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Sample records for recombinantly produced hydrophobins

  1. Novel Hydrophobin Fusion Tags for Plant-Produced Fusion Proteins

    PubMed Central

    Ritala, Anneli; Linder, Markus; Joensuu, Jussi

    2016-01-01

    Hydrophobin fusion technology has been applied in the expression of several recombinant proteins in plants. Until now, the technology has relied exclusively on the Trichoderma reesei hydrophobin HFBI. We screened eight novel hydrophobin tags, T. reesei HFBII, HFBIII, HFBIV, HFBV, HFBVI and Fusarium verticillioides derived HYD3, HYD4 and HYD5, for production of fusion proteins in plants and purification by two-phase separation. To study the properties of the hydrophobins, we used N-terminal and C-terminal GFP as a fusion partner. Transient expression of the hydrophobin fusions in Nicotiana benthamiana revealed large variability in accumulation levels, which was also reflected in formation of protein bodies. In two-phase separations, only HFBII and HFBIV were able to concentrate GFP into the surfactant phase from a plant extract. The separation efficiency of both tags was comparable to HFBI. When the accumulation was tested side by side, HFBII-GFP gave a better yield than HFBI-GFP, while the yield of HFBIV-GFP remained lower. Thus we present here two alternatives for HFBI as functional fusion tags for plant-based protein production and first step purification. PMID:27706254

  2. Scale-up of hydrophobin-assisted recombinant protein production in tobacco BY-2 suspension cells.

    PubMed

    Reuter, Lauri J; Bailey, Michael J; Joensuu, Jussi J; Ritala, Anneli

    2014-05-01

    Plant suspension cell cultures are emerging as an alternative to mammalian cells for production of complex recombinant proteins. Plant cell cultures provide low production cost, intrinsic safety and adherence to current regulations, but low yields and costly purification technology hinder their commercialization. Fungal hydrophobins have been utilized as fusion tags to improve yields and facilitate efficient low-cost purification by surfactant-based aqueous two-phase separation (ATPS) in plant, fungal and insect cells. In this work, we report the utilization of hydrophobin fusion technology in tobacco bright yellow 2 (BY-2) suspension cell platform and the establishment of pilot-scale propagation and downstream processing including first-step purification by ATPS. Green fluorescent protein-hydrophobin fusion (GFP-HFBI) induced the formation of protein bodies in tobacco suspension cells, thus encapsulating the fusion protein into discrete compartments. Cultivation of the BY-2 suspension cells was scaled up in standard stirred tank bioreactors up to 600 L production volume, with no apparent change in growth kinetics. Subsequently, ATPS was applied to selectively capture the GFP-HFBI product from crude cell lysate, resulting in threefold concentration, good purity and up to 60% recovery. The ATPS was scaled up to 20 L volume, without loss off efficiency. This study provides the first proof of concept for large-scale hydrophobin-assisted production of recombinant proteins in tobacco BY-2 cell suspensions.

  3. Fungal Hydrophobin Proteins Produce Self-Assembling Protein Films with Diverse Structure and Chemical Stability

    PubMed Central

    Lo, Victor C.; Ren, Qin; Pham, Chi L. L.; Morris, Vanessa K.; Kwan, Ann H.; Sunde, Margaret

    2014-01-01

    Hydrophobins are small proteins secreted by fungi and which spontaneously assemble into amphipathic layers at hydrophilic-hydrophobic interfaces. We have examined the self-assembly of the Class I hydrophobins EAS∆15 and DewA, the Class II hydrophobin NC2 and an engineered chimeric hydrophobin. These Class I hydrophobins form layers composed of laterally associated fibrils with an underlying amyloid structure. These two Class I hydrophobins, despite showing significant conformational differences in solution, self-assemble to form fibrillar layers with very similar structures and require a hydrophilic-hydrophobic interface to trigger self-assembly. Addition of additives that influence surface tension can be used to manipulate the fine structure of the protein films. The Class II hydrophobin NC2 forms a mesh-like protein network and the engineered chimeric hydrophobin displays two multimeric forms, depending on assembly conditions. When formed on a graphite surface, the fibrillar EAS∆15 layers are resistant to alcohol, acid and basic washes. In contrast, the NC2 Class II monolayers are dissociated by alcohol treatment but are relatively stable towards acid and base washes. The engineered chimeric Class I/II hydrophobin shows increased stability towards alcohol and acid and base washes. Self-assembled hydrophobin films may have extensive applications in biotechnology where biocompatible; amphipathic coatings facilitate the functionalization of nanomaterials.

  4. Self-assembled hydrophobin for producing water-soluble and membrane permeable fluorescent dye.

    PubMed

    Wang, Kunpeng; Xiao, Yunjie; Wang, Yanyan; Feng, Yaqing; Chen, Cheng; Zhang, Jie; Zhang, Qian; Meng, Shuxian; Wang, Zefang; Yang, Haitao

    2016-03-15

    Low water solubility and poor membrane permeability are major disadvantages that compromise applications of most fluorescent dyes. To resolve these problems, herein, using Boron-dipyrromethene (BODIPY) as a model fluorescent dye, for the first time, we provide a new strategy for the rapid and efficient production of a water-soluble and membrane-permeable dye by mixing with an amphiphilic protein named hydrophobin. Data shows BODIPY could be effectively solubilized and dispersed in 200 μg/mL hydrophobin by simple mixing and sonication. Subsequent experiments indicated that hydrophobin self-assembled into a protein film on the surface of BODIPY forming stable hydrophobin-BODIPY complexes with a size range of 10-30 nm. Furthermore, we demonstrated hydrophobin-functionalized BODIPY are toxicity free to cells. The hydrophobin-BODIPY complex could pass through both the cell plasma membrane and nuclear membrane efficiently. Our work opens a novel route to modify and functionalize fluorescent dyes and may be developed as a general strategy for broadening their applications.

  5. Self-assembled hydrophobin for producing water-soluble and membrane permeable fluorescent dye

    PubMed Central

    Wang, Kunpeng; Xiao, Yunjie; Wang, Yanyan; Feng, Yaqing; Chen, Cheng; Zhang, Jie; Zhang, Qian; Meng, Shuxian; Wang, Zefang; Yang, Haitao

    2016-01-01

    Low water solubility and poor membrane permeability are major disadvantages that compromise applications of most fluorescent dyes. To resolve these problems, herein, using Boron-dipyrromethene (BODIPY) as a model fluorescent dye, for the first time, we provide a new strategy for the rapid and efficient production of a water-soluble and membrane-permeable dye by mixing with an amphiphilic protein named hydrophobin. Data shows BODIPY could be effectively solubilized and dispersed in 200 μg/mL hydrophobin by simple mixing and sonication. Subsequent experiments indicated that hydrophobin self-assembled into a protein film on the surface of BODIPY forming stable hydrophobin-BODIPY complexes with a size range of 10–30 nm. Furthermore, we demonstrated hydrophobin-functionalized BODIPY are toxicity free to cells. The hydrophobin-BODIPY complex could pass through both the cell plasma membrane and nuclear membrane efficiently. Our work opens a novel route to modify and functionalize fluorescent dyes and may be developed as a general strategy for broadening their applications. PMID:26976627

  6. Influence of Elastin-Like Polypeptide and Hydrophobin on Recombinant Hemagglutinin Accumulations in Transgenic Tobacco Plants

    PubMed Central

    Phan, Hoang Trong; Hause, Bettina; Hause, Gerd; Arcalis, Elsa; Stoger, Eva; Maresch, Daniel; Altmann, Friedrich; Joensuu, Jussi; Conrad, Udo

    2014-01-01

    Fusion protein strategies are useful tools to enhance expression and to support the development of purification technologies. The capacity of fusion protein strategies to enhance expression was explored in tobacco leaves and seeds. C-terminal fusion of elastin-like polypeptides (ELP) to influenza hemagglutinin under the control of either the constitutive CaMV 35S or the seed-specific USP promoter resulted in increased accumulation in both leaves and seeds compared to the unfused hemagglutinin. The addition of a hydrophobin to the C-terminal end of hemagglutinin did not significantly increase the expression level. We show here that, depending on the target protein, both hydrophobin fusion and ELPylation combined with endoplasmic reticulum (ER) targeting induced protein bodies in leaves as well as in seeds. The N-glycosylation pattern indicated that KDEL sequence-mediated retention of leaf-derived hemagglutinins and hemagglutinin-hydrophobin fusions were not completely retained in the ER. In contrast, hemagglutinin-ELP from leaves contained only the oligomannose form, suggesting complete ER retention. In seeds, ER retention seems to be nearly complete for all three constructs. An easy and scalable purification method for ELPylated proteins using membrane-based inverse transition cycling could be applied to both leaf- and seed-expressed hemagglutinins. PMID:24914995

  7. Production and characterization of in planta transiently produced polygalacturanase from Aspergillus niger and its fusions with hydrophobin or ELP tags

    PubMed Central

    2014-01-01

    Background Pectinases play an important role in plant cell wall deconstruction and have potential in diverse industries such as food, wine, animal feed, textile, paper, fuel, and others. The demand for such enzymes is increasing exponentially, as are the efforts to improve their production and to implement their use in several industrial processes. The goal of this study was to examine the potential of producing polygalacturonase I from Aspergillus niger in plants and to investigate the effects of subcellular compartmentalization and protein fusions on its accumulation and activity. Results Polygalacturonase I from Aspergillus niger (AnPGI) was transiently produced in Nicotiana benthamiana by targeting it to five different cellular compartments: apoplast, endoplasmic reticulum (ER), vacuole, chloroplast and cytosol. Accumulation levels of 2.5%, 3.0%, and 1.9% of total soluble protein (TSP) were observed in the apoplast, ER, and vacuole, respectively, and specific activity was significantly higher in vacuole-targeted AnPGI compared to the same enzyme targeted to the ER or apoplast. No accumulation was found for AnPGI when targeted to the chloroplast or cytosol. Analysis of AnPGI fused with elastin-like polypeptide (ELP) revealed a significant increase in the protein accumulation level, especially when targeted to the vacuole where the protein doubles its accumulation to 3.6% of TSP, while the hydrophobin (HFBI) fusion impaired AnPGI accumulation and both tags impaired activity, albeit to different extents. The recombinant protein showed activity against polygalacturonic acid with optimum conditions at pH 5.0 and temperature from 30 to 50°C, depending on its fusion. In vivo analysis of reducing sugar content revealed a higher release of reducing sugars in plant tissue expressing recombinant AnPGI compared to wild type N. benthamiana leaves. Conclusion Our results demonstrate that subcellular compartmentalization of enzymes has an impact on both the target protein

  8. Cultivating Insect Cells To Produce Recombinant Proteins

    NASA Technical Reports Server (NTRS)

    Spaulding, Glenn; Goodwin, Thomas; Prewett, Tacey; Andrews, Angela; Francis, Karen; O'Connor, Kim

    1996-01-01

    Method of producing recombinant proteins involves growth of insect cells in nutrient solution in cylindrical bioreactor rotating about cylindrical axis, oriented horizontally and infecting cells with viruses into which genes of selected type cloned. Genes in question those encoding production of desired proteins. Horizontal rotating bioreactor preferred for use in method, denoted by acronym "HARV", described in "High-Aspect-Ratio Rotating Cell-Culture Vessel" (MSC-21662).

  9. Hydrophobin-Encapsulated Quantum Dots.

    PubMed

    Taniguchi, Shohei; Sandiford, Lydia; Cooper, Maggie; Rosca, Elena V; Ahmad Khanbeigi, Raha; Fairclough, Simon M; Thanou, Maya; Dailey, Lea Ann; Wohlleben, Wendel; von Vacano, Bernhard; de Rosales, Rafael T M; Dobson, Peter J; Owen, Dylan M; Green, Mark

    2016-02-01

    The phase transfer of quantum dots to water is an important aspect of preparing nanomaterials that are suitable for biological applications, and although numerous reports describe ligand exchange, very few describe efficient ligand encapsulation techniques. In this report, we not only report a new method of phase transferring quantum dots (QDs) using an amphiphilic protein (hydrophobin) but also describe the advantages of using a biological molecule with available functional groups and their use in imaging cancer cells in vivo and other imaging applications.

  10. High-yield production of hydrophobins RodA and RodB from Aspergillus fumigatus in Pichia pastoris.

    PubMed

    Pedersen, Mona Højgaard; Borodina, Irina; Moresco, Jacob Lange; Svendsen, Winnie Edith; Frisvad, Jens Christian; Søndergaard, Ib

    2011-06-01

    Hydrophobins are small fungal proteins with amphipatic properties and the ability to self-assemble on a hydrophobic/hydrophilic interface; thus, many technical applications for hydrophobins have been suggested. The pathogenic fungus Aspergillus fumigatus expresses the hydrophobins RodA and RodB on the surface of its conidia. RodA is known to be of importance to the pathogenesis of the fungus, while the biological role of RodB is currently unknown. Here, we report the successful expression of both hydrophobins in Pichia pastoris and present fed-batch fermentation yields of 200-300 mg/l fermentation broth. Protein bands of expected sizes were detected by SDS-PAGE and western blotting, and the identity was further confirmed by tandem mass spectrometry. Both proteins were purified using his-affinity chromatography, and the high level of purity was verified by silver-stained SDS-PAGE. Recombinant RodA as well as rRodB were able to convert a glass surface from hydrophilic to hydrophobic similar to native RodA, but only rRodB was able to decrease the hydrophobicity of a Teflon-like surface to the same extent as native RodA, while rRodA showed this ability to a lesser extent. Recombinant RodA and native RodA showed a similar ability to emulsify air in water, while recombinant RodB could also emulsify oil in water better than the control protein bovine serum albumin (BSA). This is to our knowledge the first successful expression of hydrophobins from A. fumigatus in a eukaryote host, which makes it possible to further characterize both hydrophobins. Furthermore, the expression strategy and fed-batch production using P. pastoris may be transferred to hydrophobins from other species.

  11. Hydrophobin-Based Surface Engineering for Sensitive and Robust Quantification of Yeast Pheromones

    PubMed Central

    Hennig, Stefan; Rödel, Gerhard; Ostermann, Kai

    2016-01-01

    Detection and quantification of small peptides, such as yeast pheromones, are often challenging. We developed a highly sensitive and robust affinity-assay for the quantification of the α-factor pheromone of Saccharomyces cerevisiae based on recombinant hydrophobins. These small, amphipathic proteins self-assemble into highly stable monolayers at hydrophilic-hydrophobic interfaces. Upon functionalization of solid supports with a combination of hydrophobins either lacking or exposing the α-factor, pheromone-specific antibodies were bound to the surface. Increasing concentrations of the pheromone competitively detached the antibodies, thus allowing for quantification of the pheromone. By adjusting the percentage of pheromone-exposing hydrophobins, the sensitivity of the assay could be precisely predefined. The assay proved to be highly robust against changes in sample matrix composition. Due to the high stability of hydrophobin layers, the functionalized surfaces could be repeatedly used without affecting the sensitivity. Furthermore, by using an inverse setup, the sensitivity was increased by three orders of magnitude, yielding a novel kind of biosensor for the yeast pheromone with the lowest limit of detection reported so far. This assay was applied to study the pheromone secretion of diverse yeast strains including a whole-cell biosensor strain of Schizosaccharomyces pombe modulating α-factor secretion in response to an environmental signal. PMID:27128920

  12. 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

  13. Recent Advances in Fungal Hydrophobin Towards Using in Industry.

    PubMed

    Khalesi, Mohammadreza; Gebruers, Kurt; Derdelinckx, Guy

    2015-08-01

    Fungal hydrophobin is a family of low molecular weight proteins consisting of four disulfide bridges and an extraordinary hydrophobic patch. The hydrophobic patch of hydrophobins and the molecules of gaseous CO2 may interact together and form the stable CO2-nanobubbles covered by an elastic membrane in carbonated beverages. The nanobubbles provide the required energy to provoke primary gushing. Due to the hydrophobicity of hydrophobin, this protein is used as a biosurfactant, foaming agent or encapsulating agent in food products and medicine formulations. Increasing demands for using of hydrophobins led to a challenge regarding production and purification of this product. However, the main issue to use hydrophobin in the industry is the regulatory affairs: yet there is no approved legislation for using hydrophobin in food and beverages. To comply with the legislation, establishing a consistent method for obtaining pure hydrophobins is necessary. Currently, few research teams in Europe are focusing on different aspects of hydrophobins. In this paper, an up-to-date collection of highlights from those special groups about the bio-chemical and physicochemical characteristics of hydrophobins have been studied. The recent advances of those groups concerning the production and purification, positive applications and negative function of hydrophobin are also summarised.

  14. Hydrophobin Film Structure for HFBI and HFBII and Mechanism for Accelerated Film Formation

    PubMed Central

    Magarkar, Aniket; Mele, Nawel; Abdel-Rahman, Noha; Butcher, Sarah; Torkkeli, Mika; Serimaa, Ritva; Paananen, Arja; Linder, Markus; Bunker, Alex

    2014-01-01

    Hydrophobins represent an important group of proteins from both a biological and nanotechnological standpoint. They are the means through which filamentous fungi affect their environment to promote growth, and their properties at interfaces have resulted in numerous applications. In our study we have combined protein docking, molecular dynamics simulation, and electron cryo-microscopy to gain atomistic level insight into the surface structure of films composed of two class II hydrophobins: HFBI and HFBII produced by Trichoderma reesei. Together our results suggest a unit cell composed of six proteins; however, our computational results suggest P6 symmetry, while our experimental results show P3 symmetry with a unit cell size of 56 Å. Our computational results indicate the possibility of an alternate ordering with a three protein unit cell with P3 symmetry and a smaller unit cell size, and we have used a Monte Carlo simulation of a spin model representing the hydrophobin film to show how this alternate metastable structure may play a role in increasing the rate of surface coverage by hydrophobin films, possibly indicating a mechanism of more general significance to both biology and nanotechnology. PMID:25079355

  15. Characterization of a Basidiomycota hydrophobin reveals the structural basis for a high-similarity Class I subdivision

    PubMed Central

    Gandier, Julie-Anne; Langelaan, David N.; Won, Amy; O’Donnell, Kylie; Grondin, Julie L.; Spencer, Holly L.; Wong, Philip; Tillier, Elisabeth; Yip, Christopher; Smith, Steven P.; Master, Emma R.

    2017-01-01

    Class I hydrophobins are functional amyloids secreted by fungi. They self-assemble into organized films at interfaces producing structures that include cellular adhesion points and hydrophobic coatings. Here, we present the first structure and solution properties of a unique Class I protein sequence of Basidiomycota origin: the Schizophyllum commune hydrophobin SC16 (hyd1). While the core β-barrel structure and disulphide bridging characteristic of the hydrophobin family are conserved, its surface properties and secondary structure elements are reminiscent of both Class I and II hydrophobins. Sequence analyses of hydrophobins from 215 fungal species suggest this structure is largely applicable to a high-identity Basidiomycota Class I subdivision (IB). To validate this prediction, structural analysis of a comparatively distinct Class IB sequence from a different fungal order, namely the Phanerochaete carnosa PcaHyd1, indicates secondary structure properties similar to that of SC16. Together, these results form an experimental basis for a high-identity Class I subdivision and contribute to our understanding of functional amyloid formation. PMID:28393921

  16. Characterization of a Basidiomycota hydrophobin reveals the structural basis for a high-similarity Class I subdivision.

    PubMed

    Gandier, Julie-Anne; Langelaan, David N; Won, Amy; O'Donnell, Kylie; Grondin, Julie L; Spencer, Holly L; Wong, Philip; Tillier, Elisabeth; Yip, Christopher; Smith, Steven P; Master, Emma R

    2017-04-10

    Class I hydrophobins are functional amyloids secreted by fungi. They self-assemble into organized films at interfaces producing structures that include cellular adhesion points and hydrophobic coatings. Here, we present the first structure and solution properties of a unique Class I protein sequence of Basidiomycota origin: the Schizophyllum commune hydrophobin SC16 (hyd1). While the core β-barrel structure and disulphide bridging characteristic of the hydrophobin family are conserved, its surface properties and secondary structure elements are reminiscent of both Class I and II hydrophobins. Sequence analyses of hydrophobins from 215 fungal species suggest this structure is largely applicable to a high-identity Basidiomycota Class I subdivision (IB). To validate this prediction, structural analysis of a comparatively distinct Class IB sequence from a different fungal order, namely the Phanerochaete carnosa PcaHyd1, indicates secondary structure properties similar to that of SC16. Together, these results form an experimental basis for a high-identity Class I subdivision and contribute to our understanding of functional amyloid formation.

  17. Recombinant Human Factor IX Produced from Transgenic Porcine Milk

    PubMed Central

    Lee, Meng-Hwan; Lin, Yin-Shen; Tu, Ching-Fu; Yen, Chon-Ho

    2014-01-01

    Production of biopharmaceuticals from transgenic animal milk is a cost-effective method for highly complex proteins that cannot be efficiently produced using conventional systems such as microorganisms or animal cells. Yields of recombinant human factor IX (rhFIX) produced from transgenic porcine milk under the control of the bovine α-lactalbumin promoter reached 0.25 mg/mL. The rhFIX protein was purified from transgenic porcine milk using a three-column purification scheme after a precipitation step to remove casein. The purified protein had high specific activity and a low ratio of the active form (FIXa). The purified rhFIX had 11.9 γ-carboxyglutamic acid (Gla) residues/mol protein, which approached full occupancy of the 12 potential sites in the Gla domain. The rhFIX was shown to have a higher isoelectric point and lower sialic acid content than plasma-derived FIX (pdFIX). The rhFIX had the same N-glycosylation sites and phosphorylation sites as pdFIX, but had a higher specific activity. These results suggest that rhFIX produced from porcine milk is physiologically active and they support the use of transgenic animals as bioreactors for industrial scale production in milk. PMID:24955355

  18. Expression of a Fungal Hydrophobin in the Saccharomyces cerevisiae Cell Wall: Effect on Cell Surface Properties and Immobilization

    PubMed Central

    Nakari-Setälä, Tiina; Azeredo, Joana; Henriques, Mariana; Oliveira, Rosário; Teixeira, José; Linder, Markus; Penttilä, Merja

    2002-01-01

    The aim of this work was to modify the cell surface properties of Saccharomyces cerevisiae by expression of the HFBI hydrophobin of the filamentous fungus Trichoderma reesei on the yeast cell surface. The second aim was to study the immobilization capacity of the modified cells. Fusion to the Flo1p flocculin was used to target the HFBI moiety to the cell wall. Determination of cell surface characteristics with contact angle and zeta potential measurements indicated that HFBI-producing cells are more apolar and slightly less negatively charged than the parent cells. Adsorption of the yeast cells to different commercial supports was studied. A twofold increase in the binding affinity of the hydrophobin-producing yeast to hydrophobic silicone-based materials was observed, while no improvement in the interaction with hydrophilic carriers could be seen compared to that of the parent cells. Hydrophobic interactions between the yeast cells and the support are suggested to play a major role in attachment. Also, a slight increase in the initial adsorption rate of the hydrophobin yeast was observed. Furthermore, due to the engineered cell surface, hydrophobin-producing yeast cells were efficiently separated in an aqueous two-phase system by using a nonionic polyoxyethylene detergent, C12-18EO5. PMID:12089019

  19. Quality assessment of recombinant proteins produced in plants.

    PubMed

    Medrano, Giuliana; Dolan, Maureen C; Condori, Jose; Radin, David N; Cramer, Carole L

    2012-01-01

    Plant-based expression technologies for recombinant proteins have begun to receive acceptance for pharmaceuticals and other commercial markets. Protein products derived from plants offer safer, more cost-effective, and less capital-intensive alternatives to traditional manufacturing systems using microbial fermentation or animal cell culture bioreactors. Moreover, plants are now known to be capable of expressing bioactive proteins from a diverse array of species including animals and humans. Methods development to assess the quality and performance of proteins manufactured in plants are essential to support the QA/QC demands as plant-produced protein products transition to the commercial marketplace. Within the pharmaceutical arena, process validation and acceptance criteria for biological products must comply with the Food and Drug Administration (FDA) and ICH Q6B guidelines in order to initiate the regulatory approval process. Detailed product specifications will also need to be developed and validated for plant-made proteins for the bioenergy, food, chemical synthesis, or research reagent markets.We have, therefore, developed assessment methods for important qualitative and quantitative parameters of the products and the manufacturing methods utilized in plant-based production systems. In this chapter, we describe a number of procedures to validate product identity and characteristics including mass analyses, antibody cross-reactivity, N-terminal sequencing, and bioactivity. We also address methods for routine assessment of yield, recovery, and purity. The methods presented are those developed for the synthesis and recovery of the avian cytokine, chicken interleukin-12 (ChIL-12), produced in the leaves of Nicotiana benthamiana. The ChIL-12 protein used as a model for this chapter includes a C-terminal histidine epitope (HIS-tag) and, thus, these methods may be directly applicable to other HIS-tagged proteins produced in plants. However, the overall strategy

  20. Hydrophobins as aqueous lubricant additive for a soft sliding contact.

    PubMed

    Lee, Seunghwan; Røn, Troels; Pakkanen, Kirsi I; Linder, Markus

    2015-01-01

    Two type II fungal hydrophobins, HFBI and FpHYD5, have been studied as aqueous lubricant additive at a nonpolar, compliant sliding contact (self-mated poly(dimethylsiloxane) (PDMS) contact) at two different concentrations, 0.1 mg/mL and 1.0 mg/mL. The two hydrophobins are featured as non-glycosylated (HFBI, m.w. ca. 7 kDa) vs glycosylated (FpHYD5, m.w. ca. 10 kDa) proteins. Far UV CD spectra of the two hydrophobins were very similar, suggesting overall structural similarity, but showed a noticeable difference according to the concentration. This is proposed to be related to the formation of multimers at 1.0 mg/mL. Despite 10-fold difference in the bulk concentration, the adsorbed masses of the hydrophobins onto PDMS surface obtained from the two solutions (0.1 and 1.0 mg/mL) were nearly identical, suggesting that a monolayer of the hydrophobins are formed from 0.1 mg/mL solution. PDMS-PDMS sliding interface was effectively lubricated by the hydrophobin solutions, and showed a reduction in the coefficient of friction by as much as ca. two orders of magnitude. Higher concentration solution (1.0 mg/mL) provided a superior lubrication, particularly in low-speed regime, where boundary lubrication characteristic is dominant via 'self-healing' mechanism. FpHYD5 revealed a better lubrication than HFBI presumably due to the presence of glycans and improved hydration of the sliding interface. Two type II hydrophobins function more favorably compared to a synthetic amphiphilic copolymer, PEO-PPO-PEO, with a similar molecular weight. This is ascribed to higher amount of adsorption of the hydrophobins to hydrophobic surfaces from aqueous solution.

  1. Hydrophobins are required for conidial hydrophobicity and plant root colonization in the fungal biocontrol agent Clonostachys rosea

    PubMed Central

    2014-01-01

    Background Filamentous fungi produce small cysteine rich surface active amphiphilic hydrophobins on the outer surface of cell walls that mediate interactions between the fungus and the environment. The role of hydrophobins in surface hydrophobicity, sporulation, fruit body formation, recognition and adhesion to host surface and virulence have been reported. The aim of the present study was to characterize the biological function of hydrophobins in the fungal biocontrol agent Clonostachys rosea in order to understand their potential roles in biocontrol mechanisms. Results Based on the presence of hydrophobin domains, cysteine spacing patterns and hydropathy plots, we identified three class II hydrophobin genes in C. rosea. Gene expression analysis showed basal expression of Hyd1, Hyd2 and Hyd3 in all conditions tested with the exception of induced Hyd1 expression in conidiating mycelium. Interestingly, up-regulation of Hyd1, Hyd2 and Hyd3 was found during C. rosea self interaction compared to interactions with the fungal plant pathogens Botrytis cinerea or Fusarium graminearum in dual culture assays. Phenotypic analysis of C. rosea deletion and complementation strains showed that Hyd1 and Hyd3 are jointly required for conidial hydrophobicity, although no difference in mycelia hydrophobicity was found between wild type (WT) and mutant strains. Interestingly, mutant strains showed increased growth rates, conidiation and enhanced tolerances of conidia to abiotic stresses. Antagonism tests using in vitro dual culture and detached leaf assays showed that the mutant strains were more aggressive towards B. cinerea, F. graminearum or Rhizoctonia solani, and that aggression was partly related to earlier conidial germination and enhanced tolerance of mutant strains to secreted fungal metabolites. Furthermore, in vitro Arabidopsis thaliana root colonization assays revealed reduced root colonization ability of the ΔHyd3 strain, but not for the ΔHyd1 strain. Furthermore

  2. Vaccinia virus vectors: new strategies for producing recombinant vaccines.

    PubMed Central

    Hruby, D E

    1990-01-01

    The development and continued refinement of techniques for the efficient insertion and expression of heterologous DNA sequences from within the genomic context of infectious vaccinia virus recombinants are among the most promising current approaches towards effective immunoprophylaxis against a variety of protozoan, viral, and bacterial human pathogens. Because of its medical relevance, this area is the subject of intense research interest and has evolved rapidly during the past several years. This review (i) provides an updated overview of the technology that exists for assembling recombinant vaccinia virus strains, (ii) discusses the advantages and disadvantages of these approaches, (iii) outlines the areas of outgoing research directed towards overcoming the limitations of current techniques, and (iv) provides some insight (i.e., speculation) about probable future refinements in the use of vaccinia virus as a vector. PMID:2187593

  3. Human recombinant type I collagen produced in plants.

    PubMed

    Shoseyov, Oded; Posen, Yehudit; Grynspan, Frida

    2013-07-01

    As a central element of the extracellular matrix, collagen is intimately involved in tissue development, remodeling, and repair and confers high tensile strength to tissues. Numerous medical applications, particularly, wound healing, cell therapy, bone reconstruction, and cosmetic technologies, rely on its supportive and healing qualities. Its synthesis and assembly require a multitude of genes and post-translational modifications, where even minor deviations can be deleterious or even fatal. Historically, collagen was always extracted from animal and human cadaver sources, but bare risk of contamination and allergenicity and was subjected to harsh purification conditions resulting in irreversible modifications impeding its biofunctionality. In parallel, the highly complex and stringent post-translational processing of collagen, prerequisite of its viability and proper functioning, sets significant limitations on recombinant expression systems. A tobacco plant expression platform has been recruited to effectively express human collagen, along with three modifying enzymes, critical to collagen maturation. The plant extracted recombinant human collagen type I forms thermally stable helical structures, fibrillates, and demonstrates bioactivity resembling that of native collagen. Deployment of the highly versatile plant-based biofactory can be leveraged toward mass, rapid, and low-cost production of a wide variety of recombinant proteins. As in the case of collagen, proper planning can bypass plant-related limitations, to yield products structurally and functionally identical to their native counterparts.

  4. 21 CFR 878.4494 - Absorbable poly(hydroxybutyrate) surgical suture produced by recombinant DNA technology.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Absorbable poly(hydroxybutyrate) surgical suture produced by recombinant DNA technology. 878.4494 Section 878.4494 Food and Drugs FOOD AND DRUG... recombinant DNA technology. (a) Identification. An absorbable poly(hydroxybutyrate) surgical suture is...

  5. 21 CFR 878.4494 - Absorbable poly(hydroxybutyrate) surgical suture produced by recombinant DNA technology.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Absorbable poly(hydroxybutyrate) surgical suture produced by recombinant DNA technology. 878.4494 Section 878.4494 Food and Drugs FOOD AND DRUG... recombinant DNA technology. (a) Identification. An absorbable poly(hydroxybutyrate) surgical suture is...

  6. 21 CFR 878.4494 - Absorbable poly(hydroxybutyrate) surgical suture produced by recombinant DNA technology.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Absorbable poly(hydroxybutyrate) surgical suture produced by recombinant DNA technology. 878.4494 Section 878.4494 Food and Drugs FOOD AND DRUG... recombinant DNA technology. (a) Identification. An absorbable poly(hydroxybutyrate) surgical suture is...

  7. 21 CFR 878.4494 - Absorbable poly(hydroxybutyrate) surgical suture produced by recombinant DNA technology.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... produced by recombinant DNA technology. 878.4494 Section 878.4494 Food and Drugs FOOD AND DRUG... recombinant DNA technology. (a) Identification. An absorbable poly(hydroxybutyrate) surgical suture is an... deoxyribonucleic acid (DNA) technology. The device is intended for use in general soft tissue approximation...

  8. 21 CFR 878.4494 - Absorbable poly(hydroxybutyrate) surgical suture produced by recombinant DNA technology.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... produced by recombinant DNA technology. 878.4494 Section 878.4494 Food and Drugs FOOD AND DRUG... recombinant DNA technology. (a) Identification. An absorbable poly(hydroxybutyrate) surgical suture is an... deoxyribonucleic acid (DNA) technology. The device is intended for use in general soft tissue approximation...

  9. A Highly Efficient and Simple Construction Strategy for Producing Recombinant Baculovirus Bombyx mori Nucleopolyhedrovirus

    PubMed Central

    Liu, Xingjian; Wei, Yonglong; Li, Yinü; Li, Haoyang; Yang, Xin; Yi, Yongzhu; Zhang, Zhifang

    2016-01-01

    The silkworm baculovirus expression system is widely used to produce recombinant proteins. Several strategies for constructing recombinant viruses that contain foreign genes have been reported. Here, we developed a novel defective-rescue BmNPV Bacmid (reBmBac) expression system. A CopyControl origin of replication was introduced into the viral genome to facilitate its genetic manipulation in Escherichia coli and to ensure the preparation of large amounts of high quality reBmBac DNA as well as high quality recombinant baculoviruses. The ORF1629, cathepsin and chitinase genes were partially deleted or rendered defective to improve the efficiency of recombinant baculovirus generation and the expression of foreign genes. The system was validated by the successful expression of luciferase reporter gene and porcine interferon γ. This system can be used to produce batches of recombinant baculoviruses and target proteins rapidly and efficiently in silkworms. PMID:27008267

  10. Six Hydrophobins Are Involved in Hydrophobin Rodlet Formation in Aspergillus nidulans and Contribute to Hydrophobicity of the Spore Surface

    PubMed Central

    Seidel, Constanze; Gutt, Beatrice; Röhrig, Julian; Strunk, Timo; Vincze, Paul; Walheim, Stefan; Schimmel, Thomas; Wenzel, Wolfgang; Fischer, Reinhard

    2014-01-01

    Hydrophobins are amphiphilic proteins able to self-assemble at water-air interphases and are only found in filamentous fungi. In Aspergillus nidulans two hydrophobins, RodA and DewA, have been characterized, which both localize on the conidiospore surface and contribute to its hydrophobicity. RodA is the constituent protein of very regularly arranged rodlets, 10 nm in diameter. Here we analyzed four more hydrophobins, DewB-E, in A. nidulans and found that all six hydrophobins contribute to the hydrophobic surface of the conidiospores but only deletion of rodA caused loss of the rodlet structure. Analysis of the rodlets in the dewB-E deletion strains with atomic force microscopy revealed that the rodlets appeared less robust. Expression of DewA and DewB driven from the rodA promoter and secreted with the RodA secretion signal in a strain lacking RodA, restored partly the hydrophobicity. DewA and B were able to form rodlets to some extent but never reached the rodlet structure of RodA. The rodlet-lacking rodA-deletion strain opens the possibility to systematically study rodlet formation of other natural or synthetic hydrophobins. PMID:24722460

  11. Six hydrophobins are involved in hydrophobin rodlet formation in Aspergillus nidulans and contribute to hydrophobicity of the spore surface.

    PubMed

    Grünbacher, André; Throm, Tanja; Seidel, Constanze; Gutt, Beatrice; Röhrig, Julian; Strunk, Timo; Vincze, Paul; Walheim, Stefan; Schimmel, Thomas; Wenzel, Wolfgang; Fischer, Reinhard

    2014-01-01

    Hydrophobins are amphiphilic proteins able to self-assemble at water-air interphases and are only found in filamentous fungi. In Aspergillus nidulans two hydrophobins, RodA and DewA, have been characterized, which both localize on the conidiospore surface and contribute to its hydrophobicity. RodA is the constituent protein of very regularly arranged rodlets, 10 nm in diameter. Here we analyzed four more hydrophobins, DewB-E, in A. nidulans and found that all six hydrophobins contribute to the hydrophobic surface of the conidiospores but only deletion of rodA caused loss of the rodlet structure. Analysis of the rodlets in the dewB-E deletion strains with atomic force microscopy revealed that the rodlets appeared less robust. Expression of DewA and DewB driven from the rodA promoter and secreted with the RodA secretion signal in a strain lacking RodA, restored partly the hydrophobicity. DewA and B were able to form rodlets to some extent but never reached the rodlet structure of RodA. The rodlet-lacking rodA-deletion strain opens the possibility to systematically study rodlet formation of other natural or synthetic hydrophobins.

  12. Langmuir Blodgett films of hydrophobins HFBI and HFBII

    NASA Astrophysics Data System (ADS)

    Kisko, Kaisa; Torkkeli, Mika; Vuorimaa, Elina; Lemmetyinen, Helge; Seeck, Oliver H.; Linder, Markus; Serimaa, Ritva

    2005-06-01

    Hydrophobins are small fungal proteins, which have remarkable surface-chemical properties. They self-assemble at hydrophobic/hydrophilic interfaces and work as adhesive agents and coatings. Sixteen layer Langmuir-Blodgett films of hydrophobins HFBI and HFBII from the fungus Trichoderma reesei were prepared and studied using grazing-incidence X-ray diffraction and reflectivity techniques. Both kind of films contain hexagonally ordered crystallites on the substrate with unit cell parameters of a = b = 54 Å (HFBI) and a = b = 55 Å (HFBII). The structure is similar to the structure of monolayer Langmuir-Blodgett films.

  13. Diffusion Of Hydrophobin Proteins In Solution And Interactions With A Graphite Surface

    SciTech Connect

    Mereghetti, Paolo; Wade, Rebecca C.

    2011-04-21

    Background Hydrophobins are small proteins produced by filamentous fungi that have a variety of biological functions including coating of spores and surface adhesion. To accomplish these functions, they rely on unique interface-binding properties. Using atomic-detail implicit solvent rigid-body Brownian dynamics simulations, we studied the diffusion of HFBI, a class II hydrophobin from Trichoderma reesei, in aqueous solution in the presence and absence of a graphite surface. Results In the simulations, HFBI exists in solution as a mixture of monomers in equilibrium with different types of oligomers. The oligomerization state depends on the conformation of HFBI. When a Highly Ordered Pyrolytic Graphite (HOPG) layer is present in the simulated system, HFBI tends to interact with the HOPG layer through a hydrophobic patch on the protein. Conclusions From the simulations of HFBI solutions, we identify a tetrameric encounter complex stabilized by non-polar interactions between the aliphatic residues in the hydrophobic patch on HFBI. After the formation of the encounter complex, a local structural rearrangement at the protein interfaces is required to obtain the tetrameric arrangement seen in HFBI crystals. Simulations performed with the graphite surface show that, due to a combination of a geometric hindrance and the interaction of the aliphatic sidechains with the graphite layer, HFBI proteins tend to accumulate close to the hydrophobic surface.

  14. Generation of polyclonal antibodies against recombinant human glucocerebrosidase produced in Escherichia coli.

    PubMed

    Novo, Juliana Branco; Oliveira, Maria Leonor Sarno; Magalhães, Geraldo Santana; Morganti, Ligia; Raw, Isaías; Ho, Paulo Lee

    2010-11-01

    Deficiency of the lysosomal glucocerebrosidase (GCR) enzyme results in Gaucher's disease, the most common inherited storage disorder. Treatment consists of enzyme replacement therapy by the administration of recombinant GCR produced in Chinese hamster ovary cells. The production of anti-GCR antibodies has already been described with placenta-derived human GCR that requires successive chromatographic procedures. Here, we report a practical and efficient method to obtain anti-GCR polyclonal antibodies against recombinant GCR produced in Escherichia coli and further purified by a single step through nickel affinity chromatography. The purified GCR was used to immunize BALB/c mice and the induction of anti-GCR antibodies was evaluated by enzyme-linked immunosorbent assay. The specificity of the antiserum was also evaluated by western blot analysis against recombinant GCR produced by COS-7 cells or against endogenous GCR of human cell lines. GCR was strongly recognized by the produced antibodies, either as cell-associated or as secreted forms. The detected molecular masses of 59-66 kDa are in accordance to the expected size for glycosylated GCR. The GCR produced in E. coli would facilitate the production of polyclonal (shown here) and monoclonal antibodies and their use in the characterization of new biosimilar recombinant GCRs coming in the near future.

  15. Producing recombinant therapeutic glycoproteins with enhanced sialylation using CHO-gmt4 glycosylation mutant cells

    PubMed Central

    Goh, John SY; Liu, Yingwei; Chan, Kah Fai; Wan, Corrine; Teo, Gavin; Zhang, Peiqing; Zhang, Yuanxing; Song, Zhiwei

    2014-01-01

    Recombinant glycoprotein drugs require proper glycosylation for optimal therapeutic efficacy. Glycoprotein therapeutics are rapidly removed from circulation and have reduced efficacy if they are poorly sialylated. Ricinus communis agglutinin-I (RCA-I) was found highly toxic to wild-type CHO-K1 cells and all the mutants that survived RCA-I treatment contained a dysfunctional N-acetylglucosaminyltransferase I (GnT I) gene. These mutants are named CHO-gmt4 cells. Interestingly, upon restoration of GnT I, the sialylation of a model glycoprotein, erythropoietin, produced in CHO-gmt4 cells was shown to be superior to that produced in wild-type CHO-K1 cells. This addendum summarizes the applicability of this cell line, from transient to stable expression of the recombinant protein, and from a lab scale to an industrial scale perfusion bioreactor. In addition, CHO-gmt4 cells can be used to produce glycoproteins with mannose-terminated N-glycans. Recombinant glucocerebrosidase produced by CHO-gmt4 cells will not require glycan remodeling and may be directly used to treat patients with Gaucher disease. CHO-gmt4 cells can also be used to produce other glycoprotein therapeutics which target cells expressing mannose receptors. PMID:24911584

  16. Producing recombinant therapeutic glycoproteins with enhanced sialylation using CHO-gmt4 glycosylation mutant cells.

    PubMed

    Goh, John S Y; Liu, Yingwei; Chan, Kah Fai; Wan, Corrine; Teo, Gavin; Zhang, Peiqing; Zhang, Yuanxing; Song, Zhiwei

    2014-01-01

    Recombinant glycoprotein drugs require proper glycosylation for optimal therapeutic efficacy. Glycoprotein therapeutics are rapidly removed from circulation and have reduced efficacy if they are poorly sialylated. Ricinus communis agglutinin-I (RCA-I) was found highly toxic to wild-type CHO-K1 cells and all the mutants that survived RCA-I treatment contained a dysfunctional N-acetylglucosaminyltransferase I (GnT I) gene. These mutants are named CHO-gmt4 cells. Interestingly, upon restoration of GnT I, the sialylation of a model glycoprotein, erythropoietin, produced in CHO-gmt4 cells was shown to be superior to that produced in wild-type CHO-K1 cells. This addendum summarizes the applicability of this cell line, from transient to stable expression of the recombinant protein, and from a lab scale to an industrial scale perfusion bioreactor. In addition, CHO-gmt4 cells can be used to produce glycoproteins with mannose-terminated N-glycans. Recombinant glucocerebrosidase produced by CHO-gmt4 cells will not require glycan remodeling and may be directly used to treat patients with Gaucher disease. CHO-gmt4 cells can also be used to produce other glycoprotein therapeutics which target cells expressing mannose receptors.

  17. Recombinant human N-acetylgalactosamine-6-sulfate sulfatase (GALNS) produced in the methylotrophic yeast Pichia pastoris

    PubMed Central

    Rodríguez-López, Alexander; Alméciga-Díaz, Carlos J.; Sánchez, Jhonnathan; Moreno, Jefferson; Beltran, Laura; Díaz, Dennis; Pardo, Andrea; Ramírez, Aura María; Espejo-Mojica, Angela J.; Pimentel, Luisa; Barrera, Luis A.

    2016-01-01

    Mucopolysaccharidosis IV A (MPS IV A, Morquio A disease) is a lysosomal storage disease (LSD) produced by mutations on N-acetylgalactosamine-6-sulfate sulfatase (GALNS). Recently an enzyme replacement therapy (ERT) for this disease was approved using a recombinant enzyme produced in CHO cells. Previously, we reported the production of an active GALNS enzyme in Escherichia coli that showed similar stability properties to that of a recombinant mammalian enzyme though it was not taken-up by culture cells. In this study, we showed the production of the human recombinant GALNS in the methylotrophic yeast Pichia pastoris GS115 (prGALNS). We observed that removal of native signal peptide and co-expression with human formylglycine-generating enzyme (SUMF1) allowed an improvement of 4.5-fold in the specific GALNS activity. prGALNS enzyme showed a high stability at 4 °C, while the activity was markedly reduced at 37 and 45 °C. It was noteworthy that prGALNS was taken-up by HEK293 cells and human skin fibroblasts in a dose-dependent manner through a process potentially mediated by an endocytic pathway, without any additional protein or host modification. The results show the potential of P. pastoris in the production of a human recombinant GALNS for the development of an ERT for Morquio A. PMID:27378276

  18. Suspended crystalline films of protein hydrophobin I (HFBI).

    PubMed

    Oude Vrielink, Anneloes S; Bomans, Paul H H; Vredenbregt, Edgar J D; Wirix, Maarten J M; Sommerdijk, Nico A J M; Luiten, O J; Voets, Ilja K

    2015-06-01

    Protein interfaces play an essential role in both natural and man-made materials as stabilizers, sensors, catalysts, and selective channels for ions and small molecules. Probing the molecular arrangement within such interfaces is of prime importance to understand the relation between structure and functionality. Here we report on the preparation and characterization of large area suspended crystalline films of class II hydrophobin HFBI. This small, amphiphilic globular protein readily self-assembles at the air-water interface into a 2D hexagonal lattice which can be transferred onto a holey carbon electron microscopy grid yielding large areas of hundreds of square micrometers intact hydrophobin film spun across micron-sized holes. Fourier transform analysis of low-dose electron microscopy images and selected area electron diffraction profiles reveal a unit cell dimension a=5.6±0.1nm, in agreement with reported atomic force microscopy studies on solid substrates and grazing incidence X-ray scattering experiments at the air-water interface. These findings constitute the first step towards the utilization of large-area suspended crystalline hydrophobin films as membranes for ultrapurification and chiral separation or as biological substrates for ultrafast electron diffraction.

  19. Adsorption behavior of hydrophobin and hydrophobin/surfactant mixtures at the solid-solution interface.

    PubMed

    Zhang, Xiaoli L; Penfold, Jeffrey; Thomas, Robert K; Tucker, Ian M; Petkov, Jordan T; Bent, Julian; Cox, Andrew

    2011-09-06

    The adsorption of surface-active protein hydrophobin, HFBII, and HFBII/surfactant mixtures at the solid-solution interface has been studied by neutron reflectivity, NR. At the hydrophilic silicon surface, HFBII adsorbs reversibly in the form of a bilayer at the interface. HFBII adsorption dominates the coadsorption of HFBII with cationic and anionic surfactants hexadecyltrimethyl ammonium bromide, CTAB, and sodium dodecyl sulfate, SDS, at concentrations below the critical micellar concentration, cmc, of conventional cosurfactants. For surfactant concentrations above the cmc, HFBII/surfactant solution complex formation dominates and there is little HFBII adsorption. Above the cmc, CTAB replaces HFBII at the interface, but for SDS, there is no affinity for the anionic silicon surface hence there is no resultant adsorption. HFBII adsorbs onto a hydrophobic surface (established by an octadecyl trimethyl silane, OTS, layer on silicon) irreversibly as a monolayer, similar to what is observed at the air-water interface but with a different orientation at the interface. Below the cmc, SDS and CTAB have little impact upon the adsorbed layer of HFBII. For concentrations above the cmc, conventional surfactants (CTAB and SDS) displace most of the HFBII at the interface. For nonionic surfactant C(12)E(6), the pattern of adsorption is slightly different, and although some coadsorption at the interface takes place, C(12)E(6) has little impact on the HFBII adsorption.

  20. [An effective scheme to produce recombinant uracil-DNA glycosylase of Escherichia coli for PCR diagnostics].

    PubMed

    Dmitrochenko, A E; Turiianskaia, O M; Gilep, A A; Usanov, S A; Iantsevich, A V

    2014-01-01

    An effective scheme has been developed to produce recombinant uracil-DNA glycosylase of Escherichia coli K12 intended to be used for PCR diagnostics, making it possible to achieve a high yield of the end product using a two-stage purification. The gene encoding this enzyme was cloned into the pCWori vector within the same reading frame with six residues of histidine in the C-erminal sequence. Using this vector and the E. coli DH5alpha, a host-vector expression system has been developed and conditions for protein synthesis have been optimized. To purify the protein, metal affinity chromatography with further dialysis was used to remove imidazole. The enzyme yield was no less than 60 mg of the end protein per 1 L of the culture medium. The concordance between amino acid sequences of the recombinant and native enzymes was proved by peptide mass fingerprinting and mass spectrometry. A rapid test to determine the activity of the enzyme preparation was suggested. It was found that the activity of 1.0 mg of the recombinant protein is no less than 3 x 10(3) units. The recombinant enzyme was most stable at pH 8.0 and an ionic strength of the solution equal to 200 mM; it lost its activity completely for 10 min at 60 degrees C. Storage during 1 h at 20 degrees C resulted in the loss of no more than 30% of activity. In the enzyme preparation, the activity of DNase was absent. The free energy of the unfolding of the protein globule of the recombinant uracil-DNA glycosylase is 23.1 +/- 0.2 kJ/mol. The data obtained indicate that the recombinant enzyme may be recommended for use in PCR diagnostics to prevent the appearance of false positive results caused by pollution of the reaction mixture by products of the preceding reactions.

  1. A gene responsible for prolyl-hydroxylation of moss-produced recombinant human erythropoietin

    PubMed Central

    Parsons, Juliana; Altmann, Friedrich; Graf, Manuela; Stadlmann, Johannes; Reski, Ralf; Decker, Eva L.

    2013-01-01

    Recombinant production of pharmaceutical proteins is crucial, not only for personalized medicine. While most biopharmaceuticals are currently produced in mammalian cell culture, plant-made pharmaceuticals gain momentum. Post-translational modifications in plants are similar to those in humans, however, existing differences may affect quality, safety and efficacy of the products. A frequent modification in higher eukaryotes is prolyl-4-hydroxylase (P4H)-catalysed prolyl-hydroxylation. P4H sequence recognition sites on target proteins differ between humans and plants leading to non-human posttranslational modifications of recombinant human proteins produced in plants. The resulting hydroxyprolines display the anchor for plant-specific O-glycosylation, which bears immunogenic potential for patients. Here we describe the identification of a plant gene responsible for non-human prolyl-hydroxylation of human erythropoietin (hEPO) recombinantly produced in plant (moss) bioreactors. Targeted ablation of this gene abolished undesired prolyl-hydroxylation of hEPO and thus paves the way for plant-made pharmaceuticals humanized via glyco-engineering in moss bioreactors. PMID:24145658

  2. Heterologous expression and characterization of the hydrophobin HFBI in Pichia pastoris and evaluation of its contribution to the food industry.

    PubMed

    Niu, Baolong; Wang, Dandan; Yang, Yanyan; Xu, Haijin; Qiao, Mingqiang

    2012-08-01

    The class II hydrophobin HFBI from Trichoderma reesei was heterologously expressed by Pichia pastoris using pPIC9 vector under the control of the promoter AOX1. The recombinant HFBI (rHFBI) was purified by ultrafiltration and reverse-phase high performance liquid chromatography. Tricine-SDS-PAGE and Western blotting demonstrated that rHFBI with the expected molecular weight of 7.5 kDa was secreted into the culture medium. X-ray photoelectron spectroscopy and water contact angle measurements indicated that rHFBI could lead to the conversion of the wettability of the hydrophobic siliconized glass and hydrophilic mica surfaces relying on the self-assembly membrane on hydrophobic/hydrophilic interfaces. It was demonstrated that rHFBI had the ability to stabilize oil droplets, which was far excess of the class I hydrophobin HGFI heterologously expressed in P. pastoris (rHGFI) and the typical food emulsifier sodium caseinate. In gushing experiments, it was shown that rHFBI was a strong gushing inducer in beer, whereas rHGFI did not display any signs of gushing. This provided the potential of rHFBI to be used as a novel emulsifying agent and a predictor of gushing risk.

  3. 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.

  4. Process technological effects of deletion and amplification of hydrophobins I and II in transformants of Trichoderma reesei.

    PubMed

    Bailey, M J; Askolin, S; Hörhammer, N; Tenkanen, M; Linder, M; Penttilä, M; Nakari-Setälä, T

    2002-05-01

    Transformants of the Trichoderma reeseistrains QM9414 and Rut-C30 were constructed in which the genes for the two major hydrophobin proteins, hydrophobins I (HFBI) and II (HFBII), were deleted or amplified by molecular biological techniques. Growth parameters and foam production of the transformant strains were compared with the corresponding properties of the parent strains by cultivation in laboratory bioreactors under conditions of catabolite repression (glucose medium) or induction of cellulolytic enzymes and other secondary metabolites (cellulose and lactose media). All the transformed strains exhibited vegetative growth properties similar to those of their parent. The Delta hfb2 (but not the Delta hfb1) transformant showed reduced tendency to foam, whereas both strains overproducing hydrophobins foamed extensively, particularly in the case of HFBII. Enzyme production on cellulose medium was unaltered in the Delta hfb2 transformant VTT D-99676, but both the Delta hfb2 and HFBII-overproducing transformants exhibited somewhat decreased enzyme production properties on lactose medium. Production of HFBI by the multi-copy transformant VTT D-98692 was almost 3-fold that of the parent strain QM9414. Overproduction of HFBII by the transformant VTT D-99745, obtained by transformation with three additional copies of the hfb2 gene under the cbh1 promoter, was over 5-fold compared to production by the parent strain Rut-C30. The Delta hfb2transformant VTT D-99676 produced a greatly increased number of spores on lactose medium compared with the parent strain, whereas the HFBII-overproducing transformant VTT D-99745 produced fewer spores.

