the validation set (all P < 0.05) and the whole cohort (all P < 0.001). In multivariate analysis of the entire cohort, the pretreatment CRP/Alb ratio was an independent prognostic factor for OS (HR, 1.394; 95% CI, 1.004-1.937; P = 0.048) and DMFS (HR, 1.545; 95% CI, 1.124-2.122; P = 0.007), but not for DFS (P = 0.083). The mGPS had no significant independent prognostic value for any end-point. Conclusion: CRP/Alb ratio is an useful prognostic indicator in patients with NPC, independent of disease stage. PMID:27471556

  3. Use of 125I- and 51Cr-Labeled Albumin for the Measurement of Gastrointestinal and Total Albumin Catabolism*

    PubMed Central

    Kerr, Robert M.; Bois, John J. Du; Holt, Peter R.

    1967-01-01

    A method for the simultaneous measurement of gastrointestinal protein loss and total albumin turnover entailing the use of a combination of 125iodine- and 51chromium-labeled albumin is described. Albumin turnover was calculated by the measurement of albumin-125I plasma decay and cumulative urinary excretion, and the results obtained agreed closely with previous studies utilizing albumin-131I. Gastrointestinal catabolism was calculated from the rate of fecal excretion of 51Cr and the specific activity of plasma albumin-51Cr, and these data were related to the calculated albumin turnover results. During the period of 6-14 days after administration, the ratio of specific activties of albumin-125I and -51Cr in plasma and in extravascular spaces or gastric and biliary secretions remained almost identical. Fecal excretion of 51Cr was also quite stable at this time. In six normal subjects gastrointestinal catabolism accounted for less than 10% of total albumin catabolism. Excessive gastrointestinal protein losses did not contribute to the low serum albumin in three patients with cirrhosis or in two adults with the nephrotic syndrome. Multiple mechanisms leading to hypoalbuminemia were demonstrated in other subjects with a variety of gastrointestinal disorders. Images PMID:5630419

  4. Antibodies to CD9, a Tetraspan Transmembrane Protein, Inhibit Canine Distemper Virus-Induced Cell-Cell Fusion but Not Virus-Cell Fusion

    PubMed Central

    Schmid, Erik; Zurbriggen, Andreas; Gassen, Uta; Rima, Bert; ter Meulen, Volker; Schneider-Schaulies, Jürgen

    2000-01-01

    Canine distemper virus (CDV) causes a life-threatening disease in several carnivores including domestic dogs. Recently, we identified a molecule, CD9, a member of the tetraspan transmembrane protein family, which facilitates, and antibodies to which inhibit, the infection of tissue culture cells with CDV (strain Onderstepoort). Here we describe that an anti-CD9 monoclonal antibody (MAb K41) did not interfere with binding of CDV to cells and uptake of virus. In addition, in single-step growth experiments, MAb K41 did not induce differences in the levels of viral mRNA and proteins. However, the virus release of syncytium-forming strains of CDV, the virus-induced cell-cell fusion in lytically infected cultures, and the cell-cell fusion of uninfected with persistently CDV-infected HeLa cells were strongly inhibited by MAb K41. These data indicate that anti-CD9 antibodies selectively block virus-induced cell-cell fusion, whereas virus-cell fusion is not affected. PMID:10906209

  5. Structural and kinetic analysis of the unnatural fusion protein 4-coumaroyl-CoA ligase::stilbene synthase

    SciTech Connect

    Wang, Yechun; Yi, Hankuil; Wang, Melissa; Yu, Oliver; Jez, Joseph M.

    2012-10-24

    To increase the biochemical efficiency of biosynthetic systems, metabolic engineers have explored different approaches for organizing enzymes, including the generation of unnatural fusion proteins. Previous work aimed at improving the biosynthesis of resveratrol, a stilbene associated a range of health-promoting activities, in yeast used an unnatural engineered fusion protein of Arabidopsis thaliana (thale cress) 4-coumaroyl-CoA ligase (At4CL1) and Vitis vinifera (grape) stilbene synthase (VvSTS) to increase resveratrol levels 15-fold relative to yeast expressing the individual enzymes. Here we present the crystallographic and biochemical analysis of the 4CL::STS fusion protein. Determination of the X-ray crystal structure of 4CL::STS provides the first molecular view of an artificial didomain adenylation/ketosynthase fusion protein. Comparison of the steady-state kinetic properties of At4CL1, VvSTS, and 4CL::STS demonstrates that the fusion protein improves catalytic efficiency of either reaction less than 3-fold. Structural and kinetic analysis suggests that colocalization of the two enzyme active sites within 70 {angstrom} of each other provides the basis for enhanced in vivo synthesis of resveratrol.