  5. Towards the molecular characterization of the stable producer phenotype of recombinant antibody-producing NS0 myeloma cells.

    PubMed

    Prieto, Y; Rojas, L; Hinojosa, L; González, I; Aguiar, D; de la Luz, K; Castillo, A; Pérez, R

    2011-08-01

    The loss of heterologous protein expression is one of the major problems faced by industrial cell line developers and has been reported by several authors. Therefore, the understanding of the mechanisms involved in the generation of stable and high producer cell lines is a critical issue, especially for those processes based on long term continuous cultures. We characterized two recombinant NS0 myeloma cell lines expressing Nimotuzumab, a humanized anti-human epidermal growth factor receptor (EGFR) antibody. The hR3/H7 clone is a stable producer obtained from the unstable hR3/t16 clone. The unstable clone was characterized by a bimodal distribution of intracellular immunoglobulin staining using flow cytometry. Loss of antibody production was due to the emergence of a non-producer cell subpopulation that increased with cell generation number. Immunoglobulin heavy chain (HC) and light chain (LC) ratio (HC/LC) was lower for the unstable phenotype. Proteomic maps using two dimensional gel electrophoresis (2DE) were obtained for both clones, at initial cell culture time and after 40 generations. Fifteen proteins potentially associated with the phenomenon of production stability were identified. The hR3/H7 stable clone showed an up-regulated expression pattern for most of these proteins. The regulation of recombinant antibody production by the host NS0 myeloma cell line most likely involves simultaneously cellular processes such as DNA transcription, mRNA processing, protein synthesis and folding, vesicular transport, glycolysis and energy production, according to the proteins identified in the present proteomic study.

  6. Electron-ion recombination in laser-produced plasmas using optical interferometry

    NASA Astrophysics Data System (ADS)

    Heilmann, Nathan; Peatross, Justin; Bergeson, Scott

    2011-10-01

    We are developing methods to measure electron-ion recombination in laser-produced plasmas. A high intensity fs laser pulse is focused into a gas jet and forms a plasma. A weaker probe beam first passes through a slightly mis-aligned Michelson interferometer and is also focused into the plasma. The probe ``beam'' is actually two temporally coincident but spatially offset laser beams. One of the laser beams passes through the plasma and the other does not. These beams expand and produce interference fringes in the far field, similar to a Young's double slit experiment. The spatial position of these fringes depends on the differential phase shift in the two probe beams. This differential shift is due to the electron density in the plasma, which is probed by only one beam. By measuring the fringe shift as a function of time after the plasma is formed, we should be able to measure the time-evolving electron density. At sufficiently high densities, three-body recombination will become important. In that regime, the measured recombination rate can be used to determine the electron temperature.

  7. Evolutionary analysis of hydrophobin gene family in two wood-degrading basidiomycetes, Phlebia brevispora and Heterobasidion annosum s.l.

    PubMed Central

    2013-01-01

    Background Hydrophobins are small secreted cysteine-rich proteins that play diverse roles during different phases of fungal life cycle. In basidiomycetes, hydrophobin-encoding genes often form large multigene families with up to 40 members. The evolutionary forces driving hydrophobin gene expansion and diversification in basidiomycetes are poorly understood. The functional roles of individual genes within such gene families also remain unclear. The relationship between the hydrophobin gene number, the genome size and the lifestyle of respective fungal species has not yet been thoroughly investigated. Here, we present results of our survey of hydrophobin gene families in two species of wood-degrading basidiomycetes, Phlebia brevispora and Heterobasidion annosum s.l. We have also investigated the regulatory pattern of hydrophobin-encoding genes from H. annosum s.s. during saprotrophic growth on pine wood as well as on culture filtrate from Phlebiopsis gigantea using micro-arrays. These data are supplemented by results of the protein structure modeling for a representative set of hydrophobins. Results We have identified hydrophobin genes from the genomes of two wood-degrading species of basidiomycetes, Heterobasidion irregulare, representing one of the microspecies within the aggregate H. annosum s.l., and Phlebia brevispora. Although a high number of hydrophobin-encoding genes were observed in H. irregulare (16 copies), a remarkable expansion of these genes was recorded in P. brevispora (26 copies). A significant expansion of hydrophobin-encoding genes in other analyzed basidiomycetes was also documented (1–40 copies), whereas contraction through gene loss was observed among the analyzed ascomycetes (1–11 copies). Our phylogenetic analysis confirmed the important role of gene duplication events in the evolution of hydrophobins in basidiomycetes. Increased number of hydrophobin-encoding genes appears to have been linked to the species’ ecological strategy, with

  8. Key determinants affecting sheep wool biodegradation directed by a keratinase-producing Bacillus subtilis recombinant strain.

    PubMed

    Zaghloul, Taha I; Embaby, Amira M; Elmahdy, Ahmed R

    2011-02-01

    OVAT (one variable at a time) approach was applied in this study to screen the most important physicochemical key determinants involved in the process of sheep wool biodegradation. The process was directed by a keratinase-producing Bacillus subtilis DB 100 (p5.2) recombinant strain. Data indicate that, sheep wool could be degraded efficiently in cultures incubated at 30°C, with initial pH of 7 with agitation at 150 rpm. Two times autoclaved alkali treated and undefatted chopped sheep wool is more accessible to biodegradation. B. subtilis recombinant cells could utilize sheep wool as a sole source of carbon and nitrogen. Sheep wool-based modified basal medium II, lacking NH₄Cl and yeast extract, could greatly support the growth of these bacterial cells. Sheep wool biodegradation was conducted efficiently in the absence of kanamycin consequently; high stability of the recombinant plasmid (p5.2) represents a great challenge upon scaling up this process. Three key determinants (sheep wool concentration, incubation time and inoculum size) imposing considerable constraints on the process are highlighted. Sheep wool-based tap water medium and sheep wool-based distilled water medium were formulated in this study. High levels of released end products, produced from sheep wool biodegradation are achieved upon using these two sheep wool-based water media. Data indicate that, sheep wool hydrolysate is rich in some amino acids, such as tyrosine, phenylalanine, lysine, proline, isoleucine, leucine, valine, aspartic acid and glutamic acid. Moreover, the resulting sheep wool hydrolysate contains soluble proteins of high and intermediate molecular weights. The present study demonstrates a feasible, cheap, reproducible, efficient and rapid biotechnological approach towards utilization of raw sheep wool waste through a recombinant bacterium.

  9. Adsorption of hydrophobin/β-casein mixtures at the solid-liquid interface.

    PubMed

    Tucker, I M; Petkov, J T; Penfold, J; Thomas, R K; Cox, A R; Hedges, N

    2016-09-15

    The adsorption behaviour of mixtures of the proteins β-casein and hydrophobin at the hydrophilic solid-liquid surface have been studied by neutron reflectivity. The results of measurements from sequential adsorption and co-adsorption from solution are contrasted. The adsorption properties of protein mixtures are important for a wide range of applications. Because of competing factors the adsorption behaviour of protein mixtures at interfaces is often difficult to predict. This is particularly true for mixtures containing hydrophobin as hydrophobin possesses some unusual surface properties. At β-casein concentrations ⩾0.1wt% β-casein largely displaces a pre-adsorbed layer of hydrophobin at the interface, similar to that observed in hydrophobin-surfactant mixtures. In the composition and concentration range studied here for the co-adsorption of β-casein-hydrophobin mixtures the adsorption is dominated by the β-casein adsorption. The results provide an important insight into how the competitive adsorption in protein mixtures of hydrophobin and β-casein can impact upon the modification of solid surface properties and the potential for a wide range of colloid stabilisation applications.

  10. The potential of transgenic green microalgae; a robust photobioreactor to produce recombinant therapeutic proteins.

    PubMed

    Akbari, Fariba; Eskandani, Morteza; Khosroushahi, Ahmad Yari

    2014-11-01

    Microalgae have been used in food, cosmetic, and biofuel industries as a natural source of lipids, vitamins, pigments and antioxidants for a long time. Green microalgae, as potent photobioreactors, can be considered as an economical expression system to produce recombinant therapeutical proteins at large-scale due to low cost of production and scaling-up capitalization owning to the inexpensive medium requirement, fast growth rate, and the ease of manipulation. These microalgae possess all benefit eukaryotic expression systems including the ability of post-translational modifications required for proper folding and stability of active proteins. Among the many items regarded as recombinant protein production, this review compares the different expression systems with green microalgae like Dunaliella by viewing the nuclear/chloroplast transformation challenges/benefits, related selection markers/reporter genes, and crucial factors/strategies affecting the increase of foreign protein expression in microalgae transformants. Some important factors were discussed regarding the increase of protein yielding in microalgae transformants including: transformation-associated genotypic modifications, endogenous regulatory factors, promoters, codon optimization, enhancer elements, and milking of recombinant protein.

  11. Efficient preservation in a silicon oxide matrix of Escherichia coli, producer of recombinant proteins.

    PubMed

    Desimone, Martín F; De Marzi, Mauricio C; Copello, Guillermo J; Fernández, Marisa M; Malchiodi, Emilio L; Diaz, Luis E

    2005-10-01

    The aim of this work was to study the use of silicon oxide matrices for the immobilization and preservation of recombinant-protein-producing bacteria. We immobilized Escherichia coli BL21 transformants containing different expression plasmids. One contained DNA coding for a T-cell receptor beta chain, which was expressed as inclusion bodies in the cytoplasm. The other two encoded bacterial superantigens Staphylococcal Enterotoxin G and Streptococcal Superantigen, which were expressed as soluble proteins in the periplasm. The properties of immobilization and storage stability in inorganic matrices prepared from two precursors, silicon dioxide and tetraethoxysilane, were studied. Immobilized E. coli was stored in sealed tubes at 4 and 20 degrees C and the number of viable cells and level of recombinant protein production were analyzed weekly. Different tests showed that the biochemical characteristics of immobilized E. coli remained intact. At both temperatures selected, we found that the number of bacteria in silicon dioxide-derived matrix was of the same order of magnitude (10(9) cfu ml(-1)) as before immobilization, for 2 months. After 2 weeks, cells immobilized in an alkoxide-derived matrix decreased to 10(4) cfu ml(-1) at 4 degrees C, and no viable cells were detected at 20 degrees C. We found that immobilized bacteria could be used as a starter to produce recombinant proteins with yields comparable to those obtained from glycerol stocks: 15 mg l(-1) for superantigens and 2 mg l(-1) for T-cell receptor beta chain. These results contribute to the development of methods for microbial cell preservation under field conditions.

  12. Two Novel Class II Hydrophobins from Trichoderma spp. Stimulate Enzymatic Hydrolysis of Poly(Ethylene Terephthalate) when Expressed as Fusion Proteins

    PubMed Central

    Espino-Rammer, Liliana; Ribitsch, Doris; Przylucka, Agnieszka; Marold, Annemarie; Greimel, Katrin J.; Herrero Acero, Enrique; Guebitz, Georg M.; Kubicek, Christian P.

    2013-01-01

    Poly(ethylene terephthalate) (PET) can be functionalized and/or recycled via hydrolysis by microbial cutinases. The rate of hydrolysis is however low. Here, we tested whether hydrophobins (HFBs), small secreted fungal proteins containing eight positionally conserved cysteine residues, are able to enhance the rate of enzymatic hydrolysis of PET. Species of the fungal genus Trichoderma have the most proliferated arsenal of class II hydrophobin-encoding genes among fungi. To this end, we studied two novel class II HFBs (HFB4 and HFB7) of Trichoderma. HFB4 and HFB7, produced in Escherichia coli as fusions to the C terminus of glutathione S-transferase, exhibited subtle structural differences reflected in hydrophobicity plots that correlated with unequal hydrophobicity and hydrophily, respectively, of particular amino acid residues. Both proteins exhibited a dosage-dependent stimulation effect on PET hydrolysis by cutinase from Humicola insolens, with HFB4 displaying an adsorption isotherm-like behavior, whereas HFB7 was active only at very low concentrations and was inhibitory at higher concentrations. We conclude that class II HFBs can stimulate the activity of cutinases on PET, but individual HFBs can display different properties. The present findings suggest that hydrophobins can be used in the enzymatic hydrolysis of aromatic-aliphatic polyesters such as PET. PMID:23645195

  13. Synthesis of nylon 4 from gamma-aminobutyrate (GABA) produced by recombinant Escherichia coli.

    PubMed

    Park, Si Jae; Kim, Eun Young; Noh, Won; Oh, Young Hoon; Kim, Hye Young; Song, Bong Keun; Cho, Kwang Myung; Hong, Soon Ho; Lee, Seung Hwan; Jegal, Jonggeon

    2013-07-01

    In this study, we developed recombinant Escherichia coli strains expressing Lactococcus lactis subsp. lactis Il1403 glutamate decarboxylase (GadB) for the production of GABA from glutamate monosodium salt (MSG). Syntheses of GABA from MSG were examined by employing recombinant E. coli XL1-Blue as a whole cell biocatalyst in buffer solution. By increasing the concentration of E. coli XL1-Blue expressing GadB from the OD₆₀₀ of 2-10, the concentration and conversion yield of GABA produced from 10 g/L of MSG could be increased from 4.3 to 4.8 g/L and from 70 to 78 %, respectively. Furthermore, E. coli XL1-Blue expressing GadB highly concentrated to the OD₆₀₀ of 100 produced 76.2 g/L of GABA from 200 g/L of MSG with 62.4 % of GABA yield. Finally, nylon 4 could be synthesized by the bulk polymerization using 2-pyrrolidone that was prepared from microbially synthesized GABA by the reaction with Al₂O₃ as catalyst in toluene with the yield of 96 %.

  14. Constructing a recombinant hyaluronic acid biosynthesis operon and producing food-grade hyaluronic acid in Lactococcus lactis.

    PubMed

    Sheng, Juzheng; Ling, Peixue; Wang, Fengshan

    2015-02-01

    Hyaluronic acid (HA), a natural high molecular weight polysaccharide, is produced by Streptococcus zooepidemicus. However, Streptococcus has several drawbacks including its potential to produce exotoxins, so there is demand for an alternative HA source. Here, a recombinant HA biosynthesis operon, as well as the HA biosynthesis operon of S. zooepidemicus were introduced into L. lactis using the nisin-controlled expression system, respectively. HA was successfully synthesized by recombinant L. lactis. Furthermore, overexpression of the endogenous enzymes directing the synthesis of precursor sugars was effective at increasing HA production, and increasing the supply of UDP-activated monosaccharide donors aided synthesis of monodisperse HA polysaccharides. Besides GRAS host strain (L. lactis) and NICE system, the selecting marker (lacF gene) of the recombinant strain is also food grade. Therefore, HA produced by recombinant L. lactis overcomes the problems associated with Streptococcus and provides a source of food-grading HA appropriate for widespread biotechnological applications.

  15. Specific CEA-producing colorectal carcinoma cell killing with recombinant adenoviral vector containing cytosine deaminase gene

    PubMed Central

    Shen, Li-Zong; Wu, Wen-Xi; Xu, De-Hua; Zheng, Zhong-Cheng; Liu, Xin-Yuan; Ding, Qiang; Hua, Yi-Bing; Yao, Kun

    2002-01-01

    AIM: To kill CEA positive colorectal carcinoma cells specifically using the E coli cytosine deaminase (CD) suicide gene, a new replication-deficient recombinant adenoviral vector was constructed in which CD gene was controlled under CEA promoter and its in vitro cytotoxic effects were evaluated. METHODS: Shuttle plasmid containing CD gene and regulatory sequence of the CEA gene was constructed and recombined with the right arm of adenovirus genome DNA in 293 cell strain. Dot blotting and PCR were used to identify positive plaques. The purification of adenovirus was performed with ultra-concentration in CsCl step gradients and the titration was measured with plaque formation assay. Cytotoxic effects were assayed with MTT method, The fifty percent inhibition concentration (IC50) of 5-FC was calculated using a curve-fitting parameter. The human colorectal carcinoma cell line, which was CEA-producing, and the CEA-nonproducing Hela cell line were applied in cytological tests. An established recombinant adenovirus vector AdCMVCD, in which the CD gene was controlled under CMV promoter, was used as virus control. Quantitative results were expressed as the mean ± SD of the mean. Statistical analysis was performed using ANOVA test. RESULTS: The desired recombinant adenovirus vector was named AdCEACD. The results of dot blotting and PCR showed that the recombinant adenovirus contained CEA promoter and CD gene. Virus titer was about 5.0 × 1014 pfu/L-1 after purification. The CEA-producing Lovo cells were sensitive to 5-FC and had the same cytotoxic effect after infection with AdCEACD and AdCMVCD (The IC50 values of 5-FC in parent Lovo cells, Lovo cells infected with 100 M.O.I AdCEACD and Lovo cells infected with 10 M.O.I AdCMVCD were > 15000, 216.5 ± 38.1 and 128.8 ± 25.4 μmol•L⁻¹, P < 0.001, respectively), and the cytotoxicity of 5-FC increased accordingly when the M.O.I of adenoviruses were enhanced (The value of IC50 of 5-FC was reduced to 27.9 ± 4.2 μmol•L-1

  16. Nanoscale reduction in surface friction of polymer surfaces modified with Sc3 hydrophobin from Schizophyllum commune.

    PubMed

    Misra, Rahul; Li, Jun; Cannon, Gordon C; Morgan, Sarah E

    2006-05-01

    Hydrophobins are amphipathic self-assembling proteins secreted by filamentous fungi that exhibit remarkable ability to modify synthetic surfaces. Thin coatings of Sc3 hydrophobin isolated from the wood-rotting fungus Schizophyllum commune were prepared via spin coating and adsorption techniques onto polymeric surfaces. Surface morphology and nanotribological characteristics of the films were evaluated using lateral force microscopy (LFM) and nanoindentation techniques. This paper reports the first observation of reduction in nanoscale relative surface friction of Sc3 hydrophobin protein modified polymeric surfaces. Relative friction coefficients were dramatically reduced and hydrophilicity increased for polymer surfaces modified with Sc3 hydrophobin thin films. Morphology of the protein films as well as degree of surface modification was observed to be a function of film formation technique and composition of the substrate.

  17. Purification and basic biochemical characterization of 19 recombinant plant peroxidase isoenzymes produced in Pichia pastoris☆

    PubMed Central

    Krainer, Florian W.; Pletzenauer, Robert; Rossetti, Laura; Herwig, Christoph; Glieder, Anton; Spadiut, Oliver

    2014-01-01

    The plant enzyme horseradish peroxidase (HRP) is used in several important industrial and medical applications, of which especially biosensors and diagnostic kits describe an emerging field. Although there is an increasing demand for high amounts of pure enzyme preparations, HRP is still isolated from the plant as a mixture of different isoenzymes with different biochemical properties. Based on a recent next generation sequencing approach of the horseradish transcriptome, we produced 19 individual HRP isoenzymes recombinantly in the yeast Pichia pastoris. After optimizing a previously reported 2-step purification strategy for the recombinant isoenzyme HRP C1A by substituting an unfavorable size exclusion chromatography step with an anion exchange step using a monolithic column, we purified the 19 HRP isoenzymes with varying success. Subsequent basic biochemical characterization revealed differences in catalytic activity, substrate specificity and thermal stability of the purified HRP preparations. The preparations of the isoenzymes HRP A2A and HRP A2B were found to be highly interesting candidates for future applications in diagnostic kits with increased sensitivity. PMID:24342173

  18. Identification and characterization of gushing-active hydrophobins from Fusarium graminearum and related species.

    PubMed

    Sarlin, Tuija; Kivioja, Teemu; Kalkkinen, Nisse; Linder, Markus B; Nakari-Setälä, Tiina

    2012-04-01

    Fungal infection of barley and malt, particularly by the Fusarium species, is a direct cause of spontaneous overfoaming of beer, referred to as gushing. We have shown previously that small fungal proteins, hydrophobins, act as gushing-inducing factors in beer. The aim of our present study was to isolate and characterize hydrophobins from a gushing-active fungus, Fusarium graminearum (teleomorph Gibberella zeae) and related species. We generated profile hidden Markov models (profile HMMs) for the hydrophobin classes Ia, Ib and II from the multiple sequence alignments of their known members available in public domain databases. We searched the published Fusarium graminearum genome with the Markov models. The best matching sequences and the corresponding genes were isolated from F. graminearum and the related species F. culmorum and F. poae by PCR and characterized. One each of the putative F. graminearum and F. poae hydrophobin genes were expressed in the heterologous host Trichoderma reesei. The proteins corresponding to the genes were purified and identified as hydrophobins and named GzHYD5 and FpHYD5, respectively. Concentrations of 0.003 ppm of these hydrophobins were observed to induce vigorous beer gushing.

  19. Selective isolation of hydrophobin SC3 by solid-phase extraction with polytetrafluoroethylene microparticles and subsequent mass spectrometric analysis.

    PubMed

    Kupčík, Rudolf; Zelená, Miroslava; Řehulka, Pavel; Bílková, Zuzana; Česlová, Lenka

    2016-02-01

    Hydrophobins are small proteins that play a role in a number of processes during the filamentous fungi growth and development. These proteins are characterized by the self-assembly of their molecules into an amphipathic membrane at hydrophilic-hydrophobic interfaces. Isolation and purification of hydrophobins generally present a challenge in their analysis. Hydrophobin SC3 from Schizophyllum commune was selected as a representative of class I hydrophobins in this work. A novel procedure for selective and effective isolation of hydrophobin SC3 based on solid-phase extraction with polytetrafluoroethylene microparticles loaded in a small self-made microcolumn is reported. The tailored binding of hydrophobins to polytetrafluoroethylene followed by harsh elution conditions resulted in a highly specific isolation of hydrophobin SC3 from the model mixture of ten proteins. The presented isolation protocol can have a positive impact on the analysis and utilization of these proteins including all class I hydrophobins. Hydrophobin SC3 was further subjected to reduction of its highly stable disulfide bonds and to chymotryptic digestion followed by mass spectrometric analysis. The isolation and digestion protocols presented in this work make the analysis of these highly hydrophobic and compact proteins possible.

  20. Enhanced Cutinase-Catalyzed Hydrolysis of Polyethylene Terephthalate by Covalent Fusion to Hydrophobins

    PubMed Central

    Ribitsch, Doris; Herrero Acero, Enrique; Przylucka, Agnieszka; Zitzenbacher, Sabine; Marold, Annemarie; Gamerith, Caroline; Tscheließnig, Rupert; Jungbauer, Alois; Rennhofer, Harald; Lichtenegger, Helga; Amenitsch, Heinz; Bonazza, Klaus; Kubicek, Christian P.; Guebitz, Georg M.

    2015-01-01

    Cutinases have shown potential for hydrolysis of the recalcitrant synthetic polymer polyethylene terephthalate (PET). We have shown previously that the rate of this hydrolysis can be enhanced by the addition of hydrophobins, small fungal proteins that can alter the physicochemical properties of surfaces. Here we have investigated whether the PET-hydrolyzing activity of a bacterial cutinase from Thermobifida cellulosilytica (Thc_Cut1) would be further enhanced by fusion to one of three Trichoderma hydrophobins, i.e., the class II hydrophobins HFB4 and HFB7 and the pseudo-class I hydrophobin HFB9b. The fusion enzymes exhibited decreased kcat values on soluble substrates (p-nitrophenyl acetate and p-nitrophenyl butyrate) and strongly decreased the hydrophilicity of glass but caused only small changes in the hydrophobicity of PET. When the enzyme was fused to HFB4 or HFB7, the hydrolysis of PET was enhanced >16-fold over the level with the free enzyme, while a mixture of the enzyme and the hydrophobins led only to a 4-fold increase at most. Fusion with the non-class II hydrophobin HFB9b did not increase the rate of hydrolysis over that of the enzyme-hydrophobin mixture, but HFB9b performed best when PET was preincubated with the hydrophobins before enzyme treatment. The pattern of hydrolysis by the fusion enzymes differed from that of Thc_Cut1 as the concentration of the product mono(2-hydroxyethyl) terephthalate relative to that of the main product, terephthalic acid, increased. Small-angle X-ray scattering (SAXS) analysis revealed an increased scattering contrast of the fusion proteins over that of the free proteins, suggesting a change in conformation or enhanced protein aggregation. Our data show that the level of hydrolysis of PET by cutinase can be significantly increased by fusion to hydrophobins. The data further suggest that this likely involves binding of the hydrophobins to the cutinase and changes in the conformation of its active center. PMID:25795674

  1. Lifetime and quenching of CO /a super 3 pi/ produced by recombination of CO2 ions in a helium afterglow.

    NASA Technical Reports Server (NTRS)

    Wauchop, T. S.; Broida, H. P.

    1972-01-01

    Demonstration that rapid dissociative recombination of CO2(+) in a flowing, helium afterglow is an efficient source of CO in the a super 3 pi metastable state. Ions produced by mixing CO2 with He(2 super 3 S) recombine to produce a CO metastable afterglow with a number density as great as 10 to the 9th per sq cm. Monitoring of the (a super 3 pi-X super 1 sigma) Cameron transition in CO was used to study the lifetime and quenching of CO (a super 3 pi) by CO2, N2, NO, and He. Recombination of CO2(+) also produces CO in the d super 3 delta and a' super 3 sigma states.

  2. Transplantation of genetically engineered cardiac fibroblasts producing recombinant human erythropoietin to repair the infarcted myocardium

    PubMed Central

    Ruvinov, Emil; Sharabani-Yosef, Orna; Nagler, Arnon; Einbinder, Tom; Feinberg, Micha S; Holbova, Radka; Douvdevani, Amos; Leor, Jonathan

    2008-01-01

    Background Erythropoietin possesses cellular protection properties. The aim of the present study was to test the hypothesis that in situ expression of recombinant human erythropoietin (rhEPO) would improve tissue repair in rat after myocardial infarction (MI). Methods and results RhEPO-producing cardiac fibroblasts were generated ex vivo by transduction with retroviral vector. The anti-apoptotic effect of rhEPO-producing fibroblasts was evaluated by co-culture with rat neonatal cardiomyocytes exposed to H2O2-induced oxidative stress. Annexin V/PI assay and DAPI staining showed that compared with control, rhEPO forced expression markedly attenuated apoptosis and improved survival of cultured cardiomyocytes. To test the effect of rhEPO on the infarcted myocardium, Sprague-Dawley rats were subjected to permanent coronary artery occlusion, and rhEPO-producing fibroblasts, non-transduced fibroblasts, or saline, were injected into the scar tissue seven days after infarction. One month later, immunostaining identified rhEPO expression in the implanted engineered cells but not in controls. Compared with non-transduced fibroblasts or saline injection, implanted rhEPO-producing fibroblasts promoted vascularization in the scar, and prevented cell apoptosis. By two-dimensional echocardiography and postmortem morphometry, transplanted EPO-engineered fibroblasts did not prevent left ventricular (LV) dysfunction and adverse LV remodeling 5 and 9 weeks after MI. Conclusion In situ expression of rhEPO enhances vascularization and reduces cell apoptosis in the infarcted myocardium. However, local EPO therapy is insufficient for functional improvement after MI in rat. PMID:19014419

  3. Straightforward approach to produce recombinant scorpion toxins-Pore blockers of potassium channels.

    PubMed

    Nekrasova, Oksana; Kudryashova, Ksenia; Fradkov, Arkadiy; Yakimov, Sergey; Savelieva, Maria; Kirpichnikov, Mikhail; Feofanov, Alexey

    2017-01-10

    Scorpion venom peptide blockers (KTx) of potassium channels are a valuable tool for structure-functional studies and prospective candidates for medical applications. Low yields of recombinant KTx hamper their wide application. We developed convenient and efficient bioengineering approach to a large-scale KTx production that meets increasing demands for such peptides. Maltose-binding protein was used as a carrier for cytoplasmic expression of folded disulfide-rich KTx in E. coli. TEV protease was applied for in vitro cleavage of the target peptide from the carrier. To produce KTx with retained native N-terminal sequence, the last residue of TEV protease cleavage site (CSTEV) was occupied by the native N-terminal residue of a target peptide. It was shown that decreased efficiency of hydrolysis of fusion proteins with non-canonical CSTEV can be overcome without by-product formation. Disulfide formation and folding of a target peptide occurred in cytoplasm eliminating the need for renaturation procedure in vitro. Advantages of this approach were demonstrated by producing six peptides with three disulfide bonds related to four KTx sub-families and achieving peptide yields of 12-22mg per liter of culture. The developed approach can be of general use for low-cost production of various KTx, as well as other disulfide-rich peptides and proteins.

  4. Heterologous expression of the hydrophobin FcHyd5p from Fusarium culmorum in Pichia pastoris and evaluation of its surface activity and contribution to gushing of carbonated beverages.

    PubMed

    Stübner, Matthias; Lutterschmid, Georg; Vogel, Rudi F; Niessen, Ludwig

    2010-06-30

    The class II hydrophobin FcHyd5p from Fusarium culmorum was heterologously expressed by Pichia pastoris. Transcription of the recombinant gene was confirmed by RT-PCR and expression of FcHyd5p was demonstrated using SDS-PAGE and immuno-staining with 6 x His-tag antibodies. FcHyd5p was purified and concentrated by dialysis, isoelectric focussing and the use of ultra filtration. It was demonstrated that FcHyd5p is able to change the hydrophopic properties of surfaces rendering them wettable after coating with the supernatant of recombinant P. pastoris cultures. Furthermore, due to its surface activity, FcHyd5p was able to stabilise air bubbles in aqueous solutions. The supernatant of a culture medium containing a FcHyd5p producing P. pastoris clone remained turbid for 24h and the foam stable for more than 72 h after the treatment with a homogeniser, whereas the liquid of the wild type strain clarified after 10 min and the foam disintegrated after 2h. Finally it was demonstrated, that FcHyd5p can induce spontaneous overfoaming of carbonated liquids, referred to as gushing. Addition of 2 mg freeze-dried culture supernatant from a FcHyd5p producing P. pastoris clone resulted in a lost volume of 252 ml +/- 20 per 500 ml of beer (50%) and 179 ml +/- 7 per 330 ml of carbonated water (54%), respectively. Neither untreated beer/water, nor beer/water treated with freeze-dried culture supernatant from the wild type strain showed any gushing. Furthermore, addition of 215 microg highly purified FcHyd5p resulted in a lost volume of 77 ml +/- 40 per 500 ml beer (15%).

  5. Recombinant proteinase 3 produced in different expression systems: recognition by anti-PR3 antibodies.

    PubMed

    van der Geld, Y M; Oost-Kort, W; Limburg, P C; Specks, U; Kallenberg, C G

    2000-10-20

    Anti-neutrophil cytoplasm autoantibodies (ANCA) directed against proteinase 3 (PR3) are highly sensitive and specific markers for Wegener's granulomatosis (WG). Consequently, antigen-specific assays for detection of PR3-ANCA are helpful for the diagnosis and follow-up of patients with WG. Purification of PR3 is laborious and requires large amounts of granulocytes. Therefore, several attempts have been made to produce recombinant PR3 that is recognized by PR3-ANCA. The purpose of this study was to compare the recognition of different recombinant forms of PR3 (rPR3) by anti-PR3 antibodies. Recombinant PR3 produced in E. coli (rcPR3), P. pastoris (rpPR3), insect cells using the baculovirus system (rbPR3), the human mast cell line, HMC-1 (HMC-1/PR3-S176A), or the human epithelial cell line, 293 (Delta-rPR3-S176A) as well as purified neutrophil PR3 (nPR3) were used. Recognition of these rPR3s by anti-PR3 antibodies was determined by direct and capture ELISA with 19 PR3-ANCA sera, 13 anti-PR3 mAbs and a rabbit serum raised against human PR3. In the capture ELISA rabbit anti-PR3 strongly bound nPR3 and all rPR3 products. By capture ELISA rcPR3 and rpPR3 were recognized by 11 (57%) and 13 (68%) of the 19 PR3-ANCA sera, respectively, whereas rbPR3, HMC-1/PR3-S176A, Delta-rPR3-S176A and nPR3 were recognized by all PR3-ANCA sera. By direct ELISA rabbit anti-PR3 strongly bound nPR3 and all tested rPR3 products. Using the direct ELISA none of the PR3-ANCA sera recognized rcPR3, whereas rpPR3 and rbPR3 were recognized by two (11%) and 17 (89%) of the 19 PR3-ANCA sera, respectively. All 13 anti-PR3 mAbs recognized nPR3 in the direct as well as in the capture ELISA. The rcPR3 was recognized by two mAbs in the capture ELISA but by none of the mAbs in the direct ELISA. The rpPR3 was recognized by seven mAbs in the capture ELISA and only by two mAbs in the direct ELISA. All but one of the anti-PR3 mAbs recognized rbPR3, whereas HMC-1/PR3-S176A and Delta-rPR3-S176A were recognized by

  6. Spider Silk Fibers Spun from Soluble Recombinant Silk Produced in Mammalian Cells

    NASA Astrophysics Data System (ADS)

    Lazaris, Anthoula; Arcidiacono, Steven; Huang, Yue; Zhou, Jiang-Feng; Duguay, François; Chretien, Nathalie; Welsh, Elizabeth A.; Soares, Jason W.; Karatzas, Costas N.

    2002-01-01

    Spider silks are protein-based ``biopolymer'' filaments or threads secreted by specialized epithelial cells as concentrated soluble precursors of highly repetitive primary sequences. Spider dragline silk is a flexible, lightweight fiber of extraordinary strength and toughness comparable to that of synthetic high-performance fibers. We sought to ``biomimic'' the process of spider silk production by expressing in mammalian cells the dragline silk genes (ADF-3/MaSpII and MaSpI) of two spider species. We produced soluble recombinant (rc)-dragline silk proteins with molecular masses of 60 to 140 kilodaltons. We demonstrated the wet spinning of silk monofilaments spun from a concentrated aqueous solution of soluble rc-spider silk protein (ADF-3; 60 kilodaltons) under modest shear and coagulation conditions. The spun fibers were water insoluble with a fine diameter (10 to 40 micrometers) and exhibited toughness and modulus values comparable to those of native dragline silks but with lower tenacity. Dope solutions with rc-silk protein concentrations >20% and postspinning draw were necessary to achieve improved mechanical properties of the spun fibers. Fiber properties correlated with finer fiber diameter and increased birefringence.

  7. CHO-gmt5, a novel CHO glycosylation mutant for producing afucosylated and asialylated recombinant antibodies.

    PubMed

    Haryadi, Ryan; Zhang, Peiqing; Chan, Kah Fai; Song, Zhiwei

    2013-01-01

    Engineered zinc-finger nucleases (ZFNs) are powerful tools for creating double-stranded-breaks (DSBs) in genomic DNA in a site-specific manner. These DSBs generated by ZFNs can be repaired by homology-directed repair or nonhomologous end joining, in which the latter can be exploited to generate insertion or deletion mutants. Based on published literature, we designed a pair of zinc-finger nucleases and inactivated the GDP-fucose transporter gene (Slc35c1) in a previously reported CHO mutant that has a dysfunctional CMP-sialic acid transporter gene (Slc35a1). The resulting mutant cell line, CHO-gmt5, lacks functional GDP-fucose transporter and CMP-sialic acid transporter. As a result, these cells can only produce asialylated and afucosylated glycoproteins. It is now widely recognized that removal of the core fucose from the N-glycans attached to Asn(297) of human IgG1 significantly enhances its binding to its receptor, FcγRIIIa, and thereby dramatically improves antibody-dependent cellular cytotoxicity (ADCC). Recent reports showed that removal of sialic acid from IgG1 also enhances ADCC. Therefore, CHO-gmt5 may represent a more advantageous cell line for the production of recombinant antibodies with enhanced ADCC. These cells show comparable growth rate to wild type CHO-K1 cells and uncompromised transfection efficiency, which make them desirable for use as a production line.

  8. Recombination produces coherent bacterial species clusters in both core and accessory genomes

    PubMed Central

    Croucher, Nicholas J.; Gutmann, Michael U.; Corander, Jukka; Hanage, William P.

    2015-01-01

    Background: Population samples show bacterial genomes can be divided into a core of ubiquitous genes and accessory genes that are present in a fraction of isolates. The ecological significance of this variation in gene content remains unclear. However, microbiologists agree that a bacterial species should be ‘genomically coherent’, even though there is no consensus on how this should be determined. Results: We use a parsimonious model combining diversification in both the core and accessory genome, including mutation, homologous recombination (HR) and horizontal gene transfer (HGT) introducing new loci, to produce a population of interacting clusters of strains with varying genome content. New loci introduced by HGT may then be transferred on by HR. The model fits well to a systematic population sample of 616 pneumococcal genomes, capturing the major features of the population structure with parameter values that agree well with empirical estimates. Conclusions: The model does not include explicit selection on individual genes, suggesting that crude comparisons of gene content may be a poor predictor of ecological function. We identify a clearly divergent subpopulation of pneumococci that are inconsistent with the model and may be considered genomically incoherent with the rest of the population. These strains have a distinct disease tropism and may be rationally defined as a separate species. We also find deviations from the model that may be explained by recent population bottlenecks or spatial structure.

  9. Treatment of Burn and Surgical Wounds With Recombinant Human Tropoelastin Produces New Elastin Fibers in Scars.

    PubMed

    Xie, Hua; Lucchesi, Lisa; Zheng, Bo; Ladich, Elena; Pineda, Teresa; Merten, Rose; Gregory, Cynthia; Rutten, Michael; Gregory, Kenton

    2017-02-15

    Tropoelastin (TE), the soluble precursor of insoluble elastin fibers, is produced in minimal amounts in adults. Burn injuries result in inflexible collagen-rich scars because of lack of elastin fiber formation. We studied the feasibility of using recombinant human tropoelastin to enable elastin fiber production in burn and surgical scars to improve skin flexibility. In a swine hypertrophic burn scar model, normal skin and 3 × 3-cm partial thickness thermal burns underwent dermatome resection at 1 week post burn and randomized to four subcutaneous injections of saline or TE (either 0.5, 5, or 10 mg/ml) spaced 3 days apart. Two burn sites received TE injections after wound closure (0.5 or 10 mg/ml). At 90 days, skin hardness, flexibility, and histology were evaluated. All injury sites developed hypertrophic scars. New elastin fibers were found in burn scars in all injuries injected after skin closure with low (5/5) and high (6/6) TE doses (P < .05). No elastin fibers were observed without TE treatment. No significant differences in skin hardness, flexibility, or inflammation were observed. This is the first report demonstrating that subcutaneous injections of TE into surgical and burn injuries can safely produce new elastin fibers in scars. Despite the development of new elastin fibers, skin flexibility was not improved, possibly because of insufficient elastin fiber maturation or the hypertrophic model used. The ability to restore elastin fiber formation in adult skin after burns, trauma, and surgery may improve skin regeneration and reduce disabling complications of scar formation.

  10. Chaperokine function of recombinant Hsp72 produced in insect cells using a baculovirus expression system is retained.

    PubMed

    Zheng, Hongying; Nagaraja, Ganachari M; Kaur, Punit; Asea, Edwina E; Asea, Alexzander

    2010-01-01

    Extracellular heat shock protein 72 (Hsp72; inducible form of the 70-kDa heat shock protein) plays a critical role in innate and adaptive immune responses and has shown promise as an ideal adjuvant for the optimization of antigen-specific anti-tumor vaccines. Recent studies suggest that to correctly elucidate the mechanisms by which Hsp72 exerts its beneficial effects in vitro, great care must be taken to ensure that endotoxin by-products do not invalidate the findings. In this study, we have taken advantage of the baculovirus expression vector system for production of endotoxin-free recombinant Hsp72. The coding sequence of human hsp72 was recombined into the baculovirus immediately downstream of the strong polyhedron gene promoter. Ninety-six h post-infection of Sf9 insect cells with recombinant baculovirus, maximal levels of Hsp72 protein were detected. The recombinant human Hsp72 was purified by affinity chromatography from insect cells, and purity was confirmed by SDS-PAGE and mass spectrometry. The purified human recombinant Hsp72(bv) (Hsp72 produced using the BEVS) was demonstrated to have no endotoxin contamination and was shown to have stimulated potent calcium flux in the human monocytic cell line. Furthermore, recombinant Hsp72(bv) enhanced the tolerance of neuroblastoma cells to heat stress-induced cell death and displayed classical chaperokine functions including augmentation of inflammatory cytokine productions in mouse splenocytes. The production of functional, endotoxin-free recombinant human Hsp72(bv) in insect cells is inexpensive and convenient and eliminates the need of special procedures for endotoxin depletion. Endotoxin-free recombinant human Hsp72(bv) can now be used to unlock the important role Hsp72 plays in modulating immune function.

  11. Receptor specificity and functional comparison of recombinant sea bass (Dicentrarchus labrax) gonadotropins (FSH and LH) produced in different host systems.

    PubMed

    Molés, Gregorio; Zanuy, Silvia; Muñoz, Iciar; Crespo, Berta; Martínez, Iago; Mañanós, Evaristo; Gómez, Ana

    2011-06-01

    Different yields, biopotency, and in vivo pharmacokinetics are obtained for recombinant sea bass gonadoltropins depending on the production system and DNA construct, but they show specific activation of their corresponding receptors. Gonadotropins (GTHs) are glycoprotein hormones that play a major role in the regulation of gonadal functions. Recently, we succeeded in isolating the native sea bass Fsh from sea bass pituitaries, but to ensure the availability of bioactive GTHs and no cross-contamination with other related glycoproteins, recombinant sea bass GTHs were produced using two expression systems-insect and mammalian cells-and different constructs that yielded tethered or noncovalently bound dimers. Their production levels, binding specificity to their homologous cognate receptors, and bioactivity were investigated and compared. Both expression systems were successful in the generation of bioactive recombinant GTHs, but insect Sf9 cells yielded higher amounts of recombinant proteins than mammalian Chinese Hamster Ovary (CHO) stable clones. All recombinant GTHs activated their cognate receptors without cross-ligand binding and were able to stimulate sea bass gonadal steroidogenesis in vitro, although with different biopotencies. To assess their use for in vivo applications, their half-life in sea bass plasma was evaluated. Sf9-GTHs had a lower in vivo stability compared with CHO-GTHs due to their rapid clearance from the blood circulation. Cell-dependent glycosylation could be contributing to the final in vivo stability and biopotency of these recombinant glycoproteins. In conclusion, both insect and mammalian expression systems produced bioactive sea bass recombinant gonadotropins, although with particular features useful for different proposes (e.g., antibody production or in vivo studies, respectively).

  12. Novel application of hydrophobin in medical science: a drug carrier for improving serum stability

    PubMed Central

    Zhao, Liqiang; Xu, Haijin; Li, Ying; Song, Dongmin; Wang, Xiangxiang; Qiao, Mingqiang; Gong, Min

    2016-01-01

    Multiple physiological properties of glucagon-like peptide-1 (GLP-1) ensure that it is a promising drug candidate for the treatment of type 2 diabetes. However, the in vivo half-life of GLP-1 is short because of rapid degradation by dipeptidyl peptidase-IV (DPP-IV) and renal clearance. The poor serum stability of GLP-1 has significantly limited its clinical utility, although many studies are focused on extending the serum stability of this molecule. Hydrophobin, a self-assembling protein, was first applied as drug carrier to stabilize GLP-1 against protease degradation by forming a cavity. The glucose tolerance test clarified that the complex retained blood glucose clearance activity for 72 hours suggesting that this complex might be utilized as a drug candidate administered every 2–3 days. Additionally, it was found that the mutagenesis of hydrophobin preferred a unique pH condition for self-assembly. These findings suggested that hydrophobin might be a powerful tool as a drug carrier or a pH sensitive drug-release compound. The novel pharmaceutical applications of hydrophobin might result in future widespread interest in hydrophobin. PMID:27212208

  13. Mechanism of Nonpolar Model Substances to Inhibit Primary Gushing Induced by Hydrophobin HFBI.

    PubMed

    Shokribousjein, Zahra; Riveros Galan, David; Losada-Pérez, Patricia; Wagner, Patrick; Lammertyn, Jeroen; Arghir, Iulia; Golreihan, Asefeh; Verachtert, Hubert; Aydın, Ahmet Alper; De Maeyer, Marc; Titze, Jean; Ilberg, Vladimír; Derdelinckx, Guy

    2015-05-13

    In this work, the interactions of a well-studied hydrophobin with different types of nonpolar model substances and their impact on primary gushing is evaluated. The nature, length, and degree of saturation of nonpolar molecules are key parameters defining the gushing ability or inhibition. When mixed with hydrophobins, the nonpolar molecule-hydrophobin assembly acts as a less gushing or no gushing system. This effect can be explained in the framework of a competition effect between non-polar systems and CO2 to interact with the hydrophobic patch of the hydrophobin. Interactions of these molecules with hydrophobins are promoted as a result of the similar size of the nonpolar molecules with the hydrophobic patch of the protein, at the expense of the formation of nanobubbles with CO2. In order to prove the presence of interactions and to unravel the mechanisms behind them, a complete set of experimental techniques was used. Surface sensitive techniques clearly show the presence of the interactions, whose nature is not covalent nor hydrogen bonding according to infrared spectroscopy results. Interactions were also reflected by particle size analysis in which mixtures of particles displayed larger size than their pure component counterparts. Upon mixing with nonpolar molecules, the gushing ability of the protein is significantly disrupted.

  14. Protection Conferred by recombinant Yersinia pestis Antigens Produced by a Rapid and Highly Scalable Plant Expression System

    DTIC Science & Technology

    2006-01-24

    variety of molecules have been successfully expressed in plants , including peptides (14), human proteins and enzymes (15), viral and bacterial...contaminated by the Rubisco large subunit, which is very similar in size to F1-V. Analysis of Purified Plant -Produced Antigens. Western blots were...Protection conferred by recombinant Yersinia pestis antigens produced by a rapid and highly scalable plant expression system Luca Santi*†, Anatoli

  15. Challenges in electrochemical pre-purification of recombinant proteins from green plant tissues: sgfp produced in tobacco leaves.

    PubMed

    Robić, Goran

    2013-01-01

    The use of recombinant proteins has increased greatly in recent years, as have the number of techniques and materials used for their production and purification. The principal advantage of using plants as bioreactors is the cost of the recombinant protein production, which is about 1000-fold lower as in the case of using CHO cells commonly applied in industry today. Among the different types of "green" bioreactors being studied today, there is a general consensus among scientists that production in green plant tissues such as leaves is more feasible. However, the presence of chlorophyll and phenolic compounds in plant extracts, which can precipitate and denature the proteins besides damaging separation membranes and gels, makes this technology impracticable on a commercial scale. Electrochemically produced aluminium hydroxide gel can be used to adsorb these compounds, and pre-purify recombinant synthetic green fluorescent protein (sGFP) produced in Nicotiana benthamiana leaves. Removal efficiencies of 99.7% of chlorophyll, 88.5% of phenolic compounds, and 38.5% of native proteins from the N. benthamiana extracts were achieved without removing sGFP from the extracts. Since electrochemical preparation of aluminum hydroxide gel is a cost-effective technique, its use can substantially contribute to the development of future production platforms for recombinant proteins produced in green plant tissues of pharmaceutical and industrial interest.

  16. Design of antibacterial biointerfaces by surface modification of poly (ε-caprolactone) with fusion protein containing hydrophobin and PA-1.

    PubMed

    Wang, Xiangxiang; Mao, Jiwei; Chen, Yiming; Song, Dongmin; Gao, Zhendong; Zhang, Xiuming; Bai, Yanling; Saris, Per E J; Feng, Hui; Xu, Haijin; Qiao, Mingqiang

    2017-03-01

    Class IIa bacteriocin pediocin PA-1 has broad-spectrum activity and is a well-characterized candidate food biopreservative. Here, a simple approach is designed to extend the application of pediocin PA-1 in improving the antibacterial activity of electrospun poly(caprolactone) (PCL) grafts through combining PA-1 with HGFI, which is a self-assembled protein with characteristics allowing the modulation of surface properties of other materials originated from Grifola frondosa. Saccharomyces cerevisiae was used as the host for expression of fusion protein PA-1-linker-HGFI (pH) and his-tag purification was used to purify recombinant protein pH. An antibacterial activity assay showed the fusion protein pH retained the biological property of native PA-1. Water contact angle, X-ray photoelectron spectroscopy, immunofluorescence assay and atomic force microscopy indicated the surface properties of HGFI were greatly preserved by the fusion protein pH. Finally, antibacterial activity of pH-modified PCL substrate measurements implied the fusion protein significantly improved the bacterial-resistance of the PCL film through dressing the PCL fibers with the recombinant pH protein. This work presents a new perspective on the application of hydrophobin and pediocin PA-1 in antibacterial medical devices.

  17. A class II hydrophobin gene, Trhfb3, participates in fungal asexual development of Trichoderma reesei.

    PubMed

    He, Ronglin; Li, Chen; Feng, Jun; Zhang, Dongyuan

    2016-12-23

    Hydrophobin proteins originate from filamentous fungi, which are able to self-assemble at water-air interfaces. Hydrophobins have multiple functions in fungal growth and development. In the present study, the function of the Trhfb3 gene encoding a class II hydrophobin was characterized in Trichoderma reesei The null mutant of Trhfb3 presented a wettable phenotype and a significantly reduced conidial production compared with the parent strain. The ΔTrhfb3 mutant also exhibited less biomass formation than the parent strain. In addition, Trhfb3 was expressed on carbon sources that induce lignocellulytic enzymes, with the highest expression level detected on cellobiose. The results show that Trhfb3 has a role in vegetative growth and asexual development in T. reesei.

  18. Production of specific IgY antibody to the recombinant FanC protein produced in Escherichia coli

    PubMed Central

    Nasiri, Khadijeh; Zibaee, Saeed; Nassiri, Mohammadreza; Tahmoorespur, Mojtaba; Haghparast, Alireza

    2016-01-01

    Objective(s): Enterotoxigenic Escherichia coli (ETEC) strains are one of the primary causes of diarrhea in newborn calves and in humans, pigs, and sheep. IgY technology has been identified as a promising alternative to generating a mass amount of specific antibody for use in immunotherapy and immunodiagnostics. The purpose of this study was to produce specific antibody by egg yolk antibody (IgY) to recombinant FanC protein from ETEC. Materials and Methods: FanC (K99) gene was amplified from ETEC by specific primers and polymerase chain reaction. The gene was cloned and subcloned into pTZ57R/T and pET32a (+) vectors, respectively. Recombinant vector was transferred into E. coli BL21 CodonPlus (DE3). Protein expression was investigated by 1 mM IPTG induction. Hens were immunized by the purified recombinant FanC protein. The activity and specificity of the IgY antibody were detected by dot-blotting, Western blotting, and indirect ELISA. Results: We obtained FanC specific IgYs by immunizing the hens with the recombinant FanC protein. The anti-FanC IgY showed binding specifically to the FanC protein of ETEC. Conclusion: The results emphasize that specific IgY against the recombinant FanC protein could be recommended as a candidate for passive immunization against ETEC infection in animals and humans. PMID:27746871

  19. Hydrophobins in the Life Cycle of the Ectomycorrhizal Basidiomycete Tricholoma vaccinum

    PubMed Central

    Sammer, Dominik; Gube, Matthias; Wagner, Katharina; Kothe, Erika

    2016-01-01

    Hydrophobins—secreted small cysteine-rich, amphipathic proteins—foster interactions of fungal hyphae with hydrophobic surfaces, and are involved in the formation of aerial hyphae. Phylogenetic analyses of Tricholoma vaccinum hydrophobins showed a grouping with hydrophobins of other ectomycorrhizal fungi, which might be a result of co-evolution. Further analyses indicate angiosperms as likely host trees for the last common ancestor of the genus Tricholoma. The nine hydrophobin genes in the T. vaccinum genome were investigated to infer their individual roles in different stages of the life cycle, host interaction, asexual and sexual development, and with respect to different stresses. In aerial mycelium, hyd8 was up-regulated. In silico analysis predicted three packing arrangements, i.e., ring-like, plus-like and sheet-like structure for Hyd8; the first two may assemble to rodlets of hydrophobin covering aerial hyphae, whereas the third is expected to be involved in forming a two-dimensional network of hydrophobins. Metal stress induced hydrophobin gene hyd5. In early steps of mycorrhization, induction of hyd4 and hyd5 by plant root exudates and root volatiles could be shown, followed by hyd5 up-regulation during formation of mantle, Hartig’ net, and rhizomorphs with concomitant repression of hyd8 and hyd9. During fruiting body formation, mainly hyd3, but also hyd8 were induced. Host preference between the compatible host Picea abies and the low compatibility host Pinus sylvestris could be linked to a stronger induction of hyd4 and hyd5 by the preferred host and a stronger repression of hyd8, whereas the repression of hyd9 was comparable between the two hosts. PMID:27936063

  20. Noncovalent Sidewall Functionalization of Carbon Nanotubes by Biomolecules: Single-stranded DNA and Hydrophobin

    NASA Astrophysics Data System (ADS)

    Taeger, Sebastian; Xuang, Li Yi; Günther, Katrin; Mertig, Michael

    2005-09-01

    Single-stranded DNA (ssDNA) is known to disperse individual carbon nanotubes (CNT) into aqueous suspensions. But other biomolecules are able to do so as well. We demonstrate a protein-assisted CNT dispersion by using hydrophobin. The yields of the suspensions are monitored by optical absorption spectroscopy (OAS). We perform atomic force microscopy (AFM) studies of DNA- and hydrophobin-functionalized CNT with a resolution that allows us to identify individual molecules attached to isolated CNT. We control the density of DNA on the nanotubes by the DNA:CNT ratio, and observe stable suspensions of CNT with surprisingly low surface coverages.