  6. MLL fusion proteins preferentially regulate a subset of wild-type MLL target genes in the leukemic genome

    PubMed Central

    Wang, Qian-fei; Wu, George; Mi, Shuangli; He, Fuhong; Wu, Jun; Dong, Jingfang; Luo, Roger T.; Mattison, Ryan; Kaberlein, Joseph J.; Prabhakar, Shyam; Ji, Hongkai

    2011-01-01

    MLL encodes a histone methyltransferase that is critical in maintaining gene expression during embryonic development and hematopoiesis. 11q23 translocations result in the formation of chimeric MLL fusion proteins that act as potent drivers of acute leukemia. However, it remains unclear what portion of the leukemic genome is under the direct control of MLL fusions. By comparing patient-derived leukemic cell lines, we find that MLL fusion-bound genes are a small subset of that recognized by wild-type MLL. In an inducible MLL-ENL model, MLL fusion protein binding and changes in H3K79 methylation are limited to a specific portion of the genome, whereas wild-type MLL distributes to a much larger set of gene loci. Surprisingly, among 223 MLL-ENL–bound genes, only 12 demonstrate a significant increase in mRNA expression on induction of the fusion protein. In addition to Hoxa9 and Meis1, this includes Eya1 and Six1, which comprise a heterodimeric transcription factor important in several developmental pathways. We show that Eya1 has the capacity to immortalize hematopoietic progenitor cells in vitro and collaborates with Six1 in hematopoietic transformation assays. Altogether, our data suggest that MLL fusions contribute to the development of acute leukemia through direct activation of a small set of target genes. PMID:21518926

  7. Membrane Organization and Cell Fusion During Mating in Fission Yeast Requires Multipass Membrane Protein Prm1

    PubMed Central

    Curto, M.-Ángeles; Sharifmoghadam, Mohammad Reza; Calpena, Eduardo; De León, Nagore; Hoya, Marta; Doncel, Cristina; Leatherwood, Janet; Valdivieso, M.-Henar

    2014-01-01

    The involvement of Schizosaccharomyces pombe prm1+ in cell fusion during mating and its relationship with other genes required for this process have been addressed. S. pombe prm1Δ mutant exhibits an almost complete blockade in cell fusion and an abnormal distribution of the plasma membrane and cell wall in the area of cell–cell interaction. The distribution of cellular envelopes is similar to that described for mutants devoid of the Fig1-related claudin-like Dni proteins; however, prm1+ and the dni+ genes act in different subpathways. Time-lapse analyses show that in the wild-type S. pombe strain, the distribution of phosphatidylserine in the cytoplasmic leaflet of the plasma membrane undergoes some modification before an opening is observed in the cross wall at the cell–cell contact region. In the prm1Δ mutant, this membrane modification does not take place, and the cross wall between the mating partners is not extensively degraded; plasma membrane forms invaginations and fingers that sometimes collapse/retract and that are sometimes strengthened by the synthesis of cell-wall material. Neither prm1Δ nor prm1Δ dniΔ zygotes lyse after cell–cell contact in medium containing and lacking calcium. Response to drugs that inhibit lipid synthesis or interfere with lipids is different in wild-type, prm1Δ, and dni1Δ strains, suggesting that membrane structure/organization/dynamics is different in all these strains and that Prm1p and the Dni proteins exert some functions required to guarantee correct membrane organization that are critical for cell fusion. PMID:24514900

  8. Membrane organization and cell fusion during mating in fission yeast requires multipass membrane protein Prm1.

    PubMed

    Curto, M-Ángeles; Sharifmoghadam, Mohammad Reza; Calpena, Eduardo; De León, Nagore; Hoya, Marta; Doncel, Cristina; Leatherwood, Janet; Valdivieso, M-Henar

    2014-04-01

    The involvement of Schizosaccharomyces pombe prm1(+) in cell fusion during mating and its relationship with other genes required for this process have been addressed. S. pombe prm1Δ mutant exhibits an almost complete blockade in cell fusion and an abnormal distribution of the plasma membrane and cell wall in the area of cell-cell interaction. The distribution of cellular envelopes is similar to that described for mutants devoid of the Fig1-related claudin-like Dni proteins; however, prm1(+) and the dni(+) genes act in different subpathways. Time-lapse analyses show that in the wild-type S. pombe strain, the distribution of phosphatidylserine in the cytoplasmic leaflet of the plasma membrane undergoes some modification before an opening is observed in the cross wall at the cell-cell contact region. In the prm1Δ mutant, this membrane modification does not take place, and the cross wall between the mating partners is not extensively degraded; plasma membrane forms invaginations and fingers that sometimes collapse/retract and that are sometimes strengthened by the synthesis of cell-wall material. Neither prm1Δ nor prm1Δ dniΔ zygotes lyse after cell-cell contact in medium containing and lacking calcium. Response to drugs that inhibit lipid synthesis or interfere with lipids is different in wild-type, prm1Δ, and dni1Δ strains, suggesting that membrane structure/organization/dynamics is different in all these strains and that Prm1p and the Dni proteins exert some functions required to guarantee correct membrane organization that are critical for cell fusion. PMID:24514900

  9. Internalization of a GFP-tetanus toxin C-terminal fragment fusion protein at mature mouse neuromuscular junctions.

    PubMed

    Roux, Sylvie; Colasante, Cesare; Saint Cloment, Cécile; Barbier, Julien; Curie, Thomas; Girard, Emmanuelle; Molgó, Jordi; Brûlet, Philippe

    2005-09-01

    The distribution, dynamics, internalization, and retrograde axonal traffic of a fusion protein composed of green fluorescent protein (GFP) and the atoxic C-terminal fragment of tetanus toxin (TTC) were studied after its in vivo injection. Confocal microscopy and immunogold electron microscopy revealed that the fusion protein (GFP-TTC) rapidly clustered in motor nerve terminals of the neuromuscular junction. Clathrin-coated pits, and axolemma infoldings located between active zones appeared to be involved in the internalization of the fusion protein. Biochemical analysis of detergent-extracted neuromuscular preparations showed that the GFP-TTC fusion protein was associated with lipid microdomains. We suggest that GFP-TTC clustering in these lipid microdomains favors the recruitment of other proteins involved in its endocytosis and internalization in motor nerve terminals. During its retrograde trafficking, GFP-TTC accumulated in different axonal compartments than those used by cholera toxin B-subunit suggesting that these two proteins are transported by different pathways and cargos. PMID:16023367

  10. Internalization of a GFP-tetanus toxin C-terminal fragment fusion protein at mature mouse neuromuscular junctions.

    PubMed

    Roux, Sylvie; Colasante, Cesare; Saint Cloment, Cécile; Barbier, Julien; Curie, Thomas; Girard, Emmanuelle; Molgó, Jordi; Brûlet, Philippe

    2005-12-01

    The distribution, dynamics, internalization, and retrograde axonal traffic of a fusion protein composed of green fluorescent protein (GFP)and the atoxic C-terminal fragment of tetanus toxin (TTC) were studied after its in vivo injection. Confocal microscopy and immuno-gold electron microscopy revealed that the fusion protein (GFP-TTC) rapidly clustered in motor nerve terminals of the neuromuscular junction. Clathrin-coated pits, and axolemma infoldings located between active zones appeared to be involved in the internalization of the fusion protein. Biochemical analysis of detergent-extracted neuromuscular preparations showed that the GFP-TTC fusion protein was associated with lipid microdomains. We suggest that GFP-TTC clustering in these lipid microdomains favors the recruitment of other proteins involved in its endocytosis and internalization in motor nerve terminals. During its retrograde trafficking, GFP-TTC accumulated indifferent axonal compartments than those used by cholera toxin B-subunit suggesting that these two proteins are transported by different pathways and cargos. PMID:16456925

  11. Matrix protein 2 of influenza A virus blocks autophagosome fusion with lysosomes

    PubMed Central

    Gannagé, Monique; Schmid, Dorothee; Albrecht, Randy; Dengjel, Jörn; Torossi, Tania; Rämer, Patrick C.; Lee, Monica; Strowig, Till; Arrey, Frida; Conenello, Gina; Pypaert, Marc; Andersen, Jens; García-Sastre, Adolfo; Münz, Christian

    2009-01-01

    Influenza A virus is an important human pathogen causing significant morbidity and mortality every year and threatening the human population with epidemics and pandemics. Therefore, it is important to understand the biology of this virus to develop strategies to control its pathogenicity. Here we demonstrate that live influenza A virus infection causes accumulation of autophagosomes by blocking their fusion with lysosomes. Matrix protein 2 is sufficient and necessary for this inhibition of autophagosome degradation. Macroautophagy inhibition compromises cell survival of influenza virus infected cells, but does not influence viral replication. We propose that influenza A virus, which also encodes pro-apoptotic proteins, is able to determine the death of its host cell by inducing apoptosis and blocking macroautophagy. PMID:19837376