  1. An optimized sericin-1 expression system for mass-producing recombinant proteins in the middle silk glands of transgenic silkworms.

    PubMed

    Wang, Feng; Xu, Hanfu; Yuan, Lin; Ma, Sanyuan; Wang, Yuancheng; Duan, Xiaoli; Duan, Jianping; Xiang, Zhonghuai; Xia, Qingyou

    2013-10-01

    The middle silk gland (MSG) of silkworm is thought to be a potential host for mass-producing valuable recombinant proteins. Transgenic MSG expression systems based on the usage of promoter of sericin1 gene (sericin-1 expression system) have been established to produce various recombinant proteins in MSG. However, further modifying the activity of the sericin-1 expression system to yield higher amounts of recombinant proteins is still necessary. In this study, we provide an alternative modification strategy to construct an efficient sericin-1 expression system by using the hr3 enhancer (hr3 CQ) from a Chongqing strain of the Bombyx mori nuclear polyhedrosis virus (BmNPV) and the 3'UTRs of the fibroin heavy chain (Fib-HPA), the fibroin light chain (Fib-LPA), and Sericin1 (Ser1PA) genes. We first analyzed the effects of these DNA elements on expression of luciferase, and found that the combination of hr3 CQ and Ser1PA was most effective to increase the activity of luciferase. Then, hr3 CQ and Ser1PA were used to modify the sericin1 expression system. Transgenic silkworms bearing these modified sericin1 expression vectors were generated by a piggyBac transposon mediated genetic transformation method. Our results showed that mRNA level of DsRed reporter gene in transgenic silkworms containing hr3 CQ and Ser1PA significantly increased by 9 fold to approximately 83 % of that of endogenous sericin1. As the results of that, the production of recombinant RFP increased by 16 fold to 9.5 % (w/w) of cocoon shell weight. We conclude that this modified sericin-1 expression system is efficient and will contribute to the MSG as host to mass produce valuable recombinant proteins.

  2. Haematococcus as a promising cell factory to produce recombinant pharmaceutical proteins.

    PubMed

    Saei, Amir Ata; Ghanbari, Parisa; Barzegari, Abolfazl

    2012-11-01

    The need for recombinant pharmaceutical proteins has urged scientists all over the world to search for better protein expression systems which have higher capabilities and flexibilities. Although a number of protein expression systems are now available, no system is ideal and different systems lack specific properties. Here, microalga Haematococcus is discussed as a new protein expression system which merits cheap growth medium, fast growth rate, ease of manipulation and scale-up, ease of transformation, potential of exploiting in bioreactors and ability to exert post-translational modifications to the proteins. This green single-cell plant has favorable biological and biotechnological features for production of remarkable yields of recombinant proteins with high functionality. In this review article, we highlight the favorable biotechnological characteristics of Haematococcus for lowering costs and facilitating scale-up of recombinant protein production along with its superior biological features for genetic engineering.

  3. Hydrophobins Sc3 and Sc4 gene expression in mounds, fruiting bodies and vegetative hyphae of Schizophyllum commune.

    PubMed

    Banerjee, Goutami; Robertson, Deborah L; Leonard, Thomas J

    2008-03-01

    An abnormal growth form called mound has been hypothesized to be a neoplasm in the filamentous fungus Schizophyllum commune. An alternative hypothesis is that mounds represent some unusual developmental form in the fruiting body morphogenetic pathway. Hydrophobin proteins have been found in fruiting bodies where they line the surface of gas exchange pores and function to keep the pores hydrophobic. To further determine possible relationships between mounds and fruiting bodies, mound tissue was examined for gas exchange pores and the presence of hydrophobins. Cryoscanning electron microscopic images revealed the presence of channels in mound tissue and presumptive hydrophobin rodlets similar to the air channels in fruiting bodies. Hydrophobin gene expression was also measured in mound tissue using quantitative real-time PCR and showed both monokaryotic and dikaryotic mound tissue exhibited high expression of the dikaryotic specific Sc4 hydrophobin gene. In contrast, Sc4 hydrophobin expression was barely detectable in monokaryotic fruiting bodies. The expression of Sc4 hydrophobin genes in mounds suggests mound development uses this aspect of the dikaryotic fruiting developmental pathway.

  4. Detection of recombinant human lactoferrin and lysozyme produced in a bitransgenic cow.

    PubMed

    Kaiser, Germán G; Mucci, Nicolás C; González, Vega; Sánchez, Lourdes; Parrón, José A; Pérez, María D; Calvo, Miguel; Aller, Juan F; Hozbor, Federico A; Mutto, Adrián A

    2017-03-01

    Lactoferrin and lysozyme are 2 glycoproteins with great antimicrobial activity, being part of the nonspecific defensive system of human milk, though their use in commercial products is difficult because human milk is a limited source. Therefore, many investigations have been carried out to produce those proteins in biological systems, such as bacteria, yeasts, or plants. Mammals seem to be more suitable as expression systems for human proteins, however, especially for those that are glycosylated. In the present study, we developed a bicistronic commercial vector containing a goat β-casein promoter and an internal ribosome entry site fragment between the human lactoferrin and human lysozyme genes to allow the introduction of both genes into bovine adult fibroblasts in a single transfection. Embryos were obtained by somatic cell nuclear transfer, and, after 6 transferences to recipients, 3 pregnancies and 1 viable bitransgenic calf were obtained. The presence of the vector was confirmed by fluorescent in situ hybridization of skin cells. At 13 mo of life and after artificial induction of lactation, both recombinant proteins were found in the colostrum and milk of the bitransgenic calf. Human lactoferrin concentration in the colostrum was 0.0098 mg/mL and that in milk was 0.011 mg/mL; human lysozyme concentration in the colostrum was 0.0022 mg/mL and that in milk was 0.0024 mg/mL. The molar concentration of both human proteins revealed no differences in protein production of the internal ribosome entry site upstream and downstream protein. The enzymatic activity of lysozyme in the transgenic milk was comparable to that of human milk, being 6 and 10 times higher than that of bovine lysozyme present in milk. This work represents an important step to obtain multiple proteins or enhance single protein production by using animal pharming and fewer regulatory and antibiotic-resistant foreign sequences, allowing the design of humanized milk with added biological value for

  5. Optimization of culturing conditions of recombined Escherichia coli to produce umami octopeptide-containing protein.

    PubMed

    Zhang, Yin; Wei, Xiong; Lu, Zhou; Pan, Zhongli; Gou, Xinhua; Venkitasamy, Chandrasekar; Guo, Siya; Zhao, Liming

    2017-07-15

    Using synthesized peptides to verify the taste of natural peptides was probably the leading cause for tasting disputes regarding umami peptides. A novel method was developed to prepare the natural peptide which could be used to verify the taste of umami peptide. A controversial octopeptide was selected and gene engineering was used to structure its Escherichia coli. expressing vector. A response surface method was adopted to optimize the expression conditions of the recombinant protein. The results of SDS-PAGE for the recombinant protein indicated that the recombinant expression system was successfully structured. The fitting results of the response surface experiment showed that the OD600 value was the key factor which influenced the expression of the recombinant protein. The optimal culturing process conditions predicted with the fitting model were an OD600 value of 0.5, an IPTG concentration of 0.6mM, a culturing temperature of 28.75°C and a culturing time of 5h.

  6. Characterization and biological activities of recombinant human plasminogen kringle 1-3 produced in Escherichia coli.

    PubMed

    You, Weon-Kyoo; So, Seung-Ho; Sohn, Young-Doug; Lee, Hyosil; Park, Doo-Hong; Chung, Soo-Il; Chung, Kwang-Hoe

    2004-07-01

    Angiogenesis, the formation of new capillaries from preexisting blood vessels, is involved in many pathological conditions, for example, tumorigenesis, diabetic retinopathy, and rheumatoid arthritis. Angiostatin, which contains the kringle 1-4 domains of plasminogen, is known to be a potent inhibitor of angiogenesis and a strong suppressor of various solid tumors. In this study, we expressed recombinant protein containing the kringle 1-3 domains of human plasminogen in Escherichia coli and investigated its biological activities. The protein was successfully refolded from inclusion bodies and purified at a 30% overall yield, as a single peak by HPLC. The purified recombinant protein had biochemical properties that were similar to those of the native form, which included molecular size, lysine-binding capacity, and immunoreactivity with a specific antibody. The recombinant protein was also found to strongly inhibit the proliferation of bovine capillary endothelial cells in vitro, and the formation of new capillaries on chick embryos. In addition, it suppressed the growth of primary Lewis lung carcinoma and B16 melanoma in an in vivo mouse model. Our findings suggest that the recombinant kringle 1-3 domains in a prokaryote expression system have anti-angiogenic activities, which may be useful in clinical and basic research in the field of angiogenesis.

  7. Native-sized recombinant spider silk protein produced in metabolically engineered Escherichia coli results in a strong fiber

    PubMed Central

    Xia, Xiao-Xia; Qian, Zhi-Gang; Ki, Chang Seok; Park, Young Hwan; Kaplan, David L.; Lee, Sang Yup

    2010-01-01

    Spider dragline silk is a remarkably strong fiber that makes it attractive for numerous applications. Much has thus been done to make similar fibers by biomimic spinning of recombinant dragline silk proteins. However, success is limited in part due to the inability to successfully express native-sized recombinant silk proteins (250–320 kDa). Here we show that a 284.9 kDa recombinant protein of the spider Nephila clavipes is produced and spun into a fiber displaying mechanical properties comparable to those of the native silk. The native-sized protein, predominantly rich in glycine (44.9%), was favorably expressed in metabolically engineered Escherichia coli within which the glycyl-tRNA pool was elevated. We also found that the recombinant proteins of lower molecular weight versions yielded inferior fiber properties. The results provide insight into evolution of silk protein size related to mechanical performance, and also clarify why spinning lower molecular weight proteins does not recapitulate the properties of native fibers. Furthermore, the silk expression, purification, and spinning platform established here should be useful for sustainable production of natural quality dragline silk, potentially enabling broader applications. PMID:20660779

  8. Integrated approach to produce a recombinant, His-tagged human α-galactosidase A in mammalian cells.

    PubMed

    Corchero, José Luis; Mendoza, Rosa; Lorenzo, Julia; Rodríguez-Sureda, Victor; Domínguez, Carmen; Vázquez, Esther; Ferrer-Miralles, Neus; Villaverde, Antonio

    2011-01-01

    Successful production of recombinant proteins (r-proteins) by transient gene expression (TGE) depends on several parameters (including producer cells, culture conditions, transfection procedure, or expression vector) that should be optimized when producing any recombinant product. In this work, TGE-based production of human α-galactosidase A (GLA) is described. Producer cells, expression vectors, and parameters influencing cell metabolism after transfection have been tested. The enzyme is secreted, has the right molecular weight, and is enzymatically active. Productivities of up to 30-40 mg/L have been achieved, with a simple, fast procedure. A 6 × His tag allows enzyme purification in a single step, rendering a highly pure product. We propose a TGE-based protocol able to produce up to several milligrams per liter of highly pure, active GLA in a time as short as a few days. By this, enough amounts of engineered versions of the enzyme can be easily produced to be tested in vitro or in preclinical trials.

  9. High-efficiency homologous recombination in the oil-producing alga Nannochloropsis sp.

    PubMed

    Kilian, Oliver; Benemann, Christina S E; Niyogi, Krishna K; Vick, Bertrand

    2011-12-27

    Algae have reemerged as potential next-generation feedstocks for biofuels, but strain improvement and progress in algal biology research have been limited by the lack of advanced molecular tools for most eukaryotic microalgae. Here we describe the development of an efficient transformation method for Nannochloropsis sp., a fast-growing, unicellular alga capable of accumulating large amounts of oil. Moreover, we provide additional evidence that Nannochloropsis is haploid, and we demonstrate that insertion of transformation constructs into the nuclear genome can occur by high-efficiency homologous recombination. As examples, we generated knockouts of the genes encoding nitrate reductase and nitrite reductase, resulting in strains that were unable to grow on nitrate and nitrate/nitrite, respectively. The application of homologous recombination in this industrially relevant alga has the potential to rapidly advance algal functional genomics and biotechnology.

  10. The pEAQ vector series: the easy and quick way to produce recombinant proteins in plants.

    PubMed

    Peyret, Hadrien; Lomonossoff, George P

    2013-09-01

    The pEAQ vectors are a series of plasmids designed to allow easy and quick production of recombinant proteins in plants. Their main feature is the use of the Cowpea Mosaic Virus hypertranslational "CPMV-HT" expression system, which provides high yields of recombinant protein through extremely high translational efficiency without the need for viral replication. Since their creation, the pEAQ vectors have been used to produce a wide variety of proteins in plants. Viral proteins and Virus-Like Particles (VLPs) have been of particular interest, but other types of proteins including active enzymes have also been expressed. While the pEAQ vectors have mostly been used in a transient expression context, through agroinfiltration of leaves, they have also been shown to be suitable for the production of stably transformed lines of both cell cultures and whole plants. This paper looks back on the genesis of the pEAQ vectors and reviews their use so far.

  11. Permeation studies of novel terbinafine formulations containing hydrophobins through human nails in vitro.

    PubMed

    Vejnovic, Ivana; Huonder, Cornelia; Betz, Gabriele

    2010-09-15

    Existing treatments of onychomycosis are not satisfactory. Oral therapies have many side effects and topical formulations are not able to penetrate into the human nail plate and deliver therapeutical concentrations of active agent in situ. The purpose of the present study was to determine the amount of terbinafine, which permeates through the human nail plate, from liquid formulations containing enhancers, namely hydrophobins A-C in the concentration of 0.1% (w/v). The used reference solution contained 10% (w/v) of terbinafine in 60% (v/v) ethanol/water without enhancer. Permeability studies have been performed on cadaver nails using Franz diffusion cells modified to mount nail plates and filled with 60% (v/v) ethanol/water in the acceptor chamber. Terbinafine was quantitatively determined by HPLC. The amount of terbinafine remaining in the nail was extracted by 96% ethanol from pulverized nail material after permeation experiment and presented as percentage of the dry nail weight before the milling test. Permeability coefficient (PC) of terbinafine from reference solution was determined to be 1.52E-10 cm/s. Addition of hydrophobins improved PC in the range of 3E-10 to 2E-9 cm/s. Remaining terbinafine reservoir in the nail from reference solution was 0.83% (n=2). An increase of remaining terbinafine reservoir in the nail was observed in two out of three tested formulations containing hydrophobins compared to the reference. In all cases, known minimum inhibitory concentration of terbinafine for dermatophytes (0.003 microg/ml) has been exceeded in the acceptor chamber of the diffusion cells. All tested proteins (hydrophobins) facilitated terbinafine permeation after 10 days of permeation experiment, however one of them achieved an outstanding enhancement factor of 13.05 compared to the reference. Therefore, hydrophobins can be included in the list of potential enhancers for treatment of onychomycosis.

  12. One single method to produce native and Tat-fused recombinant human α-synuclein in Escherichia coli

    PubMed Central

    2013-01-01

    Background Human α-synuclein is a small-sized, natively unfolded protein that in fibrillar form is the primary component of Lewy bodies, the pathological hallmark of Parkinson’s disease. Experimental evidence suggests that α-synuclein aggregation is the key event that triggers neurotoxicity although additional findings have proposed a protective role of α-synuclein against oxidative stress. One way to address the mechanism of this protective action is to evaluate α-synuclein-mediated protection by delivering this protein inside cells using a chimeric protein fused with the Tat-transduction domain of HIV Tat, named TAT-α-synuclein. Results A reliable protocol was designed to efficiently express and purify two different forms of human α-synuclein. The synthetic cDNAs encoding for the native α-synuclein and the fusion protein with the transduction domain of Tat protein from HIV were overexpressed in a BL21(DE3) E. coli strain as His-tagged proteins. The recombinant proteins largely localized (≥ 85%) to the periplasmic space. By using a quick purification protocol, based on recovery of periplasmic space content and metal-chelating chromatography, the recombinant α-synuclein protein forms could be purified in a single step to ≥ 95% purity. Both α-synuclein recombinant proteins form fibrils and the TAT-α-synuclein is also cytotoxic in the micromolar concentration range. Conclusions To further characterize the molecular mechanisms of α-synuclein neurotoxicity both in vitro and in vivo and to evaluate the relevance of extracellular α-synuclein for the pathogenesis and progression of Parkinson’s disease, a suitable method to produce different high-quality forms of this pathological protein is required. Our optimized expression and purification procedure offers an easier and faster means of producing different forms (i.e., both the native and the TAT-fusion form) of soluble recombinant α-synuclein than previously described procedures. PMID:23557146

  13. The Effect of Hydrophobin Protein on Conductive Properties of Carbon Nanotube Field-Effect Transistors: First Study on Sensing Mechanism.

    PubMed

    Yotprayoonsakl, Peerapong; Szilvay, Géza R; Laaksonen, Päivi; Linder, Markus B; Ahlskog, Markus

    2015-03-01

    Hydrophobin is a surface active protein having both hydrophobic and hydrophilic functional domains which has previously been used for functionalization and solubilization of graphene and carbon nanotubes. In this work, field-effect transistors based on single nanotubes have been employed for electronic detection of hydrophobin protein in phosphate buffer solution. Individual nanotubes, single- and multiwalled, are characterized by atomic force microscopy after being immersed in protein solution, showing a relatively dense coverage with hydrophobin. We have studied aspects such as nanotube length (0.3-1.2 µm) and the hysteresis effect in the gate voltage dependent conduction. When measured in ambient condition after the exposure to hydrophobin, the resistance increase has a strong dependence on the nanotube length, which we ascribe to mobility degradation and localization effects. The change could be exceptionally large when measured in-situ in solution and at suitable gate voltage conditions, which is shown to relate to the different mechanism behind the hysteresis effect.

  14. Decreased fluidity of cell membranes causes a metal ion deficiency in recombinant Saccharomyces cerevisiae producing carotenoids.

    PubMed

    Liu, Peitong; Sun, Liang; Sun, Yuxia; Shang, Fei; Yan, Guoliang

    2016-04-01

    The genome-wide transcriptional responses of S. cerevisiae to heterologous carotenoid biosynthesis were investigated using DNA microarray analysis. The results show that the genes involved in metal ion transport were specifically up-regulated in the recombinant strain, and metal ions, including Cu(2+), Fe(2+), Mn(2+), and Mg(2+), were deficient in the recombinant strain compared to the ion content of the parent strain. The decrease in metal ions was ascribed to a decrease in cell membrane (CM) fluidity caused by lower levels of unsaturated fatty acids and ergosterol. This was confirmed by the observation that metal ion levels were restored when CM fluidity was increased by supplying linoleic acid. In addition, a 24.3 % increase in the β-carotene concentration was observed. Collectively, our results suggest that heterologous production of carotenoids in S. cerevisiae can induce cellular stress by rigidifying the CM, which can lead to a deficiency in metal ions. Due to the importance of CM fluidity in cellular physiology, maintaining normal CM fluidity might be a potential approach to improving carotenoid production in genetically engineered S. cerevisiae.

  15. Rational Engineering of Recombinant Picornavirus Capsids to Produce Safe, Protective Vaccine Antigen

    PubMed Central

    Burman, Alison; Jackson, Terry; Ren, Jingshan; Loureiro, Silvia; Jones, Ian M.; Fry, Elizabeth E.; Stuart, David I.; Charleston, Bryan

    2013-01-01

    Foot-and-mouth disease remains a major plague of livestock and outbreaks are often economically catastrophic. Current inactivated virus vaccines require expensive high containment facilities for their production and maintenance of a cold-chain for their activity. We have addressed both of these major drawbacks. Firstly we have developed methods to efficiently express recombinant empty capsids. Expression constructs aimed at lowering the levels and activity of the viral protease required for the cleavage of the capsid protein precursor were used; this enabled the synthesis of empty A-serotype capsids in eukaryotic cells at levels potentially attractive to industry using both vaccinia virus and baculovirus driven expression. Secondly we have enhanced capsid stability by incorporating a rationally designed mutation, and shown by X-ray crystallography that stabilised and wild-type empty capsids have essentially the same structure as intact virus. Cattle vaccinated with recombinant capsids showed sustained virus neutralisation titres and protection from challenge 34 weeks after immunization. This approach to vaccine antigen production has several potential advantages over current technologies by reducing production costs, eliminating the risk of infectivity and enhancing the temperature stability of the product. Similar strategies that will optimize host cell viability during expression of a foreign toxic gene and/or improve capsid stability could allow the production of safe vaccines for other pathogenic picornaviruses of humans and animals. PMID:23544011

  16. Enhancing isomaltulose production by recombinant Escherichia coli producing sucrose isomerase: culture medium optimization containing agricultural wastes and cell immobilization.

    PubMed

    Li, Sha; Xu, Hong; Yu, Jianguang; Wang, Yanyuan; Feng, Xiaohai; Ouyang, Pingkai

    2013-10-01

    Isomaltulose is a structural isomer of sucrose commercially used in food industries. In this work, recombinant Escherichia coli producing sucrose isomerase (SIase) was used to convert sucrose into isomaltulose. To develop an economical industrial medium, untreated cane molasses (10.63 g l⁻¹), yeast extract (25.93 g l⁻¹), and corn steep liquor (10.45 g l⁻¹) were used as main culture compositions for SIase production. The relatively high SIase activity (14.50 ± 0.11 U mg DCW⁻¹) was obtained by the recombinant cells. To the best of our knowledge, this is the first investigation on SIase production by engineered E. coli using untreated cane molasses. The recombinant E. coli cells expressing the SIase gene were immobilized in calcium alginate gel in order to improve the efficiency of recycling. The immobilization was most effective with 2 % (w/v) sodium alginate and 3 % (w/v) calcium chloride. The optimal initial biomass for immobilization was 20 % (w/v, wet wt.), with a hardening time of 8 h for cell immobilization. The immobilized E. coli cells exhibited good stability for 30 batches with the productivity of 0.45 g isomaltulose g pellet⁻¹ h⁻¹. A continuous isomaltulose formation process using a column reactor remained stable for 40 days with 83 ± 2 % isomaltulose yield, which would be beneficial for economical production of isomaltulose.

  17. Construction of Recombinant Bacmid Containing M2e-Ctxb and Producing the Fusion Protein in Insect Cell Lines

    PubMed Central

    Mirzaei, Nima; Mokhtari Azad, Talat; Nategh, Rakhshandeh; Soleimanjahi, Hoorieh; Amirmozafari, Nour

    2014-01-01

    Background: Sequence variations in glycoproteins of influenza virus surface impel us to design new candidate vaccines yearly. Ectodomain of influenza M2 protein is a surface and highly conserved protein. M2e in influenza vaccines may eliminate the need for changing vaccine formulation every year. Objectives: In this study, a recombinant baculovirus containing M2e and cholera toxin subunit B fusion gene was generated with transposition process to express in large amounts in insect cell lines. Materials and Methods: M2e-ctxB fusion gene was created and cloned into pFastBac HT. The recombinant vector was transformed into DH10Bac cells to introduce the fusion gene into the bacmid DNA via a site-specific transposition process. The recombinant bacmid was then extracted from white colonies and further analyzed using PCR, DNA sequence analyzing, and indirect immunofluorescence assay. Results: PCR and DNA sequence analyzing results showed that the fusion gene was constructed as a single open reading frame and was successfully inserted into bacmid DNA. Moreover, indirect immunofluorescence results showed that the fusion gene was successfully expressed. Conclusions: Baculovirus expression vector system is valuable to produce M2e based influenza vaccines due to its simple utilization and ease of target gene manipulation. The expressed protein in such systems can improve the evaluating process of new vaccination strategies. PMID:24719728

  18. Transient Glyco-Engineering to Produce Recombinant IgA1 with Defined N- and O-Glycans in Plants.

    PubMed

    Dicker, Martina; Tschofen, Marc; Maresch, Daniel; König, Julia; Juarez, Paloma; Orzaez, Diego; Altmann, Friedrich; Steinkellner, Herta; Strasser, Richard

    2016-01-01

    The production of therapeutic antibodies to combat pathogens and treat diseases, such as cancer is of great interest for the biotechnology industry. The recent development of plant-based expression systems has demonstrated that plants are well-suited for the production of recombinant monoclonal antibodies with defined glycosylation. Compared to immunoglobulin G (IgG), less effort has been undertaken to express immunoglobulin A (IgA), which is the most prevalent antibody class at mucosal sites and a promising candidate for novel recombinant biopharmaceuticals with enhanced anti-tumor activity. Here, we transiently expressed recombinant human IgA1 against the VP8* rotavirus antigen in glyco-engineered ΔXT/FT Nicotiana benthamiana plants. Mass spectrometric analysis of IgA1 glycopeptides revealed the presence of complex biantennary N-glycans with terminal N-acetylglucosamine present on the N-glycosylation site of the CH2 domain in the IgA1 alpha chain. Analysis of the peptide carrying nine potential O-glycosylation sites in the IgA1 alpha chain hinge region showed the presence of plant-specific modifications including hydroxyproline formation and the attachment of pentoses. By co-expression of enzymes required for initiation and elongation of human O-glycosylation it was possible to generate disialylated mucin-type core 1 O-glycans on plant-produced IgA1. Our data demonstrate that ΔXT/FT N. benthamiana plants can be engineered toward the production of recombinant IgA1 with defined human-type N- and O-linked glycans.

  19. Transient Glyco-Engineering to Produce Recombinant IgA1 with Defined N- and O-Glycans in Plants

    PubMed Central

    Dicker, Martina; Tschofen, Marc; Maresch, Daniel; König, Julia; Juarez, Paloma; Orzaez, Diego; Altmann, Friedrich; Steinkellner, Herta; Strasser, Richard

    2016-01-01

    The production of therapeutic antibodies to combat pathogens and treat diseases, such as cancer is of great interest for the biotechnology industry. The recent development of plant-based expression systems has demonstrated that plants are well-suited for the production of recombinant monoclonal antibodies with defined glycosylation. Compared to immunoglobulin G (IgG), less effort has been undertaken to express immunoglobulin A (IgA), which is the most prevalent antibody class at mucosal sites and a promising candidate for novel recombinant biopharmaceuticals with enhanced anti-tumor activity. Here, we transiently expressed recombinant human IgA1 against the VP8* rotavirus antigen in glyco-engineered ΔXT/FT Nicotiana benthamiana plants. Mass spectrometric analysis of IgA1 glycopeptides revealed the presence of complex biantennary N-glycans with terminal N-acetylglucosamine present on the N-glycosylation site of the CH2 domain in the IgA1 alpha chain. Analysis of the peptide carrying nine potential O-glycosylation sites in the IgA1 alpha chain hinge region showed the presence of plant-specific modifications including hydroxyproline formation and the attachment of pentoses. By co-expression of enzymes required for initiation and elongation of human O-glycosylation it was possible to generate disialylated mucin-type core 1 O-glycans on plant-produced IgA1. Our data demonstrate that ΔXT/FT N. benthamiana plants can be engineered toward the production of recombinant IgA1 with defined human-type N- and O-linked glycans. PMID:26858738

  20. Protein folding and conformational stress in microbial cells producing recombinant proteins: a host comparative overview

    PubMed Central

    Gasser, Brigitte; Saloheimo, Markku; Rinas, Ursula; Dragosits, Martin; Rodríguez-Carmona, Escarlata; Baumann, Kristin; Giuliani, Maria; Parrilli, Ermenegilda; Branduardi, Paola; Lang, Christine; Porro, Danilo; Ferrer, Pau; Tutino, Maria Luisa; Mattanovich, Diethard; Villaverde, Antonio

    2008-01-01

    Different species of microorganisms including yeasts, filamentous fungi and bacteria have been used in the past 25 years for the controlled production of foreign proteins of scientific, pharmacological or industrial interest. A major obstacle for protein production processes and a limit to overall success has been the abundance of misfolded polypeptides, which fail to reach their native conformation. The presence of misfolded or folding-reluctant protein species causes considerable stress in host cells. The characterization of such adverse conditions and the elicited cell responses have permitted to better understand the physiology and molecular biology of conformational stress. Therefore, microbial cell factories for recombinant protein production are depicted here as a source of knowledge that has considerably helped to picture the extremely rich landscape of in vivo protein folding, and the main cellular players of this complex process are described for the most important cell factories used for biotechnological purposes. PMID:18394160

  1. Prokaryotic High-Level Expression System in Producing Adhesin Recombinant Protein E of Nontypeable Haemophilus influenzae

    PubMed Central

    Tavakoli, Minoo; Bouzari, Saeed; Siadat, Seyed Davar; Najar Peerayeh, Shahin; Jafari, Anis

    2015-01-01

    Background: Adhesion protein E (PE) of Haemophilus influenzae is a 16 - 18 kDa protein with 160 amino acids which causes adhesion to epithelial cells and acts as a major factor in pathogenesis. Objectives: In this study, we performed cloning, expression and purification of PE as a candidate antigen for vaccine design upon further study. Materials and Methods: At first, the pe gene of NTHi ATCC 49766 strain (483 bp) was amplified by PCR. Then, to sequence the resulted amplicon, it was cloned into TA vector (pTZ57R/T). In the next step, the sequenced gene was sub-cloned in pBAD/gIII A vector and transformed into competent Escherichia coli TOP10. For overexpression, the recombinant bacteria were grown in broth medium containing arabinose and the recombinant protein was purified using metal affinity chromatography (Ni-nitrilotriacetic acid) (Ni-NTA agarose). Finally, the protein was detected using sodium dodecyl sulfate polyacrylamide gel electrophores (SDS-PAG) and confirmed by western blotting. Results: The cloned gene was confirmed by PCR, restriction digestion and sequencing. The sequenced gene was searched for homology in GenBank and 99% similarity was found to the already deposited genes in GenBank. Then we obtained PE using Ni-NTA agarose with up to 7 mg/mL concentration. Conclusions: The pe gene was successfully cloned and confirmed by sequencing. Finally, PE was obtained with high concentration. Due to high homology and similarity among the pe gene from NTHi ATCC 49766 and other NTHi strains in GenBank, we believe that the protein is a universal antigen to be used as a vaccine design candidate and further studies to evaluate its immunogenicity is underway. PMID:26034537

  2. Fat-free yogurt made using a galactose-positive exopolysaccharide-producing recombinant strain of Streptococcus thermophilus.

    PubMed

    Robitaille, G; Tremblay, A; Moineau, S; St-Gelais, D; Vadeboncoeur, C; Britten, M

    2009-02-01

    To prevent textural defects in low-fat and fat-free yogurts, fat substitutes are routinely added to milk. In situ production of exopolysaccharides (EPS) by starter cultures is an acknowledged alternative to the addition of biothickeners. With the aim of increasing in situ EPS production, a recombinant galactose-positive EPS(+) Streptococcus thermophilus strain, RD-534-S1, was generated and compared with the parent galactose-negative EPS(+) strain RD-534. The RD-534-S1 strain produced up to 84 mg/L of EPS during a single-strain milk fermentation process, which represented 1.3 times more than the EPS produced by strain RD-534. Under conditions that mimic industrial yogurt production, the starter culture consisting of RD-534-S1 and (EPS(-)) Lactobacillus bulgaricus L210R strain (RD-534-S1/L210R) led to an EPS production increase of 1.65-fold as compared with RD-534-S1 alone. However, the amount of EPS produced did not differ from that found in yogurts produced using an isogenic starter culture that included the parent S. thermophilus strain RD-534 and Lb. bulgaricus L210R (RD-534/L210R). Moreover, the gel characteristics of set-style yogurt and the rheological properties of stirred-style yogurt produced using RD-534-S1/L210R were similar to the values obtained for yogurts made with RD-534/L210R. In conclusion, it is possible to increase the production of EPS by ropy S. thermophilus strains through genetic engineering of galactose metabolism. However, when used in combination with Lb. bulgaricus for yogurt manufacture, the EPS overproduction of recombinant strain is not significant.

  3. Recombinant L-Asparaginase from Zymomonas mobilis: A Potential New Antileukemic Agent Produced in Escherichia coli

    PubMed Central

    Pereira, Juliana Christina Castanheira Vicente; Costa-Amaral, Isabele Campos; da Costa, Elaine Sobral; Ribeiro, Maria Cecília Menks; Land, Marcelo Gerardin Poirot; Alves, Tito Lívio Moitinho; Larentis, Ariane Leites; Almeida, Rodrigo Volcan

    2016-01-01

    L-asparaginase is an enzyme used as a chemotherapeutic agent, mainly for treating acute lymphoblastic leukemia. In this study, the gene of L-asparaginase from Zymomonas mobilis was cloned in pET vectors, fused to a histidine tag, and had its codons optimized. The L-asparaginase was expressed extracellularly and intracellularly (cytoplasmically) in Escherichia coli in far larger quantities than obtained from the microorganism of origin, and sufficient for initial cytotoxicity tests on leukemic cells. The in silico analysis of the protein from Z. mobilis indicated the presence of a signal peptide in the sequence, as well as high identity to other sequences of L-asparaginases with antileukemic activity. The protein was expressed in a bioreactor with a complex culture medium, yielding 0.13 IU/mL extracellular L-asparaginase and 3.6 IU/mL intracellular L-asparaginase after 4 h of induction with IPTG. The cytotoxicity results suggest that recombinant L-asparaginase from Z. mobilis expressed extracellularly in E.coli has a cytotoxic and cytostatic effect on leukemic cells. PMID:27253887

  4. Hydrodynamic behavior of shaking flasks used for producing a recombinant protein by filamentous bacteria

    NASA Astrophysics Data System (ADS)

    Cordova Aguilar, Maria Soledad; Garcia, Monica; Trujillo-Roldan, Mauricio Alberto; Ascanio, Gabriel; Zenit, Roberto; Soto, Enrique

    2012-11-01

    Shake flasks are widely used for culture research. The agitation rate is one of the factors that determines the mass transfer. However, it has not been studied in detail. In this work, a comparison of the hydrodynamic performance for conventional, baffled and coiled spring Erlenmeyer flasks is presented. The velocity fields for a horizontal plane were measured by means of a Particle Image Velocimetry (PIV) technique and high speed videos were recorded to observe the behavior of the interface as a function of the agitation rate. It was observed not only that there is a strong dependence between the geometry and the hydrodynamics, but also there is a good agreement with the results obtained previously by Gamboa et al., in 2011, with the evaluation of the influence of culture conditions of S. lividans on protein O-glycosylation. The turbulence intensity increases with shaken rate. However, for the baffled geometry, it was observed a decrease for a critical speed, which is related with the in-phase and out-phase regions. These results can be an explanation for the variations in protein productivity as a function of the flask geometry and the differences in aggregation morphology and the pattern of O-glycosylation of the recombinant protein.

  5. Recombinant L-Asparaginase from Zymomonas mobilis: A Potential New Antileukemic Agent Produced in Escherichia coli.

    PubMed

    Einsfeldt, Karen; Baptista, Isis Cavalcante; Pereira, Juliana Christina Castanheira Vicente; Costa-Amaral, Isabele Campos; Costa, Elaine Sobral da; Ribeiro, Maria Cecília Menks; Land, Marcelo Gerardin Poirot; Alves, Tito Lívio Moitinho; Larentis, Ariane Leites; Almeida, Rodrigo Volcan

    2016-01-01

    L-asparaginase is an enzyme used as a chemotherapeutic agent, mainly for treating acute lymphoblastic leukemia. In this study, the gene of L-asparaginase from Zymomonas mobilis was cloned in pET vectors, fused to a histidine tag, and had its codons optimized. The L-asparaginase was expressed extracellularly and intracellularly (cytoplasmically) in Escherichia coli in far larger quantities than obtained from the microorganism of origin, and sufficient for initial cytotoxicity tests on leukemic cells. The in silico analysis of the protein from Z. mobilis indicated the presence of a signal peptide in the sequence, as well as high identity to other sequences of L-asparaginases with antileukemic activity. The protein was expressed in a bioreactor with a complex culture medium, yielding 0.13 IU/mL extracellular L-asparaginase and 3.6 IU/mL intracellular L-asparaginase after 4 h of induction with IPTG. The cytotoxicity results suggest that recombinant L-asparaginase from Z. mobilis expressed extracellularly in E.coli has a cytotoxic and cytostatic effect on leukemic cells.

  6. Plant cell calcium-rich environment enhances thermostability of recombinantly produced alpha-amylase from the hyperthermophilic bacterium Thermotoga maritime.

    PubMed

    Santa-Maria, Monica C; Chou, Chung-Jung; Yencho, G Craig; Haigler, Candace H; Thompson, William F; Kelly, Robert M; Sosinski, Bryon

    2009-12-01

    In the industrial processing of starch for sugar syrup and ethanol production, a liquefaction step is involved where starch is initially solubilized at high temperature and partially hydrolyzed with a thermostable and thermoactive alpha-amylase. Most amylases require calcium as a cofactor for their activity and stability, therefore calcium, along with the thermostable enzyme, are typically added to the starch mixture during enzymatic liquefaction, thereby increasing process costs. An attractive alternative would be to produce the enzyme directly in the tissue to be treated. In a proof of concept study, tobacco cell cultures were used as model system to test in planta production of a hyperthermophilic alpha-amylase from Thermotoga maritima. While comparable biochemical properties to recombinant production in Escherichia coli were observed, thermostability of the plant-produced alpha-amylase benefited significantly from high intrinsic calcium levels in the tobacco cells. The plant-made enzyme retained 85% of its initial activity after 3 h incubation at 100 degrees C, whereas the E. coli-produced enzyme was completely inactivated after 30 min under the same conditions. The addition of Ca(2+) or plant cell extracts from tobacco and sweetpotato to the E. coli-produced enzyme resulted in a similar stabilization, demonstrating the importance of a calcium-rich environment for thermostability, as well as the advantage of producing this enzyme directly in plant cells where calcium is readily available.

  7. Purification and physiochemical properties of a recombinant bovine growth hormone produced by cultured murine fibroblasts.

    PubMed

    Leung, F C; Jones, B; Steelman, S L; Rosenblum, C I; Kopchick, J J

    1986-10-01

    Mouse fibroblast cell lines which secrete bovine (b) GH have been generated. This was accomplished by cotransforming mouse L cells (thymidine kinase-negative [TK-] and adenine phosphoribosyl transferase-negative [APRT-]) with DNA molecules encoding the Rous sarcoma virus-long-terminal repeat and bGH genes along with the herpes viral TK gene and the hamster APRT gene. One stable cell line, Pd lambda-pbGH 4-13, was found to secrete approximately 75 micrograms bGH per 24 h/5.0 X 10(6) cells. Media from this cell line were collected for purification of recombinant bGH (rbGH). Purification involved (NH4)2SO4 fractionation, ion-exchange chromatography, and gel filtration on Sephacryl S-200. The rbGH was characterized by bioassay, RIA, radioreceptor assay, and sodium dodecyl sulfate gel electrophoresis. Results of these analyses were compared with those obtained with a highly purified pituitary bGH. In the rat tibia bioassay, rbGH was found to be as potent as pituitary bGH. Results from the RIA, radioreceptor assay, and sodium dodecyl sulfate gel electrophoresis and Western blot analysis also suggested that the rbGH was similar to that of pituitary origin. Amino acid composition, partial (amino-terminal) sequence, and tryptic peptide maps were also found to be similar between the rbGH and pituitary bGH preparations. The amino terminus of the rbGH showed similar heterogeneity to that of the bGH of pituitary origin. We conclude that rbGH which was synthesized, processed, and secreted from transformed mouse fibroblasts possessed almost exactly the same physiochemical properties as pituitary bGH.

  8. Expression of recombinant organophosphorus hydrolase in the original producer of the enzyme, Sphingobium fuliginis ATCC 27551.

    PubMed

    Nakayama, Kosuke; Ohmori, Takeshi; Ishikawa, Satoshi; Iwata, Natsumi; Seto, Yasuo; Kawahara, Kazuyoshi

    2016-05-01

    The plasmid encoding His-tagged organophosphorus hydrolase (OPH) cloned from Sphingobium fuliginis was modified to be transferred back to this bacterium. The replication function of S. amiense plasmid was inserted at downstream of OPH gene, and S. fuliginis was transformed with this plasmid. The transformant produced larger amount of active OPH with His-tag than E. coli.

  9. A Recombinant Human Anti-Platelet scFv Antibody Produced in Pichia pastoris for Atheroma Targeting

    PubMed Central

    Vallet-Courbin, Amelie; Larivière, Mélusine; Hocquellet, Agnès; Hemadou, Audrey; Parimala, Sarjapura-Nagaraja; Laroche-Traineau, Jeanny; Santarelli, Xavier; Clofent-Sanchez, Gisèle; Jacobin-Valat, Marie-Josée; Noubhani, Abdelmajid

    2017-01-01

    Cells of the innate and adaptive immune system are key factors in the progression of atherosclerotic plaque, leading to plaque instability and rupture, potentially resulting in acute atherothrombotic events such as coronary artery disease, cerebrovascular disease and peripheral arterial disease. Here, we describe the cloning, expression, purification, and immunoreactivity assessment of a recombinant single-chain variable fragment (scFv) derived from a human anti-αIIbβ3 antibody (HuAb) selected to target atheromatous lesions for the presence of platelets. Indeed, platelets within atheroma plaques have been shown to play a role in inflammation, in platelet-leucocyte aggregates and in thrombi formation and might thus be considered relevant biomarkers of atherosclerotic progression. The DNA sequence that encodes the anti-αIIbβ3 TEG4 scFv previously obtained from a phage-display selection on activated platelets, was inserted into the eukaryote vector (pPICZαA) in fusion with a tag sequence encoding 2 cysteines useable for specific probes grafting experiments. The recombinant protein was expressed at high yields in Pichia pastoris (30 mg/L culture). The advantage of P. pastoris as an expression system is the production and secretion of recombinant proteins in the supernatant, ruling out the difficulties encountered when scFv are produced in the cytoplasm of bacteria (low yield, low solubility and reduced affinity). The improved conditions allowed for the recovery of highly purified and biologically active scFv fragments ready to be grafted in a site-directed way to nanoparticles for the imaging of atherosclerotic plaques involving inflammatory processes and thus at high risk of instability. PMID:28125612

  10. Real time detection of anthrax spores using highly specific anti-EA1 recombinant antibodies produced by competitive panning.

    PubMed

    Love, Tracey E; Redmond, Caroline; Mayers, Carl N

    2008-05-20

    We describe a targeted approach for the production of biological recognition elements capable of fast, specific detection of anthrax spores on biosensor surfaces. The aim was to produce single chain antibodies (scFvs) to EA1, a Bacillus anthracis S-layer protein that is also present, although not identical, in related to Bacillus species. The aim of the work was to produce antibodies that would detect B. anthracis EA1 protein and intact spores with a high degree of specificity, but would not detect other Bacillus species. Existing monoclonal antibodies were evaluated and found to recognise B. anthracis EA1 and S-layer proteins from other closely related Bacillus species. Recombinant anti-EA1 scFvs were isolated from B. anthracis immune library that contained antibody genes raised against B. anthracis spores and purified exosporium. Two approaches for scFv selection were used; standard (non-competitive) panning, and competitive panning. The non-competitive biopanning strategy isolated scFvs that recognised EA1 from B. anthracis, but also cross-reacted with other Bacillus species. In contrast, the competitive panning approach used S-layer proteins from other Bacillus species to generate scFvs that were highly specific to B. anthracis EA1 and demonstrated apparent nanomolar binding affinities. Specific, real time detection of B. anthracis spores was demonstrated with these scFvs using an evanescent wave biosensor, the Resonant Mirror. The approach described can be used to generate specific antibodies to any desired target where homologous proteins also exist in closely related species, and demonstrates clear advantages to using recombinant technology to produce biological recognition elements for detection of biological threat agents.

  11. Bioinspired deposition of TiO2 thin films induced by hydrophobins.

    PubMed

    Santhiya, D; Burghard, Z; Greiner, C; Jeurgens, Lars P H; Subkowski, T; Bill, J

    2010-05-04

    The deposition of ceramic thin films from aqueous solutions at low temperature using biopolymers as templates has attracted much attention due to economic and environmental benefits. Titanium dioxide is one of the most attractive functional materials and shows a wide range of applications across vastly different areas because of its unique chemical, optical, and electrical properties. In the present work, we deposited smooth, nanocrystalline titania thin films by an aqueous deposition method on surface active and amphipathic proteins of fungal origin called hydrophobins. Initially, the hydrophobin molecules were self-assembled on a silicon substrate and characterized by angle-resolved X-ray photoelectron spectroscopy (AR-XPS), atomic force microscopy (AFM) and surface potential measurements. Thin films of titanium dioxide were deposited on the surface of hydrophobin self-assembled monolayers from aqueous titanium(IV) bis(ammonium lactate) dihydroxide solution at near-ambient conditions. The microstructure of the as-deposited films was analyzed by AFM, scanning and transmission electron microscopy, which revealed the presence of nanocrystals. The titania films were also characterized using AR-XPS and Fourier transform infrared spectroscopic (FTIR) techniques. Appropriate mechanisms involved in film deposition are suggested. Additionally, nanoindentation tests on as deposited titania films showed their high resistance against mechanical stress.

  12. Co-expression of ferrochelatase allows for complete heme incorporation into recombinant proteins produced in E. coli.

    PubMed

    Sudhamsu, Jawahar; Kabir, Mariam; Airola, Michael V; Patel, Bhumit A; Yeh, Syun-Ru; Rousseau, Denis L; Crane, Brian R

    2010-09-01

    Over-expression of heme binding proteins in Escherichia coli often results in sub-optimal heme incorporation and the amount of heme-bound protein produced usually varies with the protein of interest. Complete heme incorporation is important for biochemical characterization, spectroscopy, structural studies, and for the production of homogeneous commercial proteins with high activity. We have determined that recombinant proteins expressed in E. coli often contain less than a full complement of heme because they rather are partially incorporated with free-base porphyrin. Porphyrin-incorporated proteins have similar spectral characteristics as the desired heme-loaded targets, and thus are difficult to detect, even in purified samples. We present a straightforward and inexpensive solution to this problem that involves the co-expression of native ferrochelatase with the protein of interest. The method is shown to be effective for proteins that contain either Cys- or His-ligated hemes.

  13. A Cell Line Producing Recombinant Nerve Growth Factor Evokes Growth Responses in Intrinsic and Grafted Central Cholinergic Neurons

    NASA Astrophysics Data System (ADS)

    Ernfors, Patrik; Ebendal, Ted; Olson, Lars; Mouton, Peter; Stromberg, Ingrid; Persson, Hakan

    1989-06-01

    The rat β nerve growth factor (NGF) gene was inserted into a mammalian expression vector and cotransfected with a plasmid conferring resistance to neomycin into mouse 3T3 fibroblasts. From this transfection a stable cell line was selected that contains several hundred copies of the rat NGF gene and produces excess levels of recombinant NGF. Such genetically modified cells were implanted into the rat brain as a probe for in vivo effects of NGF on central nervous system neurons. In a model of the cortical cholinergic deficits in Alzheimer disease, we demonstrate a marked increase in the survival of, and fiber outgrowth from, grafts of fetal basal forebrain cholinergic neurons, as well as stimulation of fiber formation by intact adult intrinsic cholinergic circuits in the cerebral cortex. Adult cholinergic interneurons in intact striatum also sprout vigorously toward implanted fibroblasts. Our results suggest that this model has implications for future treatment of neurodegenerative diseases.

  14. Recombinant anthrax toxin receptor-Fc fusion proteins produced in plants protect rabbits against inhalational anthrax.

    PubMed

    Wycoff, Keith L; Belle, Archana; Deppe, Dorothée; Schaefer, Leah; Maclean, James M; Haase, Simone; Trilling, Anke K; Liu, Shihui; Leppla, Stephen H; Geren, Isin N; Pawlik, Jennifer; Peterson, Johnny W

    2011-01-01

    Inhalational anthrax, a zoonotic disease caused by the inhalation of Bacillus anthracis spores, has a ∼50% fatality rate even when treated with antibiotics. Pathogenesis is dependent on the activity of two toxic noncovalent complexes: edema toxin (EdTx) and lethal toxin (LeTx). Protective antigen (PA), an essential component of both complexes, binds with high affinity to the major receptor mediating the lethality of anthrax toxin in vivo, capillary morphogenesis protein 2 (CMG2). Certain antibodies against PA have been shown to protect against anthrax in vivo. As an alternative to anti-PA antibodies, we produced a fusion of the extracellular domain of human CMG2 and human IgG Fc, using both transient and stable tobacco plant expression systems. Optimized expression led to the CMG2-Fc fusion protein being produced at high levels: 730 mg/kg fresh leaf weight in Nicotiana benthamiana and 65 mg/kg in N. tabacum. CMG2-Fc, purified from tobacco plants, fully protected rabbits against a lethal challenge with B. anthracis spores at a dose of 2 mg/kg body weight administered at the time of challenge. Treatment with CMG2-Fc did not interfere with the development of the animals' own immunity to anthrax, as treated animals that survived an initial challenge also survived a rechallenge 30 days later. The glycosylation of the Fc (or lack thereof) had no significant effect on the protective potency of CMG2-Fc in rabbits or on its serum half-life, which was about 5 days. Significantly, CMG2-Fc effectively neutralized, in vitro, LeTx-containing mutant forms of PA that were not neutralized by anti-PA monoclonal antibodies.

  15. A Recombinant Fungal Chitin Deacetylase Produces Fully Defined Chitosan Oligomers with Novel Patterns of Acetylation.

    PubMed

    Naqvi, Shoa; Cord-Landwehr, Stefan; Singh, Ratna; Bernard, Frank; Kolkenbrock, Stephan; El Gueddari, Nour Eddine; Moerschbacher, Bruno M

    2016-11-15

    Partially acetylated chitosan oligosaccharides (paCOS) are potent biologics with many potential applications, and their bioactivities are believed to be dependent on their structure, i.e., their degrees of polymerization and acetylation, as well as their pattern of acetylation. However, paCOS generated via chemical N-acetylation or de-N-acetylation of GlcN or GlcNAc oligomers, respectively, typically display random patterns of acetylation, making it difficult to control and predict their bioactivities. In contrast, paCOS produced from chitin deacetylases (CDAs) acting on chitin oligomer substrates may have specific patterns of acetylation, as shown for some bacterial CDAs. However, compared to what we know about bacterial CDAs, we know little about the ability of fungal CDAs to produce defined paCOS with known patterns of acetylation. Therefore, we optimized the expression of a chitin deacetylase from the fungus Puccinia graminis f. sp. tritici in Escherichia coli The best yield of functional enzyme was obtained as a fusion protein with the maltose-binding protein (MBP) secreted into the periplasmic space of the bacterial host. We characterized the MBP fusion protein from P. graminis (PgtCDA) and tested its activity on different chitinous substrates. Mass spectrometric sequencing of the products obtained by enzymatic deacetylation of chitin oligomers, i.e., tetramers to hexamers, revealed that PgtCDA generated paCOS with specific acetylation patterns of A-A-D-D, A-A-D-D-D, and A-A-D-D-D-D, respectively (A, GlcNAc; D, GlcN), indicating that PgtCDA cannot deacetylate the two GlcNAc units closest to the oligomer's nonreducing end. This unique property of PgtCDA significantly expands the so far very limited library of well-defined paCOS available to test their bioactivities for a wide variety of potential applications.

  16. Quantitative Determination of Bandpasses for Producing Vegetation Indices from Recombined NEON Hyperspectral Imagery

    NASA Astrophysics Data System (ADS)

    Hulslander, D.

    2015-12-01

    Hyperspectral imaging systems can be used to produce spectral reflectance curves giving rich information about composition, relative abundances of materials, mixes and combinations. However, as each spectral return from these systems is a vector with several hundred elements, they can be very difficult to process and analyze, and problemeatic to compare within, across, and between datasets over time and space. Vegetation indices (e.g. NDVI, ARVI, EVI, et al) attempt to combine spectral features in to single-value scores. When derived from calibrated and atmospherically compensated reflectance data, these indices can be quantitatively compared. Historically, these indices have been calculated from multispectral sensor data. These sensors have a handful (4 to 16 or so) of bandbasses ranging from 20 nm to 200 nm FWHM covering specific spectral regions for a variety of reasons, including both intended applications and system limitations. Hyperspectral sensors, however, cover the spectrum with many, many narrow (5 to 10 nm) bandpasses. This allows for analyses using the full, detailed spectral curve, or combination of the bands in to regions by averaging or in to composites using transforms or other techniques. This raises the question of exactly which bands should be used and combined in what manner for ideally deriving well-known vegetation indices typically made from multispectral data. In this study we use derivatives and other curve and signal analysis techniques to analyze vegetation reflectance spectra to quantitatively define optimal bandpasses for several vegetation indices and combine the 5 nm hypserspectral bandpasses of the NEON Imaging Spectrometer to synthesize them.

  17. Novel Feruloyl Esterase from Lactobacillus fermentum NRRL B-1932 and Analysis of the Recombinant Enzyme Produced in Escherichia coli

    PubMed Central

    Bischoff, Kenneth M.; Anderson, Amber M.; Rich, Joseph O.