  12. Concordance of preclinical and clinical pharmacology and toxicology of monoclonal antibodies and fusion proteins: soluble targets

    PubMed Central

    Martin, Pauline L; Bugelski, Peter J

    2012-01-01

    Monoclonal antibodies (mAbs) and fusion proteins directed towards soluble targets make an important contribution to the treatment of disease. The purpose of this review was to correlate the clinical and preclinical data on the 14 currently approved mAbs and fusion proteins targeted to soluble targets. The principal sources used to gather data were: the peer reviewed Literature; European Medicines Agency ‘Scientific Discussions’ and United States Food and Drug Administration ‘Pharmacology/Toxicology Reviews’ and package inserts (United States Prescribing Information). Data on the following approved biopharmaceuticals were included: adalimumab, anakinra, bevacizumab, canakinumab, certolizumab pegol, denosumab, eculizumab, etanercept, golimumab, infliximab, omalizumab, ranibizumab, rilonacept and ustekinumab. Some related biopharmaceuticals in late-stage development were also included for comparison. Good concordance with human pharmacodynamics was found for both non-human primates (NHPs) receiving the human biopharmaceutical and mice receiving rodent homologues (surrogates). In contrast, there was limited concordance for human adverse effects in genetically deficient mice, mice receiving surrogates or NHPs receiving the human pharmaceutical. In summary, the results of this survey show that although both mice and NHPs have good predictive value for human pharmacodynamics, neither species have good predictive value for human adverse effects. No evidence that NHPs have superior predictive value was found. PMID:22168335

  13. Feeding transgenic plants that express a tolerogenic fusion protein effectively protects against arthritis.

    PubMed

    Hansson, Charlotta; Schön, Karin; Kalbina, Irina; Strid, Åke; Andersson, Sören; Bokarewa, Maria I; Lycke, Nils Y

    2016-04-01

    Although much explored, oral tolerance for treatment of autoimmune diseases still awaits the establishment of novel and effective vectors. We investigated whether the tolerogenic CTA1(R7K)-COL-DD fusion protein can be expressed in edible plants, to induce oral tolerance and protect against arthritis. The fusion protein was recombinantly expressed in Arabidopsis thaliana plants, which were fed to H-2(q) -restricted DBA/1 mice to assess the preventive effect on collagen-induced arthritis (CIA). The treatment resulted in fewer mice exhibiting disease and arthritis scores were significantly reduced. Immune suppression was evident in treated mice, and serum biomarkers for inflammation as well as anticollagen IgG responses were reduced. In spleen and draining lymph nodes, CD4(+) T-cell responses were reduced. Concomitant with a reduced effector T-cell activity with lower IFNγ, IL-13 and IL-17A production, we observed an increase in IL-10 production to recall antigen stimulation in vitro, suggesting reduced Th1, Th2 and Th17 activity subsequent to up-regulated IL-10 and regulatory T-cell (Treg) functions. This study shows that edible plants expressing a tolerogen were effective at stimulating CD4 T-cell tolerance and in protecting against CIA disease. Our study conveys optimism as to the potential of using edible plants for oral treatment of rheumatoid arthritis. PMID:26403330

  14. Coexpression of cellulases in Pichia pastoris as a self-processing protein fusion.

    PubMed

    de Amorim Araújo, Juliana; Ferreira, Túlio César; Rubini, Marciano Régis; Duran, Ana Gilhema Gomez; De Marco, Janice Lisboa; de Moraes, Lidia Maria Pepe; Torres, Fernando Araripe Gonçalves

    2015-12-01

    The term cellulase refers to any component of the enzymatic complex produced by some fungi, bacteria and protozoans which act serially or synergistically to catalyze the cleavage of cellulosic materials. Cellulases have been widely used in many industrial applications ranging from food industry to the production of second generation ethanol. In an effort to develop new strategies to minimize the costs of enzyme production we describe the development of a Pichia pastoris strain able to coproduce two different cellulases. For that purpose the eglII (endoglucanase II) and cbhII (cellobiohydrolase II) genes from Trichoderma reesei were fused in-frame separated by the self-processing 2A peptide sequence from the foot-and-mouth disease virus. The protein fusion construct was placed under the control of the strong inducible AOX1 promoter. Analysis of culture supernatants from methanol-induced yeast transformants showed that the protein fusion was effectively processed. Enzymatic assay showed that the processed enzymes were fully functional with the same catalytic properties of the individual enzymes produced separately. Furthermore, when combined both enzymes acted synergistically on filter paper to produce cellobiose as the main end-product. Based on these results we propose that P. pastoris should be considered as an alternative platform for the production of cellulases at competitive costs. PMID:26698316

  15. Recombinant GDNF: Tetanus toxin fragment C fusion protein produced from insect cells

    SciTech Connect

    Li, Jianhong; Chian, Ru-Ju; Ay, Ilknur; Celia, Samuel A.; Kashi, Brenda B.; Tamrazian, Eric; Matthews, Jonathan C.; Remington, Mary P.; Pepinsky, R. Blake; Fishman, Paul S.; Brown, Robert H.; Francis, Jonathan W.