    2016-01-01

    ABSTRACT A total of 33 Lactobacillus strains were screened for feruloyl esterase (FE) activity using agar plates containing ethyl ferulate as the sole carbon source, and Lactobacillus fermentum NRRL B-1932 demonstrated the strongest FE activity among a dozen species showing a clearing zone on the opaque plate containing ethyl ferulate. FE activities were monitored using high-performance liquid chromatography with an acetonitrile-trifluoroacetic acid gradient. To produce sufficient purified FE from L. fermentum strain NRRL B-1932 (LfFE), the cDNA encoding LfFE (Lffae) was amplified and cloned by using available closely related genome sequences and overexpressed in Escherichia coli. A 29.6-kDa LfFE protein was detected from the protein extract of E. coli BL21(pLysS) carrying pET28bLffae upon IPTG (isopropyl-β-d-thiogalactopyranoside) induction. The recombinant LfFE containing a polyhistidine tag was purified by nickel-nitrilotriacetic acid affinity resin. The purified LfFE showed strong activities against several artificial substrates, including p-nitrophenyl acetate and 4-methylumbelliferyl p-trimethylammoniocinnamate chloride. The optimum pH and temperature of the recombinant LfFE were around 6.5 and 37°C, respectively, as determined using either crude or purified recombinant LfFE. This study will be essential for the production of the LfFE in E. coli on a larger scale that could not be readily achieved by L. fermentum fermentation. IMPORTANCE The production of feruloyl esterase (FE) from Lactobacillus fermentum NRRL B-1932 reported in this study will have immense potential commercial applications not only in biofuel production but also in pharmaceutical, polymer, oleo chemical, cosmetic additive, and detergent industries, as well as human health-related applications, including food flavoring, functional foods, probiotic agents, preventive medicine, and animal feed. Given the essential role FE plays in the production of hydroxycinnamic acids and ferulic acid

  18. Validation of the manufacturing process used to produce long-acting recombinant factor IX Fc fusion protein.

    PubMed

    McCue, J; Osborne, D; Dumont, J; Peters, R; Mei, B; Pierce, G F; Kobayashi, K; Euwart, D

    2014-07-01

    Recombinant factor IX Fc (rFIXFc) fusion protein is the first of a new class of bioengineered long-acting factors approved for the treatment and prevention of bleeding episodes in haemophilia B. The aim of this work was to describe the manufacturing process for rFIXFc, to assess product quality and to evaluate the capacity of the process to remove impurities and viruses. This manufacturing process utilized a transferable and scalable platform approach established for therapeutic antibody manufacturing and adapted for production of the rFIXFc molecule. rFIXFc was produced using a process free of human- and animal-derived raw materials and a host cell line derived from human embryonic kidney (HEK) 293H cells. The process employed multi-step purification and viral clearance processing, including use of a protein A affinity capture chromatography step, which binds to the Fc portion of the rFIXFc molecule with high affinity and specificity, and a 15 nm pore size virus removal nanofilter. Process validation studies were performed to evaluate identity, purity, activity and safety. The manufacturing process produced rFIXFc with consistent product quality and high purity. Impurity clearance validation studies demonstrated robust and reproducible removal of process-related impurities and adventitious viruses. The rFIXFc manufacturing process produces a highly pure product, free of non-human glycan structures. Validation studies demonstrate that this product is produced with consistent quality and purity. In addition, the scalability and transferability of this process are key attributes to ensure consistent and continuous supply of rFIXFc.

  19. [Producing recombinant adenovirus encoding green fluorescent protein (Ad-GFP) by suspension cultured HEK-293 N3S cells].

    PubMed

    Tian, Bo; Wu, Bin; Zhang, Qun-Wei; Bi, Jian-Jin; Wang, Lan; Zhu, Bao-Zhen; Geng, Yue; Wu, Zu-Ze

    2007-09-01

    Adenovirus vectors are one of the most promising gene transfer systems. They are of great value for gene therapy because these vectors achieve temporal high-level transgene expression and high gene transfer efficiency. To meet increasing needs of adenovirus vectors for gene therapy programs, parallel development of efficient, scalable and reproducible production processes is required. Perfusion cultivation of 293 cells is one of the most commonly used methods to produce adenovirus vectors and it is suitable for industrialized production specially. Experimental studies had been carried out to produce recombinant adenovirus containing the green fluorescent protein gene (Ad-GFP) by perfusion cultivation of HEK-293 N3S cells in a 5L stirring bioreactors. Perfusion rate was 1-2 volume/day. To infect the 293 N3S cells with Ad-GFP at the density of (2-4) x 10(6) cells/ ml. The time of collecting cells was 48 hours post infection. After three rounds of freeze/thaw and centrifugation, the crude viral lysates were stored at--80 degrees C until use. Then to get the Ad-GFP products by 2 x CsCl-gradient purification. The purity of the products was determined by the A260/A280 ratio and a high performance liquid chromatography (HPLC) assay. The infective titer was determined by a TCID50 assay. The culture term was 10-12 days. The infectious titer, the number of virus particle and the ratio of infectious titer to virus particle for the product were 1.0 x 10(11) IU/mL, 1.68 x 10(12) VP/mL and 6.0% IU/VP respectively. The A260/A280 ratio was 1.33, and the purity determined by HPLC was 99.2%. The cell specific productivity was around 1000 IU/cell. By perfusion cultivation of 293 N3S cells in a 5L stirring bioreactors, we established the production process for Ad-GFP, which paves a way to produce other recombinant adenovirus for gene therapy.

  20. Causes of proteolytic degradation of secreted recombinant proteins produced in methylotrophic yeast Pichia pastoris: case study with recombinant ovine interferon-tau.

    PubMed

    Sinha, Jayanta; Plantz, Bradley A; Inan, Mehmet; Meagher, Michael M

    2005-01-05

    It was observed that during fermentative production of recombinant ovine interferon-tau (r-oIFN-tau) in Pichia pastoris, a secreted recombinant protein, the protein was degraded increasingly after 48 h of induction and the rate of degradation increased towards the end of fermentation at 72 h, when the fermentation was stopped. Proteases, whose primary source was the vacuoles, was found in increasing levels in the cytoplasm and in the fermentation broth after 48 h of induction and reached maximal values when the batch was completed at 72 h. Protease levels at various cell fractions as well as in the culture supernatant were lower when glycerol was used as the carbon source instead of methanol. It can be concluded that methanol metabolism along with cell lysis towards the end of fermentation contributes to increased proteolytic activity and eventual degradation of recombinant protein.

  1. Rapid, scalable, and low-cost purification of recombinant adeno-associated virus produced by baculovirus expression vector system

    PubMed Central

    Buclez, Pierre-Olivier; Dias Florencio, Gabriella; Relizani, Karima; Beley, Cyriaque; Garcia, Luis; Benchaouir, Rachid

    2016-01-01

    Recombinant adeno-associated viruses (rAAV) are largely used for gene transfer in research, preclinical developments, and clinical trials. Their broad in vivo biodistribution and long-term efficacy in postmitotic tissues make them good candidates for numerous gene transfer applications. Upstream processes able to produce large amounts of rAAV were developed, particularly those using baculovirus expression vector system. In parallel, downstream processes present a large panel of purification methods, often including multiple and time consuming steps. Here, we show that simple tangential flow filtration, coupled with an optimized iodixanol-based isopycnic density gradient, is sufficient to purify several liters of crude lysate produced by baculovirus expression vector system in only one working day, leading to high titers and good purity of rAAV products. Moreover, we show that the viral vectors retain their in vitro and in vivo functionalities. Our results demonstrate that simple, rapid, and relatively low-cost methods can easily be implemented for obtaining a high-quality grade of gene therapy products based on rAAV technology. PMID:27226971

  2. High-level expression and purification of recombinant human growth hormone produced in soluble form in Escherichia coli.

    PubMed

    Levarski, Zdenko; Šoltýsová, Andrea; Krahulec, Ján; Stuchlík, Stanislav; Turňa, Ján

    2014-08-01

    Human growth hormone (hGH) was one of the first recombinant proteins approved for the treatment of human growth disorders. Its small size (191 amino acids), possession of only 2 disulphide bonds and absence of posttranslational modifications make Escherichia coli the host of choice for its production on any scale. In this work, we have utilized an efficient T7 based expression system to produce high levels of soluble thioredoxin-hGH (Trx-hGH) fusion protein. We outline a relatively simple three step purification process employing two immobilized metal-affinity chromatography and one anion-exchange steps and removal of fusion partner by enterokinase cleavage yielding native hGH. The ability of cell populations to produce quantities of up to 1 g/L of the soluble Trx-hGH fusion protein has been tested in flask cultivations as well as in batch and fed-batch bioreactor runs. The sequence and structure of derived hGH were confirmed by mass spectrometry and circular dichroism and its native function, to induce cell proliferation, was confirmed by employing a Nb2 cell line proliferation assay.

  3. Effect of recombinant Lactococcus lactis producing myelin peptides on neuroimmunological changes in rats with experimental allergic encephalomyelitis.

    PubMed

    Kasarełło, K; Szczepankowska, A; Kwiatkowska-Patzer, B; Lipkowski, A W; Gadamski, R; Sulejczak, D; Łachwa, M; Biały, M; Bardowski, J

    2016-01-01

    Multiple sclerosis (MS) is a human autoimmune neurodegenerative disease with an unknown etiology. Despite various therapies, there is no effective cure for MS. Since the mechanism of the disease is based on autoreactive T-cell responses directed against myelin antigens, oral tolerance is a promising approach for the MS treatment. Here, the experiments were performed to assess the impact of oral administration of recombinant Lactococcus lactis producing encephalogenic fragments of three myelin proteins: myelin basic protein, proteolipid protein, and myelin oligodendrocyte glycoprotein, on neuroimmunological changes in rats with experimental allergic encephalomyelitis (EAE) - an animal model of MS. Lactococcus lactis whole-cell lysates were administered intragastrically at two doses (103 and 106 colony forming units) in a twenty-fold feeding regimen to Lewis rats with EAE. Spinal cord slices were subjected to histopathological analysis and morphometric evaluation, and serum levels of cytokines (IL-1b, IL-10, TNF-α and IFN-γ) were measured. Results showed that administration of the L. lactis preparations at the tested doses to rats with EAE, diminished the histopathological changes observed in EAE rats and reduced the levels of serum IL-1b, IL-10 and TNF-α, previously increased by evoking EAE. This suggests that oral delivery of L. lactis producing myelin peptide fragments could be an alternative strategy to induce oral tolerance for the treatment of MS.

  4. Escherichia coli can produce recombinant chitinase in the soil to control the pathogenesis by Fusarium oxysporum without colonization.

    PubMed

    Chung, Soohee; Kim, Sang-Dal

    2007-03-01

    Fusarium wilt of cucumbers was effectively controlled by Escherichia coli expressing an endochitinase gene (chiA), and the rate was as effective (60.0%) as the wildtype strain S. proteamaculans 3095 (55.0%) where the gene was cloned. However, live cells of soil inoculated E. coli host harboring the chiA gene did not proliferate but declined 100-fold from 108 CFU during the first week and showed less than 10 cells after day 14, suggesting that E. coli was able to express and produce the chitinase enzyme to the soil even as the population was gradually decreasing. Because the majority of the strains was alive for only a short period of time and the Fusarium-affected seedlings showed symptoms of wilting within 7-10 days, it seems that the pathogen control was decided early after the introduction of the biocontrol agent, eliminating the survival of the antagonist. These results indicated that soil inoculated E. coli could sufficiently express and produce the recombinant protein to control the pathogen, and root or soil colonization of the antagonist might not be a significant factor in determining the efficacy of biological control.

  5. Medical devices; general and plastic surgery devices; classification of absorbable poly(hydroxybutyrate) surgical suture produced by recombinant DNA technology. Final rule.

    PubMed

    2007-08-03

    The Food and Drug Administration (FDA) is classifying the absorbable poly(hydroxybutyrate) surgical suture produced by recombinant deoxyribonucleic acid (DNA) technology into class II (special controls). The special control that will apply to the device is the guidance document entitled "Class II Special Controls Guidance Document: Absorbable Poly(hydroxybutyrate) Surgical Suture Produced by Recombinant DNA Technology." The agency is classifying these devices into class II (special controls) in order to provide a reasonable assurance of safety and effectiveness of these devices. Elsewhere in this issue of the Federal Register, FDA is announcing the availability of the guidance document that will serve as the special control for this device.

  6. Recombinant Nox4 cytosolic domain produced by a cell or cell-free base systems exhibits constitutive diaphorase activity

    SciTech Connect

    Nguyen, Minh Vu Chuong; Zhang, Leilei; Lhomme, Stanislas; Mouz, Nicolas

    2012-03-16

    Highlights: Black-Right-Pointing-Pointer A comparison of two bacterial cell and cell-free protein expression systems is presented. Black-Right-Pointing-Pointer Soluble and active truncated Nox4 proteins are produced. Black-Right-Pointing-Pointer Nox4 has a constitutive diaphorase activity which is independent of cytosolic factors. Black-Right-Pointing-Pointer Isoform Nox4B is unable to initiate the first electronic transfer step. Black-Right-Pointing-Pointer Findings contribute to the understanding of the mechanism of Nox4 oxidase activity. -- Abstract: The membrane protein NADPH (nicotinamide adenine dinucleotide phosphate) oxidase Nox4 constitutively generates reactive oxygen species differing from other NADPH oxidases activity, particularly in Nox2 which needs a stimulus to be active. Although the precise mechanism of production of reactive oxygen species by Nox2 is well characterized, the electronic transfer throughout Nox4 remains unclear. Our study aims to investigate the initial electronic transfer step (diaphorase activity) of the cytosolic tail of Nox4. For this purpose, we developed two different approaches to produce soluble and active truncated Nox4 proteins. We synthesized soluble recombinant proteins either by in vitro translation or by bacteria induction. While proteins obtained by bacteria induction demonstrate an activity of 4.4 {+-} 1.7 nmol/min/nmol when measured against iodonitro tetrazolium chloride and 20.5 {+-} 2.8 nmol/min/nmol with cytochrome c, the soluble proteins produced by cell-free expression system exhibit a diaphorase activity with a turn-over of 26 {+-} 2.6 nmol/min/nmol when measured against iodonitro tetrazolium chloride and 48 {+-} 20.2 nmol/min/nmol with cytochrome c. Furthermore, the activity of the soluble proteins is constitutive and does not need any stimulus. We also show that the cytosolic tail of the isoform Nox4B lacking the first NADPH binding site is unable to demonstrate any diaphorase activity pointing out the

  7. Control of specific carbon dioxide production in a fed-batch culture producing recombinant protein using a soft sensor.

    PubMed

    Gustavsson, Robert; Lukasser, Cornelia; Mandenius, Carl-Fredrik

    2015-04-20

    The feeding of a fed-batch cultivation producing recombinant protein was controlled by a soft sensor set-up. It was assumed that the control approach could be based on the cell's production of carbon dioxide and that this parameter indicates the metabolic state occurring at induced protein expression. The soft sensor used the on-line signals from a carbon dioxide analyser and a near-infrared (NIR) probe for biomass to estimate the specific production rate qCO2. Control experiments were carried out with various qCO2 set-points where we observe that the feeding of nutrients to the culture could easily be controlled and resulted in a decreased variability compared to uncontrolled cultivations. We therefore suggest that this control approach could serve as an alternative to other commonly applied methods such as controlling the cell's overflow metabolism of acetate or the cell's specific growth rate. However, further studies of the internal metabolic fluxes of CO2 during protein expression would be recommended for a more precise characterization of the relationship between qCO2 and protein expression in order to fully interpret the control behaviour.

  8. Recombinant bovine dihydrofolate reductase produced by mutagenesis and nested PCR of murine dihydrofolate reductase cDNA.

    PubMed

    Cody, Vivian; Mao, Qilong; Queener, Sherry F

    2008-11-01

    Recent reports of the slow-tight binding inhibition of bovine liver dihydrofolate reductase (bDHFR) in the presence of polyphenols isolated from green tea leaves has spurred renewed interest in the biochemical properties of bDHFR. Earlier studies were done with native bDHFR but in order to validate models of polyphenol binding to bDHFR, larger quantities of bDHFR are necessary to support structural studies. Bovine DHFR differs from its closest sequence homologue, murine DHFR, by 19 amino acids. To obtain the bDHFR cDNA, murineDHFR cDNA was transformed by a series of nested PCRs to reproduce the amino acid coding sequence for bovine DHFR. The bovine liver DHFR cDNA has an open reading frame of 561 base pairs encoding a protein of 187 amino acids that has a high level of conservation at the primary sequence level with other DHFR enzymes, and more so for the amino acid residues in the active site of the mammalian DHFR enzymes. Expression of the bovine DHFR cDNA in bacterial cells produced a stable recombinant protein with high enzymatic activity and kinetic properties similar to those previously reported for the native protein.

  9. Techno-economic analysis of a conceptual biofuel production process from bioethylene produced by photosynthetic recombinant cyanobacteria

    SciTech Connect

    Markham, Jennifer N.; Tao, Ling; Davis, Ryan; Voulis, Nina; Angenent, Largus T.; Ungerer, Justin; Yu, Jianping

    2016-08-25

    Ethylene is a petrochemical produced in large volumes worldwide. It serves as a building block for a wide variety of plastics, textiles, and chemicals, and can be converted into liquid transportation fuels. There is great interest in the development of technologies that produce ethylene from renewable resources, such as biologically derived CO2 and biomass. One of the metabolic pathways used by microbes to produce ethylene is via an ethylene-forming enzyme (EFE). By expressing a bacterial EFE gene in a cyanobacterium, ethylene has been produced through photosynthetic carbon fixation. Here, we present a conceptual design and techno-economic analysis of a process of biofuel production based on the upgradation of ethylene generated by the recombinant cyanobacterium. This analysis focuses on potential near-term to long-term cost projections for the integrated process of renewable fuels derived from ethylene. The cost projections are important in showing the potential of this technology and determining research thrusts needed to reach target goals. The base case for this analysis is a midterm projection using tubular photobioreactors for cyanobacterial growth and ethylene production, cryogenic distillation for ethylene separation and purification, a two-step Ziegler oligomerization process with subsequent hydrotreatment and upgradation for fuel production, and a wastewater treatment process that utilizes anaerobic digestion of cyanobacterial biomass. The minimum fuel selling price (MFSP) for the midterm projection is 15.07 per gallon gasoline equivalent (GGE). Near-term and long-term projections are 28.66 per GGE and 5.36 per GGE, respectively. Single- and multi-point sensitivity analyses are conducted to determine the relative effect that chosen variables could have on the overall costs. This analysis identifies several key variables for improving the overall process economics and outlines strategies to guide future research directions. Finally, the

  10. Techno-economic analysis of a conceptual biofuel production process from bioethylene produced by photosynthetic recombinant cyanobacteria

    SciTech Connect

    Markham, Jennifer N.; Tao, Ling; Davis, Ryan; Voulis, Nina; Angenent, Largus T.; Ungerer, Justin; Yu, Jianping

    2016-01-01

    Ethylene is a petrochemical produced in large volumes worldwide. It serves as a building block for a wide variety of plastics, textiles, and chemicals, and can be converted into liquid transportation fuels. There is great interest in the development of technologies that produce ethylene from renewable resources, such as biologically derived CO2 and biomass. One of the metabolic pathways used by microbes to produce ethylene is via an ethylene-forming enzyme (EFE). By expressing a bacterial EFE gene in a cyanobacterium, ethylene has been produced through photosynthetic carbon fixation. Here, we present a conceptual design and techno-economic analysis of a process of biofuel production based on the upgradation of ethylene generated by the recombinant cyanobacterium. This analysis focuses on potential near-term to long-term cost projections for the integrated process of renewable fuels derived from ethylene. The cost projections are important in showing the potential of this technology and determining research thrusts needed to reach target goals. The base case for this analysis is a midterm projection using tubular photobioreactors for cyanobacterial growth and ethylene production, cryogenic distillation for ethylene separation and purification, a two-step Ziegler oligomerization process with subsequent hydrotreatment and upgradation for fuel production, and a wastewater treatment process that utilizes anaerobic digestion of cyanobacterial biomass. The minimum fuel selling price (MFSP) for the midterm projection is $15.07 per gallon gasoline equivalent (GGE). Near-term and long-term projections are $28.66 per GGE and $5.36 per GGE, respectively. Single- and multi-point sensitivity analyses are conducted to determine the relative effect that chosen variables could have on the overall costs. This analysis identifies several key variables for improving the overall process economics and outlines strategies to guide future research directions. The productivity of ethylene

  11. Techno-economic analysis of a conceptual biofuel production process from bioethylene produced by photosynthetic recombinant cyanobacteria

    DOE PAGES

    Markham, Jennifer N.; Tao, Ling; Davis, Ryan; ...

    2016-08-25

    Ethylene is a petrochemical produced in large volumes worldwide. It serves as a building block for a wide variety of plastics, textiles, and chemicals, and can be converted into liquid transportation fuels. There is great interest in the development of technologies that produce ethylene from renewable resources, such as biologically derived CO2 and biomass. One of the metabolic pathways used by microbes to produce ethylene is via an ethylene-forming enzyme (EFE). By expressing a bacterial EFE gene in a cyanobacterium, ethylene has been produced through photosynthetic carbon fixation. Here, we present a conceptual design and techno-economic analysis of a processmore » of biofuel production based on the upgradation of ethylene generated by the recombinant cyanobacterium. This analysis focuses on potential near-term to long-term cost projections for the integrated process of renewable fuels derived from ethylene. The cost projections are important in showing the potential of this technology and determining research thrusts needed to reach target goals. The base case for this analysis is a midterm projection using tubular photobioreactors for cyanobacterial growth and ethylene production, cryogenic distillation for ethylene separation and purification, a two-step Ziegler oligomerization process with subsequent hydrotreatment and upgradation for fuel production, and a wastewater treatment process that utilizes anaerobic digestion of cyanobacterial biomass. The minimum fuel selling price (MFSP) for the midterm projection is 15.07 per gallon gasoline equivalent (GGE). Near-term and long-term projections are 28.66 per GGE and 5.36 per GGE, respectively. Single- and multi-point sensitivity analyses are conducted to determine the relative effect that chosen variables could have on the overall costs. This analysis identifies several key variables for improving the overall process economics and outlines strategies to guide future research directions. Finally, the

  12. Purification and characterization of Streptococcus sobrinus dextranase produced in recombinant Escherichia coli and sequence analysis of the dextranase gene.

    PubMed Central

    Wanda, S Y; Curtiss, R

    1994-01-01

    The plasmid (pYA902) with the dextranase (dex) gene of Streptococcus sobrinus UAB66 (serotype g) produces a C-terminal truncated dextranase enzyme (Dex) with a multicomplex mass form which ranges from 80 to 130 kDa. The Escherichia coli-produced enzyme was purified and characterized, and antibodies were raised in rabbits. Purified dextranase has a native-form molecular mass of 160 to 260 kDa and specific activity of 4,000 U/mg of protein. Potential immunological cross-reactivity between dextranase and the SpaA protein specified by various recombinant clones was studied by using various antisera and Western blot (immunoblot) analysis. No cross-reactivity was observed. Optimal pH (5.3) and temperature (39 degrees C) and the isoelectric points (3.56, 3.6, and 3.7) were determined and found to be similar to those for dextranase purified from S. sobrinus. The dex DNA restriction map was determined, and several subclones were obtained. The nucleotide sequence of the dex gene was determined by using subclones pYA993 and pYA3009 and UAB66 chromosomal DNA. The open reading frame for dex was 4,011 bp, ending with a stop codon TAA. A ribosome-binding site and putative promoter preceding the start codon were identified. The deduced amino acid sequence of Dex revealed the presence of a signal peptide of 30 amino acids. The cleavage site for the signal sequence was determined by N-terminal amino acid sequence analysis for Dex produced in E. coli chi 2831(pYA902). The C terminus consists of a serine- and threonine-rich region followed by the peptide LPKTGD, 3 charged amino acids, 19 amino acids with a strongly hydrophobic character, and a charged pentapeptide tail, which are proposed to correspond to the cell wall-spanning region, the LPXTGX consensus sequence, and the membrane-anchoring domains of surface-associated proteins of gram-positive cocci. Images PMID:8021165

  13. Significance of the class II hydrophobin FgHyd5p for the life cycle of Fusarium graminearum.

    PubMed

    Minenko, Ekaterina; Vogel, Rudi F; Niessen, Ludwig

    2014-04-01

    Hydrophobins are small secreted proteins ubiquitously found in filamentous fungi. Some hydrophobins were shown to have functions in fungal development, while others lack known function. Class II hydrophobins from Fusarium graminearum and Fusarium culmorum are characterized by formation of low stability aggregates and their solubility in organic solvents. They are economically relevant to the brewing industry because they can induce beer gushing. Since cellular functions of Hyd5p's are still unknown, we analyzed the influence of FgHyd5p on growth and morphology of F. graminearum using FgΔhyd5 knock-out mutants expressing sGFP under the control of the hyd5 promoter and compared them with the performance of the parent wild type strain. Results demonstrate that FgHyd5p does not affect the colony and hyphal morphology. FgHyd5p affects the hydrophobicity of aerial mycelia but had no obvious function in penetration of hyphae through the water air interface. The hydrophobin affects the morphology of conidia, but not their fitness. Different sources of carbon and nitrogen as well as different pH have no effect on the expression of the hyd5 gene, which was demonstrated to be expressed upon growth of F. graminearum on hydrophobic surfaces.

  14. TALEN-Mediated Homologous Recombination Produces Site-Directed DNA Base Change and Herbicide-Resistant Rice.

    PubMed

    Li, Ting; Liu, Bo; Chen, Chih Ying; Yang, Bing

    2016-05-20

    Over the last decades, much endeavor has been made to advance genome editing technology due to its promising role in both basic and synthetic biology. The breakthrough has been made in recent years with the advent of sequence-specific endonucleases, especially zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats (CRISPRs) guided nucleases (e.g., Cas9). In higher eukaryotic organisms, site-directed mutagenesis usually can be achieved through non-homologous end-joining (NHEJ) repair to the DNA double-strand breaks (DSBs) caused by the exogenously applied nucleases. However, site-specific gene replacement or genuine genome editing through homologous recombination (HR) repair to DSBs remains a challenge. As a proof of concept gene replacement through TALEN-based HR in rice (Oryza sativa), we successfully produced double point mutations in rice acetolactate synthase gene (OsALS) and generated herbicide resistant rice lines by using TALENs and donor DNA carrying the desired mutations. After ballistic delivery into rice calli of TALEN construct and donor DNA, nine HR events with different genotypes of OsALS were obtained in T0 generation at the efficiency of 1.4%-6.3% from three experiments. The HR-mediated gene edits were heritable to the progeny of T1 generation. The edited T1 plants were as morphologically normal as the control plants while displayed strong herbicide resistance. The results demonstrate the feasibility of TALEN-mediated genome editing in rice and provide useful information for further genome editing by other nuclease-based genome editing platforms.

  15. Scaled-up manufacturing of recombinant antibodies produced by plant cells in a 200-L orbitally-shaken disposable bioreactor.

    PubMed

    Raven, Nicole; Rasche, Stefan; Kuehn, Christoph; Anderlei, Tibor; Klöckner, Wolf; Schuster, Flora; Henquet, Maurice; Bosch, Dirk; Büchs, Jochen; Fischer, Rainer; Schillberg, Stefan

    2015-02-01

    Tobacco BY-2 cells have emerged as a promising platform for the manufacture of biopharmaceutical proteins, offering efficient protein secretion, favourable growth characteristics and cultivation in containment under a controlled environment. The cultivation of BY-2 cells in disposable bioreactors is a useful alternative to conventional stainless steel stirred-tank reactors, and orbitally-shaken bioreactors could provide further advantages such as simple bag geometry, scalability and predictable process settings. We carried out a scale-up study, using a 200-L orbitally-shaken bioreactor holding disposable bags, and BY-2 cells producing the human monoclonal antibody M12. We found that cell growth and recombinant protein accumulation were comparable to standard shake flask cultivation, despite a 200-fold difference in cultivation volume. Final cell fresh weights of 300-387 g/L and M12 yields of ∼20 mg/L were achieved with both cultivation methods. Furthermore, we established an efficient downstream process for the recovery of M12 from the culture broth. The viscous spent medium prevented clarification using filtration devices, but we used expanded bed adsorption (EBA) chromatography with SP Sepharose as an alternative for the efficient capture of the M12 antibody. EBA was introduced as an initial purification step prior to protein A affinity chromatography, resulting in an overall M12 recovery of 75-85% and a purity of >95%. Our results demonstrate the suitability of orbitally-shaken bioreactors for the scaled-up cultivation of plant cell suspension cultures and provide a strategy for the efficient purification of antibodies from the BY-2 culture medium.

  16. BslA is a self-assembling bacterial hydrophobin that coats the Bacillus subtilis biofilm

    PubMed Central

    Hobley, Laura; Ostrowski, Adam; Rao, Francesco V.; Bromley, Keith M.; Porter, Michael; Prescott, Alan R.; MacPhee, Cait E.; van Aalten, Daan M. F.; Stanley-Wall, Nicola R.

    2013-01-01

    Biofilms represent the predominant mode of microbial growth in the natural environment. Bacillus subtilis is a ubiquitous Gram-positive soil bacterium that functions as an effective plant growth-promoting agent. The biofilm matrix is composed of an exopolysaccharide and an amyloid fiber-forming protein, TasA, and assembles with the aid of a small secreted protein, BslA. Here we show that natively synthesized and secreted BslA forms surface layers around the biofilm. Biophysical analysis demonstrates that BslA can self-assemble at interfaces, forming an elastic film. Molecular function is revealed from analysis of the crystal structure of BslA, which consists of an Ig-type fold with the addition of an unusual, extremely hydrophobic “cap” region. A combination of in vivo biofilm formation and in vitro biophysical analysis demonstrates that the central hydrophobic residues of the cap are essential to allow a hydrophobic, nonwetting biofilm to form as they control the surface activity of the BslA protein. The hydrophobic cap exhibits physiochemical properties remarkably similar to the hydrophobic surface found in fungal hydrophobins; thus, BslA is a structurally defined bacterial hydrophobin. We suggest that biofilms formed by other species of bacteria may have evolved similar mechanisms to provide protection to the resident bacterial community. PMID:23904481

  17. Molecular simulation of hydrophobin adsorption at an oil-water interface.

    PubMed

    Cheung, David L

    2012-06-12

    Hydrophobins are small, amphiphilic proteins expressed by strains of filamentous fungi. They fulfill a number of biological functions, often related to adsorption at hydrophobic interfaces, and have been investigated for a number of applications in materials science and biotechnology. In order to understand the biological function and applications of these proteins, a microscopic picture of the adsorption of these proteins at interfaces is needed. Using molecular dynamics simulations with a chemically detailed coarse-grained potential, the behavior of typical hydrophobins at the water-octane interface is studied. Calculation of the interfacial adsorption strengths indicates that the adsorption is essentially irreversible, with adsorption strengths of the order of 100 k(B)T (comparable to values determined for synthetic nanoparticles but significantly larger than small molecule surfactants and biomolecules). The protein structure at the interface is unchanged at the interface, which is consistent with the biological function of these proteins. Comparison of native proteins with pseudoproteins that consist of uniform particles shows that the surface structure of these proteins has a large effect on the interfacial adsorption strengths, as does the flexibility of the protein.

  18. A simple MALDI plate functionalization by Vmh2 hydrophobin for serial multi-enzymatic protein digestions.

    PubMed

    Longobardi, Sara; Gravagnuolo, Alfredo Maria; Funari, Riccardo; Della Ventura, Bartolomeo; Pane, Francesca; Galano, Eugenio; Amoresano, Angela; Marino, Gennaro; Giardina, Paola

    2015-01-01

    The development of efficient and rapid methods for the identification with high sequence coverage of proteins is one of the most important goals of proteomic strategies today. The on-plate digestion of proteins is a very attractive approach, due to the possibility of coupling immobilized-enzymatic digestion with direct matrix-assisted laser desorption/ionization (MALDI)-time of flight (TOF)-mass spectrometry (MS) analysis. The crucial step in the development of on-plate immobilization is however the functionalization of the solid surface. Fungal self-assembling proteins, the hydrophobins, are able to efficiently functionalize surfaces. We have recently shown that such modified plates are able to absorb either peptides or proteins and are amenable to MALDI-TOF-MS analysis. In this paper, the hydrophobin-coated MALDI sample plates were exploited as a lab-on-plate for noncovalent immobilization of enzymes commonly used in protein identification/characterization, such as trypsin, V8 protease, PNGaseF, and alkaline phosphatase. Rapid and efficient on-plate reactions were performed to achieve high sequence coverage of model proteins, particularly when performing multiple enzyme digestions. The possibility of exploiting this direct on-plate MALDI-TOF/TOF analysis has been investigated on model proteins and, as proof of concept, on entire whey milk proteome.

  19. Peptaibol, secondary-metabolite, and hydrophobin pattern of commercial biocontrol agents formulated with species of the Trichoderma harzianum complex.

    PubMed

    Degenkolb, Thomas; Fog Nielsen, Kristian; Dieckmann, Ralf; Branco-Rocha, Fabiano; Chaverri, Priscila; Samuels, Gary J; Thrane, Ulf; von Döhren, Hans; Vilcinskas, Andreas; Brückner, Hans

    2015-04-01

    The production of bioactive polypeptides (peptaibiotics) in vivo is a sophisticated adaptation strategy of both mycoparasitic and saprotrophic Trichoderma species for colonizing and defending their natural habitats. This feature is of major practical importance, as the detection of peptaibiotics in plant-protective Trichoderma species, which are successfully used against economically relevant bacterial and fungal plant pathogens, certainly contributes to a better understanding of these complex antagonistic interactions. We analyzed five commercial biocontrol agents (BCAs), namely Canna(®) , Trichosan(®) , Vitalin(®) , Promot(®) WP, and TrichoMax(®) , formulated with recently described species of the Trichoderma harzianum complex, viz. T. afroharzianum, T. simmonsii, and T. guizhouense. By using the well-established, HPLC/MS-based peptaibiomics approach, it could unequivocally be demonstrated that all of these formulations contained new and recurrent peptaibols, i.e., peptaibiotics carrying an acetylated N-terminus, the C-terminus of which is reduced to a 1,2-amino alcohol. Their chain lengths, including the amino alcohol, were 11, 14, and 18 residues, respectively. Peptaibols were also to be the dominating secondary metabolites in plate cultures of the four strains obtained from four of the Trichoderma- based BCAs, contributing 95% of the UHPLC-UV/VIS peak areas and 99% of the total ion count MS peak area from solid media. Furthermore, species-specific hydrophobins, as well as non-peptaibiotic secondary metabolites, were detected, the latter being known for their antifungal, siderophore, or plant-growth-promoting activities. Notably, none of the isolates produced low-molecular weight mycotoxins.

  20. Human leukocytic pyrogen test for detection of pyrogenic material in growth hormone produced by recombinant Escherichia coli.

    PubMed Central

    Dinarello, C A; O'Connor, J V; LoPreste, G; Swift, R L

    1984-01-01

    Human growth hormone is biosynthetically produced in recombinant strains of Escherichia coli as methionyl human growth hormone (met-hGH). When purified from the bacterial culture, met-hGH is biologically active in established assays for growth hormone. Therefore, a phase I trial of met-hGH was carried out in healthy human adults; during the first trial, however, signs, symptoms, and clinical laboratory tests characteristic of an acute-phase response to pyrogenic agents was observed. Prior testing of the met-hGH preparation used in the phase I trial did not reveal evidence of toxicity, and the U.S. Pharmacopeial Convention rabbit pyrogen test, as well as the Limulus amoebocyte lysate (LAL) test, had not detected significant levels of exogenous pyrogens or endotoxin. In addition, standard inhibition studies with added endotoxin showed no inhibition by the LAL test. When this preparation of met-hGH was incubated with human blood mononuclear cells, leukocytic pyrogen (LP) was released into the supernatant medium, suggesting that the preparation contained pyrogenic material. Various lots of met-hGH based on different purification and formulating methods were tested by the human LP assay for contaminating pyrogens. The results of these tests aided in the identification of procedures for met-hGH preparations which did not induce LP in vitro. Thus, subsequent lots of met-hGH which had passed the LP test were used in repeat clinical studies, and no inflammatory or pyrogenic reactions were observed. When the LP test was used, experiments revealed that the original lot of met-hGH was contaminated with endotoxin which had not been detected in the LAL or rabbit pyrogen tests. Lyophilization in glycine-phosphate buffer had resulted in a 10- to 20-fold reduction of endotoxin reactivity in the LAL test and the U.S. Pharmacopeial Convention rabbit pyrogen test. These data provide a probable explanation for the negative result from the LAL and rabbit pyrogen test in the initial lot

  1. Recombination occurs within minutes of replication blockage by RTS1 producing restarted forks that are prone to collapse

    PubMed Central

    Nguyen, Michael O; Jalan, Manisha; Morrow, Carl A; Osman, Fekret; Whitby, Matthew C

    2015-01-01

    The completion of genome duplication during the cell cycle is threatened by the presence of replication fork barriers (RFBs). Following collision with a RFB, replication proteins can dissociate from the stalled fork (fork collapse) rendering it incapable of further DNA synthesis unless recombination intervenes to restart replication. We use time-lapse microscopy and genetic assays to show that recombination is initiated within ∼10 min of replication fork blockage at a site-specific barrier in fission yeast, leading to a restarted fork within ∼60 min, which is only prevented/curtailed by the arrival of the opposing replication fork. The restarted fork is susceptible to further collapse causing hyper-recombination downstream of the barrier. Surprisingly, in our system fork restart is unnecessary for maintaining cell viability. Seemingly, the risk of failing to complete replication prior to mitosis is sufficient to warrant the induction of recombination even though it can cause deleterious genetic change. DOI: http://dx.doi.org/10.7554/eLife.04539.001 PMID:25806683

  2. Origin of the CMS gene locus in rapeseed cybrid mitochondria: active and inactive recombination produces the complex CMS gene region in the mitochondrial genomes of Brassicaceae.

    PubMed

    Oshima, Masao; Kikuchi, Rie; Imamura, Jun; Handa, Hirokazu

    2010-01-01

    CMS (cytoplasmic male sterile) rapeseed is produced by asymmetrical somatic cell fusion between the Brassica napus cv. Westar and the Raphanus sativus Kosena CMS line (Kosena radish). The CMS rapeseed contains a CMS gene, orf125, which is derived from Kosena radish. Our sequence analyses revealed that the orf125 region in CMS rapeseed originated from recombination between the orf125/orfB region and the nad1C/ccmFN1 region by way of a 63 bp repeat. A precise sequence comparison among the related sequences in CMS rapeseed, Kosena radish and normal rapeseed showed that the orf125 region in CMS rapeseed consisted of the Kosena orf125/orfB region and the rapeseed nad1C/ccmFN1 region, even though Kosena radish had both the orf125/orfB region and the nad1C/ccmFN1 region in its mitochondrial genome. We also identified three tandem repeat sequences in the regions surrounding orf125, including a 63 bp repeat, which were involved in several recombination events. Interestingly, differences in the recombination activity for each repeat sequence were observed, even though these sequences were located adjacent to each other in the mitochondrial genome. We report results indicating that recombination events within the mitochondrial genomes are regulated at the level of specific repeat sequences depending on the cellular environment.

  3. Molecular farming of human cytokines and blood products from plants: challenges in biosynthesis and detection of plant-produced recombinant proteins.

    PubMed

    da Cunha, Nicolau B; Vianna, Giovanni R; da Almeida Lima, Thaina; Rech, Elíbio

    2014-01-01

    Plants have emerged as an attractive alternative to the traditional mammalian cell cultures or microbial cell-based systems system for the production of valuable recombinant proteins. Through recombinant DNA technology, plants can be engineered to produce large quantities of pharmaceuticals and industrial proteins of high quality at low costs. The recombinant production, by transgenic plants, of therapeutic proteins normally present in human plasma, such as cytokines, coagulation factors, anticoagulants, and immunoglobulins, represents a response to the ongoing challenges in meeting the demand for therapeutic proteins to treat serious inherited or acquired bleeding and immunological diseases. As the clinical utilization of fractionated plasma molecules is limited by high production costs, using recombinant biopharmaceuticals derived from plants represents a feasible alternative to provide efficient treatment. Plant-derived pharmaceuticals also reduce the potential risks to patients of infection with pathogens or unwanted immune responses due to immunogenic antigens. In this review, we summarize the recent advances in molecular farming of cytokines. We also examine the technological basis, upcoming challenges, and perspectives for the biosynthesis and detection of these molecules in different plant production platforms.

  4. One-pot synthesis of a gold nanoparticle-Vmh2 hydrophobin nanobiocomplex for glucose monitoring

    NASA Astrophysics Data System (ADS)

    Politi, Jane; De Stefano, Luca; Rea, Ilaria; Gravagnuolo, Alfredo Maria; Giardina, Paola; Methivier, Christophe; Casale, Sandra; Spadavecchia, Jolanda

    2016-05-01

    HydrophobinVmh2 is a small amphiphilic protein, which self-assembles on different surfaces and naturally interacts with glucose. Here, we report on the synthesis of a nanobiocomplex made of polyethylene glycol, Vmh2 and gold nanoparticles by a one-step process and on its ability to recognise glucose in an aqueous solution at 0.3-0.6-1.2 mg ml-1 concentrations. Even though the Vmh2 proteins are intrinsically bonded to the gold core, effective glucose interaction monitoring was demonstrated by using dynamic light scattering, ultraviolet-visible, polarization-modulated infrared reflection-absorption and x-ray photoelectron spectroscopies. Experimental results highlighted an affinity constant of 7.3 ± 0.3 mg ml-1 between the nanobiosystem and the sugar, and a detection sensitivity of 0.13 ± 0.06 a.u./mg ml-1.

  5. Bioactive modification of silicon surface using self-assembled hydrophobins from Pleurotus ostreatus.

    PubMed

    De Stefano, L; Rea, I; De Tommasi, E; Rendina, I; Rotiroti, L; Giocondo, M; Longobardi, S; Armenante, A; Giardina, P

    2009-10-01

    A crystalline silicon surface can be made biocompatible and chemically stable by a self-assembled biofilm of proteins, the hydrophobins (HFBs) purified from the fungus Pleurotus ostreatus. The protein-modified silicon surface shows an improvement in wettability and is suitable for immobilization of other proteins. Two different proteins were successfully immobilized on the HFBs-coated chips: the bovine serum albumin and an enzyme, a laccase, which retains its catalytic activity even when bound on the chip. Variable-angle spectroscopic ellipsometry (VASE), water contact angle (WCA), and fluorescence measurements demonstrated that the proposed approach in silicon surface bioactivation is a feasible strategy for the fabrication of a new class of hybrid devices.

  6. Bioactive modification of silicon surface using self-assembled hydrophobins from Pleurotus ostreatus

    NASA Astrophysics Data System (ADS)

    de Stefano, L.; Rea, I.; de Tommasi, E.; Rendina, I.; Rotiroti, L.; Giocondo, M.; Longobardi, S.; Armenante, A.; Giardina, P.

    2009-10-01

    A crystalline silicon surface can be made biocompatible and chemically stable by a self-assembled biofilm of proteins, the hydrophobins (HFBs) purified from the fungus Pleurotus ostreatus. The protein-modified silicon surface shows an improvement in wettability and is suitable for immobilization of other proteins. Two different proteins were successfully immobilized on the HFBs-coated chips: the bovine serum albumin and an enzyme, a laccase, which retains its catalytic activity even when bound on the chip. Variable-angle spectroscopic ellipsometry (VASE), water contact angle (WCA), and fluorescence measurements demonstrated that the proposed approach in silicon surface bioactivation is a feasible strategy for the fabrication of a new class of hybrid devices.

  7. Genes encoding homologous antigens in taeniid cestode parasites: Implications for development of recombinant vaccines produced in Escherichia coli.

    PubMed

    Gauci, Charles; Lightowlers, Marshall W

    2013-01-01

    Recombinant vaccine antigens are being evaluated for their ability to protect livestock animals against cysticercosis and related parasitic infections. Practical use of some of these vaccines is expected to reduce parasite transmission, leading to a reduction in the incidence of neurocysticercosis and hydatid disease in humans. We recently showed that an antigen (TSOL16), expressed in Escherichia coli, confers high levels of protection against Taenia solium cysticercosis in pigs, which provides a strategy for control of T. solium parasite transmission. Here, we discuss the characteristics of this antigen that may affect the utility of TSOL16 and related antigens for development as recombinant vaccines. We also report that genes encoding antigens closely related to TSOL16 from T. solium also occur in other related species of parasites. These highly homologous antigens have the potential to be used as vaccines and may provide protection against related species of Taenia that cause infection in other hosts.

  8. Fusion of the Dhfr/Mtx and IR/MAR gene amplification methods produces a rapid and efficient method for stable recombinant protein production.

    PubMed

    Noguchi, Chiemi; Araki, Yoshio; Miki, Daisuke; Shimizu, Noriaki

    2012-01-01

    Amplification of the dihydrofolate reductase gene (Dhfr) by methotrexate (Mtx) exposure is commonly used for recombinant protein expression in Chinese hamster ovary (CHO) cells. However, this method is both time- and labor-intensive, and the high-producing cells that are generated are frequently unstable in culture. Another gene amplification method is based on using a plasmid bearing a mammalian replication initiation region (IR) and a matrix attachment region (MAR), which result in the spontaneous initiation of gene amplification in transfected cells. The IR/MAR and Dhfr/Mtx methods of gene amplification are based on entirely different principles. In this study, we combine these two methods to yield a novel method, termed the IR/MAR-Dhfr fusion method, which was used to express three proteins, the Fc receptor, GFP, and recombinant antibody. The fusion method resulted in a dramatic increase in expression of all three proteins in two CHO sub-lines, DXB-11, and DG44. The IR/MAR-Dhfr fusion amplified the genes rapidly and efficiently, and produced larger amounts of antibody than the Dhfr/Mtx or IR/MAR methods alone. While the amplified structure produced by the Dhfr/Mtx method was highly unstable, and the antibody production rate rapidly decreased with the culture time of the cells, the IR/MAR-Dhfr fusion method resulted in stable amplification and generated clonal cells that produced large amounts of antibody protein over a long period of time. In summary, the novel IR/MAR-Dhfr fusion method enables isolation of stable cells that produce larger amounts of a target recombinant protein more rapidly and easily than either the Dhfr/Mtx or IR/MAR methods alone.

  9. Fusion to Hydrophobin HFBI Improves the Catalytic Performance of a Cytochrome P450 System

    PubMed Central

    Schulz, Sebastian; Schumacher, Dominik; Raszkowski, Daniel; Girhard, Marco; Urlacher, Vlada B.

    2016-01-01

    Cytochrome P450 monooxygenases (P450) are heme-containing enzymes that oxidize a broad range of substrates in the presence of molecular oxygen and NAD(P)H. For their activity, most P450s rely on one or two redox proteins responsible for the transfer of electrons from the cofactor NAD(P)H to the heme. One of the challenges when using P450s in vitro, especially when non-physiological redox proteins are applied, is the inefficient transfer of electrons between the individual proteins resulting in non-productive consumption of NAD(P)H – referred to as uncoupling. Herein, we describe the improvement of the coupling efficiency between a P450 and its redox partner – diflavin reductase – by fusing both enzymes individually to the hydrophobin HFBI – a small self-assembling protein of the fungus Trichoderma reesei. The separated monooxygenase (BMO) and reductase (BMR) domains of P450 BM3 from Bacillus megaterium were chosen as a P450-reductase model system and individually fused to HFBI. The fusion proteins could be expressed in soluble form in Escherichia coli. When HFBI-fused BMO and BMR were mixed in vitro, substantially higher coupling efficiencies were measured as compared with the respective non-fused enzymes. Consequently, myristic acid conversion increased up to 20-fold (after 6 h) and 5-fold (after 24 h). Size exclusion chromatography demonstrated that in vitro the hydrophobin-fused enzymes build multimeric protein assemblies. Thus, the higher activity is hypothesized to be due to HFBI-mediated self-assembly arranging BMO and BMR in close spatial proximity in aqueous solution. PMID:27458582

  10. Characterization and downstream mannose phosphorylation of human recombinant α-L-iduronidase produced in Arabidopsis complex glycan-deficient (cgl) seeds.

    PubMed

    He, Xu; Pierce, Owen; Haselhorst, Thomas; von Itzstein, Mark; Kolarich, Daniel; Packer, Nicolle H; Gloster, Tracey M; Vocadlo, David J; Qian, Yi; Brooks, Doug; Kermode, Allison R

    2013-12-01

    Mucopolysaccharidosis (MPS) I is a lysosomal storage disease caused by a deficiency of α-L-iduronidase (IDUA) (EC 3.2.1.76); enzyme replacement therapy is the conventional treatment for this genetic disease. Arabidopsis cgl mutants are characterized by a deficiency of the activity of N-acetylglucosaminyl transferase I (EC 2.4.1.101), the first enzyme in the pathway of hybrid and complex N-glycan biosynthesis. To develop a seed-based platform for the production of recombinant IDUA for potential treatment of MPS I, cgl mutant seeds were generated to express human IDUA at high yields and to avoid maturation of the N-linked glycans on the recombinant human enzyme. Enzyme kinetic data showed that cgl-IDUA has similar enzymatic properties to the commercial recombinant IDUA derived from cultured Chinese hamster ovary (CHO) cells (Aldurazyme™). The N-glycan profile showed that cgl-derived IDUA contained predominantly high-mannose-type N-glycans (94.5%), and the residual complex/hybrid N-glycan-containing enzyme was efficiently removed by an additional affinity chromatography step. Furthermore, purified cgl-IDUA was amenable to sequential in vitro processing by soluble recombinant forms of the two enzymes that mediate the addition of the mannose-6-phosphate (M6P) tag in mammalian cells-UDP-GlcNAc:lysosomal enzyme N-acetylglucosamine (GlcNAc)-1-phosphotransferase-and GlcNAc-1-phosphodiester α-N-acetylglucosaminidase (the 'uncovering enzyme'). Arabidopsis seeds provide an alternative system for producing recombinant lysosomal enzymes for enzyme replacement therapy; the purified enzymes can be subjected to downstream processing to create the M6P, a recognition marker essential for efficient receptor-mediated uptake into lysosomes of human cells.

  11. Surface-water Interface Induces Conformational Changes Critical for Protein Adsorption: Implications for Monolayer Formation of EAS Hydrophobin

    PubMed Central

    Ley, Kamron; Christofferson, Andrew; Penna, Matthew; Winkler, Dave; Maclaughlin, Shane; Yarovsky, Irene

    2015-01-01

    The class I hydrophobin EAS is part of a family of small, amphiphilic fungal proteins best known for their ability to self-assemble into stable monolayers that modify the hydrophobicity of a surface to facilitate further microbial growth. These proteins have attracted increasing attention for industrial and biomedical applications, with the aim of designing surfaces that have the potential to maintain their clean state by resisting non-specific protein binding. To gain a better understanding of this process, we have employed all-atom molecular dynamics to study initial stages of the spontaneous adsorption of monomeric EAS hydrophobin on fully hydroxylated silica, a commonly used industrial and biomedical substrate. Particular interest has been paid to the Cys3-Cys4 loop, which has been shown to exhibit disruptive behavior in solution, and the Cys7-Cys8 loop, which is believed to be involved in the aggregation of EAS hydrophobin at interfaces. Specific and water mediated interactions with the surface were also analyzed. We have identified two possible binding motifs, one which allows unfolding of the Cys7-Cys8 loop due to the surfactant-like behavior of the Cys3-Cys4 loop, and another which has limited unfolding due to the Cys3-Cys4 loop remaining disordered in solution. We have also identified intermittent interactions with water which mediate the protein adsorption to the surface, as well as longer lasting interactions which control the diffusion of water around the adsorption site. These results have shown that EAS behaves in a similar way at the air-water and surface-water interfaces, and have also highlighted the need for hydrophilic ligand functionalization of the silica surface in order to prevent the adsorption of EAS hydrophobin. PMID:26636091

  12. Surface-water Interface Induces Conformational Changes Critical for Protein Adsorption: Implications for Monolayer Formation of EAS Hydrophobin.

    PubMed

    Ley, Kamron; Christofferson, Andrew; Penna, Matthew; Winkler, Dave; Maclaughlin, Shane; Yarovsky, Irene

    2015-01-01

    The class I hydrophobin EAS is part of a family of small, amphiphilic fungal proteins best known for their ability to self-assemble into stable monolayers that modify the hydrophobicity of a surface to facilitate further microbial growth. These proteins have attracted increasing attention for industrial and biomedical applications, with the aim of designing surfaces that have the potential to maintain their clean state by resisting non-specific protein binding. To gain a better understanding of this process, we have employed all-atom molecular dynamics to study initial stages of the spontaneous adsorption of monomeric EAS hydrophobin on fully hydroxylated silica, a commonly used industrial and biomedical substrate. Particular interest has been paid to the Cys3-Cys4 loop, which has been shown to exhibit disruptive behavior in solution, and the Cys7-Cys8 loop, which is believed to be involved in the aggregation of EAS hydrophobin at interfaces. Specific and water mediated interactions with the surface were also analyzed. We have identified two possible binding motifs, one which allows unfolding of the Cys7-Cys8 loop due to the surfactant-like behavior of the Cys3-Cys4 loop, and another which has limited unfolding due to the Cys3-Cys4 loop remaining disordered in solution. We have also identified intermittent interactions with water which mediate the protein adsorption to the surface, as well as longer lasting interactions which control the diffusion of water around the adsorption site. These results have shown that EAS behaves in a similar way at the air-water and surface-water interfaces, and have also highlighted the need for hydrophilic ligand functionalization of the silica surface in order to prevent the adsorption of EAS hydrophobin.

  13. Temperature effects on product-quality-related enzymes in batch CHO cell cultures producing recombinant tPA.

    PubMed

    Clark, Kevin J R; Chaplin, Frank W R; Harcum, Sarah W

    2004-01-01

    Culture conditions that affect product quality are important to the successful operation and optimization of bioreactors. Previous studies have demonstrated that enzymes, such as proteases and sialidases, accumulate in batch bioreactors. These enzymes are known to be detrimental to the quality of recombinant glycoproteins. Bioreactor temperature has been used to control cell growth and recombinant protein production rates. However, the effect of culture temperature on the production of proteases and sialidases has not been investigated. In this study, Chinese hamster ovary cells were cultured with a temperature profile that decreased from 37 to 34 degrees C over 8 days and with a constant temperature of 37 degrees C. Analysis of extracellular protease activity indicated that two major proteases were present (50 and 69 kDa). The 50 kDa protease activity decreased similarly with time for both culture conditions. The 69 kDa protease activity increased with time for both culture conditions. The constant-temperature cultures had significantly lower 69 kDa protease levels compared to the ramped-temperature cultures in the early stationary phase. Intracellular sialidase activity was present in both cultures. The intracellular sialidase activity increased dramatically for both culture conditions immediately after the cells were inoculated into fresh medium. The initial peak in intracellular sialidase activity was followed by a first-order decay. The intracellular sialidase activities for the two culture conditions were not significantly different. The production of recombinant tissue type plasminogen activator was not significantly different for the two culture conditions. Thus, the previously hypothesized advantages that lower culture temperatures have reduced protease activity and improved productivity do not appear to be universal.

  14. [Characterization of hepatitis C virus structural proteins and HCV-like particles produced in recombinant baculovirus infected insect cells].

    PubMed

    Belzhelarskaia, S N; Koroleva, N N; Popenko, V V; Drutsa, V L; Orlova, O V; Rubtsov, P M; Kochetkov, S N

    2010-01-01

    Three proteins, namely: "core" protein C and glycoproteins E1 and E2, are main structural proteins forming a hepatitis C vius (HCV) virion. The virus structure and assembly, a role of the structural proteins in virion morphogenesis remain unknown because of the lack of an efficient culture system for HCV to be grown in vitro. Using recombinant baculoviruses expressing HCV structural protein genes in insect cells the specific structural proteins at the level of 25-35% relative to a common cell protein content, heterodimers of the glcoproteins, and HCV-like particles have been obtained. It has been demonstrated that recombinant proteins C, E1, and E2 go through the posttranslation modification, the glycoproteins form the non-covalent heterodimer, and HCV-like particles are located in endoplasmatic reticulum membrains of infected cells. An ability of the expressed proteins for forming E1E2 dimers and HCV-like particles was used for studying the role of E1 protein glcosylation upon expression and processing of the glycoproteins.

  15. Co-cultivation of Aspergillus nidulans Recombinant Strains Produces an Enzymatic Cocktail as Alternative to Alkaline Sugarcane Bagasse Pretreatment

    PubMed Central

    Lima, Matheus S.; Damasio, André R. de L.; Crnkovic, Paula M.; Pinto, Marcelo R.; da Silva, Ana M.; da Silva, Jean C. R.; Segato, Fernando; de Lucas, Rosymar C.; Jorge, João A.; Polizeli, Maria de L. T. de M.

    2016-01-01

    Plant materials represent a strategic energy source because they can give rise to sustainable biofuels through the fermentation of their carbohydrates. A clear example of a plant-derived biofuel resource is the sugar cane bagasse exhibiting 60–80% of fermentable sugars in its composition. However, the current methods of plant bioconversion employ severe and harmful chemical/physical pretreatments raising biofuel cost production and environmental degradation. Replacing these methods with co-cultivated enzymatic cocktails is an alternative. Here we propose a pretreatment for sugarcane bagasse using a multi-enzymatic cocktail from the co-cultivation of four Aspergillus nidulans recombinant strains. The co-cultivation resulted in the simultaneous production of GH51 arabinofuranosidase (AbfA), GH11 endo-1,4-xylanase (XlnA), GH43 endo-1,5-arabinanase (AbnA) and GH12 xyloglucan specific endo-β-1,4-glucanase (XegA). This core set of recombinant enzymes was more efficient than the alternative alkaline method in maintaining the cellulose integrity and exposing this cellulose to the following saccharification process. Thermogravimetric and differential thermal analysis revealed residual byproducts on the alkali pretreated biomass, which were not found in the enzymatic pretreatment. Therefore, the enzymatic pretreatment was residue-free and seemed to be more efficient than the applied alkaline method, which makes it suitable for bioethanol production. PMID:27199917

  16. Transgenic cows that produce recombinant human lactoferrin in milk are not protected from experimental Escherichia coli intramammary infection.

    PubMed

    Hyvönen, P; Suojala, L; Orro, T; Haaranen, J; Simola, O; Røntved, C; Pyörälä, S

    2006-11-01

    This is the first study describing an experimental mastitis model using transgenic cows expressing recombinant human lactoferrin (rhLf) in their milk. The aim of the study was to investigate the concentrations in milk and protective effects of bovine and recombinant human lactoferrin in experimental Escherichia coli mastitis. Experimental intramammary infection was induced in one udder quarter of seven first-lactating rhLf-transgenic cows and six normal cows, using an E. coli strain isolated from cows with clinical mastitis and known to be susceptible to Lf in vitro. Clinical signs were recorded during the experimental period, concentrations of human and bovine Lf and indicators of inflammation and bacterial counts were determined for milk, and concentrations of acute-phase proteins and tumor necrosis factor alpha were determined for sera and milk. Serum cortisol and blood hematological and biochemical parameters were also determined. Expression levels of rhLf in the milk of transgenic cows remained constant throughout the experiment (mean, 2.9 mg/ml). The high Lf concentrations in the milk of transgenic cows did not protect them from intramammary infection. All cows became infected and developed clinical mastitis. The rhLf-transgenic cows showed milder systemic signs and lower serum cortisol and haptoglobin concentrations than did controls. This may be explained by lipopolysaccharide-neutralizing and immunomodulatory effects of the high Lf concentrations in their milk. However, Lf does not seem to be a very efficient protein for genetic engineering to enhance the mastitis resistance of dairy cows.

  17. Recombinant Scorpine Produced Using SUMO Fusion Partner in Escherichia coli Has the Activities against Clinically Isolated Bacteria and Inhibits the Plasmodium falciparum Parasitemia In Vitro

    PubMed Central

    Gu, Yaping; Zhou, Huayun; Cao, Jun; Gao, Qi

    2014-01-01

    Scorpine, a small cationic peptide from the venom of Pandinus imperator, which has been shown to have anti-bacterial and anti-plasmodial activities, has potential important applications in the pharmaceutical industries. However, the isolation of scorpine from natural sources is inefficient and time-consuming. Here, we first report the expression and purification of recombinant scorpine in Escherichia coli, using small ubiquitin-related modifier (SUMO) fusion partner. The fusion protein was expressed in soluble form in E. coli, and expression was verified by SDS-PAGE and western blotting analysis. The fusion protein was purified to 90% purity by nickel–nitrilotriacetic acid (Ni2+–NTA) resin chromatography. After the SUMO-scorpine fusion protein was cleaved by the SUMO protease, the cleaved sample was reapplied to a Ni2+–NTA column. Tricine/SDS-PAGE gel results indicated that Scorpine had been purified successfully to more than 95% purity. The recombinantly expressed Scorpine showed anti-bacterial activity against two standard bacteria including Staphylococcus aureus ATCC 29213 and Acinetobacter baumannii ATCC 19606, and clinically isolated bacteria including S. aureus S, S. aureus R, A. baumannii S, and A. baumannii R. It also produced 100% reduction in Plasmodium falciparum parasitemia in vitro. Thus, the expression strategy presented in this study allowed convenient high yield and easy purification of recombinant Scorpine for pharmaceutical applications in the future. PMID:25068263

  18. Recombinant Adenovirus Delivery of Calreticulin–ESAT-6 Produces an Antigen-Specific Immune Response but no Protection Against a Mycobacterium Tuberculosis Challenge

    PubMed Central

    Esparza-González, S. C.; Troy, A.; Troudt, J.; Loera-Arias, M. J.; Villatoro-Hernández, J.; Torres-López, E.; Ancer-Rodríguez, J.; Gutiérrez-Puente, Y.; Muñoz-Maldonado, G.; Saucedo-Cárdenas, O.; Montes-de-Oca-Luna, R.; Izzo, A.

    2015-01-01

    Bacillus Calmette–Guerin (BCG) has failed to efficaciously control the worldwide spread of the disease. New vaccine development targets virulence antigens of Mycobacterium tuberculosis that are deleted in Mycobacterium bovis BCG. Immunization with ESAT-6 and CFP10 provides protection against M. tuberculosis in a murine infection model. Further, previous studies have shown that calreticulin increases the cell-mediated immune responses to antigens. Therefore, to test whether calreticulin enhances the immune response against M. tuberculosis antigens, we fused ESAT-6 to calreticulin and constructed a recombinant replication-deficient adenovirus to express the resulting fusion protein (AdCRT–ESAT-6). The adjuvant effect of calreticulin was assayed by measuring cytokine responses specific to ESAT-6. Recombinant adenovirus expressing the fusion protein produced higher levels of interferon-γ and tumour necrosis factor-α in response to ESAT-6. This immune response was not improved by the addition of CFP-10 to the CRT-ESAT-6 fusion protein (AdCRT–ESAT-6–CFP10). Mice immunized with these recombinant adenoviruses did not decrease the mycobacterial burden after low-dose aerosol infection with M. tuberculosis. We conclude that calreticulin can be used as an adjuvant to enhance the immune response against mycobacterial antigens, but it is not enough to protect against tuberculosis. PMID:22010821

  19. Multiple microfermentor battery: a versatile tool for use with automated parallel cultures of microorganisms producing recombinant proteins and for optimization of cultivation protocols.

    PubMed

    Frachon, Emmanuel; Bondet, Vincent; Munier-Lehmann, Hélène; Bellalou, Jacques

    2006-08-01

    A multiple microfermentor battery was designed for high-throughput recombinant protein production in Escherichia coli. This novel system comprises eight aerated glass reactors with a working volume of 80 ml and a moving external optical sensor for measuring optical densities at 600 nm (OD600) ranging from 0.05 to 100 online. Each reactor can be fitted with miniature probes to monitor temperature, dissolved oxygen (DO), and pH. Independent temperature regulation for each vessel is obtained with heating/cooling Peltier devices. Data from pH, DO, and turbidity sensors are collected on a FieldPoint (National Instruments) I/O interface and are processed and recorded by a LabVIEW program on a personal computer, which enables feedback control of the culture parameters. A high-density medium formulation was designed, which enabled us to grow E. coli to OD600 up to 100 in batch cultures with oxygen-enriched aeration. Accordingly, the biomass and the amount of recombinant protein produced in a 70-ml culture were at least equivalent to the biomass and the amount of recombinant protein obtained in a Fernbach flask with 1 liter of conventional medium. Thus, the microfermentor battery appears to be well suited for automated parallel cultures and process optimization, such as that needed for structural genomics projects.

  20. Efficient selection of high-producing subclones during gene amplification of recombinant Chinese hamster ovary cells by flow cytometry and cell sorting.

    PubMed

    Borth, N; Zeyda, M; Kunert, R; Katinger, H

    The screening procedure for high-producing cell lines is extremely time- and labor-intensive and costly, and is at present guided by an empirical approach based on individual experience. Flow cytometry and cell sorting, with its ability to analyze and separate single cells, an ideal method in the selection of such rare cells. The isolation of recombinant cell lines is especially difficult due to repeated gene amplification, which introduces high mutational variation into the population. We have established and evaluated a modification of a previous method that traps secreted product on the surface of the secreting cell, thus allowing direct analysis of single cell specific production rates. This method was used to select for high-producing subclones of a recombinant Chinese hamster ovary (CHO) cell line producing a human antibody against HIV-1 by repeated rounds of gene amplification and cell sorting. This cell line has been amplified in previous investigations, so that the amount of work and testing required by traditional methods can be compared with the protocol described herein. Forty-five 96-well plates were necessary to obtain a high-producing subclone by limited dilution methods, whereas only five plates were required when cell sorting was used. The specific production rate of the best clone obtained by sorting, however, was five times that of the clone obtained by traditional methods. In contrast to the clones obtained by limited dilution, which consisted of several populations of low- and high-producing cells even at high methotrexate concentrations (6.4 microM), the clones isolated by sorting were already homogeneous at 0.8 microM methotrexate.

  1. Correction: BTI-Tnao38, a new cell line derived from Trichoplusia ni, is permissive for AcMNPV infection and produces high levels of recombinant proteins.

    PubMed

    Hashimoto, Yoshi; Zhang, Sheng; Zhang, Shiying; Chen, Yun-Ru; Blissard, Gary W

    2012-04-24

    After publication we discovered an error in the identification of the origin of the cell line reported in our article in BMC Biotechnology (2010, 10:50), entitled "Ao38, a new cell line from eggs of the black witch moth, Ascalapha odorata (Lepidoptera: Noctuidae), is permissive for AcMNPV infection and produces high levels of recombinant proteins". Upon analysis of primary A. odorata cultures, we found that they were contaminated with cells of Trichoplusia ni origin. The origin of the Ao38 cell line was determined as T. ni using three marker genes and the Ao38 cell line was renamed BTI-Tnao38. References to the origin of the cell line as Ascalapha odorata should be replaced with "a cell line of Trichoplusia ni origin". The absence of TNCL virus detection in the BTI-Tnao38 (Ao38) cell line was confirmed using a highly sensitive RT-PCR protocol capable of detecting TNCL virus RNA at approximately 0.018 copies/cell. Because of these observations, we have revised the title of the original article to "Correction: BTI-Tnao38, a new cell line derived from Trichoplusia ni, is permissive for AcMNPV infection and produces high levels of recombinant proteins" and two additional authors were added to reflect their contributions to the analysis of this cell line.

  2. Heterologous production and detection of recombinant directing 2-deoxystreptamine (DOS) in the non-aminoglycoside-producing host Streptomyces venezuelae YJ003.

    PubMed

    Kurumbang, Nagendra Prasad; Oh, Tae-Jin; Liou, Kwangkyoung; Sohng, Jae Kyung

    2008-05-01

    2-Deoxystreptamine is a core aglycon that is vital to backbone formation in various aminoglycosides. This core structure can be modified to develop hybrid types of aminoglycoside antibiotics. We obtained three genes responsible for 2-deoxystreptamine production, neo7, neo6, and neo5, which encode 2-deoxy-scyllo-inosose synthase, L-glutamine: 2-deoxy-scyllo-inosose aminotransferase, and dehydrogenase, respectively, from the neomycin gene cluster. These genes were cloned into pIBR25, a Streptomyces expression vector, resulting in pNDOS. The recombinant pNDOS was transformed into a non-aminoglycoside-producing host, Streptomyces venezuelae YJ003, for heterologous expression. Based on comparisons of the retention time on LC-ESI/MS and ESIMS data with those of the 2-deoxystreptamine standard, a compound produced by S. venezuelae YJ003/pNDOS was found to be 2-deoxystreptamine.

  3. Use of a Chimeric Hsp70 to Enhance the Quality of Recombinant Plasmodium falciparum S-Adenosylmethionine Decarboxylase Protein Produced in Escherichia coli.

    PubMed

    Makhoba, Xolani Henry; Burger, Adélle; Coertzen, Dina; Zininga, Tawanda; Birkholtz, Lyn-Marie; Shonhai, Addmore

    2016-01-01

    S-adenosylmethionine decarboxylase (PfAdoMetDC) from Plasmodium falciparum is a prospective antimalarial drug target. The production of recombinant PfAdoMetDC for biochemical validation as a drug target is important. The production of PfAdoMetDC in Escherichia coli has been reported to result in unsatisfactory yields and poor quality product. The co-expression of recombinant proteins with molecular chaperones has been proposed as one way to improve the production of the former in E. coli. E. coli heat shock proteins DnaK, GroEL-GroES and DnaJ have previously been used to enhance production of some recombinant proteins. However, the outcomes were inconsistent. An Hsp70 chimeric protein, KPf, which is made up of the ATPase domain of E. coli DnaK and the substrate binding domain of P. falciparum Hsp70 (PfHsp70) has been previously shown to exhibit chaperone function when it was expressed in E. coli cells whose resident Hsp70 (DnaK) function was impaired. We proposed that because of its domain constitution, KPf would most likely be recognised by E. coli Hsp70 co-chaperones. Furthermore, because it possesses a substrate binding domain of plasmodial origin, KPf would be primed to recognise recombinant PfAdoMetDC expressed in E. coli. First, using site-directed mutagenesis, followed by complementation assays, we established that KPf with a mutation in the hydrophobic residue located in its substrate binding cavity was functionally compromised. We further co-expressed PfAdoMetDC with KPf, PfHsp70 and DnaK in E. coli cells either in the absence or presence of over-expressed GroEL-GroES chaperonin. The folded and functional status of the produced PfAdoMetDC was assessed using limited proteolysis and enzyme assays. PfAdoMetDC co-expressed with KPf and PfHsp70 exhibited improved activity compared to protein co-expressed with over-expressed DnaK. Our findings suggest that chimeric KPf may be an ideal Hsp70 co-expression partner for the production of recombinant plasmodial

  4. Biochemical and immunological characterization of a recombinantly-produced antifungal cysteine proteinase inhibitor from green kiwifruit (Actinidia deliciosa).

    PubMed

    Popovic, Milica; Andjelkovic, Uros; Burazer, Lidija; Lindner, Buko; Petersen, Arnd; Gavrovic-Jankulovic, Marija

    2013-10-01

    Plant proteinase inhibitors are considered important defense molecules against insect and pathogen attack. The cysteine proteinase inhibitor (CPI) from green kiwifruit (Actinidia deliciosa) belongs to the cystatin family and shows potent antifungal activity (in vitro and in vivo). However, the low abundance of this molecule in fruit (6μg/g of fresh fruit) seems to limit further investigations on the interaction between phytocystatin and photopathogenic fungi. In this paper the cDNA of the kiwi CPI was expressed in Escherichia coli. Fifteen N-terminal amino acids were identified by Edman degradation, and 77% of the rCPI primary structure was confirmed by mass fingerprint. The structural homology of recombinant CPI (rCPI) to its natural counterpart has been clearly demonstrated in immunological assays (immunoblot and ELISA inhibition). Biological activity of rCPI was demonstrated in inhibition assay with cysteine proteinase papain (EC50 2.78nM). In addition, rCPI reveals antifungal properties toward pathogenic fungi (Alternaria radicina and Botrytis cinerea), which designates it as an interesting model protein for the exploration of plant phytocystatins - pathogen interactions. Understanding the molecular mechanisms of natural plant resistance could lead to the development of ecologically safe fungicides for controlling post-harvest diseases and maintaining food quality.

  5. Characterization of a plant-produced recombinant human secretory IgA with broad neutralizing activity against HIV

    PubMed Central

    Paul, Matthew; Reljic, Rajko; Klein, Katja; Drake, Pascal MW; van Dolleweerd, Craig; Pabst, Martin; Windwarder, Markus; Arcalis, Elsa; Stoger, Eva; Altmann, Friedrich; Cosgrove, Catherine; Bartolf, Angela; Baden, Susan; Ma, Julian K-C

    2014-01-01

    Recombinant Secretory IgA (SIgA) complexes have the potential to improve antibody-based passive immunotherapeutic approaches to combat many mucosal pathogens. In this report, we describe the expression, purification and characterization of a human SIgA format of the broadly neutralizing anti-HIV monoclonal antibody (mAb) 2G12, using both transgenic tobacco plants and transient expression in Nicotiana benthamiana as expression hosts (P2G12 SIgA). The resulting heterodecameric complexes accumulated in intracellular compartments in leaf tissue, including the vacuole. SIgA complexes could not be detected in the apoplast. Maximum yields of antibody were 15.2 μg/g leaf fresh mass (LFM) in transgenic tobacco and 25 μg/g LFM after transient expression, and assembly of SIgA complexes was superior in transgenic tobacco. Protein L purified antibody specifically bound HIV gp140 and neutralised tier 2 and tier 3 HIV isolates. Glycoanalysis revealed predominantly high mannose structures present on most N-glycosylation sites, with limited evidence for complex glycosylation or processing to paucimannosidic forms. O-glycan structures were not identified. Functionally, P2G12 SIgA, but not IgG, effectively aggregated HIV virions. Binding of P2G12 SIgA was observed to CD209 / DC-SIGN, but not to CD89 / FcalphaR on a monocyte cell line. Furthermore, P2G12 SIgA demonstrated enhanced stability in mucosal secretions in comparison to P2G12 IgG mAb. PMID:25484063

  6. Development and Characterization of Recombinant Antibody Fragments That Recognize and Neutralize In Vitro Stx2 Toxin from Shiga Toxin-Producing Escherichia coli

    PubMed Central

    Luz, Daniela; Chen, Gang; Maranhão, Andrea Q.; Rocha, Leticia B.; Sidhu, Sachdev; Piazza, Roxane M. F.

    2015-01-01

    Background Stx toxin is a member of the AB5 family of bacterial toxins: the active A subunit has N-glycosidase activity against 28S rRNA, resulting in inhibition of protein synthesis in eukaryotic cells, and the pentamer ligand B subunits (StxB) bind to globotria(tetra)osylceramide receptors (Gb3/Gb4) on the cell membrane. Shiga toxin-producing Escherichia coli strains (STEC) may produce Stx1 and/or Stx2 and variants. Strains carrying Stx2 are considered more virulent and related to the majority of outbreaks, besides being usually associated with hemolytic uremic syndrome in humans. The development of tools for the detection and/or neutralization of these toxins is a turning point for early diagnosis and therapeutics. Antibodies are an excellent paradigm for the design of high-affinity, protein-based binding reagents used for these purposes. Methods and Findings In this work, we developed two recombinant antibodies; scFv fragments from mouse hybridomas and Fab fragments by phage display technology using a human synthetic antibody library. Both fragments showed high binding affinity to Stx2, and they were able to bind specifically to the GKIEFSKYNEDDTF region of the Stx2 B subunit and to neutralize in vitro the cytotoxicity of the toxin up to 80%. Furthermore, the scFv fragments showed 79% sensitivity and 100% specificity in detecting STEC strains by ELISA. Conclusion In this work, we developed and characterized two recombinant antibodies against Stx2, as promising tools to be used in diagnosis or therapeutic approaches against STEC, and for the first time, we showed a human monovalent molecule, produced in bacteria, able to neutralize the cytotoxicity of Stx2 in vitro. PMID:25790467

  7. One-step enzymatic hydrolysis of starch using a recombinant strain of Saccharomyces cerevisiae producing alpha-amylase, glucoamylase and pullulanase.

    PubMed

    Janse, B J; Pretorius, I S

    1995-03-01

    A recombinant strain of Saccharomyces cerevisiae was constructed that contained the genes encoding a bacterial alpha-amylase (AMY1), a yeast glucoamylase (STA2) and a bacterial pullulanase (pulA). The Bacillus amyloliquefaciens alpha-amylase and S. cerevisiae var. diastaticus glucoamylase genes were expressed in S. cerevisiae using their native promoters and the encoded enzymes secreted under direction of their native leader sequences. In contrast, the Klebsiella pneumoniae pullulanase gene was placed under the control of the yeast alcohol dehydrogenase gene promoter (ADC1P) and secreted using the yeast mating pheromone alpha-factor secretion signal (MF alpha 1S). Transcription termination of the pullulanase gene was effected by the yeast tryptophan synthase gene terminator (TRP5T), whereas termination of the glucoamylase and alpha-amylase genes was directed by their native terminators. Pullulanase (PUL1) produced by recombinant yeasts containing ADC1P MF alpha 1S pulA TRP5T (designated PUL1) was further characterized and compared to its bacterial counterpart (PulA). The different genes were introduced into S. cerevisiae in different combinations and the various amylolytic Saccharomyces transformants compared to Schwanniomyces occidentalis. Introduction of PUL1 into a S. cerevisiae strain containing both STA2 and AMY1, resulted in 99% assimilation of starch.

  8. Spontaneous hybrids between native and exotic Rubus in the Western United States produce offspring both by apomixis and by sexual recombination.

    PubMed

    Clark, L V; Jasieniuk, M

    2012-11-01

    Facultative asexual reproduction is a trait commonly found in invasive species. With a combination of sexual and asexual reproductive modes, such species may adapt to new environments via sexual recombination during range expansion, while at the same time having the benefits of asexuality such as the maintenance of fitness effects that depend upon heterozygosity. In the Western United States, native species of Rubus (Rosaceae) reproduce sexually whereas exotic naturalized Rubus species reproduce by pseudogamous apomixis. We hypothesized that new asexual lineages of Rubus could arise from hybridization in this range. To detect hybridization between native and exotic Rubus, we genotyped 579 individuals collected across California, Oregon and Washington with eight nuclear microsatellites and two chloroplast markers. Principal Coordinate Analysis and Bayesian clustering revealed a limited amount of hybridization of the native R. ursinus with the exotic R. armeniacus and R. pensilvanicus, as well as cultivated varieties. Genetic distances between these hybrids and their offspring indicated that both R. ursinus × R. armeniacus and R. ursinus × R. pensilvanicus produced a mix of apomictic and sexual seeds, with sexual seeds being more viable. Although neither of these hybrid types is currently considered invasive, they model the early stages of evolution of new invasive lineages, given the potential for fixed heterosis and the generation of novel genotypes. The hybrids also retain the ability to increase their fitness via sexual recombination and natural selection. Mixed reproductive systems such as those described here may be an important step in the evolution of asexual invasive species.

  9. The Arthroderma benhamiae Hydrophobin HypA Mediates Hydrophobicity and Influences Recognition by Human Immune Effector Cells

    PubMed Central

    Heddergott, Christoph; Bruns, Sandra; Nietzsche, Sandor; Leonhardt, Ines; Kurzai, Oliver; Kniemeyer, Olaf

    2012-01-01

    Dermatophytes are the most common cause of superficial mycoses in humans and animals. They can coexist with their hosts for many years without causing significant symptoms but also cause highly inflammatory diseases. To identify mechanisms involved in the modulation of the host response during infection caused by the zoophilic dermatophyte Arthroderma benhamiae, cell wall-associated surface proteins were studied. By two-dimensional gel electrophoresis, we found that a hydrophobin protein designated HypA was the dominant cell surface protein. HypA was also detected in the supernatant during the growth and conidiation of the fungus. The A. benhamiae genome harbors only a single hydrophobin gene, designated hypA. A hypA deletion mutant was generated, as was a complemented hypA mutant strain (hypAC). In contrast to the wild type and the complemented strain, the hypA deletion mutant exhibited “easily wettable” mycelia and conidia, indicating the loss of surface hydrophobicity of both morphotypes. Compared with the wild type, the hypA deletion mutant triggered an increased activation of human neutrophil granulocytes and dendritic cells, characterized by an increased release of the immune mediators interleukin-6 (IL-6), IL-8, IL-10, and tumor necrosis factor alpha (TNF-α). For the first time, we observed the formation of neutrophil extracellular traps against dermatophytes, whose level of formation was increased by the ΔhypA mutant compared with the wild type. Furthermore, conidia of the ΔhypA strain were killed more effectively by neutrophils. Our data suggest that the recognition of A. benhamiae by the cellular immune defense system is notably influenced by the presence of the surface rodlet layer formed by the hydrophobin HypA. PMID:22408226

  10. The Arthroderma benhamiae hydrophobin HypA mediates hydrophobicity and influences recognition by human immune effector cells.

    PubMed

    Heddergott, Christoph; Bruns, Sandra; Nietzsche, Sandor; Leonhardt, Ines; Kurzai, Oliver; Kniemeyer, Olaf; Brakhage, Axel A

    2012-05-01

    Dermatophytes are the most common cause of superficial mycoses in humans and animals. They can coexist with their hosts for many years without causing significant symptoms but also cause highly inflammatory diseases. To identify mechanisms involved in the modulation of the host response during infection caused by the zoophilic dermatophyte Arthroderma benhamiae, cell wall-associated surface proteins were studied. By two-dimensional gel electrophoresis, we found that a hydrophobin protein designated HypA was the dominant cell surface protein. HypA was also detected in the supernatant during the growth and conidiation of the fungus. The A. benhamiae genome harbors only a single hydrophobin gene, designated hypA. A hypA deletion mutant was generated, as was a complemented hypA mutant strain (hypA(C)). In contrast to the wild type and the complemented strain, the hypA deletion mutant exhibited "easily wettable" mycelia and conidia, indicating the loss of surface hydrophobicity of both morphotypes. Compared with the wild type, the hypA deletion mutant triggered an increased activation of human neutrophil granulocytes and dendritic cells, characterized by an increased release of the immune mediators interleukin-6 (IL-6), IL-8, IL-10, and tumor necrosis factor alpha (TNF-α). For the first time, we observed the formation of neutrophil extracellular traps against dermatophytes, whose level of formation was increased by the ΔhypA mutant compared with the wild type. Furthermore, conidia of the ΔhypA strain were killed more effectively by neutrophils. Our data suggest that the recognition of A. benhamiae by the cellular immune defense system is notably influenced by the presence of the surface rodlet layer formed by the hydrophobin HypA.

  11. Productivity and quality of recombinant proteins produced by stable CHO cell clones can be predicted by transient expression in HEK cells.

    PubMed

    Diepenbruck, Carolin; Klinger, Matthias; Urbig, Thomas; Baeuerle, Patrick; Neef, Rüdiger

    2013-06-01

    Selection of lead candidates in drug discovery is a complex and time-consuming process. Here, we describe an approach that allows prediction of the productivity and quality of recombinant proteins by stable producer cell clones with the help of transient transfection studies. This is exemplified for three distinct bispecific T cell engager (BiTE(®))-a new class of single-chain antibody-based therapeutics showing very promising results in the treatment of cancer. BiTE(®) titers of transiently transfected HEK cells showed a striking correlation with titers of selected stable CHO cell clones. Likewise, the percentage of the monomeric BiTE(®) fraction in cell culture supernatants correlated well between transiently expressing HEK and stably expressing CHO cell clones. This validates the use of transient transfection studies for the selection of biopharmaceutical lead candidates with desired pharmaceutical properties.

  12. The amphiphilic hydrophobin Vmh2 plays a key role in one step synthesis of hybrid protein-gold nanoparticles.

    PubMed

    Politi, Jane; De Stefano, Luca; Longobardi, Sara; Giardina, Paola; Rea, Ilaria; Methivier, Christophe; Pradier, Claire-Marie; Casale, Sandra; Spadavecchia, Jolanda

    2015-12-01

    We report a simple and original method to synthesize gold nanoparticles in which a fungal protein, the hydrophobin Vmh2 from Pleurotus ostreatus and dicarboxylic acid-terminated polyethylene-glycol (PEG) has been used as additional components in a one step process, leading to hybrid protein-metal nanoparticles (NPs). The nanoparticles have been characterized by ultra-violet/visible, infrared and X-ray photoelectron spectroscopies, dynamic light scattering and also by electron microscopy imaging. The results of these analytical techniques highlight nanometric sized, stable, hybrid complexes of about 12 nm, with outer surface rich in functional chemical groups. Interaction with protein and antibodies has also been exploited.

  13. Optimizing promoters and secretory signal sequences for producing ethanol from inulin by recombinant Saccharomyces cerevisiae carrying Kluyveromyces marxianus inulinase.

    PubMed

    Hong, Soo-Jeong; Kim, Hyo Jin; Kim, Jin-Woo; Lee, Dae-Hee; Seo, Jin-Ho

    2015-02-01

    Inulin is a polyfructan that is abundant in plants such as Jerusalem artichoke, chicory and dahlia. Inulinase can easily hydrolyze inulin to fructose, which is consumed by microorganisms. Generally, Saccharomyces cerevisiae, an industrial workhorse strain for bioethanol production, is known for not having inulinase activity. The inulinase gene from Kluyveromyces marxianus (KmINU), with the ability of converting inulin to fructose, was introduced into S. cerevisiae D452-2. The inulinase gene was fused to three different types of promoter (GPD, PGK1, truncated HXT7) and secretory signal sequence (KmINU, MFα1, SUC2) to generate nine expression cassettes. The inulin fermentation performance of the nine transformants containing different promoter and signal sequence combinations for inulinase production were compared to select an optimized expression system for efficient inulin fermentation. Among the nine inulinase-producing transformants, the S. cerevisiae carrying the PGK1 promoter and MFα1 signal sequence (S. cerevisiae D452-2/p426PM) showed not only the highest specific KmINU activity, but also the best inulin fermentation capability. Finally, a batch fermentation of the selected S. cerevisiae D452-2/p426PM in a bioreactor with 188.2 g/L inulin was performed to produce 80.2 g/L ethanol with 0.43 g ethanol/g inulin of ethanol yield and 1.22 g/L h of ethanol productivity.

  14. Construction of a Recombinant Leuconostoc mesenteroides CJNU 0147 Producing 1,4-Dihydroxy-2-Naphthoic Acid, a Bifidogenic Growth Factor.

    PubMed

    Eom, Ji-Eun; Moon, Gi-Seong

    2015-01-01

    1,4-Dihydroxy-2-naphthoic acid (DHNA), a precursor of menaquinone (vitamin K2), has an effect on growth stimulation of bifidobacteria and prevention of osteoporosis, making it a promising functional food material. Therefore, we tried to clone the menB gene encoding DHNA synthase from Leuconostoc mesenteroides CJNU 0147. Based on the genome sequence of Leu. mesenteroides ATCC 8293 (GenBank accession no., CP000414), a primer set (Leu_menBfull_F and Leu_menBfull_R) was designed for the PCR amplification of menB gene of CJNU 0147. A DNA fragment (1,190 bp), including the menB gene, was amplified, cloned into pGEM-T Easy vector, and sequenced. The deduced amino acid sequence of MenB (DHNA synthase) protein of CJNU 0147 had a 98% similarity to the corresponding protein of ATCC 8293. The menB gene was subcloned into pCW4, a lactic acid bacteria - E. coli shuttle vector, and transferred to CJNU 0147. The transcription of menB gene of CJNU 0147 (pCW4::menB) was increased, when compared with those of CJNU 0147 (pCW4) and CJNU 0147 (-). The DHNA was produced from it at a detectable level, indicating that the cloned menB gene of CJNU 0147 encoded a DHNA synthase which is responsible for the production of DHNA, resulting in an increase of bifidogenic growth stimulation activity.

  15. Recruitment of Class I Hydrophobins to the Air:Water Interface Initiates a Multi-step Process of Functional Amyloid Formation*

    PubMed Central

    Morris, Vanessa K.; Ren, Qin; Macindoe, Ingrid; Kwan, Ann H.; Byrne, Nolene; Sunde, Margaret

    2011-01-01

    Class I fungal hydrophobins form amphipathic monolayers composed of amyloid rodlets. This is a remarkable case of functional amyloid formation in that a hydrophobic:hydrophilic interface is required to trigger the self-assembly of the proteins. The mechanism of rodlet formation and the role of the interface in this process have not been well understood. Here, we have studied the effect of a range of additives, including ionic liquids, alcohols, and detergents, on rodlet formation by two class I hydrophobins, EAS and DewA. Although the conformation of the hydrophobins in these different solutions is not altered, we observe that the rate of rodlet formation is slowed as the surface tension of the solution is decreased, regardless of the nature of the additive. These results suggest that interface properties are of critical importance for the recruitment, alignment, and structural rearrangement of the amphipathic hydrophobin monomers. This work gives insight into the forces that drive macromolecular assembly of this unique family of proteins and allows us to propose a three-stage model for the interface-driven formation of rodlets. PMID:21454575

  16. Interspecific variability of class II hydrophobin GEO1 in the genus Geosmithia.

    PubMed

    Frascella, Arcangela; Bettini, Priscilla P; Kolařík, Miroslav; Comparini, Cecilia; Pazzagli, Luigia; Luti, Simone; Scala, Felice; Scala, Aniello

    2014-11-01

    The genus Geosmithia Pitt (Ascomycota: Hypocreales) comprises cosmopolite fungi living in the galleries built by phloeophagous insects. Following the characterization in Geosmithia species 5 of the class II hydrophobin GEO1 and of the corresponding gene, the presence of the geo1 gene was investigated in 26 strains derived from different host plants and geographic locations and representing the whole phylogenetic diversity of the genus. The geo1 gene was detected in all the species tested where it maintained the general organization shown in Geosmithia species 5, comprising three exons and two introns. Size variations were found in both introns and in the first exon, the latter being due to the presence of an intragenic tandem repeat sequence corresponding to a stretch of glycine residues in the deduced proteins. At the amino acid level the deduced proteins had 44.6 % identity and no major differences in the biochemical parameters (pI, GRAVY index, hydropathy plots) were found. GEO1 release in the fungal culture medium was also assessed by turbidimetric assay and SDS-PAGE, and showed high variability between species. The phylogeny based on the geo1 sequences did not correspond to that generated from a neutral marker (ITS rDNA), suggesting that sequence similarities could be influenced by other factors than phylogenetic relatedness, such as the intimacy of the symbiosis with insect vectors. The hypothesis of a strong selection pressure on the geo1 gene was sustained by the low values (<1) of non synonymous to synonymous nucleotide substitutions ratios (Ka/Ks), which suggest that purifying selection might act on this gene. These results are compatible with either a birth-and-death evolution scenario or horizontal transfer of the gene between Geosmithia species.

  17. Molecular dynamics of the "hydrophobic patch" that immobilizes hydrophobin protein HFBII on silicon.

    PubMed

    Moldovan, Clara; Thompson, Damien

    2011-09-01

    The experimentally-observed stable, electrically-conducting interface formed between hydrophobin protein HFBII and silicon provides a model system for the Bio/ICT interfaces required for bionanoelectronics. The present work used molecular dynamics (MD) computer simulations to investigate the atom-scale details of the assembly and structure of the HFBII/silicon interface, using models on the order of 40,000 atoms to compute energy profiles for the full protein interacting with a bare Si(111) substrate in aqueous solution. Five nanoseconds of free, equilibrated dynamics were performed for six models with initial protein:silicon separations ranging from 1.2 to 0.2 nanometers in steps of 0.2 nm. Three of the models formed extensive protein:silicon van der Waals's interfacial contacts. The model with 0.2 nm starting separation serves as an illustrative example of the dynamic interface created, whereby hydrophobic patch residues cycle between flat and more protruding patch conformations that favor respectively close inter-patch and close patch-surface contacts, with protein:surface separations cycling between 0.2 and 0.4 nm over the 5 ns of dynamics. Analysis of residue-based binding energies at the interface reveal three leucines Leu19, Leu21 and Leu63, together with isoleucine Ile22 and alanine Ala61, as the primary drivers towards adhesion on bare silicon, providing the atom-scale details of HFBII’s hydrophobic patch which in turn provides leads for the engineering of more tightly-coupled interfaces.

  18. Recombinant gonadotropins.

    PubMed

    Lathi, R B; Milki, A A

    2001-10-01

    Recombinant DNA technology makes it possible to produce large amounts of human gene products for pharmacologic applications, supplanting the need for human tissues. The genes for the alpha and beta subunits of follicle-stimulating hormone (FSH), luteinizing hormone (LH), and human chorionic gonadotropin (hCG) have been characterized and cloned. Recombinant FSH (rFSH) has been shown to be safe and effective in the treatment of fertility disorders. In comparison with the urinary gonadotropin products, human menopausal gonadotropins (HMG), and urinary follitropins (uFSH), rFSH is more potent and better tolerated by patients. Recombinant HCG appears to be as efficacious as urinary HCG with the benefit of improved local tolerance. Recombinant LH (rLH) is likely to be recommended as a supplement to rFSH for ovulation induction in hypogonadotropic women. It may also benefit in vitro fertilization patients undergoing controlled ovarian hyperstimulation with rFSH combined with pituitary suppression, with a gonadotropin-releasing hormone agonist or antagonist.

  19. Identification and characterization of a -1 reading frameshift in the heavy chain constant region of an IgG1 recombinant monoclonal antibody produced in CHO cells

    PubMed Central

    Lian, Zhirui; Wu, Qindong; Wang, Tongtong

    2016-01-01

    ABSTRACT Frameshifts lead to complete alteration of the intended amino acid sequences, and therefore may affect the biological activities of protein therapeutics and pose potential immunogenicity risks. We report here the identification and characterization of a novel -1 frameshift variant in a recombinant IgG1 therapeutic monoclonal antibody (mAb) produced in Chinese hamster ovary cells during the cell line selection studies. The variant was initially observed as an atypical post-monomer fragment peak in size exclusion chromatography. Characterization of the fragment peak using intact and reduced liquid chromatography-mass spectrometry (LC-MS) analyses determined that the fragment consisted of a normal light chain disulfide-linked to an aberrant 26 kDa fragment that could not be assigned to any HC fragment even after considering common modifications. Further analysis using LC-MS/MS peptide mapping revealed that the aberrant fragment contained the expected HC amino acid sequence (1-232) followed by a 20-mer novel sequence corresponding to expression of heavy chain DNA sequence in the -1 reading frame. Examination of the DNA sequence around the frameshift initiation site revealed that a mononucleotide repeat GGGGGG located in the IgG1 HC constant region was most likely the structural root cause of the frameshift. Rapid identification of the frameshift allowed us to avoid use of a problematic cell line containing the frameshift as the production cell line. The frameshift reported here may be observed in other mAb products and the hypothesis-driven analytical approaches employed here may be valuable for rapid identification and characterization of frameshift variants in other recombinant proteins. PMID:26652198

  20. Human anti-varicella-zoster virus (VZV) recombinant monoclonal antibody produced after Zostavax immunization recognizes the gH/gL complex and neutralizes VZV infection.

    PubMed

    Birlea, Marius; Owens, Gregory P; Eshleman, Emily M; Ritchie, Alanna; Traktinskiy, Igor; Bos, Nathan; Seitz, Scott; Azarkh, Yevgeniy; Mahalingam, Ravi; Gilden, Don; Cohrs, Randall J

    2013-01-01

    Varicella-zoster virus (VZV) is a ubiquitous, highly cell-associated, and exclusively human neurotropic alphaherpesvirus. VZV infection is initiated by membrane fusion, an event dependent in part on VZV glycoproteins gH and gL. Consistent with its location on the virus envelope, the gH/gL complex is a target of neutralizing antibodies produced after virus infection. One week after immunizing a 59-year-old VZV-seropositive man with Zostavax, we sorted his circulating blood plasma blasts and amplified expressed immunoglobulin variable domain sequences by single-cell PCR. Sequence analysis identified two plasma blast clones, one of which was used to construct a recombinant monoclonal antibody (rec-RC IgG). The rec-RC IgG colocalized with VZV gE on the membranes of VZV-infected cells and neutralized VZV infection in tissue culture. Mass spectrometric analysis of proteins immunoprecipitated by rec-RC IgG identified both VZV gH and gL. Transfection experiments showed that rec-RC IgG recognized a VZV gH/gL protein complex but not individual gH or gL proteins. Overall, our recombinant monoclonal anti-VZV antibody effectively neutralizes VZV and recognizes a conformational epitope within the VZV gH/L protein complex. An unlimited supply of this antibody provides the opportunity to analyze membrane fusion events that follow virus attachment and to identify multiple epitopes on VZV-specific proteins.

  1. Effector properties and glycosylation patterns of recombinant human anti-D-IgG1 antibodies produced by human PER.C6(®) cells.

    PubMed

    Olovnikova, N I; Grigorieva, O V; Petrov, A V

    2012-12-01

    Creation of effective monoclonal anti-D immunoglobulin for prevention of hemolytic disease of the newborn remains an unsolved problem because there is still no producer cell strain providing stable production and adequate glycosylation of antibodies. Recombinant anti-D have been obtained on the basis of human PER.C6(®) cells and characterized. Anti-D antibodies expressed in PER.C6(®) exhibited lower hemolytic activity in antibody-dependent cytotoxicity (ADCC) reaction mediated by low-affinity Fcγ receptors in comparison with identical antibodies of lymphoblastoid origin. Monoclonal antibodies produced by PER.C6(®) are completely fucosylated and desialylated, i.e. are characterized by abnormal glycosylation. Addition of kifunensine (α-mannosidase I inhibitor) to the medium led to production of antibodies with high hemolytic activity. Reduced activity of monoclonal antibodies in PER.C6(®) cells and the effect of kifunensine (causing synthesis of defucosylated glycans) suggest that the absence of fucose is the key factor responsible for Fc affinity for low-affinity receptors.

  2. Production of Extracellular Traps against Aspergillus fumigatus In Vitro and in Infected Lung Tissue Is Dependent on Invading Neutrophils and Influenced by Hydrophobin RodA

    PubMed Central

    Aimanianda, Vishukumar; Nietzsche, Sandor; Thywißen, Andreas; Jeron, Andreas; Latgé, Jean-Paul; Brakhage, Axel A.; Gunzer, Matthias

    2010-01-01

    Aspergillus fumigatus is the most important airborne fungal pathogen causing life-threatening infections in immunocompromised patients. Macrophages and neutrophils are known to kill conidia, whereas hyphae are killed mainly by neutrophils. Since hyphae are too large to be engulfed, neutrophils possess an array of extracellular killing mechanisms including the formation of neutrophil extracellular traps (NETs) consisting of nuclear DNA decorated with fungicidal proteins. However, until now NET formation in response to A. fumigatus has only been demonstrated in vitro, the importance of neutrophils for their production in vivo is unclear and the molecular mechanisms of the fungus to defend against NET formation are unknown. Here, we show that human neutrophils produce NETs in vitro when encountering A. fumigatus. In time-lapse movies NET production was a highly dynamic process which, however, was only exhibited by a sub-population of cells. NETosis was maximal against hyphae, but reduced against resting and swollen conidia. In a newly developed mouse model we could then demonstrate the existence and measure the kinetics of NET formation in vivo by 2-photon microscopy of Aspergillus-infected lungs. We also observed the enormous dynamics of neutrophils within the lung and their ability to interact with and phagocytose fungal elements in situ. Furthermore, systemic neutrophil depletion in mice almost completely inhibited NET formation in lungs, thus directly linking the immigration of neutrophils with NET formation in vivo. By using fungal mutants and purified proteins we demonstrate that hydrophobin RodA, a surface protein making conidia immunologically inert, led to reduced NET formation of neutrophils encountering Aspergillus fungal elements. NET-dependent killing of Aspergillus-hyphae could be demonstrated at later time-points, but was only moderate. Thus, these data establish that NET formation occurs in vivo during host defence against A. fumigatus, but suggest

  3. Production of extracellular traps against Aspergillus fumigatus in vitro and in infected lung tissue is dependent on invading neutrophils and influenced by hydrophobin RodA.

    PubMed

    Bruns, Sandra; Kniemeyer, Olaf; Hasenberg, Mike; Aimanianda, Vishukumar; Nietzsche, Sandor; Thywissen, Andreas; Jeron, Andreas; Latgé, Jean-Paul; Brakhage, Axel A; Gunzer, Matthias

    2010-04-29

    Aspergillus fumigatus is the most important airborne fungal pathogen causing life-threatening infections in immunocompromised patients. Macrophages and neutrophils are known to kill conidia, whereas hyphae are killed mainly by neutrophils. Since hyphae are too large to be engulfed, neutrophils possess an array of extracellular killing mechanisms including the formation of neutrophil extracellular traps (NETs) consisting of nuclear DNA decorated with fungicidal proteins. However, until now NET formation in response to A. fumigatus has only been demonstrated in vitro, the importance of neutrophils for their production in vivo is unclear and the molecular mechanisms of the fungus to defend against NET formation are unknown. Here, we show that human neutrophils produce NETs in vitro when encountering A. fumigatus. In time-lapse movies NET production was a highly dynamic process which, however, was only exhibited by a sub-population of cells. NETosis was maximal against hyphae, but reduced against resting and swollen conidia. In a newly developed mouse model we could then demonstrate the existence and measure the kinetics of NET formation in vivo by 2-photon microscopy of Aspergillus-infected lungs. We also observed the enormous dynamics of neutrophils within the lung and their ability to interact with and phagocytose fungal elements in situ. Furthermore, systemic neutrophil depletion in mice almost completely inhibited NET formation in lungs, thus directly linking the immigration of neutrophils with NET formation in vivo. By using fungal mutants and purified proteins we demonstrate that hydrophobin RodA, a surface protein making conidia immunologically inert, led to reduced NET formation of neutrophils encountering Aspergillus fungal elements. NET-dependent killing of Aspergillus-hyphae could be demonstrated at later time-points, but was only moderate. Thus, these data establish that NET formation occurs in vivo during host defence against A. fumigatus, but suggest

  4. Bactericidal properties of the antimicrobial peptide Ib-AMP4 from Impatiens balsamina produced as a recombinant fusion-protein in Escherichia coli.

    PubMed

    Fan, Xiaobo; Schäfer, Holger; Reichling, Jürgen; Wink, Michael

    2013-10-01

    Antimicrobial peptides (AMPs) represent a novel class of powerful natural antimicrobial agents. As AMPs are bactericidal, production of AMPs in recombinant bacteria is far from trivial. We report the production of Impatiens balsamina antimicrobial peptide 4 (Ib-AMP4, originally isolated from Impatiens balsamina) in Escherichia coli as a fusion protein and investigate Ib-AMP4's antimicrobial effects on human pathogens. A plasmid vector pET32a-Trx-Ib-AMP4 was constructed and transferred into E. coli. After induction, a soluble fusion protein was expressed successfully. The Ib-AMP4 peptide was obtained with a purity of over 90% after nickel affinity chromatography, ultrafiltration, enterokinase cleavage and sephadex size exclusion chromatography. For maximum activity, Ib-AMP4, which possesses two disulfide bonds, required activation with 5 μg/mL H2 O2 . Antimicrobial assays showed that Ib-AMP4 could efficiently target clinical multiresistant isolates including methicillin-resistant Staphylococcus aureus and extended-spectrum β-lactamase-producing E. coli. Time kill experiments revealed that Ib-AMP4 is bactericidal within 10 min after application. Haemolysis and cytotoxicity assays implied selectivity towards bacteria, an important prerequisite for clinical applications. Ib-AMP4 might be an interesting candidate for clinical studies involving patients with septicemia or for coating clinical devices, such as catheters. The method described here may be applicable for expression and purification of other AMPs with multiple disulfide bridges.

  5. IL-10-IFN-γ Double Producers CD4+ T Cells Are Induced by Immunization with an Amastigote Stage Specific Derived Recombinant Protein of Trypanosoma Cruzi

    PubMed Central

    Flores-García, Yevel; Rosales-Encina, José Luis; Satoskar, Abhay R.; Talamás-Rohana, Patricia

    2011-01-01

    During the acute phase of infection, T. cruzi replicates extensively and releases immunomodulatory molecules that delay parasite-specific responses mediated by effector T cells. This mechanism of evasion allows the parasite to spread in the host. Parasite molecules that regulate the host immune response during Chagas'disease have not been fully identified. GPI-anchored mucins, glycoinositolphospholipids, and glycoproteins comprise some of the most abundant T. cruzi surface molecules. IL-10 IFN-γ-secreting CD4+ T cells are activated during chronic infections and are responsible for prolonged persistence of parasite and for host protection against severe inflammatory responses. In this work we evaluated the role of rMBP::SSP4 protein of T. cruzi, a recombinant protein derived from a GPI anchored antigen, SSP4, as an immunomodulator molecule, finding that it was able to induce high concentrations of IL-10 and IFN-γ both in vivo and in vitro; during this last condition, both cytokines were produced by IL-10-IFN-γ-secreting CD4+ T cells. PMID:21927578

  6. Scintillation proximity assay for human DNA topoisomerase I using recombinant biotinyl-fusion protein produced in baculovirus-infected insect cells.

    PubMed

    Lerner, C G; Saiki, A Y

    1996-09-05

    DNA topoisomerases are well-established targets of important therapeutic agents which include the antibacterial quinolones and anticancer camptothecins. Screens for new classes of topoisomerase inhibitors generally employ methods, such as gel electrophoresis, which are not readily amenable to a rapid high-throughput format. We describe here a high-throughput assay to screen for inhibitors of human DNA topoisomerase I based on the scintillation proximity assay. The assay employs recombinant biotinyl-topoisomerase I fusion protein, a hybrid protein which contains a domain that is biotinylated during in vivo expression. The hybrid topoisomerase I fusion protein is found to be biotinylated, active, and nuclear-localized when produced in insect cells using a baculovirus expression system. The biotinyl-topoisomerase I fusion protein can be captured from crude nuclear extracts by immobilization on streptavidin-coated scintillation proximity assay beads. The assay detects binding of 3H-labeled DNA to the bead-immobilized enzyme by scintillation counting. The method is also able to detect stabilization of covalent protein-DNA complexes by camptothecin, an inhibitor previously shown to stabilize covalent intermediates that form during catalysis.

  7. Improvement in the stability and functionality of Nicotiana tabacum produced recombinant TRAIL through employment of endoplasmic reticulum expression and ascorbate buffer mediated extraction strategies

    PubMed Central

    Heidari, Hamid Reza; Bandehpour, Mojgan; Vahidi, Hossein; Barar, Jaleh; Kazemi, Bahram; Naderi-Manesh, Hossein

    2014-01-01

    Introduction: In order to employ Nicotiana tabacum cells as a profitable natural bioreactor for production of bio-functional "Soluble human TRAIL" (ShTRAIL), endoplasmic reticulum (ER) targeted expression and innovative extraction procedures were exploited. Methods: At first, the ShTRAIL encoding gene was sub-cloned into designed H2 helper vector to equip it with potent TMV omega leader sequences, ER sorting signal peptide, poly-histidine tag and ER retention signal peptide (KDEL). Then, the ER targeted ShTRAIL cassette was sequentially sub-cloned into "CaMV-35S" helper and "pGreen-0179" final expression vectors. Afterward, Agrobacterium mediated transformation method was adopted to express the ShTRAIL in the ER of N. tabacum . Next, the ShTRAIL protein was extracted through both phosphate and innovative ascorbate extraction buffers. Subsequently, oligomerization state of the ShTRAIL was evaluated through cross-linking assay and western blot analysis. Then, semi-quantitative western blot analysis was performed to estimate the ShTRAIL production. Finally, biological activity of the ShTRAIL was evaluated through MTT assay. Results: The phosphate buffer extracted ShTRAIL was produced in dimmer form, whereas the ShTRAIL extracted with ascorbate buffer generated trimer form. The ER targeted ShTRAIL strategy increased the ShTRAIL’s production level up to about 20 μg/g of fresh weight of N. tabacum . MTT assay indicated that ascorbate buffer extracted ShTRAIL could prohibit proliferation of A549 cell line. Conclusion: Endoplasmic reticulum expression and reductive ascorbate buffer extraction procedure can be employed to enhance the stability and overall production level of bio-functional recombinant ShTRAIL from transgenic N. tabacum cells. PMID:25337465

  8. On-line casein micelle disruption for downstream purification of recombinant human myelin basic protein produced in the milk of transgenic cows.