    2009-07-31

    Glial cell line-derived neurotrophic factor (GDNF) has potent survival-promoting effects on CNS motor neurons in experimental animals. Its therapeutic efficacy in humans, however, may have been limited by poor bioavailability to the brain and spinal cord. With a view toward improving delivery of GDNF to CNS motor neurons in vivo, we generated a recombinant fusion protein comprised of rat GDNF linked to the non-toxic, neuron-binding fragment of tetanus toxin. Recombinant GDNF:TTC produced from insect cells was a soluble homodimer like wild-type GDNF and was bi-functional with respect to GDNF and TTC activity. Like recombinant rat GDNF, the fusion protein increased levels of immunoreactive phosphoAkt in treated NB41A3-hGFR{alpha}-1 neuroblastoma cells. Like TTC, GDNF:TTC bound to immobilized ganglioside GT1b in vitro with high affinity and selectivity. These results support further testing of recombinant GDNF:TTC as a non-viral vector to improve delivery of GDNF to brain and spinal cord in vivo.

  16. Enhanced HIV-1 neutralization by a CD4-VH3-IgG1 fusion protein

    SciTech Connect

    Meyuhas, Ronit; Noy, Hava; Fishman, Sigal; Margalit, Alon; Montefiori, David C.; Gross, Gideon

    2009-08-21

    HIV-1 gp120 is an alleged B cell superantigen, binding certain VH3+ human antibodies. We reasoned that a CD4-VH3 fusion protein could possess higher affinity for gp120 and improved HIV-1 inhibitory capacity. To test this we produced several human IgG1 immunoligands harboring VH3. Unlike VH3-IgG1 or VH3-CD4-IgG1, CD4-VH3-IgG1 bound gp120 considerably stronger than CD4-IgG1. CD4-VH3-IgG1 exhibited {approx}1.5-2.5-fold increase in neutralization of two T-cell laboratory-adapted strains when compared to CD4-IgG1. CD4-VH3-IgG1 improved neutralization of 7/10 clade B primary isolates or pseudoviruses, exceeding 20-fold for JR-FL and 13-fold for Ba-L. It enhanced neutralization of 4/8 clade C viruses, and had negligible effect on 1/4 clade A pseudoviruses. We attribute this improvement to possible pairing of VH3 with CD4 D1 and stabilization of an Ig Fv-like structure, rather than to superantigen interactions. These novel findings support the current notion that CD4 fusion proteins can act as better HIV-1 entry inhibitors with potential clinical implications.

  17. Chemokines, costimulatory molecules and fusion proteins for the immunotherapy of solid tumors

    PubMed Central

    Lechner, Melissa G; Russell, Sarah M; Bass, Rikki S; Epstein, Alan L

    2011-01-01

    In this article, the role of chemokines and costimulatory molecules in the immunotherapy of experimental murine solid tumors and immunotherapy used in ongoing clinical trials are presented. Chemokine networks regulate physiologic cell migration that may be disrupted to inhibit antitumor immune responses or coopted to promote tumor growth and metastasis in cancer. Recent studies highlight the potential use of chemokines in cancer immunotherapy to improve innate and adaptive cell interactions and to recruit immune effector cells into the tumor microenvironment. Another critical component of antitumor immune responses is antigen priming and activation of effector cells. Reciprocal expression and binding of costimulatory molecules and their ligands by antigen-presenting cells and naive lymphocytes ensures robust expansion, activity and survival of tumor-specific effector cells in vivo. Immunotherapy approaches using agonist antibodies or fusion proteins of immunomodulatory molecules significantly inhibit tumor growth and boost cell-mediated immunity. To localize immune stimulation to the tumor site, a series of fusion proteins consisting of a tumor-targeting monoclonal antibody directed against tumor necrosis and chemokines or costimulatory molecules were generated and tested in tumor-bearing mice. While several of these reagents were initially shown to have therapeutic value, combination therapies with methods to delete suppressor cells had the greatest effect on tum