    PubMed

    Al-Ghobashy, Medhat A; Williams, Martin A K; Brophy, Brigid; Laible, Götz; Harding, David R K

    2009-06-01

    Downstream purification of a model recombinant protein (human myelin basic protein) from milk of transgenic cows is described. The recombinant protein was expressed as a His tagged fusion protein in the milk of transgenic cows and was found associated with the casein micellar phase. While difficulties in obtaining good recoveries were found when employing conventional micelle disruption procedures, direct capture using the cation exchanger SP Sepharose Big Beads was found successful in the extraction of the recombinant protein. Early breakthrough suggested a slow release of the recombinant protein from the micelles and dictated micelle disruption in order to obtain good yields. A new approach for deconstruction of the calcium core of the casein micelles, employing the interaction between the micellar calcium and the active sites of the cation exchanger resin was developed. Milk samples were loaded to the column in aliquots with a column washing step after each aliquot. This sequential loading approach successfully liberated the recombinant protein from the micelles and was found superior to the conventional sample loading approach. It increased the recovery by more than 25%, reduced fouling due to milk components and improved the column hydrodynamic properties as compared to the conventional sample loading approach. Hardware and software modifications to the chromatography system were necessary in order to keep the whole process automated. A second purification step using a Ni2+ affinity column was used to isolate the recombinant protein at purity more than 90% and a recovery percentage of 78%.

  9. (1)H, (13)C and (15)N resonance assignments of the RodA hydrophobin from the opportunistic pathogen Aspergillus fumigatus.

    PubMed

    Pille, Ariane; Kwan, Ann H; Cheung, Ivan; Hampsey, Matthew; Aimanianda, Vishukumar; Delepierre, Muriel; Latge, Jean-Paul; Sunde, Margaret; Guijarro, J Iñaki

    2015-04-01

    Hydrophobins are fungal proteins characterised by their amphipathic properties and an idiosyncratic pattern of eight cysteine residues involved in four disulphide bridges. The soluble form of these proteins spontaneously self-assembles at hydrophobic/hydrophilic interfaces to form an amphipathic monolayer. The RodA hydrophobin of the opportunistic pathogen Aspergillus fumigatus forms an amyloid layer with a rodlet morphology that covers the surface of fungal spores. This rodlet layer bestows hydrophobicity to the spores facilitating their dispersal in the air and rendering the conidia inert relative to the human immune system. As a first step in the analysis of the solution structure and self-association of RodA, we report the (1)H, (13)C and (15)N resonance assignments of the soluble monomeric form of RodA.

  10. Granulocyte/macrophage-colony stimulating factor produced by recombinant avian poxviruses enriches the regional lymph nodes with antigen-presenting cells and acts as an immunoadjuvant.

    PubMed

    Kass, E; Panicali, D L; Mazzara, G; Schlom, J; Greiner, J W

    2001-01-01

    Recombinant avian poxviruses [fowlpox and canarypox (ALVAC)], restricted for replication in nonavian cell substrates and expressing granulocyte/macrophage-colony stimulating factor (avipox-GM-CSF), were evaluated for their ability to enrich an immunization site with antigen-presenting cells (APCs) and, in turn, function as biological vaccine adjuvants. Avipox-GM-CSF administered as a single s.c. injection significantly enhanced the percentage and absolute number of APCs in the regional lymph nodes that drain the injection site. Both the magnitude and duration of the cellular and phenotypic increases within the lymph nodes induced by the avipox-GM-CSF viruses were significantly (P < 0.05) greater than those measured in mice treated with four daily injections of recombinant GM-CSF protein. Temporal studies revealed that the APC enrichment of regional lymph nodes was sustained for 21-28 days after injection of the recombinant avipox virus expressing GM-CSF and, moreover, three injections of the recombinant virus could be given without any appreciable loss of in vivo bioactivity. Mice expressing human carcinoembryonic antigen (CEA) as a transgene (CEA.Tg) developed CEA-specific humoral and cell-mediated immunity after being immunized with avipox-CEA. The coadministration of recombinant avipox viruses expressing CEA and GM-CSF significantly enhanced CEA-specific host immunity with an accompanying immunotherapeutic response in tumor-bearing CEA.Tg mice. The optimal use of avipox-GM-CSF, in terms of dose and dose schedule, especially when used with different immunogens, remains to be determined. Nonetheless, the present findings demonstrate: (a) the effective delivery of GM-CSF to an immunization site using a recombinant avian poxvirus; (b) the compatibility of delivering an antigen and GM-CSF in replication-defective viruses to enhance antigen-specific immunity; and (c) the combined use of recombinant avipox viruses expressing CEA and GM-CSF to generate antitumor

  11. Self-assembly of MPG1, a hydrophobin protein from the rice blast fungus that forms functional amyloid coatings, occurs by a surface-driven mechanism

    PubMed Central

    Pham, Chi L. L.; Rey, Anthony; Lo, Victor; Soulès, Margaux; Ren, Qin; Meisl, Georg; Knowles, Tuomas P. J.; Kwan, Ann H.; Sunde, Margaret

    2016-01-01

    Rice blast is a devastating disease of rice caused by the fungus Magnaporthe oryzae and can result in loss of a third of the annual global rice harvest. Two hydrophobin proteins, MPG1 and MHP1, are highly expressed during rice blast infections. These hydrophobins have been suggested to facilitate fungal spore adhesion and to direct the action of the enzyme cutinase 2, resulting in penetration of the plant host. Therefore a mechanistic understanding of the self-assembly properties of these hydrophobins and their interaction with cutinase 2 is crucial for the development of novel antifungals. Here we report details of a study of the structure, assembly and interactions of these proteins. We demonstrate that, in vitro, MPG1 assembles spontaneously into amyloid structures while MHP1 forms a non-fibrillar film. The assembly of MPG1 only occurs at a hydrophobic:hydrophilic interface and can be modulated by MHP1 and other factors. We further show that MPG1 assemblies can much more effectively retain cutinase 2 activity on a surface after co-incubation and extensive washing compared with other protein coatings. The assembly and interactions of MPG1 and MHP1 at hydrophobic surfaces thereby provide the basis for a possible mechanism by which the fungus can develop appropriately at the infection interface. PMID:27142249

  12. Anti-bacterial activity of recombinant human β-defensin-3 secreted in the milk of transgenic goats produced by somatic cell nuclear transfer.

    PubMed

    Liu, Jun; Luo, Yan; Ge, Hengtao; Han, Chengquan; Zhang, Hui; Wang, Yongsheng; Su, Jianmin; Quan, Fusheng; Gao, Mingqing; Zhang, Yong

    2013-01-01

    The present study was conducted to determine whether recombinant human β-defensin-3 (rHBD3) in the milk of transgenic goats has an anti-bacterial activity against Escherichia coli (E. coli), Staphylococcus aureus (S. aureus) and Streptococcus agalactiae (S. agalactiae) that could cause mastitis. A HBD3 mammary-specific expression vector was transfected by electroporation into goat fetal fibroblasts which were used to produce fourteen healthy transgenic goats by somatic cell nuclear transfer. The expression level of rHBD3 in the milk of the six transgenic goats ranged from 98 to 121 µg/ml at 15 days of lactation, and was maintained at 90-111 µg/ml during the following 2 months. Milk samples from transgenic goats showed an obvious inhibitory activity against E. coli, S. aureus and S. agalactiae in vitro. The minimal inhibitory concentrations of rHBD3 in milk against E. coli, S. aureus and S. agalactiae were 9.5-10.5, 21.8-23.0 and 17.3-18.5 µg/mL, respectively, which was similar to those of the HBD3 standard (P>0.05). The in vivo anti-bacterial activities of rHBD3 in milk were examined by intramammary infusion of viable bacterial inoculums. We observed that 9/10 and 8/10 glands of non-transgenic goats infused with S. aureus and E. coli became infected. The mean numbers of viable bacteria went up to 2.9×10(3) and 95.4×10(3) CFU/ml at 48 h after infusion, respectively; the mean somatic cell counts (SCC) in infected glands reached up to 260.4×10(5) and 622.2×10(5) cells/ml, which were significantly higher than the SCC in uninfected goat glands. In contrast, no bacteria was presented in glands of transgenic goats and PBS-infused controls, and the SSC did not significantly change throughout the period. Moreover, the compositions and protein profiles of milk from transgenic and non-transgenic goats were identical. The present study demonstrated that HBD3 were an effective anti-bacterial protein to enhance the mastitis resistance of dairy animals.

  13. Dissociative recombination in aeronomy

    NASA Technical Reports Server (NTRS)

    Fox, J. L.

    1989-01-01

    The importance of dissociative recombination in planetary aeronomy is summarized, and two examples are discussed. The first is the role of dissociative recombination of N2(+) in the escape of nitrogen from Mars. A previous model is updated to reflect new experimental data on the electronic states of N produced in this process. Second, the intensity of the atomic oxygen green line on the nightside of Venus is modeled. Use is made of theoretical rate coefficients for production of O (1S) in dissociative recombination from different vibrational levels of O2(+).

  14. Activated recombinant adenovirus proteinases

    DOEpatents

    Anderson, C.W.; Mangel, W.F.

    1999-08-10

    This application describes methods and expression constructs for producing activatable recombinant adenovirus proteinases. Purified activatable recombinant adenovirus proteinases and methods of purification are described. Activated adenovirus proteinases and methods for obtaining activated adenovirus proteinases are further included. Isolated peptide cofactors of adenovirus proteinase activity, methods of purifying and identifying peptide cofactors are also described. Antibodies immunoreactive with adenovirus proteinases, immunospecific antibodies, and methods for preparing them are also described. Other related methods and materials are also described. 29 figs.

  15. Activated recombinant adenovirus proteinases

    DOEpatents

    Anderson, Carl W.; Mangel, Walter F.

    1999-08-10

    This application describes methods and expression constructs for producing activatable recombinant adenovirus proteinases. Purified activatable recombinant adenovirus proteinases and methods of purification are described. Activated adenovirus proteinases and methods for obtaining activated adenovirus proteinases are further included. Isolated peptide cofactors of adenovirus proteinase activity, methods of purifying and identifying said peptide cofactors are also described. Antibodies immunoreactive with adenovirus proteinases, immunospecific antibodies, and methods for preparing them are also described. Other related methods and materials are also described.

  16. Vmh2 hydrophobin layer entraps glucose: A quantitative characterization by label-free optical and gravimetric methods

    NASA Astrophysics Data System (ADS)

    Della Ventura, B.; Rea, I.; Caliò, A.; Giardina, P.; Gravagnuolo, A. M.; Funari, R.; Altucci, C.; Velotta, R.; De Stefano, L.

    2016-02-01

    Hydrophobins (HFBs) are peculiar proteins which self-assemble at hydrophilic-hydrophobic interfaces into amphipathic membranes, and some of them (class I HFB) are able to form much more stable amyloid-like layers. This feature makes them suitable for many purposes, particularly when stable surface functionalization is required, also in view of their versatility in binding different kinds of molecules. For instance, it has been shown that Vmh2 from Pleurotus ostreatus (a class I HFB) is able to bind molecules like glucose, thus offering the perspective of using Vmh2 as a surface functionalization tool in bio-hybrid devices. In this paper a quantitative analysis of glucose interaction with the Vmh2 layer is reported; in particular, it is shown that Vmh2 layer swells by almost doubling its thickness as a result of glucose diffusion and each Vmh2 monomer is able to bind approximately 30 glucose molecules. These results have been achieved by self-assembling multi-layers of Vmh2 on a gold substrate and, subsequently, measuring both the mass of the bound glucose and the thickness of the resulting layer through two different and complementary techniques: quartz crystal-microbalance and ellipsometry. The data provided by the two techniques are in a satisfactory agreement and offer a plausible description of the mechanisms underlying the interaction of glucose with Vmh2 layer. This facile and versatile coating is of interest for biomedical applications of gold surfaces and particles.

  17. Application of recombinant human leukemia inhibitory factor (LIF) produced in rice (Oryza sativa L.) for maintenance of mouse embryonic stem cells.

    PubMed

    Youngblood, Bradford A; Alfano, Randall; Pettit, Steve C; Zhang, Deshui; Dallmann, H Garry; Huang, Ning; Macdonald, Clinton C

    2014-02-20

    Embryonic and induced pluripotent stem cells have the ability to differentiate into any somatic cell type, and thus have potential to treat a number of diseases that are currently incurable. Application of these cells for clinical or industrial uses would require an increase in production to yield adequate numbers of viable cells. However, the relatively high costs of cytokines and growth factors required for maintenance of stem cells in the undifferentiated state have the potential to limit translational research. Leukemia inhibitory factor (LIF), a member of the IL-6 cytokine family, is a key regulator in the maintenance of naïve states for both human and mouse stem cells. In this study, we describe a new recombinant human LIF (rhLIF) using a plant-based (rice) expression system. We found that rice-derived rhLIF possessed the same specific activity as commercial Escherichia coli-derived LIF and was capable of supporting mouse embryonic stem cell proliferation in the undifferentiated state as evidenced from pluripotency marker level analysis. Retention of the pluripotent state was found to be indistinguishable between rice-derived rhLIF and other recombinant LIF proteins currently on the market.

  18. DNA sequences, recombinant DNA molecules and processes for producing the A and B subunits of cholera toxin and preparations containing so-obtained subunit or subunits

    SciTech Connect

    Harford, N.; De Wilde, M.

    1987-05-19

    A recombinant DNA molecule is described comprising at least a portion coding for subunits A and B of cholera toxin, or a fragment or derivative of the portion wherein the fragment or derivative codes for a polypeptide have an activity which can induce an immune response to subunit A; can induce an immune response to subunit A and cause epithelial cell penetration and the enzymatic effect leading to net loss of fluid into the gut lumen; can bind to the membrane receptor for the B subunit of cholera toxin; can induce an immune response to subunit B; can induce an immune response to subunit B and bind to the membrane receptor; or has a combination of the activities.

  19. Genetic Recombination

    ERIC Educational Resources Information Center

    Whitehouse, H. L. K.

    1973-01-01

    Discusses the mechanisms of genetic recombination with particular emphasis on the study of the fungus Sordaria brevicollis. The study of recombination is facilitated by the use of mutants of this fungus in which the color of the ascospores is affected. (JR)

  20. Heterologous expression of newly identified galectin-8 from sea urchin embryos produces recombinant protein with lactose binding specificity and anti-adhesive activity

    PubMed Central

    Karakostis, Kostantinos; Costa, Caterina; Zito, Francesca; Matranga, Valeria

    2015-01-01

    Galectin family members specifically bind beta-galactoside derivatives and are involved in different cellular events, including cell communication, signalling, apoptosis, and immune responses. Here, we report a tandem-repeat type galectin from the Paracentrotus lividus sea urchin embryo, referred to as Pl-GAL-8. The 933nt sequence encodes a protein of 34.73 kDa, containing the conserved HFNPRF and WGxExR motifs in the two highly similar carbohydrate-recognition domains (CRD). The three-dimensional protein structure model of the N-CRD confirms the high evolutionary conservation of carbohydrate binding sites. The temporal gene expression is regulated during development and transcripts localize at the tip of the archenteron at gastrula stage, in a subset of the secondary mesenchyme cells that differentiate into blastocoelar (immune) cells. Functional studies using a recombinant Pl-GAL-8 expressed in bacteria demonstrate its hemo-agglutinating activity on human red blood cells through the binding to lactose, as well as its ability in inhibiting the adhesion of human Hep-G2 cells to the substrate. The recent implications in autoimmune diseases and inflammatory disorders make Gal-8 an attractive candidate for therapeutic purposes. Our results offer a solid basis for addressing the use of the new Pl-GAL-8 in functional and applicative studies, respectively in the developmental and biomedical fields. PMID:26640155

  1. Enzyme replacement therapy for Morquio A: an active recombinant N-acetylgalactosamine-6-sulfate sulfatase produced in Escherichia coli BL21.

    PubMed

    Rodríguez, Alexander; Espejo, Angela J; Hernández, Alejandra; Velásquez, Olga L; Lizaraso, Lina M; Cordoba, Henry A; Sánchez, Oscar F; Alméciga-Díaz, Carlos J; Barrera, Luis A

    2010-11-01

    Mucopolysaccharidosis IVA (MPS IVA) is an autosomal recessive disorder caused by N-acetylgalactosamine-6-sulfate sulfatase (GALNS) deficiency. Currently no effective therapies exist for MPS IVA. In this work, production of a recombinant GALNS enzyme (rGALNS) in Escherichia coli BL21 strain was studied. At shake scale, the effect of glucose concentration on microorganism growth, and microorganism culture and induction times on rGALNS production were evaluated. At bench scale, the effect of aeration and agitation on microorganism growth, and culture and induction times were evaluated. The highest enzyme activity levels at shake scale were observed in 12 h culture after 2-4 h induction. At bench scale the highest enzyme activity levels were observed after 2 h induction. rGALNS amounts in inclusion bodies fraction were up to 17-fold higher than those observed in the soluble fraction. However, the highest levels of active enzyme were found in the soluble fraction. Western blot analysis showed the presence of a 50-kDa band, in both soluble and inclusion bodies fractions. These results show for the first time the feasibility and potential of production of active rGALNS in a prokaryotic system for development of enzyme replacement therapy for MPS IVA disease.

  2. Introduction to dissociative recombination

    NASA Technical Reports Server (NTRS)

    Guberman, Steven L.; Mitchell, J. Brian A.

    1989-01-01

    Dissociative recombination (DR) of molecular ions with electrons has important consequences in many areas of physical science. Ab-initio calculations coupled with resonant scattering theory and multichannel quantum defect studies have produced detailed results illuminating the role of ion vibrational excitation, the quantum yields of the DR products, and the role of Rydberg states. The theoretical and experimental results are discussed.

  3. Recombinant, rice-produced yeast phytase shows the ability to hydrolyze phytate derived from seed-based feed, and extreme stability during ensilage treatment.

    PubMed

    Hamada, Akira; Yamaguchi, Ken-Ichi; Harada, Michiko; Horiguchi, Ken-Ichi; Takahashi, Toshiyoshi; Honda, Hideo

    2006-06-01

    When fresh rice leaves producing yeast Schwanniomyces occidentalis phytase were grounded and mixed with the whole extract of seed-based feed for pigs, the release of orthophosphate increased significantly. More specifically, phytate, a major source of phosphorus in the seeds, was hydrolyzed by heterologous phytase. Moreover, when transgenic rice plants were ensiled for up to 12 weeks, no decrease in the phytase activity of the heterologous enzyme was observed. This result strongly suggests that transgenic rice plants producing yeast phytase can be stored as silage without any loss of enzyme activity until usage as a feed additive.

  4. New skin test for detection of bovine tuberculosis on the basis of antigen-displaying polyester inclusions produced by recombinant Escherichia coli.

    PubMed

    Chen, Shuxiong; Parlane, Natalie A; Lee, Jason; Wedlock, D Neil; Buddle, Bryce M; Rehm, Bernd H A

    2014-04-01

    The tuberculin skin test for diagnosing tuberculosis (TB) in cattle lacks specificity if animals are sensitized to environmental mycobacteria, as some antigens in purified protein derivative (PPD) prepared from Mycobacterium bovis are present in nonpathogenic mycobacteria. Three immunodominant TB antigens, ESAT6, CFP10, and Rv3615c, are present in members of the pathogenic Mycobacterium tuberculosis complex but absent from the majority of environmental mycobacteria. These TB antigens have the potential to enhance skin test specificity. To increase their immunogenicity, these antigens were displayed on polyester beads by translationally fusing them to a polyhydroxyalkanoate (PHA) synthase which mediated formation of antigen-displaying inclusions in recombinant Escherichia coli. The most common form of these inclusions is poly(3-hydroxybutyric acid) (PHB). The respective fusion proteins displayed on these PHB inclusions (beads) were identified using tryptic peptide fingerprinting analysis in combination with matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). The surface exposure and accessibility of antigens were assessed by enzyme-linked immunosorbent assay (ELISA). Polyester beads displaying all three TB antigens showed greater reactivity with TB antigen-specific antibody than did beads displaying only one TB antigen. This was neither due to cross-reactivity of antibodies with the other two antigens nor due to differences in protein expression levels between beads displaying single or three TB antigens. The triple-antigen-displaying polyester beads were used for skin testing of cattle and detected all cattle experimentally infected with M. bovis with no false-positive reactions observed in those sensitized to environmental mycobacteria. The results suggested applicability of TB antigen-displaying polyester inclusions as diagnostic reagents for distinguishing TB-infected from noninfected animals.

  5. Making recombinant extracellular matrix proteins.

    PubMed

    Ruggiero, Florence; Koch, Manuel

    2008-05-01

    A variety of approaches to understand extracellular matrix protein structure and function require production of recombinant proteins. Moreover, the expression of heterologous extracellular matrix proteins, in particular collagens, using the recombinant technology is of major interest to the biomedical industry. Although extracellular matrix proteins are large, modular and often multimeric, most of them have been successfully produced in various expression systems. This review provides important factors, including the design of the construct, the cloning strategies, the expression vectors, the transfection method and the host cell systems, to consider in choosing a reliable and cost-effective way to make recombinant extracellular matrix proteins. Advantages and drawbacks of each system have been appraised. Protocols that may ease efficient recombinant production of extracellular matrix are described. Emphasis is placed on the recombinant collagen production. Members of the collagen superfamily exhibit specific structural features and generally require complex post-translational modifications to retain full biological activity that make more arduous their recombinant production.

  6. The fibrous form of intracellular inclusion bodies in recombinant variant fibrinogen-producing cells is specific to the hepatic fibrinogen storage disease-inducible variant fibrinogen.

    PubMed

    Arai, Shinpei; Ogiwara, Naoko; Mukai, Saki; Takezawa, Yuka; Sugano, Mitsutoshi; Honda, Takayuki; Okumura, Nobuo

    2017-02-04

    Fibrinogen storage disease (FSD) is a rare disorder that is characterized by the accumulation of fibrinogen in hepatocytes and induces liver injury. Six mutations in the γC domain (γG284R, γT314P, γD316N, the deletion of γG346-Q350, γG366S, and γR375W) have been identified for FSD. Our group previously established γ375W fibrinogen-producing Chinese hamster ovary (CHO) cells and observed aberrant large granular and fibrous forms of intracellular inclusion bodies. The aim of this study was to investigate whether fibrous intracellular inclusion bodies are specific to FSD-inducible variant fibrinogen. Thirteen expression vectors encoding the variant γ-chain were stably or transiently transfected into CHO cells expressing normal fibrinogen Aα- and Bβ-chains or HuH-7 cells, which were then immunofluorescently stained. Six CHO and HuH-7 cell lines that transiently produced FSD-inducible variant fibrinogen presented the fibrous (3.2-22.7 and 2.1-24.5%, respectively) and large granular (5.4-25.5 and 7.7-23.9%) forms of intracellular inclusion bodies. Seven CHO and HuH-7 cell lines that transiently produced FSD-non-inducible variant fibrinogen only exhibit the large granular form. These results demonstrate that transiently transfected variant fibrinogen-producing CHO cells and inclusion bodies of the fibrous form may be useful in non-invasive screening for FSD risk factors for FSD before its onset.

  7. Production of cellulosic ethanol and enzyme from waste fiber sludge using SSF, recycling of hydrolytic enzymes and yeast, and recombinant cellulase-producing Aspergillus niger.

    PubMed

    Cavka, Adnan; Alriksson, Björn; Rose, Shaunita H; van Zyl, Willem H; Jönsson, Leif J

    2014-08-01

    Bioethanol and enzymes were produced from fiber sludges through sequential microbial cultivations. After a first simultaneous saccharification and fermentation (SSF) with yeast, the bioethanol concentrations of sulfate and sulfite fiber sludges were 45.6 and 64.7 g/L, respectively. The second SSF, which included fresh fiber sludges and recycled yeast and enzymes from the first SSF, resulted in ethanol concentrations of 38.3 g/L for sulfate fiber sludge and 24.4 g/L for sulfite fiber sludge. Aspergillus niger carrying the endoglucanase-encoding Cel7B gene of Trichoderma reesei was grown in the spent fiber sludge hydrolysates. The cellulase activities obtained with spent hydrolysates of sulfate and sulfite fiber sludges were 2,700 and 2,900 nkat/mL, respectively. The high cellulase activities produced by using stillage and the significant ethanol concentrations produced in the second SSF suggest that onsite enzyme production and recycling of enzyme are realistic concepts that warrant further attention.

  8. Recombinant allergens

    PubMed Central

    Jutel, Marek; Solarewicz-Madejek, Katarzyna; Smolinska, Sylwia

    2012-01-01

    Allergen specific immunotherapy (SIT) is the only known causative treatment of allergic diseases. Recombinant allergen-based vaccination strategies arose from a strong need to both to improve safety and enhance efficacy of SIT. In addition, new vaccines can be effective in allergies including food allergy or atopic dermatitis, which poorly respond to the current treatment with allergen extracts. A number of successful clinical studies with both wild-type and hypoallergenic derivatives of recombinant allergens vaccines have been reported for the last decade. They showed high efficacy and safety profile as well as very strong modulation of T and B cell responses to specific allergens. PMID:23095874

  9. Identification, characterization, and recombinant expression of epidermicin NI01, a novel unmodified bacteriocin produced by Staphylococcus epidermidis that displays potent activity against Staphylococci.

    PubMed

    Sandiford, Stephanie; Upton, Mathew

    2012-03-01

    We describe the discovery, purification, characterization, and expression of an antimicrobial peptide, epidermicin NI01, which is an unmodified bacteriocin produced by Staphylococcus epidermidis strain 224. It is a highly cationic, hydrophobic, plasmid-encoded peptide that exhibits potent antimicrobial activity toward a wide range of pathogenic Gram-positive bacteria including methicillin-resistant Staphylococcus aureus (MRSA), enterococci, and biofilm-forming S. epidermidis strains. Purification of the peptide was achieved using a combination of hydrophobic interaction, cation exchange, and high-performance liquid chromatography (HPLC). Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) analysis yielded a molecular mass of 6,074 Da, and partial sequence data of the peptide were elucidated using a combination of tandem mass spectrometry (MS/MS) and de novo sequencing. The draft genome sequence of the producing strain was obtained using 454 pyrosequencing technology, thus enabling the identification of the structural gene using the de novo peptide sequence data previously obtained. Epidermicin NI01 contains 51 residues with four tryptophan and nine lysine residues, and the sequence showed approximately 50% identity to peptides lacticin Z, lacticin Q, and aureocin A53, all of which belong to a new family of unmodified type II-like bacteriocins. The peptide is active in the nanomolar range against S. epidermidis, MRSA isolates, and vancomycin-resistant enterococci. Other unique features displayed by epidermicin include a high degree of protease stability and the ability to retain antimicrobial activity over a pH range of 2 to 10, and exposure to the peptide does not result in development of resistance in susceptible isolates. In this study we also show the structural gene alone can be cloned into Escherichia coli strain BL21(DE3), and expression yields active peptide.

  10. Overexpression of Protein Disulfide Isomerase DsbC Stabilizes Multiple-Disulfide-Bonded Recombinant Protein Produced and Transported to the Periplasm in Escherichia coli

    PubMed Central

    Kurokawa, Yoichi; Yanagi, Hideki; Yura, Takashi

    2000-01-01

    Dsb proteins (DsbA, DsbB, DsbC, and DsbD) catalyze formation and isomerization of protein disulfide bonds in the periplasm of Escherichia coli. By using a set of Dsb coexpression plasmids constructed recently, we analyzed the effects of Dsb overexpression on production of horseradish peroxidase (HRP) isozyme C that contains complex disulfide bonds and tends to aggregate when produced in E. coli. When transported to the periplasm, HRP was unstable but was markedly stabilized upon simultaneous overexpression of the set of Dsb proteins (DsbABCD). Whereas total HRP production increased severalfold upon overexpression of at least disulfide-bonded isomerase DsbC, maximum transport of HRP to the periplasm seemed to require overexpression of all DsbABCD proteins, suggesting that excess Dsb proteins exert synergistic effects in assisting folding and transport of HRP. Periplasmic production of HRP also increased when calcium, thought to play an essential role in folding of nascent HRP polypeptide, was added to the medium with or without Dsb overexpression. These results suggest that Dsb proteins and calcium play distinct roles in periplasmic production of HRP, presumably through facilitating correct folding. The present Dsb expression plasmids should be useful in assessing and dissecting periplasmic production of proteins that contain multiple disulfide bonds in E. coli. PMID:10966415

  11. 'Super-perfect' enzymes: Structural stabilities and activities of recombinant triose phosphate isomerases from Pyrococcus furiosus and Thermococcus onnurineus produced in Escherichia coli.

    PubMed

    Sharma, Prerna; Guptasarma, Purnananda

    2015-05-08

    Triose phosphate isomerases (TIMs) are considered to be 'kinetically perfect' enzymes, limited in their activity only by the rates of diffusion of substrate and product molecules. Most studies conducted thus far have been on mesophile-derived TIMs. Here, we report studies of two extremophile-derived TIMs produced in Escherichia coli: (i) TonTIM, sourced from the genome of the thermophile archaeon, Thermococcus onnurineus, and (ii) PfuTIM, sourced from the genome of the hyperthermophile archaeon, Pyrococcus furiosus (PfuTIM). Although these enzymes are presumed to have evolved to function optimally at temperatures close to the boiling point of water, we find that TonTIM and PfuTIM display second-order rate-constants of activity (k(cat)/K(m) values) comparable to mesophile-derived TIMs, at 25 °C. At 90 °C, TonTIM and PfuTIM reach maximum velocities of reaction of ∼ 10(6)-10(7) μmol/s/mg, and display k(cat)/K(m) values in the range of ∼ 10(10)-10(11) M(-1) s(-1), which are three orders of magnitude higher than those reported for mesophile TIMs. Further, the two enzymes display no signs of having undergone any structural unfolding at 90 °C. Such enzymes could thus probably be called 'super-perfect' enzymes.

  12. Spectrum Recombination.

    ERIC Educational Resources Information Center

    Greenslade, Thomas B., Jr.

    1984-01-01

    Describes several methods of executing lecture demonstrations involving the recombination of the spectrum. Groups the techniques into two general classes: bringing selected portions of the spectrum together using lenses or mirrors and blurring the colors by rapid movement or foreshortening. (JM)

  13. Production and secretion of Aspergillus nidulans catalase B in filamentous fungi driven by the promoter and signal peptide of the Cladosporium fulvum hydrophobin gene hcf-1.

    PubMed

    Johnson, Hannah; Whiteford, James R; Eckert, Sabine E; Spanu, Pietro D

    2003-11-01

    We describe here the use of sequences from the hydrophobin gene hcf-1 of Cladosporium fulvum to construct pCatBex, a vector for high-level expression and secretion of CatB, a catalase from Aspergillus nidulans. Transformation of C. fulvum with pCatBex results in a 60-fold increase in the mycelial activity in the fungus and the appearance of up to 5.4 mkat/l of catalase in the growth medium. The levels of catalase in the supernatant increased dramatically following removal of nitrogen from the medium. Conversely, the overall specific activity of catalase in the cytoplasm did not change appreciably. This indicates that nitrogen depletion induces greater secretion of protein. The vector pCatBex also directs the expression and secretion of CatB in Magnaporthe grisea and may be a useful vector for the expression of genes in other filamentous fungi.

  14. Are hydrophobins and/or non-specific lipid transfer proteins responsible for gushing in beer? New hypotheses on the chemical nature of gushing inducing factors.

    PubMed

    Hippeli, Susanne; Elstner, Erich F

    2002-01-01

    Gushing of beer is characterised by the fact that immediately after opening a bottle a great number of fine bubbles are created throughout the volume of beer and ascend quickly under foam formation, which flows out of the bottle. This infuriating gushing phenomenon has been, and still is, a problem of world-wide importance to the brewing industry. It is generally assumed that the causes of malt-derived gushing are due to the use of "weathered" barley or wheat and the growth of moulds in the field, during storage and malting. We now develop a hypothesis connecting several lines of evidence from different laboratories. These results indicate that the fungal hydrophobins, hydrophobic components of conidiospores or aerial mycelia, are gushing-inducing factors. Furthermore, increased formation of ns-LTPs (non-specific lipid transfer proteins), synthesised in grains as response to fungal infection, and their modification during the brewing process may be responsible for malt-derived gushing.

  15. Atom Recombination on Surface

    NASA Astrophysics Data System (ADS)

    Kim, Young Chai

    Upon high speed re-entry of the Space Shuttle Orbiter (SSO) through the earth's atmosphere, oxygen and nitrogen atoms produced in the shock wave in front of the SSO recombine on the surface of the SSO, releasing heat. To minimize the rise of surface temperature due to the reaction, surface material of the SSO should have a low recombination probability, gamma, of atoms impinging on it. To design such material, it is necessary to understand the mechanism of atom recombination. With this in mind, gamma values were measured for recombination of O, N, and H atoms in a diffusion tube reactor between 700 and 1250 K (HT), 300 and 700 K (MT), and at 194 K (LT) on silica. The rate of recombination was first order with respect to the atom concentration from LT to HT. The Arrhenius plots, gamma vs. 1/T, were very complex. All observations are explained by assuming a surface with a small fraction of active sites that irreversibly bind chemisorbed atoms. Everything happens as if the active sites were surrounded by collection zones within which all atoms striking the surface are adsorbed reversibly with an assumed sticking probability of unity. These atoms then diffuse on the surface. Some of them reach the active sites where they can recombine with the chemisorbed atoms. At LT, all atoms striking the surface reach the active sites. As a result of desorption at MT, the collection zones shrink with increasing temperature. At HT, only atoms striking active sites directly from the gas phase lead to recombination. An analytical solution of the diffusion-reaction problem obtained for a model where the active sites are distributed uniformly fits with the experimental data from LT to HT. The two novel features of this work are the identification of the active sites on silica for recombination of H on silica at HT as surface OH groups and the suggestion that another kind of active site is responsible for recombination of O and N atoms at HT as well as for H atoms at LT and MT. Although

  16. A Method to Produce and Purify Full-Length Recombinant Alpha Dystroglycan: Analysis of N- and O-Linked Monosaccharide Composition in CHO Cells with or without LARGE Overexpression

    PubMed Central

    Yoon, Jung Hae; Xu, Rui; Martin, Paul

    2013-01-01

    α dystroglycan (αDG) is part of the dystrophin-associated glycoprotein (DAG) complex, a series of cytoskeletal, transmembrane, and membrane-associated proteins that serve to link the extracellular matrix (ECM) surrounding individual skeletal myofibers to the intracellular F-actin cytoskeleton. Glycosylation and ECM protein binding to αDG are regulated by a number of genes that, when defective, give rise to congenital or limb-girdle forms of muscular dystrophy termed dystroglycanopathies. One such dystroglycanopathy gene is LARGE. Here, we describe a method to produce and purify full-length, furin-resistant, recombinant αDG from CHO cells and CHO cells overexpressing LARGE (CHO-LARGE). In addition, we analyze the O- and N-linked monosaccharide composition of such proteins. αDG purified from CHO-LARGE cells had increased molar content of xylose and fucose relative to CHO, while no significant changes were found in N-linked monosaccharides. Glucuronic acid could not be quantified by the methods used. These studies describe a method to produce and purify the milligram amounts of αDG needed for certain biochemical methods, including monosaccharide analysis. Key words: Dystroglycan, muscular dystrophy, xylose, fucose, laminin, LARGE Correspondence: Paul.Martin@nationwidechildrens.org PMID:23390591

  17. Recombinant DNA production of spider silk proteins

    PubMed Central

    Tokareva, Olena; Michalczechen-Lacerda, Valquíria A; Rech, Elíbio L; Kaplan, David L

    2013-01-01

    Spider dragline silk is considered to be the toughest biopolymer on Earth due to an extraordinary combination of strength and elasticity. Moreover, silks are biocompatible and biodegradable protein-based materials. Recent advances in genetic engineering make it possible to produce recombinant silks in heterologous hosts, opening up opportunities for large-scale production of recombinant silks for various biomedical and material science applications. We review the current strategies to produce recombinant spider silks. PMID:24119078

  18. Recombinant DNA production of spider silk proteins.

    PubMed

    Tokareva, Olena; Michalczechen-Lacerda, Valquíria A; Rech, Elíbio L; Kaplan, David L

    2013-11-01

    Spider dragline silk is considered to be the toughest biopolymer on Earth due to an extraordinary combination of strength and elasticity. Moreover, silks are biocompatible and biodegradable protein-based materials. Recent advances in genetic engineering make it possible to produce recombinant silks in heterologous hosts, opening up opportunities for large-scale production of recombinant silks for various biomedical and material science applications. We review the current strategies to produce recombinant spider silks.

  19. Adoptive transfer of experimental allergic encephalomyelitis after in vitro treatment with recombinant murine interleukin-12. Preferential expansion of interferon-gamma-producing cells and increased expression of macrophage-associated inducible nitric oxide synthase as immunomodulatory mechanisms.

    PubMed Central

    Waldburger, K. E.; Hastings, R. C.; Schaub, R. G.; Goldman, S. J.; Leonard, J. P.

    1996-01-01

    In an adoptive transfer model of experimental allergic encephalomyelitis, stimulation of lymph node cells with proteolipid protein and recombinant murine interleukin (rmIL)-12 before cell transfer accelerated the onset and exacerbates clinical disease. In vitro stimulation with proteolipid protein in the presence of rmIL-12 was associated with an increase in interferon-gamma-producing cells and a decrease in IL-4-producing cells, indicating a preferential expansion of Th1 effector cells. This was supported by the finding that severe disease with rapid onset could be transferred with as few as 10 x 10(6) rmIL-12-stimulated lymph node cells. Immunohistochemical analysis confirmed that the accelerated onset of disease after in vitro stimulation with rmIL-12 coincided with an acute inflammatory response in the central nervous system. At peak disease, both control and rmIL-12 treatment groups exhibited extensive cellular infiltration with characteristic perivascular cuffing. No notable differences in either the cellular composition or cytokine expression within the lesions were seen between groups. However, the frequency of macrophages that stained positively for inducible nitric oxide synthase was increased in animals challenged with rmIL-12-treated lymph node cells. The results suggest that, in addition to promoting the preferential expansion of interferon-gamma-producing cells by rmIL-12 in vitro, secondary in vivo effects leading to macrophage activation and inducible nitric oxide synthase expression may contribute to the severe and protracted course of central nervous system inflammation in this model. Images Figure 2 PMID:8579100

  20. Recombinant protein expression plasmids optimized for industrial E. coli fermentation and plant systems produce biologically active human insulin-like growth factor-1 in transgenic rice and tobacco plants.

    PubMed

    Panahi, Mitra; Alli, Zaman; Cheng, Xiongying; Belbaraka, Loubaba; Belgoudi, Jaafar; Sardana, Ravinder; Phipps, Jenny; Altosaar, Illimar

    2004-06-01

    Human insulin-like growth factor-1 (hIGF-1) is a growth factor with clinical significance in medicine. The therapeutic potential of recombinant hIGF-1 (rthIGF-1) stems from the fact that hIGF-1 resembles insulin in many aspects of physiology. The expression of hIGF-1 in transgenic tobacco and rice plants using different expression cassettes is reported here. In the present study, two coding sequences were tested, one with the original human sequence, but partially optimized for expression in E. coli and the other with a plant-codon-optimized sequence that was expected to give a higher level of expression in plant systems. Three different hIGF-1 recombinant expression constructs were generated. All expression constructs utilized the maize ubiquitin 1 promoter with or without a signal sequence. Analyses conducted using a hIGF-1 specific ELISA kit showed all transgenic plants produced hIGF-1 and the accumulated hIGF-1 increased from the E. coli codon bias to higher levels when the hIGF-1 coding sequence was codon-optimized to match that of the maize zeamatin protein--the most transcribed gene in maize endosperm suspension cells. Further analyses that compared the functionality of the bacterial signal peptide Lam B in plants showed that this leader peptide led to lower expression levels when compared to transgenic plants that did not contain this sequence. This indicated that this expression construct was functional without removal of the bacterial signal sequence. The maize ubiquitin 1 promoter was found to be more active in rice plants than tobacco plants indicating that in this case, there was a class preference that was biased towards a monocot host. Biological analyses conducted using protein extracts from transgenic plants showed that the rthIGF-1 was effective in stimulating the in vitro growth and proliferation of human SH-SY5Y neuroblastoma cells. This indicated that the plant-produced rthIGF-1 was stable and biologically active. As some plants have been

  1. Ethanol production by recombinant hosts

    DOEpatents

    Ingram, Lonnie O.; Beall, David S.; Burchhardt, Gerhard F. H.; Guimaraes, Walter V.; Ohta, Kazuyoshi; Wood, Brent E.; Shanmugam, Keelnatham T.

    1995-01-01

    Novel plasmids comprising genes which code for the alcohol dehydrogenase and pyruvate decarboxylase are described. Also described are recombinant hosts which have been transformed with genes coding for alcohol dehydrogenase and pyruvate. By virtue of their transformation with these genes, the recombinant hosts are capable of producing significant amounts of ethanol as a fermentation product. Also disclosed are methods for increasing the growth of recombinant hosts and methods for reducing the accumulation of undesirable metabolic products in the growth medium of these hosts. Also disclosed are recombinant host capable of producing significant amounts of ethanol as a fermentation product of oligosaccharides and plasmids comprising genes encoding polysaccharases, in addition to the genes described above which code for the alcohol dehydrogenase and pyruvate decarboxylase. Further, methods are described for producing ethanol from oligomeric feedstock using the recombinant hosts described above. Also provided is a method for enhancing the production of functional proteins in a recombinant host comprising overexpressing an adhB gene in the host. Further provided are process designs for fermenting oligosaccharide-containing biomass to ethanol.

  2. Ethanol production by recombinant hosts

    DOEpatents

    Fowler, David E.; Horton, Philip G.; Ben-Bassat, Arie

    1996-01-01

    Novel plasmids comprising genes which code for the alcohol dehydrogenase and pyruvate decarboxylase are described. Also described are recombinant hosts which have been transformed with genes coding for alcohol dehydrogenase and pyruvate. By virtue of their transformation with these genes, the recombinant hosts are capable of producing significant amounts of ethanol as a fermentation product. Also disclosed are methods for increasing the growth of recombinant hosts and methods for reducing the accumulation of undesirable metabolic products in the growth medium of these hosts. Also disclosed are recombinant host capable of producing significant amounts of ethanol as a fermentation product of oligosaccharides and plasmids comprising genes encoding polysaccharases, in addition to the genes described above which code for the alcohol dehydrogenase and pyruvate decarboxylase. Further, methods are described for producing ethanol from oligomeric feedstock using the recombinant hosts described above. Also provided is a method for enhancing the production of functional proteins in a recombinant host comprising overexpressing an adhB gene in the host. Further provided are process designs for fermenting oligosaccharide-containing biomass to ethanol.

  3. Recombinant microorganisms for increased production of organic acids

    DOEpatents

    Yi, Jian; Kleff, Susanne; Guettler, Michael V

    2013-04-30

    Disclosed are recombinant microorganisms for producing organic acids. The recombinant microorganisms express a polypeptide that has the enzymatic activity of an enzyme that is utilized in the pentose phosphate cycle. The recombinant microorganism may include recombinant Actinobacillus succinogenes that has been transformed to express a Zwischenferment (Zwf) gene. The recombinant microorganisms may be useful in fermentation processes for producing organic acids such as succinic acid and lactic acid. Also disclosed are novel plasmids that are useful for transforming microorganisms to produce recombinant microorganisms that express enzymes such as Zwf.

  4. Recombinant microorganisms for increased production of organic acids

    DOEpatents

    Yi, Jian [East Lansing, MI; Kleff, Susanne [East Lansing, MI; Guettler, Michael V [Holt, MI

    2012-02-21

    Disclosed are recombinant microorganisms for producing organic acids. The recombinant microorganisms express a polypeptide that has the enzymatic activity of an enzyme that is utilized in the pentose phosphate cycle. The recombinant microorganism may include recombinant Actinobacillus succinogenes that has been transformed to express a Zwischenferment (Zwf) gene. The recombinant microorganisms may be useful in fermentation processes for producing organic acids such as succinic acid and lactic acid. Also disclosed are novel plasmids that are useful for transforming microorganisms to produce recombinant microorganisms that express enzymes such as Zwf.

  5. Antigenic and immunogenic properties of recombinant hemagglutinin proteins from H1N1 A/Brisbane/59/07 and B/Florida/04/06 when produced in various protein expression systems.

    PubMed

    Santiago, Felix W; Lambert Emo, Kris; Fitzgerald, Theresa; Treanor, John J; Topham, David J

    2012-06-29

    Antibodies directed against the influenza hemagglutinin (HA) protein largely mediate virus neutralization and confer protection against infection. Consequently, many studies and assays of influenza vaccines are focused on HA-specific immune responses. Recombinant HA (rHA) proteins can be produced in a number of protein expression and cell culture systems. These range from baculovirus infection of insect cell cultures, to transient transfection of plants, to stably transfected human cell lines. Furthermore, the rHA proteins may contain genetic modifications, such as histidine tags or trimerization domains, intended to ease purification or enhance protein stability. However, no systematic study of these different forms of the HA protein have been conducted. It is not clear which, if any, of these different protein expression systems or structural modifications improve or diminish the biological behavior of the proteins as immunogens or antigens in immune assays. Therefore we set out to perform systematic evaluation of rHA produced in different proteins expression systems and with varied modifications. Five rHA proteins based on recent strains of seasonal influenza A and five based on influenza B HA were kindly provided by the Biodefense and Emerging Infections Reagent Repository (BEIR). These proteins were evaluated in a combination of biochemical and structural assays, in vitro humoral and cellular immune assays, and in an animal vaccination model. Marked differences in the behavior of the individual proteins was evident suggesting that they are not equal when being used to detect an immune response. They were, nevertheless, similar at eliciting neutralizing antibody responses.

  6. In vitro stimulation of Balb/c and C57 BL/6 splenocytes by a recombinantly produced banana lectin isoform results in both a proliferation of T cells and an increased secretion of interferon-gamma.

    PubMed

    Stojanović, Marijana M; Zivković, Irena P; Petrusić, Vladimir Z; Kosec, Dusko J; Dimitrijević, Rajna D; Jankov, Ratko M; Dimitrijević, Ljiljana A; Gavrović-Jankulović, Marija D

    2010-01-01

    Lectins are widely used in many types of assay but some lectins such as banana lectin (BanLec) are recognised as potent immunostimulators. Although BanLec's structure and binding characteristics are now familiar, its immunostimulatory potential has not yet been fully explored. The synthesis by recombinant technology of a BanLec isoform (rBanLec) whose binding properties are similar to its natural counterpart has made it possible to overcome the twin problems of natural BanLec's microheterogeneity and low availability. This study's aim is to explore the immunostimulatory potential of rBanLec in the murine model. Analyses of the responses of Balb/c- and C57 BL/6-originated splenocytes to in vitro rBanLec stimulation were performed to examine the dependency of rBanLec's immunostimulatory potential upon the splenocytes' genetic background. It is shown that the responses of Balb/c- and C57 BL/6-originated splenocytes to rBanLec stimulation differ both qualitatively and in intensity. The hallmarks of the induced responses are T lymphocyte proliferation and intensive interferon-gamma secretion. Both phenomena are more marked in Balb/c-originated cultures; Balb/c-originated lymphocytes produce interleukin (IL)-4 and IL-10 following rBanLec stimulation. Our results demonstrate that any responses to rBanLec stimulation are highly dependent upon genetic background; they suggest that genetic background must be an important consideration in any further investigations using animal models or when exploring rBanLec's potential human applications.

  7. The recombination epoch revisited

    NASA Technical Reports Server (NTRS)

    Krolik, Julian H.

    1989-01-01

    Previous studies of cosmological recombination have shown that this process produces as a by-product a highly superthermal population of Ly-alpha photons which retard completion of recombination. Cosmological redshifting was thought to determine the frequency distribution of the photons, while two-photon decay of hydrogen's 2s state was thought to control their numbers. It is shown here that frequency diffusion due to photon scattering dominate the cosmological redshift in the frequency range near line center which fixes the ratio of ground state to excited state population, while incoherent scattering into the far-red damping wing effectively destroys Ly-alpha photons as a rate which is competitive with two-photon decay. The former effect tends to hold back recombination, while the latter tends to accelerate it; the net results depends on cosmological parameters, particularly the combination Omega(b) h/sq rt (2q0), where Omega(b) is the fraction of the critical density provided by baryons.

  8. Protein body-inducing fusions for high-level production and purification of recombinant proteins in plants.

    PubMed

    Conley, Andrew J; Joensuu, Jussi J; Richman, Alex; Menassa, Rima

    2011-05-01

    For the past two decades, therapeutic and industrially important proteins have been expressed in plants with varying levels of success. The two major challenges hindering the economical production of plant-made recombinant proteins include inadequate accumulation levels and the lack of efficient purification methods. To address these limitations, several fusion protein strategies have been recently developed to significantly enhance the production yield of plant-made recombinant proteins, while simultaneously assisting in their subsequent purification. Elastin-like polypeptides are thermally responsive biopolymers composed of a repeating pentapeptide 'VPGXG' sequence that are valuable for the purification of recombinant proteins. Hydrophobins are small fungal proteins capable of altering the hydrophobicity of their respective fusion partner, thus enabling efficient purification by surfactant-based aqueous two-phase systems. Zera, a domain of the maize seed storage protein γ-zein, can induce the formation of protein storage bodies, thus facilitating the recovery of fused proteins using density-based separation methods. These three novel protein fusion systems have also been shown to enhance the accumulation of a range of different recombinant proteins, while concurrently inducing the formation of protein bodies. The packing of these fusion proteins into protein bodies may exclude the recombinant protein from normal physiological turnover. Furthermore, these systems allow for quick, simple and inexpensive nonchromatographic purification of the recombinant protein, which can be scaled up to industrial levels of protein production. This review will focus on the similarities and differences of these artificial storage organelles, their biogenesis and their implication for the production of recombinant proteins in plants and their subsequent purification.

  9. Recombinant Vaccinia Virus: Immunization against Multiple Pathogens

    NASA Astrophysics Data System (ADS)

    Perkus, Marion E.; Piccini, Antonia; Lipinskas, Bernard R.; Paoletti, Enzo

    1985-09-01

    The coding sequences for the hepatitis B virus surface antigen, the herpes simplex virus glycoprotein D, and the influenza virus hemagglutinin were inserted into a single vaccinia virus genome. Rabbits inoculated intravenously or intradermally with this polyvalent vaccinia virus recombinant produced antibodies reactive to all three authentic foreign antigens. In addition, the feasibility of multiple rounds of vaccination with recombinant vaccinia virus was demonstrated.

  10. Recombinant Collagenlike Proteins

    NASA Technical Reports Server (NTRS)

    Fertala, Andzej

    2007-01-01

    A group of collagenlike recombinant proteins containing high densities of biologically active sites has been invented. The method used to express these proteins is similar to a method of expressing recombinant procollagens and collagens described in U. S. Patent 5,593,859, "Synthesis of human procollagens and collagens in recombinant DNA systems." Customized collagenous proteins are needed for biomedical applications. In particular, fibrillar collagens are attractive for production of matrices needed for tissue engineering and drug delivery. Prior to this invention, there was no way of producing customized collagenous proteins for these and other applications. Heretofore, collagenous proteins have been produced by use of such biological systems as yeasts, bacteria, and transgenic animals and plants. These products are normal collagens that can also be extracted from such sources as tendons, bones, and hides. These products cannot be made to consist only of biologically active, specific amino acid sequences that may be needed for specific applications. Prior to this invention, it had been established that fibrillar collagens consist of domains that are responsible for such processes as interaction with cells, binding of growth factors, and interaction with a number of structural proteins present in the extracellular matrix. A normal collagen consists of a sequence of domains that can be represented by a corresponding sequence of labels, e.g., D1D2D3D4. A collagenlike protein of the present invention contains regions of collagen II that contain multiples of a single domain (e.g., D1D1D1D1 or D4D4D4D4) chosen for its specific biological activity. By virtue of the multiplicity of the chosen domain, the density of sites having that specific biological activity is greater than it is in a normal collagen. A collagenlike protein according to this invention can thus be made to have properties that are necessary for tissue engineering.

  11. Human Insulin from Recombinant DNA Technology

    NASA Astrophysics Data System (ADS)

    Johnson, Irving S.

    1983-02-01

    Human insulin produced by recombinant DNA technology is the first commercial health care product derived from this technology. Work on this product was initiated before there were federal guidelines for large-scale recombinant DNA work or commercial development of recombinant DNA products. The steps taken to facilitate acceptance of large-scale work and proof of the identity and safety of such a product are described. While basic studies in recombinant DNA technology will continue to have a profound impact on research in the life sciences, commercial applications may well be controlled by economic conditions and the availability of investment capital.

  12. Expression of recombinant antibodies.

    PubMed

    Frenzel, André; Hust, Michael; Schirrmann, Thomas

    2013-01-01

    Recombinant antibodies are highly specific detection probes in research, diagnostics, and have emerged over the last two decades as the fastest growing class of therapeutic proteins. Antibody generation has been dramatically accelerated by in vitro selection systems, particularly phage display. An increasing variety of recombinant production systems have been developed, ranging from Gram-negative and positive bacteria, yeasts and filamentous fungi, insect cell lines, mammalian cells to transgenic plants and animals. Currently, almost all therapeutic antibodies are still produced in mammalian cell lines in order to reduce the risk of immunogenicity due to altered, non-human glycosylation patterns. However, recent developments of glycosylation-engineered yeast, insect cell lines, and transgenic plants are promising to obtain antibodies with "human-like" post-translational modifications. Furthermore, smaller antibody fragments including bispecific antibodies without any glycosylation are successfully produced in bacteria and have advanced to clinical testing. The first therapeutic antibody products from a non-mammalian source can be expected in coming next years. In this review, we focus on current antibody production systems including their usability for different applications.

  13. Expression of Recombinant Antibodies

    PubMed Central

    Frenzel, André; Hust, Michael; Schirrmann, Thomas

    2013-01-01

    Recombinant antibodies are highly specific detection probes in research, diagnostics, and have emerged over the last two decades as the fastest growing class of therapeutic proteins. Antibody generation has been dramatically accelerated by in vitro selection systems, particularly phage display. An increasing variety of recombinant production systems have been developed, ranging from Gram-negative and positive bacteria, yeasts and filamentous fungi, insect cell lines, mammalian cells to transgenic plants and animals. Currently, almost all therapeutic antibodies are still produced in mammalian cell lines in order to reduce the risk of immunogenicity due to altered, non-human glycosylation patterns. However, recent developments of glycosylation-engineered yeast, insect cell lines, and transgenic plants are promising to obtain antibodies with “human-like” post-translational modifications. Furthermore, smaller antibody fragments including bispecific antibodies without any glycosylation are successfully produced in bacteria and have advanced to clinical testing. The first therapeutic antibody products from a non-mammalian source can be expected in coming next years. In this review, we focus on current antibody production systems including their usability for different applications. PMID:23908655

  14. Novel hydrophobic surface binding protein, HsbA, produced by Aspergillus oryzae.

    PubMed

    Ohtaki, Shinsaku; Maeda, Hiroshi; Takahashi, Toru; Yamagata, Youhei; Hasegawa, Fumihiko; Gomi, Katsuya; Nakajima, Tasuku; Abe, Keietsu

    2006-04-01

    Hydrophobic surface binding protein A (HsbA) is a secreted protein (14.5 kDa) isolated from the culture broth of Aspergillus oryzae RIB40 grown in a medium containing polybutylene succinate-co-adipate (PBSA) as a sole carbon source. We purified HsbA from the culture broth and determined its N-terminal amino acid sequence. We found a DNA sequence encoding a protein whose N terminus matched that of purified HsbA in the A. ozyzae genomic sequence. We cloned the hsbA genomic DNA and cDNA from A. oryzae and constructed a recombinant A. oryzae strain highly expressing hsbA. Orthologues of HsbA were present in animal pathogenic and entomopathogenic fungi. Heterologously synthesized HsbA was purified and biochemically characterized. Although the HsbA amino acid sequence suggests that HsbA may be hydrophilic, HsbA adsorbed to hydrophobic PBSA surfaces in the presence of NaCl or CaCl(2). When HsbA was adsorbed on the hydrophobic PBSA surfaces, it promoted PBSA degradation via the CutL1 polyesterase. CutL1 interacts directly with HsbA attached to the hydrophobic QCM electrode surface. These results suggest that when HsbA is adsorbed onto the PBSA surface, it recruits CutL1, and that when CutL1 is accumulated on the PBSA surface, it stimulates PBSA degradation. We previously reported that when the A. oryzae hydrophobin RolA is bound to PBSA surfaces, it too specifically recruits CutL1. Since HsbA is not a hydrophobin, A. oryzae may use several types of proteins to recruit lytic enzymes to the surface of hydrophobic solid materials and promote their degradation.

  15. Improving baculovirus recombination

    PubMed Central

    Zhao, Yuguang; Chapman, David A. G.; Jones, Ian M.

    2003-01-01

    Recombinant baculoviruses have established themselves as a favoured technology for the high-level expression of recombinant proteins. The construction of recombinant viruses, however, is a time consuming step that restricts consideration of the technology for high throughput developments. Here we use a targeted gene knockout technology to inactivate an essential viral gene that lies adjacent to the locus used for recombination. Viral DNA prepared from the knockout fails to initiate an infection unless rescued by recombination with a baculovirus transfer vector. Modified viral DNA allows 100% recombinant virus formation, obviates the need for further virus purification and offers an efficient means of mass parallel recombinant formation. PMID:12527795

  16. Current Drive in Recombining Plasma

    SciTech Connect

    P.F. Schmit and N.J. Fisch

    2012-05-15

    The Langevin equations describing the average collisional dynamics of suprathermal particles in nonstationary plasma remarkably admit an exact analytical solution in the case of recombining plasma. The current density produced by arbitrary particle fluxes is derived including the effect of charge recombination. Since recombination has the effect of lowering the charge density of the plasma, thus reducing the charged particle collisional frequencies, the evolution of the current density can be modified substantially compared to plasma with fixed charge density. The current drive efficiency is derived and optimized for discrete and continuous pulses of current, leading to the discovery of a nonzero "residual" current density that persists indefinitely under certain conditions, a feature not present in stationary plasmas.

  17. Using Crossover Breakpoints in Recombinant Inbred Lines to Identify Quantitative Trait Loci Controlling the Global Recombination Frequency

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Recombination is a crucial component of evolution and breeding, producing new genetic combinations on which selection can act. Rates of recombination vary tremendously, not only between species but also within species and for specific chromosomal segments. In this study, by examining recombination...

  18. Recognition of a core fragment of Beauveria bassiana hydrophobin gene promoter (P hyd1) and its special use in improving fungal biocontrol potential

    PubMed Central

    Wang, Zheng-Liang; Ying, Sheng-Hua; Feng, Ming-Guang

    2013-01-01

    To identify a suitable promoter for use in engineering fungal entomopathogens to improve heterologous gene expression and fungal biocontrol potential, a 1798 bp promoter (Phyd1) upstream of Beauveria bassiana class I hydrophobin gene (hyd1) was optimized by upstream truncation and site-directed mutation. A truncated 1290 bp fragment (Phyd1-t1) drove eGFP expression in B. bassiana much more efficiently than full-length Phyd1. Further truncating Phyd1-t1 to 1179, 991 and 791 bp or mutating one of the binding domains of three transcription factors in Phyd1-t1 reduced significantly the expression of eGFP (enhanced green fluorescence protein). Under Phyd1-t1 control, eGFP was expressed more abundantly in conidiogenic cells and conidia than in mycelia. Therefore, Phyd1-t1 was used to integrate a bacterium-derived, insect midgut-specific toxin (vip3Aa1) gene into B. bassiana, yielding a transgenic strain (BbHV8) expressing 9.8-fold more toxin molecules in conidia than a counterpart strain (BbV28) expressing the toxin under the control of PgpdA, a promoter widely used for gene expression in fungi. Consequently, BbHV8 showed much higher per os virulence to Spodoptera litura larvae than BbV28 in standardized bioassays with normal conidia for both cuticle penetration and ingestion or heat-killed conidia for ingestion only. Conclusively, Phyd1-t1 is a useful tool for enhancing beneficial protein expression, such as vip3Aa1, in fungal conidia, which are the active ingredients of mycoinsecticides. PMID:22639846

  19. Recognition of a core fragment ofBeauveria bassiana hydrophobin gene promoter (P hyd1) and its special use in improving fungal biocontrol potential.

    PubMed

    Wang, Zheng-Liang; Ying, Sheng-Hua; Feng, Ming-Guang

    2013-01-01

    To identify a suitable promoter for use in engineering fungal entomopathogens to improve heterologous gene expression and fungal biocontrol potential, a 1798 bp promoter (P hyd1) upstream of Beauveria bassiana class I hydrophobin gene (hyd1) was optimized by upstream truncation and site-directed mutation. A truncated 1290 bp fragment (P hyd1-t1) drove eGFP expression in B. bassiana much more efficiently than full-length P hyd1. Further truncating P hyd1-t1 to 1179, 991 and 791 bp or mutating one of the binding domains of three transcription factors in P hyd1-t1 reduced significantly the expression of eGFP (enhanced green fluorescence protein). Under P hyd1-t1 control, eGFP was expressed more abundantly in conidiogenic cells and conidia than in mycelia. Therefore, P hyd1-t1 was used to integrate a bacterium-derived, insect midgut-specific toxin (vip3Aa1) gene into B. bassiana, yielding a transgenic strain (BbHV8) expressing 9.8-fold more toxin molecules in conidia than a counterpart strain (BbV28) expressing the toxin under the control of P gpdA, a promoter widely used for gene expression in fungi. Consequently, BbHV8 showed much higher per os virulence to Spodoptera litura larvae than BbV28 in standardized bioassays with normal conidia for both cuticle penetration and ingestion or heat-killed conidia for ingestion only. Conclusively, P hyd1-t1 is a useful tool for enhancing beneficial protein expression, such as vip3Aa1, in fungal conidia, which are the active ingredients of mycoinsecticides.

  20. Large-scale detection of recombination in nucleotide sequences

    NASA Astrophysics Data System (ADS)

    Chan, Cheong Xin; Beiko, Robert G.; Ragan, Mark A.

    2008-01-01

    Genetic recombination following a genetic transfer event can produce heterogeneous phylogenetic histories within sets of genes that share a common ancestral origin. Delineating recombination events will enhance our understanding in genome evolution. However, the task of detecting recombination is not trivial due to effect of more-recent evolutionary changes that can obscure such event from detection. In this paper, we demonstrate the use of a two-phase strategy for detecting recombination events on a large-scale dataset.

  1. Enhanced co-production of hydrogen and poly-(R)-3-hydroxybutyrate by recombinant PHB producing E. coli over-expressing hydrogenase 3 and acetyl-CoA synthetase.

    PubMed

    Wang, Rui-Yan; Shi, Zhen-Yu; Chen, Jin-Chun; Wu, Qiong; Chen, Guo-Qiang

    2012-09-01

    Recombinant Escherichia coli was constructed for co-production of hydrogen and polyhydroxybutyrate (PHB) due to its rapid growth and convenience of genetic manipulation. In particular, anaerobic metabolic pathways dedicated to co-production of hydrogen and PHB were established due to the advantages of directing fluxes away from toxic compounds such as formate and acetate to useful products. Here, recombinant E. coli expressing hydrogenase 3 and/or acetyl-CoA synthetase showed improved PHB and hydrogen production when grown with or without acetate as a carbon source. When hydrogenase 3 was over-expressed, hydrogen yield was increased from 14 to 153 mmol H(2)/mol glucose in a mineral salt (MS) medium with glucose as carbon source, accompanied by an increased PHB yield from 0.55 to 5.34 mg PHB/g glucose in MS medium with glucose and acetate as carbon source.

  2. Recombinant glucose uptake system

    DOEpatents

    Ingrahm, Lonnie O.; Snoep, Jacob L.; Arfman, Nico

    1997-01-01

    Recombinant organisms are disclosed that contain a pathway for glucose uptake other than the pathway normally utilized by the host cell. In particular, the host cell is one in which glucose transport into the cell normally is coupled to PEP production. This host cell is transformed so that it uses an alternative pathway for glucose transport that is not coupled to PEP production. In a preferred embodiment, the host cell is a bacterium other than Z. mobilis that has been transformed to contain the glf and glk genes of Z. mobilis. By uncoupling glucose transport into the cell from PEP utilization, more PEP is produced for synthesis of products of commercial importance from a given quantity of biomass supplied to the host cells.

  3. Recombinant protein production technology

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Recombinant protein production is an important technology for antibody production, biochemical activity study, and structural determination during the post-genomic era. Limiting factors in recombinant protein production include low-level protein expression, protein precipitation, and loss of protein...

  4. Therapeutic Recombinant Monoclonal Antibodies

    ERIC Educational Resources Information Center

    Bakhtiar, Ray

    2012-01-01

    During the last two decades, the rapid growth of biotechnology-derived techniques has led to a myriad of therapeutic recombinant monoclonal antibodies with significant clinical benefits. Recombinant monoclonal antibodies can be obtained from a number of natural sources such as animal cell cultures using recombinant DNA engineering. In contrast to…

  5. Photoionization and Recombination

    NASA Technical Reports Server (NTRS)

    Nahar, Sultana N.

    2000-01-01

    Theoretically self-consistent calculations for photoionization and (e + ion) recombination are described. The same eigenfunction expansion for the ion is employed in coupled channel calculations for both processes, thus ensuring consistency between cross sections and rates. The theoretical treatment of (e + ion) recombination subsumes both the non-resonant recombination ("radiative recombination"), and the resonant recombination ("di-electronic recombination") processes in a unified scheme. In addition to the total, unified recombination rates, level-specific recombination rates and photoionization cross sections are obtained for a large number of atomic levels. Both relativistic Breit-Pauli, and non-relativistic LS coupling, calculations are carried out in the close coupling approximation using the R-matrix method. Although the calculations are computationally intensive, they yield nearly all photoionization and recombination parameters needed for astrophysical photoionization models with higher precision than hitherto possible, estimated at about 10-20% from comparison with experimentally available data (including experimentally derived DR rates). Results are electronically available for over 40 atoms and ions. Photoionization and recombination of He-, and Li-like C and Fe are described for X-ray modeling. The unified method yields total and complete (e+ion) recombination rate coefficients, that can not otherwise be obtained theoretically or experimentally.

  6. A recombinant 63-kDa form of Bacillus anthracis protective antigen produced in the yeast Saccharomyces cerevisiae provides protection in rabbit and primate inhalational challenge models of anthrax infection.

    PubMed

    Hepler, Robert W; Kelly, Rosemarie; McNeely, Tessie B; Fan, Hongxia; Losada, Maria C; George, Hugh A; Woods, Andrea; Cope, Leslie D; Bansal, Alka; Cook, James C; Zang, Gina; Cohen, Steven L; Wei, Xiaorong; Keller, Paul M; Leffel, Elizabeth; Joyce, Joseph G; Pitt, Louise; Schultz, Loren D; Jansen, Kathrin U; Kurtz, Myra

    2006-03-06

    Infection by Bacillus anthracis is preventable by prophylactic vaccination with several naturally derived and recombinant vaccine preparations. Existing data suggests that protection is mediated by antibodies directed against the protective antigen (PA) component of the anthrax toxin complex. PA is an 83-kDa protein cleaved in vivo to yield a biologically active 63-kDa protein. In an effort to evaluate the potential of yeast as an expression system for the production of recombinant PA, and to determine if the yeast-purified rPA63 can protect from a lethal inhalational challenge, the sequence of the 63-kDa form of PA was codon-optimized and expressed in the yeast Saccharomyces cerevisiae. Highly purified rPA63 isolated from Saccharomyces under denaturing conditions demonstrated reduced biological activity in a macrophage-killing assay compared to non-denatured rPA83 purified from Escherichia coli. Rabbits and non-human primates (NHP) immunized with rPA63 and later challenged with a lethal dose of B. anthracis spores were generally protected from infection. These results indicate that epitopes present in the 63-kDa from of PA can protect rabbits and non-human primates from a lethal spore challenge, and further suggest that a fully functional rPA63 is not required in order to provide these epitopes.

  7. Genetic recombination and molecular evolution.

    PubMed

    Charlesworth, B; Betancourt, A J; Kaiser, V B; Gordo, I

    2009-01-01

    Reduced rates of genetic recombination are often associated with reduced genetic variability and levels of adaptation. Several different evolutionary processes, collectively known as Hill-Robertson (HR) effects, have been proposed as causes of these correlates of recombination. Here, we use DNA sequence polymorphism and divergence data from the noncrossing over dot chromosome of Drosophila to discriminate between two of the major forms of HR effects: selective sweeps and background selection. This chromosome shows reduced levels of silent variability and reduced effectiveness of selection. We show that neither model fits the data on variability. We propose that, in large genomic regions with restricted recombination, HR effects among nonsynonymous mutations undermine the effective strength of selection, so that their background selection effects are weakened. This modified model fits the data on variability and also explains why variability in very large nonrecombining genomes is not completely wiped out. We also show that HR effects of this type can produce an individual selection advantage to recombination, as well as greatly reduce the mean fitness of nonrecombining genomes and genomic regions.

  8. Co-factor activated recombinant adenovirus proteinases

    DOEpatents

    Anderson, C.W.; Mangel, W.F.

    1996-08-06

    This application describes methods and expression constructs for producing activatable recombinant adenovirus proteinases. Purified activatable recombinant adenovirus proteinases and methods of purification are described. Activated adenovirus proteinases and methods for obtaining activated adenovirus proteinases are further included. Isolated peptide cofactors of adenovirus proteinase activity, methods of purifying and identifying the peptide cofactors are also described. Antibodies immunoreactive with adenovirus proteinases, immunospecific antibodies, and methods for preparing them are also described. Other related methods and materials are also described. 29 figs.

  9. Co-factor activated recombinant adenovirus proteinases

    DOEpatents

    Anderson, Carl W.; Mangel, Walter F.

    1996-08-06

    This application describes methods and expression constructs for producing activatable recombinant adenovirus proteinases. Purified activatable recombinant adenovirus proteinases and methods of purification are described. Activated adenovirus proteinases and methods for obtaining activated adenovirus proteinases are further included. Isolated peptide cofactors of adenovirus proteinase activity, methods of purifying and identifying said peptide cofactors are also described. Antibodies immunoreactive with adenovirus proteinases, immunospecific antibodies, and methods for preparing them are also described. Other related methods and materials are also described.

  10. A Plant-Produced Bacteriophage Tailspike Protein for the Control of Salmonella

    PubMed Central

    Miletic, Sean; Simpson, David J.; Szymanski, Christine M.; Deyholos, Michael K.; Menassa, Rima

    2016-01-01

    The receptor binding domain of the tailspike protein Gp9 from the P22 bacteriophage was recently shown to reduce Salmonella colonization in the chicken gut. In this study, we transiently expressed the receptor binding domain of the Gp9 tailspike protein in Nicotiana benthamiana, and targeted it to the endoplasmic reticulum (ER) or to the chloroplasts. Gp9 was also fused to either an elastin-like polypeptide (ELP) or hydrophobin I tag, which were previously described to improve accumulation levels of recombinant proteins. The highest levels of recombinant protein accumulation occurred when unfused Gp9 was targeted to the ER. Lower levels of chloroplast-targeted Gp9 were also detected. ELP-fused Gp9 was purified and demonstrated to bind to Salmonella enterica serovar Typhimurium in vitro. Upon oral administration of lyophilized leaves expressing Gp9-ELP to newly hatched chickens, we found that this tailspike protein has the potential to be used as a therapeutic to control Salmonella contamination in chickens. PMID:26779243

  11. Recombination of cluster ions

    NASA Technical Reports Server (NTRS)

    Johnsen, Rainer

    1993-01-01

    Some of our recent work on molecular band emissions from recombination of molecular dimer ions (N4(+) and CO(+) CO) is discussed. Much of the experimental work was done by Y. S. Cao; the results on N4(+) recombination have been published. A brief progress report is given on our ongoing measurements of neutral products of recombination using the flowing-afterglow Langmuir-probe technique in conjunction with laser-induced fluorescence.

  12. Recombinant baculoviruses for insect control.

    PubMed

    Inceoglu, A B; Kamita, S G; Hinton, A C; Huang, Q; Severson, T F; Kang, K; Hammock, B D

    2001-10-01

    Baculoviruses are double-stranded DNA viruses which are highly selective for several insect groups. They are valuable natural control agents, but their utility in many agricultural applications has been limited by their slow speed of kill and narrow host specificity. Baculoviruses have been genetically modified to express foreign genes under powerful promoters in order to accelerate their speed of kill. In our and other laboratories, the expression of genes coding for insect juvenile hormone esterases and various peptide neurotoxins has resulted in recombinant baculoviruses with promise as biological insecticides. These viruses are efficacious in the laboratory, greenhouse and field and dramatically reduce damage caused by insect feeding. The recombinant viruses synergize and are synergized by classical pesticides such as pyrethroids. Since they are highly selective for pest insects, they can be used without disrupting biological control. Because the recombinant virus produces fewer progeny in infected larvae than the wild-type virus, they are rapidly out-competed in the ecosystem. The viruses can be used effectively with crops expressing endotoxins of Bacillus thuringiensis. They can be produced industrially but also by village industries, indicating that they have the potential to deliver sustainable pest control in developing countries. It remains to be seen, however, whether the current generation of recombinant baculoviruses will be competitive with the new generation of synthetic chemical pesticides. Current research clearly indicates, though, that the use of biological vectors of genes for insect control will find a place in agriculture. Baculoviruses will also prove valuable in testing the potential utility of proteins and peptides for insect control.

  13. Implications of recombination for HIV diversity.

    PubMed

    Ramirez, Bertha Cecilia; Simon-Loriere, Etienne; Galetto, Roman; Negroni, Matteo

    2008-06-01

    The human immunodeficiency virus (HIV) population is characterised by extensive genetic variability that results from high error and recombination rates of the reverse transcription process, and from the fast turnover of virions in HIV-infected individuals. Among the viral variants encountered at the global scale, recombinant forms are extremely abundant. Some of these recombinants (known as circulating recombinant forms) become fixed and undergo rapid expansion in the population. The reasons underlying their epidemiological success remain at present poorly understood and constitute a fascinating area for future research to improve our understanding of immune escape, pathogenicity and transmission. Recombinant viruses are generated during reverse transcription as a consequence of template switching between the two genetically different genomic RNAs present in a heterozygous virus. Recombination can thereby generate shortcuts in evolution by producing mosaic reverse transcription products of parental genomes. Therefore, in a single infectious cycle multiple mutations that are positively selected can be combined or, conversely, negatively selected mutations can be removed. Recombination is therefore involved in different aspects of HIV evolution, adaptation to its host, and escape from antiviral treatments.

  14. Electron Recombination in a Dense Hydrogen Plasma

    SciTech Connect

    Jana, M.R.; Johnstone, C.; Kobilarcik, T.; Koizumi, G.M.; Moretti, A.; Popovic, M.; Tollestrup, A.V.; Yonehara, K.; Leonova, M.A.; Schwarz, T.A.; Chung, M.; /Unlisted /IIT, Chicago /Fermilab /MUONS Inc., Batavia /Turin Polytechnic

    2012-05-01

    A high pressure hydrogen gas filled RF cavity was subjected to an intense proton beam to study the evolution of the beam induced plasma inside the cavity. Varying beam intensities, gas pressures and electric fields were tested. Beam induced ionized electrons load the cavity, thereby decreasing the accelerating gradient. The extent and duration of this degradation has been measured. A model of the recombination between ionized electrons and ions is presented, with the intent of producing a baseline for the physics inside such a cavity used in a muon accelerator. Analysis of the data taken during the summer of 2011 shows that self recombination takes place in pure hydrogen gas. The decay of the number of electrons in the cavity once the beam is turned off indicates self recombination rather than attachment to electronegative dopants or impurities. The cross section of electron recombination grows for larger clusters of hydrogen and so at the equilibrium of electron production and recombination in the cavity, processes involving H{sub 5}{sup +} or larger clusters must be taking place. The measured recombination rates during this time match or exceed the analytic predicted values. The accelerating gradient in the cavity recovers fully in time for the next beam pulse of a muon collider. Exactly what the recombination rate is and how much the gradient degrades during the 60 ns muon collider beam pulse will be extrapolated from data taken during the spring of 2012.

  15. Recombinant clostridia that fix CO2 and CO and uses thereof

    DOEpatents

    Papoutsakis, Eleftherios T.; Al-Hinai, Mohab Ali; Jones, Shawn Williams; Indurthi, Dinesh Chanukya; Mitchell, Daniel Knox; Fast, Alan

    2014-06-24

    The present invention relates a recombinant Clostridium expressing one or more heterologous Wood-Ljungdahl (WL) genes. In particular, the recombinant Clostridium produces a metabolite at an increased level. The present invention also relates to a method for producing a metabolite by the recombinant Clostridium.

  16. Interplay between modifications of chromatin and meiotic recombination hotspots.

    PubMed

    Brachet, Elsa; Sommermeyer, Vérane; Borde, Valérie

    2012-02-01

    Meiotic recombination lies at the heart of sexual reproduction. It is essential for producing viable gametes with a normal haploid genomic content and its dysfunctions can be at the source of aneuploidies, such as the Down syndrome, or many genetic disorders. Meiotic recombination also generates genetic diversity that is transmitted to progeny by shuffling maternal and paternal alleles along chromosomes. Recombination takes place at non-random chromosomal sites called 'hotspots'. Recent evidence has shown that their location is influenced by properties of chromatin. In addition, many studies in somatic cells have highlighted the need for changes in chromatin dynamics to allow the process of recombination. In this review, we discuss how changes in the chromatin landscape may influence the recombination map, and reciprocally, how recombination events may lead to epigenetic modifications at sites of recombination, which could be transmitted to progeny.

  17. Evolution of Resistance in Culex quinquefasciatus (Say) Selected With a Recombinant Bacillus thuringiensis Strain-Producing Cyt1Aa and Cry11Ba, and the Binary Toxin, Bin, From Lysinibacillus sphaericus.

    PubMed

    Wirth, Margaret C; Walton, William E; Federici, Brian A

    2015-09-01

    Fourth instars of Culex quinquefasciatus (Say) (Diptera: Culicidae) were selected with a recombinant bacterial strain synthesizing the mosquitocidal proteins from Lysinibacillus sphaericus (Bin) and Cry11Ba and Cyt1Aa from Bacillus thuringiensis. Selection was initiated in Generation 1 with a concentration of 0.04 μg/ml, which rose to a maximum selection concentration of 8.0 μg/ml in Generation 14, followed by an unexpected, rapid increase in mortality in Generation 15. Subsequently, a selection concentration of 0.8 μg/ml was determined to be survivable. During this same period, resistance rose to nearly 1,000-fold (by Generation 12) and declined to 18.8-fold in Generation 19. Resistance remained low and fluctuated between 5.3 and 7.3 up to Generation 66. The cross-resistance patterns and interactions among the component proteins were analyzed to identify possible causes of this unusual pattern of evolution. Poor activity in the mid-range concentrations and lower-than-expected synergistic interactions were identified as potential sources of the early resistance. These findings should be considered in the development of genetically engineered strains intended to control nuisance and vector mosquitoes.

  18. 92-kD gelatinase is produced by eosinophils at the site of blister formation in bullous pemphigoid and cleaves the extracellular domain of recombinant 180-kD bullous pemphigoid autoantigen.

    PubMed Central

    Ståhle-Bäckdahl, M; Inoue, M; Guidice, G J; Parks, W C

    1994-01-01

    Eosinophils are prominent in bullous pemphigoid (BP), and proteases secreted from these and other inflammatory cells may induce disruption of the basement membrane. We used in situ hybridization and immunohistochemistry to localize the sites of 92-kD gelatinase expression in BP lesions. In all samples (20/20), a strong signal for gelatinase mRNA was detected only in eosinophils and was most pronounced where these cells accumulated at the floor of forming blisters. No other cells were positive for enzyme mRNA. Both eosinophils and neutrophils, however, contained immunoreactive 92-kD gelatinase indicating that active expression occurred only in eosinophils. Degranulated eosinophils were also seen near blisters, and as demonstrated by gelatin zymography, immunoblotting, and ELISA, 92-kD gelatinase protein was prominent in BP blister fluid. No other gelatinolytic activity was specifically detected in BP fluid, and only small amounts of 92-kD gelatinase were present in suction blister fluids. As demonstrated in vitro, 92-kD gelatinase cleaved the extracellular, collagenous domain of recombinant 180-kD BP autoantigen (BP180, BPAG2, HD4, type XVII collagen), a transmembrane molecule of the epidermal hemidesmosome. Our results suggest that production and release 92-kD gelatinase by eosinophils contributes significantly to tissue damage in BP. Images PMID:8182134

  19. Evolution of Resistance in Culex quinquefasciatus (Say) Selected With a Recombinant Bacillus thuringiensis Strain-Producing Cyt1Aa and Cry11Ba, and the Binary Toxin, Bin, From Lysinibacillus sphaericus

    PubMed Central

    Wirth, Margaret C.; Walton, William E.; Federici, Brian A.

    2015-01-01

    Fourth instars of Culex quinquefasciatus (Say) (Diptera: Culicidae) were selected with a recombinant bacterial strain synthesizing the mosquitocidal proteins from Lysinibacillus sphaericus (Bin) and Cry11Ba and Cyt1Aa from Bacillus thuringiensis. Selection was initiated in Generation 1 with a concentration of 0.04 μg/ml, which rose to a maximum selection concentration of 8.0 μg/ml in Generation 14, followed by an unexpected, rapid increase in mortality in Generation 15. Subsequently, a selection concentration of 0.8 μg/ml was determined to be survivable. During this same period, resistance rose to nearly 1,000-fold (by Generation 12) and declined to 18.8-fold in Generation 19. Resistance remained low and fluctuated between 5.3 and 7.3 up to Generation 66. The cross-resistance patterns and interactions among the component proteins were analyzed to identify possible causes of this unusual pattern of evolution. Poor activity in the mid-range concentrations and lower-than-expected synergistic interactions were identified as potential sources of the early resistance. These findings should be considered in the development of genetically engineered strains intended to control nuisance and vector mosquitoes. PMID:26336254

  20. Recombinant Baculovirus Isolation.

    PubMed

    King, Linda A; Hitchman, Richard; Possee, Robert D

    2016-01-01

    Although there are several different methods available of making recombinant baculovirus expression vectors (reviewed in Chapter 3 ), all require a stage in which insect cells are transfected with either the virus genome alone (Bac-to-Bac(®) or BaculoDirect™, Invitrogen) or virus genome and transfer vector. In the latter case, this allows the natural process of homologous recombination to transfer the foreign gene, under control of the polyhedrin or other baculovirus gene promoter, from the transfer vector to the virus genome to create the recombinant virus. Previously, many methods required a plaque-assay to separate parental and recombinant virus prior to amplification and use of the recombinant virus. Fortunately, this step is no longer required for most systems currently available. This chapter provides an overview of the historical development of increasingly more efficient systems for the isolation of recombinant baculoviruses (Chapter 3 provides a full account of the different systems and transfer vectors available). The practical details cover: transfection of insect cells with either virus DNA or virus DNA and plasmid transfer vector; a reliable plaque-assay method that can be used to separate recombinant virus from parental (nonrecombinant) virus where this is necessary; methods for the small-scale amplification of recombinant virus; and subsequent titration by plaque-assay or real-time polymerase chain reaction (PCR). Methods unique to the Bac-to-Bac(®) system are also covered and include the transformation of bacterial cells and isolation of bacmid DNA ready for transfection of insect cells.

  1. Recombinant DNA technology in apple.

    PubMed

    Gessler, Cesare; Patocchi, Andrea

    2007-01-01

    This review summarizes the achievements of almost 20 years of recombinant DNA technology applied to apple, grouping the research results into the sections: developing the technology, insect resistance, fungal disease resistance, self-incompatibility, herbicide resistance, fire blight resistance, fruit ripening, allergens, rooting ability, and acceptance and risk assessment. The diseases fire blight, caused by Erwinia amylovora, and scab, caused by Venturia inaequalis, were and still are the prime targets. Shelf life improvement and rooting ability of rootstocks are also relevant research areas. The tools to create genetically modified apples of added value to producers, consumers, and the environment are now available.

  2. Homologous recombination using bacterial artificial chromosomes.

    PubMed

    Lai, Cary; Fischer, Tobias; Munroe, Elizabeth

    2015-02-02

    This protocol introduces the technique of homologous recombination in bacteria to insert a linear DNA fragment into bacterial artificial chromosomes (BACs). Homologous recombination allows the modification of large DNA molecules, in contrast with conventional restriction endonuclease-based strategies, which cleave large DNAs into numerous fragments and are unlikely to permit the precise targeting afforded by recombination-based approaches. The method uses a phage lambda-derived recombination system (using exo, beta, and gam) as well as other enzymatic activities provided by the host (Escherichia coli). In the method described here, a DNA fragment encoding enhanced cyan fluorescent protein is inserted immediately after the start codon of the gene encoding choline acetyltransferase ("ChAT"), the final enzyme in acetylcholine biosynthesis, using homologous recombination between sequences that are present both on the introduced DNA fragment and in the target BAC. The desired recombination products are identified via positive selection for resistance to kanamycin. In principle, the resulting modified BAC could be used to produce transgenic mice that express this fluorescent protein in cholinergic neurons. The approach described here could be used to insert any DNA fragment.

  3. Multiphoton Assisted Recombination

    NASA Astrophysics Data System (ADS)

    Shuman, E. S.; Jones, R. R.; Gallagher, T. F.

    2008-12-01

    We have observed multiphoton assisted recombination in the presence of a 38.8 GHz microwave field. Stimulated emission of up to ten microwave photons results in energy transfer from continuum electrons, enabling recombination. The maximum electron energy loss is far greater than the 2Up predicted by the standard “simpleman’s” model. The data are well reproduced by both an approximate analytic expression and numerical simulations in which the combined Coulomb and radiation fields are taken into account.

  4. Influence of protein formulation and carrier solution on asymmetrical flow field-flow fractionation: a case study of the plant-produced recombinant anthrax protective antigen pp-PA83.

    PubMed

    Palais, Caroline; Chichester, Jessica A; Manceva, Slobodanka; Yusibov, Vidadi; Arvinte, Tudor

    2015-02-01

    Asymmetrical flow field-flow fractionation (afFFF) was used to investigate the properties of a plant-produced anthrax toxin protective antigen, pp-PA83. The afFFF fractogram consisted of two main peaks with molar masses similar to the molecular mass of pp-PA83 monomer. afFFF carrier solutions strongly influenced the ratio and the intensity of the two main peaks. These differences indicate that conformation changes in the pp-PA83 molecule occurred during the afFFF analysis. Similar fractograms were obtained for different pp-PA83 formulations when the afFFF carrier solution and the protein formulation were the same (or very similar). The data show that in specific cases, afFFF could be used to study protein conformation and document the importance of studying the influence of the carrier solution on afFFF.

  5. Using crossover breakpoints in recombinant inbred lines to identify quantitative trait loci controlling the global recombination frequency.

    PubMed

    Esch, Elisabeth; Szymaniak, Jessica M; Yates, Heather; Pawlowski, Wojciech P; Buckler, Edward S

    2007-11-01

    Recombination is a crucial component of evolution and breeding, producing new genetic combinations on which selection can act. Rates of recombination vary tremendously, not only between species but also within species and for specific chromosomal segments. In this study, by examining recombination events captured in recombinant inbred mapping populations previously created for maize, wheat, Arabidopsis, and mouse, we demonstrate that substantial variation exists for genomewide crossover rates in both outcrossed and inbred plant and animal species. We also identify quantitative trait loci (QTL) that control this variation. The method that we developed and employed here holds promise for elucidating factors that regulate meiotic recombination and for creation of hyperrecombinogenic lines, which can help overcome limited recombination that hampers breeding progress.

  6. Utilization of Site-Specific Recombination in Biopharmaceutical Production.

    PubMed

    Ahmadi, Maryam; Damavandi, Narges; Akbari Eidgahi, Mohammad Reza; Davami, Fatemeh

    2016-01-01

    Mammalian expression systems, due to their capacity in post-translational modification, are preferred systems for biopharmaceutical protein production. Several recombinant protein systems have been introduced to the market, most of which are under clinical development. In spite of significant improvements such as cell line engineering, introducing novel expression methods, gene silencing and process development, expression level is unpredictable and unstable because of the random location of integration in the genome. Site-specific recombination techniques are capable of producing stable and high producer clonal cells; therefore, they are gaining more importance in the biopharmaceutical production. Site-specific recombination methods increase the recombinant protein production by specifically inserting a vector at a locus with specific expression trait. The present review focused on the latest developments in site-specific recombination techniques, their specific features and comparisons.

  7. Utilization of Site-Specific Recombination in Biopharmaceutical Production

    PubMed Central

    Ahmadi, Maryam; Damavandi, Narges; Akbari, Mohammad Reza; Davami, Fatemeh

    2016-01-01

    Mammalian expression systems, due to their capacity in post-translational modification, are preferred systems for biopharmaceutical protein production. Several recombinant protein systems have been introduced to the market, most of which are under clinical development. In spite of significant improvements such as cell line engineering, introducing novel expression methods, gene silencing and process development, expression level is unpredictable and unstable because of the random location of integration in the genome. Site-specific recombination techniques are capable of producing stable and high producer clonal cells; therefore, they are gaining more importance in the biopharmaceutical production. Site-specific recombination methods increase the recombinant protein production by specifically inserting a vector at a locus with specific expression trait. The present review focused on the latest developments in site-specific recombination techniques, their specific features and comparisons. PMID:26602035

  8. Regulation of Meiotic Recombination

    SciTech Connect

    Gregory p. Copenhaver

    2011-11-09

    Meiotic recombination results in the heritable rearrangement of DNA, primarily through reciprocal exchange between homologous chromosome or gene conversion. In plants these events are critical for ensuring proper chromosome segregation, facilitating DNA repair and providing a basis for genetic diversity. Understanding this fundamental biological mechanism will directly facilitate trait mapping, conventional plant breeding, and development of genetic engineering techniques that will help support the responsible production and conversion of renewable resources for fuels, chemicals, and the conservation of energy (1-3). Substantial progress has been made in understanding the basal recombination machinery, much of which is conserved in organisms as diverse as yeast, plants and mammals (4, 5). Significantly less is known about the factors that regulate how often and where that basal machinery acts on higher eukaryotic chromosomes. One important mechanism for regulating the frequency and distribution of meiotic recombination is crossover interference - or the ability of one recombination event to influence nearby events. The MUS81 gene is thought to play an important role in regulating the influence of interference on crossing over. The immediate goals of this project are to use reverse genetics to identify mutants in two putative MUS81 homologs in the model plant Arabidopsis thaliana, characterize those mutants and initiate a novel forward genetic screen for additional regulators of meiotic recombination. The long-term goal of the project is to understand how meiotic recombination is regulated in higher eukaryotes with an emphasis on the molecular basis of crossover interference. The ability to monitor recombination in all four meiotic products (tetrad analysis) has been a powerful tool in the arsenal of yeast geneticists. Previously, the qrt mutant of Arabidopsis, which causes the four pollen products of male meiosis to remain attached, was developed as a facile system

  9. Recombinant Protein Production and Insect Cell Culture and Process

    NASA Technical Reports Server (NTRS)

    Spaulding, Glenn F. (Inventor); Goodwin, Thomas J. (Inventor); OConnor, Kim C. (Inventor); Francis, Karen M. (Inventor); Andrews, Angela D. (Inventor); Prewett, Tracey L. (Inventor)

    1997-01-01

    A process has been developed for recombinant production of selected polypeptides using transformed insect cells cultured in a horizontally rotating culture vessel modulated to create low shear conditions. A metabolically transformed insect cell line is produced using the culture procedure regardless of genetic transformation. The recombinant polypeptide can be produced by an alternative process using virtually infected or stably transformed insect cells containing a gene encoding the described polypeptide. The insect cells can also be a host for viral production.

  10. Recombinant protein production and insect cell culture and process

    NASA Technical Reports Server (NTRS)

    Spaulding, Glenn (Inventor); Prewett, Tacey (Inventor); Goodwin, Thomas (Inventor); Francis, Karen (Inventor); Andrews, Angela (Inventor); Oconnor, Kim (Inventor)

    1993-01-01

    A process has been developed for recombinant production of selected polypeptides using transformed insect cells cultured in a horizontally rotating culture vessel modulated to create low shear conditions. A metabolically transformed insect cell line is produced using the culture procedure regardless of genetic transformation. The recombinant polypeptide can be produced by an alternative process using the cultured insect cells as host for a virus encoding the described polypeptide such as baculovirus. The insect cells can also be a host for viral production.

  11. Fundamental Studies of Recombinant Hydrogenases

    SciTech Connect

    Adams, Michael W

    2014-01-25

    This research addressed the long term goals of understanding the assembly and organization of hydrogenase enzymes, of reducing them in size and complexity, of determining structure/function relationships, including energy conservation via charge separation across membranes, and in screening for novel H2 catalysts. A key overall goal of the proposed research was to define and characterize minimal hydrogenases that are produced in high yields and are oxygen-resistant. Remarkably, in spite of decades of research carried out on hydrogenases, it is not possible to readily manipulate or design the enzyme using molecular biology approaches since a recombinant form produced in a suitable host is not available. Such resources are essential if we are to understand what constitutes a “minimal” hydrogenase and design such catalysts with certain properties, such as resistance to oxygen, extreme stability and specificity for a given electron donor. The model system for our studies is Pyrococcus furiosus, a hyperthermophile that grows optimally at 100°C, which contains three different nickel-iron [NiFe-] containing hydrogenases. Hydrogenases I and II are cytoplasmic while the other, MBH, is an integral membrane protein that functions to both evolve H2 and pump protons. Three important breakthroughs were made during the funding period with P. furiosus soluble hydrogenase I (SHI). First, we produced an active recombinant form of SHI in E. coli by the co-expression of sixteen genes using anaerobically-induced promoters. Second, we genetically-engineered P. furiosus to overexpress SHI by an order of magnitude compared to the wild type strain. Third, we generated the first ‘minimal’ form of SHI, one that contained two rather than four subunits. This dimeric form was stable and active, and directly interacted with a pyruvate-oxidizing enzyme with any intermediate electron carrier. The research resulted in five peer-reviewed publications.

  12. Production of a recombinant full-length prion protein in a soluble form without refolding or detergents.

    PubMed

    Arii, Yasuhiro; Oshiro, Satoshi; Wada, Keita; Fukuoka, Shin-ichi

    2011-01-01

    Recombinant prion protein has been produced in insoluble form and refolded following solubilization with denaturants. It is, however, preferable to use a soluble recombinant protein prepared without artificial solubilization. In this study, a soluble recombinant prion protein was produced in Escherichia coli cells by coexpression of neuregulin I-β1 and purified to high purity.

  13. Dissociative recombination in planetary ionospheres

    NASA Technical Reports Server (NTRS)

    Fox, J. L.

    1993-01-01

    Ionization in planetary atmospheres can be produced by solar photoionization, photoelectron impact ionization, and, in auroral regions, by impact of precipitating particles. This ionization is lost mainly in dissociative recombination (DR) of molecular ions. Although atomic ions cannot undergo DR, they can be transformed locally through ion-molecule reactions into molecular ions, or they may be transported vertically or horizontally to regions of the atmosphere where such transformations are possible. Because DR reactions tend to be very exothermic, they can be an important source of kinetically or internally excited fragments. In interplanetary thermospheres, the neutral densities decrease exponentially with altitude. Below the homopause (or turbopause), the atmosphere is assumed to be throughly mixed by convection and/or turbulence. Above the homopause, diffusion is the major transport mechanism, and each species is distributed according to its mass, with the logarithmic derivative of the density with repect to altitude given approximately by -1/H, where H = kT/mg is the scale height. In this expression, T is the neutral temperature, g is the local acceleratiion of gravity, and m is the mass of the species. Thus lighter species become relatively more abundant, and heavier species less abundant, as the altitude increases. This variation of the neutral composition can lead to changes in the ion composition; furthermore, as the neutral densities decrease, dissociative recombination becomes more important relative to ion-neutral reactions as a loss mechanism for molecular ions.

  14. Recombinant expression of hydroxylated human collagen in Escherichia coli.

    PubMed

    Rutschmann, Christoph; Baumann, Stephan; Cabalzar, Jürg; Luther, Kelvin B; Hennet, Thierry

    2014-05-01

    Collagen is the most abundant protein in the human body and thereby a structural protein of considerable biotechnological interest. The complex maturation process of collagen, including essential post-translational modifications such as prolyl and lysyl hydroxylation, has precluded large-scale production of recombinant collagen featuring the biophysical properties of endogenous collagen. The characterization of new prolyl and lysyl hydroxylase genes encoded by the giant virus mimivirus reveals a method for production of hydroxylated collagen. The coexpression of a human collagen type III construct together with mimivirus prolyl and lysyl hydroxylases in Escherichia coli yielded up to 90 mg of hydroxylated collagen per liter culture. The respective levels of prolyl and lysyl hydroxylation reaching 25 % and 26 % were similar to the hydroxylation levels of native human collagen type III. The distribution of hydroxyproline and hydroxylysine along recombinant collagen was also similar to that of native collagen as determined by mass spectrometric analysis of tryptic peptides. The triple helix signature of recombinant hydroxylated collagen was confirmed by circular dichroism, which also showed that hydroxylation increased the thermal stability of the recombinant collagen construct. Recombinant hydroxylated collagen produced in E. coli supported the growth of human umbilical endothelial cells, underlining the biocompatibility of the recombinant protein as extracellular matrix. The high yield of recombinant protein expression and the extensive level of prolyl and lysyl hydroxylation achieved indicate that recombinant hydroxylated collagen can be produced at large scale for biomaterials engineering in the context of biomedical applications.

  15. Producing biofuels using polyketide synthases

    DOEpatents

    Katz, Leonard; Fortman, Jeffrey L; Keasling, Jay D

    2013-04-16

    The present invention provides for a non-naturally occurring polyketide synthase (PKS) capable of synthesizing a carboxylic acid or a lactone, and a composition such that a carboxylic acid or lactone is included. The carboxylic acid or lactone, or derivative thereof, is useful as a biofuel. The present invention also provides for a recombinant nucleic acid or vector that encodes such a PKS, and host cells which also have such a recombinant nucleic acid or vector. The present invention also provides for a method of producing such carboxylic acids or lactones using such a PKS.

  16. Recombinant renewable polyclonal antibodies.

    PubMed

    Ferrara, Fortunato; D'Angelo, Sara; Gaiotto, Tiziano; Naranjo, Leslie; Tian, Hongzhao; Gräslund, Susanne; Dobrovetsky, Elena; Hraber, Peter; Lund-Johansen, Fridtjof; Saragozza, Silvia; Sblattero, Daniele; Kiss, Csaba; Bradbury, Andrew R M

    2015-01-01

    Only a small fraction of the antibodies in a traditional polyclonal antibody mixture recognize the target of interest, frequently resulting in undesirable polyreactivity. Here, we show that high-quality recombinant polyclonals, in which hundreds of different antibodies are all directed toward a target of interest, can be easily generated in vitro by combining phage and yeast display. We show that, unlike traditional polyclonals, which are limited resources, recombinant polyclonal antibodies can be amplified over one hundred million-fold without losing representation or functionality. Our protocol was tested on 9 different targets to demonstrate how the strategy allows the selective amplification of antibodies directed toward desirable target specific epitopes, such as those found in one protein but not a closely related one, and the elimination of antibodies recognizing common epitopes, without significant loss of diversity. These recombinant renewable polyclonal antibodies are usable in different assays, and can be generated in high throughput. This approach could potentially be used to develop highly specific recombinant renewable antibodies against all human gene products.

  17. The dissociative recombination of ?

    NASA Astrophysics Data System (ADS)

    Laubé, S.; Lehfaoui, L.; Rowe, B. R.; Mitchell, J. B. A.

    1998-09-01

    The dissociative recombination rate coefficient for 0953-4075/31/18/016/img2 has been measured at 300 K using a flowing afterglow Langmuir probe-mass spectrometer apparatus. A value of 0953-4075/31/18/016/img3 has been found.

  18. Recombineering linear BACs.

    PubMed

    Chen, Qingwen; Narayanan, Kumaran

    2015-01-01

    Recombineering is a powerful genetic engineering technique based on homologous recombination that can be used to accurately modify DNA independent of its sequence or size. One novel application of recombineering is the assembly of linear BACs in E. coli that can replicate autonomously as linear plasmids. A circular BAC is inserted with a short telomeric sequence from phage N15, which is subsequently cut and rejoined by the phage protelomerase enzyme to generate a linear BAC with terminal hairpin telomeres. Telomere-capped linear BACs are protected against exonuclease attack both in vitro and in vivo in E. coli cells and can replicate stably. Here we describe step-by-step protocols to linearize any BAC clone by recombineering, including inserting and screening for presence of the N15 telomeric sequence, linearizing BACs in vivo in E. coli, extracting linear BACs, and verifying the presence of hairpin telomere structures. Linear BACs may be useful for functional expression of genomic loci in cells, maintenance of linear viral genomes in their natural conformation, and for constructing innovative artificial chromosome structures for applications in mammalian and plant cells.

  19. Recombinant DNA for Teachers.

    ERIC Educational Resources Information Center

    Duvall, James G., III

    1992-01-01

    A science teacher describes his experience at a workshop to learn to teach the Cold Spring Harbor DNA Science Laboratory Protocols. These protocols lead students through processes for taking E. coli cells and transforming them into a new antibiotic resistant strain. The workshop featured discussions of the role of DNA recombinant technology in…

  20. Recombinant antibodies and their use in biosensors.

    PubMed

    Zeng, Xiangqun; Shen, Zhihong; Mernaugh, Ray

    2012-04-01

    Inexpensive, noninvasive immunoassays can be used to quickly detect disease in humans. Immunoassay sensitivity and specificity are decidedly dependent upon high-affinity, antigen-specific antibodies. Antibodies are produced biologically. As such, antibody quality and suitability for use in immunoassays cannot be readily determined or controlled by human intervention. However, the process through which high-quality antibodies can be obtained has been shortened and streamlined by use of genetic engineering and recombinant antibody techniques. Antibodies that traditionally take several months or more to produce when animals are used can now be developed in a few weeks as recombinant antibodies produced in bacteria, yeast, or other cell types. Typically most immunoassays use two or more antibodies or antibody fragments to detect antigens that are indicators of disease. However, a label-free biosensor, for example, a quartz-crystal microbalance (QCM) needs one antibody only. As such, the cost and time needed to design and develop an immunoassay can be substantially reduced if recombinant antibodies and biosensors are used rather than traditional antibody and assay (e.g. enzyme-linked immunosorbant assay, ELISA) methods. Unlike traditional antibodies, recombinant antibodies can be genetically engineered to self-assemble on biosensor surfaces, at high density, and correctly oriented to enhance antigen-binding activity and to increase assay sensitivity, specificity, and stability. Additionally, biosensor surface chemistry and physical and electronic properties can be modified to further increase immunoassay performance above and beyond that obtained by use of traditional methods. This review describes some of the techniques investigators have used to develop highly specific and sensitive, recombinant antibody-based biosensors for detection of antigens in simple or complex biological samples.

  1. Nascent Vibrational/Rotational Distribution Produced by Hydrogen Atom Recombination.

    DTIC Science & Technology

    1988-02-01

    12. PERSONAL AUTHOR(S) WoiAn0 13a.TYP OF E OT 13b. TIME COVERED , 14. PA.TE OF REPORT (~r, Month, Day) i5P4Ca COUNT 13.TP F EOT - FROM TO / Albury10 I...UNCLASSIFIED V%-’.- . ~ -d,𔃿.. 5. 5 ~ ~ .2-~’~- ’~ ~% %J’ % ’ ’ P ’.k PREFACE I This program was initiated with support from J. Pollard and R. Cohen. L. Friesen ...Probabilities for Final Vibrational/Rotational State Formed from Initial ’Resonance State v - 13, J - 8 for Initial E /k - 50 K ......... 16 2

  2. Method for producing aldehyde from CO.sub.2

    SciTech Connect

    Liao, James C.; Atsumi, Shota

    2015-09-29

    The invention provides recombinant microorganisms capable of producing isobutyraldehyde using CO.sub.2 as a carbon source. The invention further provides methods of preparing and using such microorganisms to produce isobutyraldehyde.

  3. Sex recombination, and reproductive fitness: an experimental study using Paramecium

    SciTech Connect

    Nyberg, D.

    1982-08-01

    The effect of sex and recombination on reproductive fitness are measured using five wild stocks of Paramecium primaurelia. Among the wild stocks there were highly significant differences in growth rates. No hybrid had as low a fitness as the least fit parental stock. Recombination produced genotypes of higher fitness than those of either parent only in the cross between the two stocks of lowest fitness. The increase in variance of fitness as a result of recombination was almost exclusively attributable to the generation lines with low fitness. The fitness consequences of sexuality and mate choice were stock specific; some individuals leaving the most descendants by inbreeding, others by outcrossing. For most crosses the short-term advantage of sex, if any, accrue from the fusion of different gametes (hybrid vigor) and not from recombination. Since the homozygous genotype with the highest fitnes left the most progeny by inbreeding (no recombination), the persistence of conjugation in P. primaurelia is paradoxical. (JMT)

  4. Streptomyces as host for recombinant production of Mycobacterium tuberculosis proteins.

    PubMed

    Vallin, Carlos; Ramos, Astrid; Pimienta, Elsa; Rodríguez, Caridad; Hernández, Tairí; Hernández, Ivones; Del Sol, Ricardo; Rosabal, Grisel; Van Mellaert, Lieve; Anné, Jozef

    2006-01-01

    The 45/47 kDa APA protein (Rv1860) of Mycobacterium tuberculosis was produced by Streptomyces lividans. The recombinant protein could be recovered from the culture medium of an S. lividans clone containing the apa gene under control of the promoter and signal sequence of the Streptomyces coelicolor agarase gene. The recombinant protein production was further scaled-up using fermentation conditions. The APA protein was subsequently purified from the culture supernatant by means of immunochromatography. About 80 mg of recombinant protein were obtained per liter of culture media. In vivo tests with the APA protein purified from S. lividans TK24/pRGAPA1 revealed that the recombinant protein was antigenic and could induce high titers of specific antibodies in the mouse biological model. Results obtained concerning heterologous production of APA, its immunogenic and antigenic capacity, demonstrated the potential of S. lividans as a valuable host for the production of recombinant proteins from M. tuberculosis.

  5. Site directed recombination

    DOEpatents

    Jurka, Jerzy W.

    1997-01-01

    Enhanced homologous recombination is obtained by employing a consensus sequence which has been found to be associated with integration of repeat sequences, such as Alu and ID. The consensus sequence or sequence having a single transition mutation determines one site of a double break which allows for high efficiency of integration at the site. By introducing single or double stranded DNA having the consensus sequence flanking region joined to a sequence of interest, one can reproducibly direct integration of the sequence of interest at one or a limited number of sites. In this way, specific sites can be identified and homologous recombination achieved at the site by employing a second flanking sequence associated with a sequence proximal to the 3'-nick.

  6. Cellulase producing microorganism ATCC 55702

    DOEpatents

    Dees, H. Craig

    1997-01-01

    Bacteria which produce large amounts of cellulase--containing cell-free fermentate have been identified. The original bacterium (ATCC 55703) was genetically altered using nitrosoguanidine (MNNG) treatment to produce the enhanced cellulase producing bacterium (ATCC 55702), which was identified through replicate plating. ATCC 55702 has improved characteristics and qualifies for the degradation of cellulosic waste materials for fuel production, food processing, textile processing, and other industrial applications. ATCC 55702 is an improved bacterial host for genetic manipulations using recombinant DNA techniques, and is less likely to destroy genetic manipulations using standard mutagenesis techniques.

  7. Cellulase producing microorganism ATCC 55702

    DOEpatents

    Dees, H.C.

    1997-12-30

    Bacteria which produce large amounts of cellulase--containing cell-free fermentate have been identified. The original bacterium (ATCC 55703) was genetically altered using nitrosoguanidine (MNNG) treatment to produce the enhanced cellulase producing bacterium (ATCC 55702), which was identified through replicate plating. ATCC 55702 has improved characteristics and qualifies for the degradation of cellulosic waste materials for fuel production, food processing, textile processing, and other industrial applications. ATCC 55702 is an improved bacterial host for genetic manipulations using recombinant DNA techniques, and is less likely to destroy genetic manipulations using standard mutagenesis techniques. 5 figs.

  8. Cell biology of mitotic recombination.

    PubMed

    Lisby, Michael; Rothstein, Rodney

    2015-03-02

    Homologous recombination provides high-fidelity DNA repair throughout all domains of life. Live cell fluorescence microscopy offers the opportunity to image individual recombination events in real time providing insight into the in vivo biochemistry of the involved proteins and DNA molecules as well as the cellular organization of the process of homologous recombination. Herein we review the cell biological aspects of mitotic homologous recombination with a focus on Saccharomyces cerevisiae and mammalian cells, but will also draw on findings from other experimental systems. Key topics of this review include the stoichiometry and dynamics of recombination complexes in vivo, the choreography of assembly and disassembly of recombination proteins at sites of DNA damage, the mobilization of damaged DNA during homology search, and the functional compartmentalization of the nucleus with respect to capacity of homologous recombination.

  9. Cell Biology of Mitotic Recombination

    PubMed Central

    Lisby, Michael; Rothstein, Rodney

    2015-01-01

    Homologous recombination provides high-fidelity DNA repair throughout all domains of life. Live cell fluorescence microscopy offers the opportunity to image individual recombination events in real time providing insight into the in vivo biochemistry of the involved proteins and DNA molecules as well as the cellular organization of the process of homologous recombination. Herein we review the cell biological aspects of mitotic homologous recombination with a focus on Saccharomyces cerevisiae and mammalian cells, but will also draw on findings from other experimental systems. Key topics of this review include the stoichiometry and dynamics of recombination complexes in vivo, the choreography of assembly and disassembly of recombination proteins at sites of DNA damage, the mobilization of damaged DNA during homology search, and the functional compartmentalization of the nucleus with respect to capacity of homologous recombination. PMID:25731763

  10. Dissection of Mycobacterium tuberculosis antigens using recombinant DNA.

    PubMed Central

    Young, R A; Bloom, B R; Grosskinsky, C M; Ivanyi, J; Thomas, D; Davis, R W

    1985-01-01

    A recombinant DNA strategy has been used systematically to survey the Mycobacterium tuberculosis genome for sequences that encode specific antigens detected by monoclonal antibodies. M. tuberculosis genomic DNA fragments with randomly generated endpoints were used to construct a large lambda gt11 recombinant DNA expression library. Sufficient numbers of recombinants were produced to contain inserts whose endpoints occur at nearly every base pair in the pathogen genome. Protein antigens specified by linear segments of pathogen DNA and produced by the recombinant phage of Escherichia coli were screened with monoclonal antibody probes. This approach was coupled with an improved detection method for gene isolation using antibodies to clonally isolate DNA sequences that specify polypeptide components of M. tuberculosis. The methodology described here, which is applicable to other pathogens, offers possibilities for the development of more sensitive and specific immunodiagnostic and seroepidemiological tests for tuberculosis and, ultimately, for the development of more effective vaccines. Images PMID:2581251

  11. Nonreplicating vaccinia vector efficiently expresses recombinant genes.

    PubMed

    Sutter, G; Moss, B

    1992-11-15

    Modified vaccinia Ankara (MVA), a highly attenuated vaccinia virus strain that has been safety tested in humans, was evaluated for use as an expression vector. MVA has multiple genomic deletions and is severely host cell restricted: it grows well in avian cells but is unable to multiply in human and most other mammalian cells tested. Nevertheless, we found that replication of viral DNA appeared normal and that both early and late viral proteins were synthesized in human cells. Proteolytic processing of viral structural proteins was inhibited, however, and only immature virus particles were detected by electron microscopy. We constructed an insertion plasmid with the Escherichia coli lacZ gene under the control of the vaccinia virus late promoter P11, flanked by sequences of MVA DNA, to allow homologous recombination at the site of a naturally occurring 3500-base-pair deletion within the MVA genome. MVA recombinants were isolated and propagated in permissive avian cells and shown to express the enzyme beta-galactosidase upon infection of nonpermissive human cells. The amount of enzyme made was similar to that produced by a recombinant of vaccinia virus strain Western Reserve, which also had the lacZ gene under control of the P11 promoter, but multiplied to high titers. Since recombinant gene expression is unimpaired in nonpermissive human cells, MVA may serve as a highly efficient and exceptionally safe vector.

  12. Recombinant CBM-fusion technology - Applications overview.

    PubMed

    Oliveira, Carla; Carvalho, Vera; Domingues, Lucília; Gama, Francisco M

    2015-01-01

    Carbohydrate-binding modules (CBMs) are small components of several enzymes, which present an independent fold and function, and specific carbohydrate-binding activity. Their major function is to bind the enzyme to the substrate enhancing its catalytic activity, especially in the case of insoluble substrates. The immense diversity of CBMs, together with their unique properties, has long raised their attention for many biotechnological applications. Recombinant DNA technology has been used for cloning and characterizing new CBMs. In addition, it has been employed to improve the purity and availability of many CBMs, but mainly, to construct bi-functional CBM-fused proteins for specific applications. This review presents a comprehensive summary of the uses of CBMs recombinantly produced from heterologous organisms, or by the original host, along with the latest advances. Emphasis is given particularly to the applications of recombinant CBM-fusions in: (a) modification of fibers, (b) production, purification and immobilization of recombinant proteins, (c) functionalization of biomaterials and (d) development of microarrays and probes.

  13. Green factory: plants as bioproduction platforms for recombinant proteins.

    PubMed

    Xu, Jianfeng; Dolan, Maureen C; Medrano, Giuliana; Cramer, Carole L; Weathers, Pamela J

    2012-01-01

    Molecular farming, long considered a promising strategy to produce valuable recombinant proteins not only for human and veterinary medicine, but also for agriculture and industry, now has some commercially available products. Various plant-based production platforms including whole-plants, aquatic plants, plant cell suspensions, and plant tissues (hairy roots) have been compared in terms of their advantages and limits. Effective recombinant strategies are summarized along with descriptions of scalable culture systems and examples of commercial progress and success.

  14. Recombinant Protein Production by In Vivo Polymer Inclusion Display ▿

    PubMed Central

    Grage, Katrin; Peters, Verena; Rehm, Bernd H. A.

    2011-01-01

    A novel approach to produce purified recombinant proteins was established. The target protein is produced as polyhydroxyalkanoate (PHA) synthase fusion protein, which mediates intracellular formation of PHA inclusions displaying the target protein. After isolation of the PHA inclusions, the pure target protein was released by simple enterokinase digestion. PMID:21803888

  15. Recombineering: genetic engineering in bacteria using homologous recombination.

    PubMed

    Thomason, Lynn C; Sawitzke, James A; Li, Xintian; Costantino, Nina; Court, Donald L

    2014-04-14

    The bacterial chromosome and bacterial plasmids can be engineered in vivo by homologous recombination using PCR products and synthetic oligonucleotides as substrates. This is possible because bacteriophage-encoded recombination proteins efficiently recombine sequences with homologies as short as 35 to 50 bases. Recombineering allows DNA sequences to be inserted or deleted without regard to location of restriction sites. This unit first describes preparation of electrocompetent cells expressing the recombineering functions and their transformation with dsDNA or ssDNA. It then presents support protocols that describe several two-step selection/counter-selection methods of making genetic alterations without leaving any unwanted changes in the targeted DNA, and a method for retrieving onto a plasmid a genetic marker (cloning by retrieval) from the Escherichia coli chromosome or a co-electroporated DNA fragment. Additional protocols describe methods to screen for unselected mutations, removal of the defective prophage from recombineering strains, and other useful techniques.

  16. Bacterial Recombineering: Genome Engineering via Phage-Based Homologous Recombination.

    PubMed

    Pines, Gur; Freed, Emily F; Winkler, James D; Gill, Ryan T

    2015-11-20

    The ability to specifically modify bacterial genomes in a precise and efficient manner is highly desired in various fields, ranging from molecular genetics to metabolic engineering and synthetic biology. Much has changed from the initial realization that phage-derived genes may be employed for such tasks to today, where recombineering enables complex genetic edits within a genome or a population. Here, we review the major developments leading to recombineering becoming the method of choice for in situ bacterial genome editing while highlighting the various applications of recombineering in pushing the boundaries of synthetic biology. We also present the current understanding of the mechanism of recombineering. Finally, we discuss in detail issues surrounding recombineering efficiency and future directions for recombineering-based genome editing.

  17. Recombinant levels of Escherichia coli K-12 mutants deficient in various replication, recombination, or repair genes.

    PubMed Central

    Zieg, J; Maples, V F; Kushner, S R

    1978-01-01

    Escherichia coli strains containing mutations in lexA, rep, uvrA, uvrD, uvrE, lig, polA, dam, or xthA were constructed and tested for conjugation and transduction proficiencies and ability to form Lac+ recombinants in an assay system utilizing a nontandem duplication of two partially deleted lactose operons (lacMS286phi80dIIlacBK1). lexA and rep mutants were as deficient (20% of wild type) as recB and recC strains in their ability to produce Lac+ progeny. All the other strains exhibited increased frequencies of Lac+ recombinant formation, compared with wild type, ranging from 2- to 13-fold. Some strains showed markedly increased conjugation proficiency (dam uvrD) compared to wild type, while others appeared deficient (polA107). Some differences in transduction proficiency were also observed. Analysis of the Lac+ recombinants formed by the various mutants indicated that they were identical to the recombinants formed by a wild-type strain. The results indicate that genetic recombination in E. coli is a highly regulated process involving multiple gene products. PMID:350859

  18. Orientation Dependence in Homologous Recombination

    PubMed Central

    Yamamoto, K.; Takahashi, N.; Fujitani, Y.; Yoshikura, H.; Kobayashi, I.

    1996-01-01

    Homologous recombination was investigated in Escherichia coli with two plasmids, each carrying the homologous region (two defective neo genes, one with an amino-end deletion and the other with a carboxyl-end deletion) in either direct or inverted orientation. Recombination efficiency was measured in recBC sbcBC and recBC sbcA strains in three ways. First, we measured the frequency of cells carrying neo(+) recombinant plasmids in stationary phase. Recombination between direct repeats was much more frequent than between inverted repeats in the recBC sbcBC strain but was equally frequent in the two substrates in the recBC sbcA strain. Second, the fluctuation test was used to exclude bias by a rate difference between the recombinant and parental plasmids and led to the same conclusion. Third, direct selection for recombinants just after transformation with or without substrate double-strand breaks yielded essentially the same results. Double-strand breaks elevated recombination in both the strains and in both substrates. These results are consistant with our previous findings that the major route of recombination in recBC sbcBC strains generates only one recombinant DNA from two DNAs and in recBC sbcA strains generates two recombinant DNAs from two DNAs. PMID:8722759

  19. Scaling and fractal behaviour underlying meiotic recombination.

    PubMed

    Waxman, D; Stoletzki, N

    2010-01-01

    In this paper we investigate some of the mathematical properties of meiotic recombination. Working within the framework of a genetic model with n loci, where alpha alleles are possible at each locus, we find that the proportion of all possible diploid parental genotypes that can produce a particular haploid gamete is exp[-n log(alpha(2)/[2alpha-1])]. We show that this proportion connects recombination with a fractal geometry of dimension log(2alpha-1)/log(alpha). The fractal dimension of a geometric object manifests itself when it is measured at increasingly smaller length scales. Decreasing the length scale of a geometric object is found to be directly analogous, in a genetics problem, to specifying a multilocus haplotype at a larger number of loci, and it is here that the fractal dimension reveals itself.

  20. Cosmological recombination: feedback of helium photons and its effect on the recombination spectrum

    NASA Astrophysics Data System (ADS)

    Chluba, J.; Sunyaev, R. A.

    2010-02-01

    In this paper, we consider the reprocessing of high-frequency photons emitted by HeII and HeI during the epoch of cosmological recombination by HeI and HI. We demonstrate that, in comparison to computations which neglect all feedback processes, the number of cosmological recombination photons that are related to the presence of helium in the early Universe could be increased by ~40-70 per cent. Our computations imply that per helium nucleus ~3-6 additional photons could be produced. Therefore, a total of ~12-14 helium-related photons per helium atom are emitted during cosmological recombination. This is an important addition to cosmological recombination spectrum which in the future may render it slightly easier to determine the primordial abundance of helium using differential measurements of the cosmic microwave background (CMB) energy spectrum. Also, since these photons are the only witnesses of the feedback process at high redshift, observing them in principle offers a way to check our understanding of the recombination physics. Here, most interestingly, the feedback of HeII photons on HeI leads to the appearance of several additional, rather narrow spectral features in the HeI recombination spectrum at low frequencies. Consequently, the signatures of helium-related features in the CMB spectral distortion from cosmological recombination at some given frequency can exceed the average level of ~17 per cent several times. We find that in particular the bands around ν ~ 10, ~35, ~80 and ~200GHz seem to be affected strongly. In addition, we computed the changes in the cosmological ionization history, finding that only the feedback of primary HeI photons on the dynamics of HeII -> HeI recombination has an effect, producing a change of ΔNe/Ne ~ +0.17 per cent at z ~ 2300. This result seems to be ~2-3 times smaller than the one obtained in earlier computations for this process, however, the difference will not be very important for the analysis of future CMB data.

  1. Project Produce

    ERIC Educational Resources Information Center

    Wolfinger, Donna M.

    2005-01-01

    The grocery store produce section used to be a familiar but rather dull place. There were bananas next to the oranges next to the limes. Broccoli was next to corn and lettuce. Apples and pears, radishes and onions, eggplants and zucchinis all lay in their appropriate bins. Those days are over. Now, broccoli may be next to bok choy, potatoes beside…

  2. Homologous recombination prevents methylation-induced toxicity in Escherichia coli.

    PubMed

    Nowosielska, Anetta; Smith, Stephen A; Engelward, Bevin P; Marinus, M G

    2006-01-01

    Methylating agents such as N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and methyl methane sulfonate (MMS) produce a wide variety of N- and O-methylated bases in DNA, some of which can block replication fork progression. Homologous recombination is a mechanism by which chromosome replication can proceed despite the presence of lesions. The two major recombination pathways, RecBCD and RecFOR, which repair double-strand breaks (DSBs) and single-strand gaps respectively, are needed to protect against toxicity with the RecBCD system being more important. We find that recombination-deficient cell lines, such as recBCD recF, and ruvC recG, are as sensitive to the cytotoxic effects of MMS and MNNG as the most base excision repair (BER)-deficient (alkA tag) isogenic mutant strain. Recombination and BER-deficient double mutants (alkA tag recBCD) were more sensitive to MNNG and MMS than the single mutants suggesting that homologous recombination and BER play essential independent roles. Cells deleted for the polA (DNA polymerase I) or priA (primosome) genes are as sensitive to MMS and MNNG as alkA tag bacteria. Our results suggest that the mechanism of cytotoxicity by alkylating agents includes the necessity for homologous recombination to repair DSBs and single-strand gaps produced by DNA replication at blocking lesions or single-strand nicks resulting from AP-endonuclease action.

  3. Purification of recombinant ovalbumin from inclusion bodies of Escherichia coli.

    PubMed

    Upadhyay, Vaibhav; Singh, Anupam; Panda, Amulya K

    2016-01-01

    Recombinant ovalbumin expressed in bacterial host is essentially free from post-translational modifications and can be useful in understanding the structure-function relationship of the protein. In this study, ovalbumin was expressed in Escherichia coli in the form of inclusion bodies. Ovalbumin inclusion bodies were solubilized using urea and refolded by decreasing the urea concentration by dilution. Refolded protein was purified by anion exchange chromatography. Overall recovery of purified recombinant ovalbumin from inclusion bodies was about 30% with 98% purity. Purified recombinant ovalbumin was characterized by mass spectrometry, circular dichroism and fluorescence spectroscopy. Recombinant ovalbumin was shown to be resistant to trypsin using protease resistance assay. This indicated proper refolding of ovalbumin from inclusion bodies of E. coli. This method provides a simple way of producing ovalbumin free of post-translational modifications.

  4. Break-induced replication and recombinational telomere elongation in yeast.

    PubMed

    McEachern, Michael J; Haber, James E

    2006-01-01

    When a telomere becomes unprotected or if only one end of a chromosomal double-strand break succeeds in recombining with a template sequence, DNA can be repaired by a recombination-dependent DNA replication process termed break-induced replication (BIR). In budding yeasts, there are two BIR pathways, one dependent on the Rad51 recombinase protein and one Rad51 independent; these two repair processes lead to different types of survivors in cells lacking the telomerase enzyme that is required for normal telomere maintenance. Recombination at telomeres is triggered by either excessive telomere shortening or disruptions in the function of telomere-binding proteins. Telomere elongation by BIR appears to often occur through a "roll and spread" mechanism. In this process, a telomeric circle produced by recombination at a dysfunctional telomere acts as a template for a rolling circle BIR event to form an elongated telomere. Additional BIR events can then copy the elongated sequence to all other telomeres.

  5. Extensive Interallelic Polymorphisms Drive Meiotic Recombination into a Crossover Pathway

    PubMed Central

    Dooner, Hugo K.

    2002-01-01

    Recombinants isolated from most meiotic intragenic recombination experiments in maize, but not in yeast, are borne principally on crossover chromosomes. This excess of crossovers is not explained readily by the canonical double-strand break repair model of recombination, proposed to account for a large body of yeast data, which predicts that crossovers (COs) and noncrossovers (NCOs) should be recovered equally. An attempt has been made here to identify general rules governing the recovery of the CO and NCO classes of intragenic recombinants in maize. Recombination was analyzed in bz heterozygotes between a variety of mutations derived from the same or different progenitor alleles. The mutations include point mutations, transposon insertions, and transposon excision footprints. Consequently, the differences between the bz heteroalleles ranged from just two nucleotides to many nucleotides, indels, and insertions. In this article, allelic pairs differing at only two positions are referred to as dimorphic to distinguish them from polymorphic pairs, which differ at multiple positions. The present study has revealed the following effects at these bz heteroalleles: (1) recombination between polymorphic heteroalleles produces mostly CO chromosomes; (2) recombination between dimorphic heteroalleles produces both CO and NCO chromosomes, in ratios apparently dependent on the nature of the heteroalleles; and (3) in dimorphic heterozygotes, the two NCO classes are recovered in approximately equal numbers when the two mutations are point mutations but not when one or both mutations are insertions. These observations are discussed in light of a recent version of the double-strand break repair model of recombination that postulates separate pathways for the formation of CO and NCO products. PMID:12034905

  6. Auger recombination in sodium iodide

    NASA Astrophysics Data System (ADS)

    McAllister, Andrew; Kioupakis, Emmanouil; Åberg, Daniel; Schleife, André

    2014-03-01

    Scintillators are an important tool used to detect high energy radiation - both in the interest of national security and in medicine. However, scintillator detectors currently suffer from lower energy resolutions than expected from basic counting statistics. This has been attributed to non-proportional light yield compared to incoming radiation, but the specific mechanism for this non-proportionality has not been identified. Auger recombination is a non-radiative process that could be contributing to the non-proportionality of scintillating materials. Auger recombination comes in two types - direct and phonon-assisted. We have used first-principles calculations to study Auger recombination in sodium iodide, a well characterized scintillating material. Our findings indicate that phonon-assisted Auger recombination is stronger in sodium iodide than direct Auger recombination. Computational resources provided by LLNL and NERSC. Funding provided by NA-22.

  7. Extensive recombination-induced disruption of genetic interactions is highly deleterious but can be partially reversed by small numbers of secondary recombination events.

    PubMed

    Monjane, Adérito L; Martin, Darren P; Lakay, Francisco; Muhire, Brejnev M; Pande, Daniel; Varsani, Arvind; Harkins, Gordon; Shepherd, Dionne N; Rybicki, Edward P

    2014-07-01

    of producing hybrid genomes with decreased fitness due to the disruption of favorable genetic interactions. Using two synthetic maize streak virus genome chimeras containing alternating genome segments derived from two natural viral strains, we examined both the fitness costs of extreme degrees of recombination (both chimeras had 182 recombination breakpoints) and the capacity of secondary recombination events to recoup these costs. After the severely defective chimeras were introduced together into a suitable host, viruses with between 1 and 3 secondary recombination events arose, which had greatly increased replication and infective capacities. This indicates that even in extreme cases where recombination-induced genetic disruptions are almost lethal, and 91 consecutive secondary recombination events would be required to reconstitute either one of the parental viruses, moderate degrees of fitness recovery can be achieved through relatively small numbers of secondary recombination events.

  8. Modification by chloramphenicol of diethyl sulphate-induced male recombination frequency in Drosophila melanogaster.

    PubMed

    Miglani, G S; Kaur, N P

    1995-06-01

    To study the effect of chloramphenicol (CPL, an inhibitor of protein synthesis) on diethyl sulphate (DES, a potent mutagen) induced male recombination frequency, the F1 (+/aristaless dumpy black cinnabar, al dp b cn) larvae of D. melanogaster were given a pre- or post-treatment of CPL with DES during the first or second half of larval life. In order to determine sensitivity of different germ cell stages to the induction and modification of male recombination frequency, five 3-day broods were taken from every F1 male. DES showed toxic effect on egg-to-adult development. DES was found to be a potent recombinogen. Several cases of non-reciprocal male recombination were recorded. The most frequent recombinant phenotype observed was b cn followed by cn and al. Majority of the recombinants appeared in clusters suggesting their pre-meiotic origin. DES produced male recombination at a stage where only primary spermatocytes were present in the larval testes. CPL when given as a pre- or post-treatment with DES revealed highest frequency of male recombination in broods that represented effect of treatment on spermatogonia predominantly. CPL enhanced the overall level of male recombination produced by DES in both pre- and post-treatments. The results suggested the role of protein synthesis in induction of male recombination in D. melanogaster. In addition, the present experiments give a methodology of enhancing the frequency of chemically-induced male recombination.

  9. Recombinant Origin of the Retrovirus XMRV

    PubMed Central

    Paprotka, Tobias; Delviks-Frankenberry, Krista A.; Cingöz, Oya; Martinez, Anthony; Kung, Hsing-Jien; Tepper, Clifford G.; Hu, Wei-Shau; Fivash, Matthew J.; Coffin, John M.; Pathak, Vinay K.

    2012-01-01

    The retrovirus XMRV (xenotropic murine leukemia virus-related virus) has been detected in human prostate tumors and in blood samples from patients with chronic fatigue syndrome, but these findings have not been replicated. We hypothesized that an understanding of when and how XMRV first arose might help explain the discrepant results. We studied human prostate cancer cell lines CWR22Rv1 and CWR-R1, which produce XMRV virtually identical to the viruses recently found in patient samples, as well as their progenitor human prostate tumor xenograft (CWR22) that had been passaged in mice. We detected XMRV infection in the two cell lines and in the later passage xenografts, but not in the early passages. Importantly, we found that the host mice contained two proviruses, PreXMRV-1 and PreXMRV-2, which share 99.92% identity with XMRV over >3.2-kb stretches of their genomes. We conclude that XMRV was not present in the original CWR22 tumor but was generated by recombination of two proviruses during tumor passaging in mice. The probability that an identical recombinant was generated independently is negligible (~10-12); our results suggest that the association of XMRV with human disease is due to contamination of human samples with virus originating from this recombination event. PMID:21628392

  10. Recombinant lectins: an array of tailor-made glycan-interaction biosynthetic tools.

    PubMed

    Oliveira, Carla; Teixeira, José A; Domingues, Lucília

    2013-03-01

    Lectins are a heterogeneous group of proteins found in plants, animals and microorganisms, which possess at least one non-catalytic domain that binds reversibly to specific mono- or oligosaccharides. The range of lectins and respective biological activities is unsurprising given the immense diversity and complexity of glycan structures and the multiple modes of interaction with proteins. Recombinant DNA technology has been traditionally used for cloning and characterizing newly discovered lectins. It has also been employed as a means of producing pure and sequence-defined lectins for different biotechnological applications. This review focuses on the production of recombinant lectins in heterologous organisms, and highlighting the Escherichia coli and Pichia pastoris expression systems, which are the most employed. The choice of expression host depends on the lectin. Non-glycosylated recombinant lectins are produced in E. coli and post-translational modified recombinant lectins are produced in eukaryotic organisms, namely P. pastoris and non-microbial hosts such as mammalian cells. Emphasis is given to the applications of the recombinant lectins especially (a) in cancer diagnosis and/or therapeutics, (b) as anti-microbial, anti-viral, and anti-insect molecules or (c) in microarrays for glycome profiling. Most reported applications are from recombinant plant lectins. These applications benefit from the tailor-made design associated with recombinant production and will aid in unraveling the complex biological mechanisms of glycan-interactions, bringing recombinant lectins to the forefront of glycobiology. In conclusion, recombinant lectins are developing into valuable biosynthetic tools for biomedical research.

  11. Delayed recombination and standard rulers

    SciTech Connect

    De Bernardis, Francesco; Melchiorri, Alessandro; Bean, Rachel; Galli, Silvia; Silk, Joseph I.; Verde, Licia

    2009-02-15

    Measurements of baryonic acoustic oscillations (BAOs) in galaxy surveys have been recognized as a powerful tool for constraining dark energy. However, this method relies on the knowledge of the size of the acoustic horizon at recombination derived from cosmic microwave background (CMB) anisotropy measurements. This estimate is typically derived assuming a standard recombination scheme; additional radiation sources can delay recombination altering the cosmic ionization history and the cosmological inferences drawn from CMB and BAO data. In this paper we quantify the effect of delayed recombination on the determination of dark energy parameters from future BAO surveys such as the Baryon Oscillation Spectroscopic Survey and the Wide-Field Multi-Object Spectrograph. We find the impact to be small but still not negligible. In particular, if recombination is nonstandard (to a level still allowed by CMB data), but this is ignored, future surveys may incorrectly suggest the presence of a redshift-dependent dark energy component. On the other hand, in the case of delayed recombination, adding to the analysis one extra parameter describing deviations from standard recombination does not significantly degrade the error bars on dark energy parameters and yields unbiased estimates. This is due to the CMB-BAO complementarity.

  12. Testing for recombinant erythropoietin.

    PubMed

    Delanghe, Joris R; Bollen, Mathieu; Beullens, Monique

    2008-03-01

    Erythropoietin (Epo) is a glycoprotein hormone that promotes the production of red blood cells. Recombinant human Epo (rhEpo) is illicitly used to improve performance in endurance sports. Doping in sports is discouraged by the screening of athletes for rhEpo. Both direct tests (indicating the presence of exogeneous Epo isoforms) and indirect tests (indicating hematological changes induced by exogenous Epo administration) can be used for Epo detection. At present, the test adopted by the World Anti Doping Agency is based on a combination of isoelectric focusing and double immunoblotting, and distinguishes between endogenous and rhEpo. However, the adopted monoclonal anti-Epo antibodies are not monospecific. Therefore, the test can occasionally lead to the false-positive detection of rhEpo (epoetin-beta) in post-exercise, protein-rich urine, or in case of contamination of the sample with microorganisms. An improved preanalytical care may counteract a lot of these problems. Adaptation of the criteria may be helpful to further refine direct Epo testing. Indirect tests have the disadvantage that they require blood instead of urine samples, but they can be applied to detect a broader range of performance improving techniques which are illicitly used in sports.

  13. [Detection and antigenic characteristics of the recombinant nucleocapsid proteins of Lassa and Marburg viruses].

    PubMed

    Vladyko, A S; Scheslenok, E P; Fomina, E G; Semizhon, P A; Ignat'ev, G M; Shkolina, T V; Kras'ko, A G; Semenov, S F; Vinokurov, N V

    2012-01-01

    Two plasmid vectors, which allow the recombinant polypeptides of Lassa and Marburg viruses to be expressed in prokaryotic cells E. coli strain BL21 (DE3), were produced. The two recombinant polypeptides are able to bind specific antibodies. This provides an opportunity to use them as antigenic components of immunoassay diagnostic test kits.

  14. Utilizing Protein-lean Co-products from Corn Containing Recombinant Pharmaceutical Proteins for Ethanol Production

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Protein-lean fractions of corn (maize) containing recombinant (r) pharmaceutical proteins were used to produce fuel ethanol and residual r-proteins in the co-product, distillers dry grains with solubles (DDGS), were determined. Transgenic corn lines containing recombinant green fluorescence protein ...

  15. Protein expression in yeast as an approach to production of recombinant malaria antigens.

    PubMed

    Bathurst, I C

    1994-01-01

    The selection of a system suitable for expression of recombinant malaria antigens for vaccine development is, in the final analysis, empirical. However, experience gained with both malaria antigens and other recombinant proteins has provided helpful guidelines. Recombinant DNA technology has been successfully applied to the development of vaccines against a number of human diseases. For example, recombinant DNA-derived hepatitis B virus surface antigen has been produced from both prokaryotic and eukaryotic systems. Yeast has been demonstrated to be an excellent host for the expression of recombinant proteins with uses in diagnostics, therapeutics, and vaccine production. Both intracellular and secretory systems have been developed and optimized for the production of high levels of recombinant proteins. Recombinant DNA technology, and in particular yeast expression systems, have been successfully used to produce malaria antigens, several of which have been protective in various animal models. In contrast, attempts to produce sufficient quantities of antigens for a malaria vaccine from in vitro cultures of the malaria parasite have been unsuccessful. Recombinant proteins can be produced and purified from yeast in large quantities and at low cost, each being requirements for a vaccine to be used in a global vaccination program against malaria.

  16. Genetic Analysis of Recombinant Inbred Lines For Sorghum Bicolor x Perennial S. Propinquum.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    From an annual S. bicolor x perennial S. propinquum F2 population used in early-generation genetic analysis, we have produced and describe here a recombinant inbred line (RIL) population of 161 F5 genotypes that segregates for rhizomatousness and many other traits. The genetic map of the recombinant...

  17. RECOMBINATIONS OF MUTANT PHAGES OF BACILLUS MEGATHERIUM 899A

    PubMed Central

    Murphy, James S.

    1953-01-01

    A group of mutant phages stemming from the virus of B. megatherium 899a (lysogenic), growing on a sensitive B. megatherium strain (KM), have been studied with respect to their recombination reactions. All these mutants and many of their recombinations can be recognized by a characteristic plaque morphology. A similar group of phages have been isolated directly from a culture of B. megatherium 899a in this laboratory. Previous work has shown that when two different plaque mutant phages both infect essentially all the bacteria in a culture, a characteristic per cent of recombinants is produced. This percentage depends on the two recombinants used, each pair having its own value. Hershey and coworkers (2–5) have demonstrated with coli-phage T2, that the percentages of recombination found can be handled mathematically and that they demonstrate the existence of a relationship between the mutations entirely comparable to crossover percentages as used in gene locus maps in genetics. This has been found to hold true for the phages studied in the present work. Only one "linkage group" has been detected and all the mutants studied showed low percentages of recombination (0.8 to 7.6). B. megatherium 899a phage and some of its mutants have been examined with an electron microscope and no differences have been detected between the different mutant strains. PMID:13109115

  18. Homologous recombination catalyzed by a nuclear extract from Xenopus oocytes.

    PubMed Central

    Lehman, C W; Carroll, D

    1991-01-01

    Xenopus laevis oocytes efficiently recombine linear DNA injected into their nuclei (germinal vesicles). This process requires homologous sequences at or near the molecular ends. Here we report that a cell-free extract made from germinal vesicles is capable of accomplishing the complete recombination reaction in vitro. Like the in vivo process, the extract converts the overlapping ends of linear substrate molecules into covalently closed products. Establishment of this cell-free system has allowed examination of the cofactors required for recombination. The first step involves a 5'----3' exonuclease activity that requires a divalent cation but not NTPs. Completion of recombination requires a hydrolyzable NTP; maximal product formation occurs in the presence of millimolar levels of ATP or dATP. At submillimolar levels of all four dNTPs, homologous recombination is inefficient, and a side reaction produces end-joined products. This cell-free system should facilitate a step-by-step understanding of an homologous recombination pathway that operates not only in Xenopus laevis oocytes but also in cells from a wide variety of organisms. Images PMID:1961753

  19. Effect of varying epistasis on the evolution of recombination.

    PubMed

    Kouyos, Roger D; Otto, Sarah P; Bonhoeffer, Sebastian

    2006-06-01

    Whether recombination decelerates or accelerates a population's response to selection depends, at least in part, on how fitness-determining loci interact. Realistically, all genomes likely contain fitness interactions both with positive and with negative epistasis. Therefore, it is crucial to determine the conditions under which the potential beneficial effects of recombination with negative epistasis prevail over the detrimental effects of recombination with positive epistasis. Here, we examine the simultaneous effects of diverse epistatic interactions with different strengths and signs in a simplified model system with independent pairs of interacting loci and selection acting only on the haploid phase. We find that the average form of epistasis does not predict the average amount of linkage disequilibrium generated or the impact on a recombination modifier when compared to results using the entire distribution of epistatic effects and associated single-mutant effects. Moreover, we show that epistatic interactions of a given strength can produce very different effects, having the greatest impact when selection is weak. In summary, we observe that the evolution of recombination at mutation-selection balance might be driven by a small number of interactions with weak selection rather than by the average epistasis of all interactions. We illustrate this effect with an analysis of published data of Saccharomyces cerevisiae. Thus to draw conclusions on the evolution of recombination from experimental data, it is necessary to consider the distribution of epistatic interactions together with the associated selection coefficients.

  20. Three Decades of Recombinant DNA.

    ERIC Educational Resources Information Center

    Palmer, Jackie

    1985-01-01

    Discusses highlights in the development of genetic engineering, examining techniques with recombinant DNA, legal and ethical issues, GenBank (a national database of nucleic acid sequences), and other topics. (JN)

  1. Controlled Release from Recombinant Polymers

    PubMed Central

    Price, Robert; Poursaid, Azadeh; Ghandehari, Hamidreza

    2014-01-01

    Recombinant polymers provide a high degree of molecular definition for correlating structure with function in controlled release. The wide array of amino acids available as building blocks for these materials lend many advantages including biorecognition, biodegradability, potential biocompatibility, and control over mechanical properties among other attributes. Genetic engineering and DNA manipulation techniques enable the optimization of structure for precise control over spatial and temporal release. Unlike the majority of chemical synthetic strategies used, recombinant DNA technology has allowed for the production of monodisperse polymers with specifically defined sequences. Several classes of recombinant polymers have been used for controlled drug delivery. These include, but are not limited to, elastin-like, silk-like, and silk-elastinlike proteins, as well as emerging cationic polymers for gene delivery. In this article, progress and prospects of recombinant polymers used in controlled release will be reviewed. PMID:24956486

  2. Recombination device for storage batteries

    DOEpatents

    Kraft, H.; Ledjeff, K.

    1984-01-01

    A recombination device including a gas-tight enclosure connected to receive the discharge gases from a rechargeable storage battery. Catalytic material for the recombination of hydrogen and oxygen to form water is supported within the enclosure. The enclosure is sealed from the atmosphere by a liquid seal including two vertical chambers interconnected with an inverted U-shaped overflow tube. The first chamber is connected at its upper portion to the enclosure and the second chamber communicates at its upper portion with the atmosphere. If the pressure within the enclosure differs as overpressure or vacuum by more than the liquid level, the liquid is forced into one of the two chambers and the overpressure is vented or the vacuum is relieved. The recombination device also includes means for returning recombined liquid to the battery and for absorbing metal hydrides.

  3. Recombination device for storage batteries

    DOEpatents

    Kraft, Helmut; Ledjeff, Konstantin

    1985-01-01

    A recombination device including a gas-tight enclosure connected to receive he discharge gases from a rechargeable storage battery. Catalytic material for the recombination of hydrogen and oxygen to form water is supported within the enclosure. The enclosure is sealed from the atmosphere by a liquid seal including two vertical chambers interconnected with an inverted U-shaped overflow tube. The first chamber is connected at its upper portion to the enclosure and the second chamber communicates at its upper portion with the atmosphere. If the pressure within the enclosure differs as overpressure or vacuum by more than the liquid level, the liquid is forced into one of the two chambers and the overpressure is vented or the vacuum is relieved. The recombination device also includes means for returning recombined liquid to the battery and for absorbing metal hydrides.

  4. Perovskite photovoltaics: Slow recombination unveiled

    NASA Astrophysics Data System (ADS)

    Moser, Jacques-E.

    2017-01-01

    One of the most salient features of hybrid lead halide perovskites is the extended lifetime of their photogenerated charge carriers. This property has now been shown experimentally to originate from a slow, thermally activated recombination process.

  5. Controlled release from recombinant polymers.

    PubMed

    Price, Robert; Poursaid, Azadeh; Ghandehari, Hamidreza

    2014-09-28

    Recombinant polymers provide a high degree of molecular definition for correlating structure with function in controlled release. The wide array of amino acids available as building blocks for these materials lend many advantages including biorecognition, biodegradability, potential biocompatibility, and control over mechanical properties among other attributes. Genetic engineering and DNA manipulation techniques enable the optimization of structure for precise control over spatial and temporal release. Unlike the majority of chemical synthetic strategies used, recombinant DNA technology has allowed for the production of monodisperse polymers with specifically defined sequences. Several classes of recombinant polymers have been used for controlled drug delivery. These include, but are not limited to, elastin-like, silk-like, and silk-elastinlike proteins, as well as emerging cationic polymers for gene delivery. In this article, progress and prospects of recombinant polymers used in controlled release will be reviewed.

  6. Stable recombination hotspots in birds.

    PubMed

    Singhal, Sonal; Leffler, Ellen M; Sannareddy, Keerthi; Turner, Isaac; Venn, Oliver; Hooper, Daniel M; Strand, Alva I; Li, Qiye; Raney, Brian; Balakrishnan, Christopher N; Griffith, Simon C; McVean, Gil; Przeworski, Molly

    2015-11-20

    The DNA-binding protein PRDM9 has a critical role in specifying meiotic recombination hotspots in mice and apes, but it appears to be absent from other vertebrate species, including birds. To study the evolution and determinants of recombination in species lacking the gene that encodes PRDM9, we inferred fine-scale genetic maps from population resequencing data for two bird species: the zebra finch, Taeniopygia guttata, and the long-tailed finch, Poephila acuticauda. We found that both species have recombination hotspots, which are enriched near functional genomic elements. Unlike in mice and apes, most hotspots are shared between the two species, and their conservation seems to extend over tens of millions of years. These observations suggest that in the absence of PRDM9, recombination targets functional features that both enable access to the genome and constrain its evolution.

  7. Influenza Vaccine, Inactivated or Recombinant

    MedlinePlus

    ... die from flu, and many more are hospitalized.Flu vaccine can:keep you from getting flu, make flu ... inactivated or recombinant influenza vaccine?A dose of flu vaccine is recommended every flu season. Children 6 months ...

  8. CATALYTIC RECOMBINATION OF RADIOLYTIC GASES IN THORIUM OXIDE SLURRIES

    DOEpatents

    Morse, L.E.

    1962-08-01

    A method for the coinbination of hydrogen and oxygen in aqueous thorium oxide-uranium oxide slurries is described. A small amount of molybdenum oxide catalyst is provided in the slurry. This catalyst is applicable to the recombination of hydrogen and/or deuterium and oxygen produced by irradiation of the slurries in nuclear reactors. (AEC)

  9. Producing dicarboxylic acids using polyketide synthases

    DOEpatents

    Katz, Leonard; Fortman, Jeffrey L.; Keasling, Jay D.

    2015-05-26

    The present invention provides for a polyketide synthase (PKS) capable of synthesizing a dicarboxylic acid (diacid). Such diacids include diketide-diacids and triketide-diacids. The invention includes recombinant nucleic acid encoding the PKS, and host cells comprising the PKS. The invention also includes methods for producing the diacids.

  10. Producing dicarboxylic acids using polyketide synthases

    SciTech Connect

    Katz, Leonard; Fortman, Jeffrey L; Keasling, Jay D

    2013-10-29

    The present invention provides for a polyketide synthase (PKS) capable of synthesizing a dicarboxylic acid (diacid). Such diacids include diketide-diacids and triketide-diacids. The invention includes recombinant nucleic acid encoding the PKS, and host cells comprising the PKS. The invention also includes methods for producing the diacids.

  11. Combinatorics in Recombinational Population Genomics

    NASA Astrophysics Data System (ADS)

    Parida, Laxmi

    The work that I will discuss is motivated by the need for understanding, and processing, the manifestations of recombination events in chromosome sequences. In this talk, we focus on two related problems. First, we explore the very general problem of reconstructability of pedigree history. How plausible is it to unravel the history of a complete unit (chromosome) of inheritance? The second problem deals with reconstructing the recombinational history of a collection of chromosomes.

  12. Production of recombinant Agaricus bisporus tyrosinase in Saccharomyces cerevisiae cells.

    PubMed

    Lezzi, Chiara; Bleve, Gianluca; Spagnolo, Stefano; Perrotta, Carla; Grieco, Francesco

    2012-12-01

    It has been demonstrated that Agaricus bisporus tyrosinase is able to oxidize various phenolic compounds, thus being an enzyme of great importance for a number of biotechnological applications. The tyrosinase-coding PPO2 gene was isolated by reverse-transcription polymerase chain reaction (RT-PCR) using total RNA extracted from the mushroom fruit bodies as template. The gene was sequenced and cloned into pYES2 plasmid, and the resulting pY-PPO2 recombinant vector was then used to transform Saccharomyces cerevisiae cells. Native polyacrylamide gel electrophoresis followed by enzymatic activity staining with L-3,4-dihydroxyphenylalanine (L-DOPA) indicated that the recombinant tyrosinase is biologically active. The recombinant enzyme was overexpressed and biochemically characterized, showing that the catalytic constants of the recombinant tyrosinase were higher than those obtained when a commercial tyrosinase was used, for all the tested substrates. The present study describes the recombinant production of A. bisporus tyrosinase in active form. The produced enzyme has similar properties to the one produced in the native A. bisporus host, and its expression in S. cerevisiae provides good potential for protein engineering and functional studies of this important enzyme.

  13. Recombinant protein expression in Nicotiana.

    PubMed

    Matoba, Nobuyuki; Davis, Keith R; Palmer, Kenneth E

    2011-01-01

    Recombinant protein pharmaceuticals are now widely used in treatment of chronic diseases, and several recombinant protein subunit vaccines are approved for human and veterinary use. With growing demand for complex protein pharmaceuticals, such as monoclonal antibodies, manufacturing capacity is becoming limited. There is increasing need for safe, scalable, and economical alternatives to mammalian cell culture-based manufacturing systems, which require substantial capital investment for new manufacturing facilities. Since a seminal paper reporting immunoglobulin expression in transgenic plants was published in 1989, there have been many technological advances in plant expression systems to the present time where production of proteins in leaf tissues of nonfood crops such as Nicotiana species is considered a viable alternative. In particular, transient expression systems derived from recombinant plant viral vectors offer opportunities for rapid expression screening, construct optimization, and expression scale-up. Extraction of recombinant proteins from Nicotiana leaf tissues can be achieved by collection of secreted protein fractions, or from a total protein extract after grinding the leaves with buffer. After separation from solids, the major purification challenge is contamination with elements of the photosynthetic complex, which can be solved by application of a variety of facile and proven strategies. In conclusion, the technologies required for safe, efficient, scalable manufacture of recombinant proteins in Nicotiana leaf tissues have matured to the point where several products have already been tested in phase I clinical trials and will soon be followed by a rich pipeline of recombinant vaccines, microbicides, and therapeutic proteins.

  14. Delayed recombination and cosmic parameters

    SciTech Connect

    Galli, Silvia; Melchiorri, Alessandro; Bean, Rachel; Silk, Joseph

    2008-09-15

    Current cosmological constraints from cosmic microwave background anisotropies are typically derived assuming a standard recombination scheme, however additional resonance and ionizing radiation sources can delay recombination, altering the cosmic ionization history and the cosmological inferences drawn from the cosmic microwave background data. We show that for recent observations of the cosmic microwave background anisotropy, from the Wilkinson microwave anisotropy probe satellite mission (WMAP) 5-year survey and from the arcminute cosmology bolometer array receiver experiment, additional resonance radiation is nearly degenerate with variations in the spectral index, n{sub s}, and has a marked effect on uncertainties in constraints on the Hubble constant, age of the universe, curvature and the upper bound on the neutrino mass. When a modified recombination scheme is considered, the redshift of recombination is constrained to z{sub *}=1078{+-}11, with uncertainties in the measurement weaker by 1 order of magnitude than those obtained under the assumption of standard recombination while constraints on the shift parameter are shifted by 1{sigma} to R=1.734{+-}0.028. From the WMAP5 data we obtain the following constraints on the resonance and ionization sources parameters: {epsilon}{sub {alpha}}<0.39 and {epsilon}{sub i}<0.058 at 95% c.l.. Although delayed recombination limits the precision of parameter estimation from the WMAP satellite, we demonstrate that this should not be the case for future, smaller angular scales measurements, such as those by the Planck satellite mission.

  15. Rational development of an attenuated recombinant cyprinid herpesvirus 3 vaccine using prokaryotic mutagenesis and in vivo bioluminescent imaging

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Cyprinid herpesvirus 3 (CyHV-3) is causing severe economic losses worldwide in the carp industry, and a safe and efficacious attenuated vaccine compatible with mass vaccination is needed. We produced single deleted recombinants using prokaryotic mutagenesis. When producing a recombinant lacking open...

  16. Recombinant synthesis of hyaluronan by Agrobacterium sp.

    PubMed

    Mao, Zichao; Chen, Rachel Ruizhen

    2007-01-01

    Hyaluronan (HA) is a sugar polymer of a repeating disaccharide, beta1-3 D-N-acetylglucosamine (GlcNAc) beta1-4 D-glucuronic acid (GlcA). It finds applications in numerous biomedical procedures such as ophthalmic surgery and osteoarthritis treatment. Until recently, the only commercial sources were extraction of rooster combs and from fermentation of pathogenic Streptococcus. In this work, we demonstrate that metabolic engineering strategies enable the recombinant synthesis of hyaluronan in a safe microorganism. Agrobacterium sp. ATCC 31749 is a commercial production strain for a food polymer, Curdlan. A broad host range expression vector was successfully developed to express the 3 kb HA synthase gene from Pasteurella multocida, along with a kfiD gene encoding UDP-glucose dehydrogenase from Escherichia coli K5 strain. Coexpression of these two heterologous enzymes enables Agrobacterium to produce HA. Hyaluronan was accumulated up to 0.3 g/L in shaker flask cultivation. The molecular weight of the polymer from various Agrobacterium strains is in the range of 0.7-2 MD. To our knowledge, this is the first successful recombinant hyaluronan synthesis in a Gram-negative bacterium that naturally produces a food product. The ease of genetic modifications provides future opportunities to tailor properties of polymers for specific applications.

  17. Mismatch repair of heteroduplex DNA intermediates of extrachromosomal recombination in mammalian cells.

    PubMed Central

    Deng, W P; Nickoloff, J A

    1994-01-01

    Previous work indicated that extrachromosomal recombination in mammalian cells could be explained by the single-strand annealing (SSA) model. This model predicts that extrachromosomal recombination leads to nonconservative crossover products and that heteroduplex DNA (hDNA) is formed by annealing of complementary single strands. Mismatched bases in hDNA may subsequently be repaired to wild-type or mutant sequences, or they may remain unrepaired and segregate following DNA replication. We describe a system to examine the formation and mismatch repair of hDNA in recombination intermediates. Our results are consistent with extrachromosomal recombination occurring via SSA and producing crossover recombinant products. As predicted by the SSA model, hDNA was present in double-strand break-induced recombination intermediates. By placing either silent or frameshift mutations in the predicted hDNA region, we have shown that mismatches are efficiently repaired prior to DNA replication. Images PMID:8264607

  18. Repeatability and heritability of divergent recombination frequencies in the Iowa Stiff Stalk Synthetic (Zea mays L.).

    PubMed

    Hadad, R G; Pfeiffer, T W; Poneleit, C G

    1996-10-01

    Variability in recombination frequency was reported in the Iowa Stiff Stalk Synthetic. The objectives of the present research were to verify the differences in recombination frequency among individuals in the Iowa Stiff Stalk Synthetic maize population and to determine if the recombination frequency differences persisted among the S1 progeny. Testcrosses to measure male recombination frequency on three chromosomes (4, su1-c2; 5, a2-bt1-pr1; 9, sh1-bz1-wx1) were repeated for eight S0 individuals. Recombination frequencies were repeatably divergent among those individuals which were selected based on high or low recombination frequencies on specific chromosomes. Individuals which had been selected for long and short total map distances across the three chromosome regions produced repeatably divergent recombination frequencies only at the su1-c2 region. The recombination frequencies of the S1 lines, derived from the S0 individuals which had the most divergent recombination frequencies on a single chromosome, were significantly different. The broadsense heritability estimates derived from the regression of six S1 lines on six S0 individuals ranged from 0.69 to 0.20 for the five chromosome regions. We conclude that genetic differences for recombination frequency exist in this population and that modification by selection should be possible.

  19. Variability of recombination frequencies in the Iowa Stiff Stalk Synthetic (Zea mays L.).

    PubMed

    Fatmi, A; Poneleit, C G; Pfeiffer, T W

    1993-08-01

    Variability in recombination frequency has been reported in several plant populations. The objectives of the present research were to establish the range in variability in recombination among genotypes in the important corn population Iowa Stiff Stalk Synthetic and to identify individual genotypes which produced increased or decreased recombination frequencies. Approximately 150 individual S0 plants were testcrossed to measure male recombination frequency on three chromosomes: 4, sul-c2; 5, a2-btl-pr1; and 9, sh1-bz1-wx1. Although the variance component for individuals accounted for only 20-33% of the total variation, highly significant variability among individuals was present at all chromosome regions. Thus the environmental effects did not prevent measurement of differences between S0 individuals. At each chromosome region, individual genotypes with recombination frequencies at least two standard deviations above or below the population mean were isolated. Reports in the literature suggest that the variability reported here for the BSSS population should be representative of that present in other corn breeding populations. Recombination frequencies were positively correlated between adjacent regions of chromosome 9 and also between adjacent regions of chromosome 5. Recombination frequencies were positively correlated between both regions on chromosome 5 with the su1-c2 region of chromosome 4. Negative correlations were observed between chromosome 9 recombination and recombination in each region of chromosomes 4 and 5. Thus rankings of S0 individual recombination frequencies were not consistent for all three chromosomes.

  20. Recombination at the DNA level. Abstracts

    SciTech Connect

    Not Available

    1984-01-01

    Abstracts of papers in the following areas are presented: (1) chromosome mechanics; (2) yeast systems; (3) mammalian homologous recombination; (4) transposons; (5) Mu; (6) plant transposons/T4 recombination; (7) topoisomerase, resolvase, and gyrase; (8) Escherichia coli general recombination; (9) recA; (10) repair; (11) eucaryotic enzymes; (12) integration and excision of bacteriophage; (13) site-specific recombination; and (14) recombination in vitro. (ACR)

  1. Recombination Drives Vertebrate Genome Contraction

    PubMed Central

    Nam, Kiwoong; Ellegren, Hans

    2012-01-01

    Selective and/or neutral processes may govern variation in DNA content and, ultimately, genome size. The observation in several organisms of a negative correlation between recombination rate and intron size could be compatible with a neutral model in which recombination is mutagenic for length changes. We used whole-genome data on small insertions and deletions within transposable elements from chicken and zebra finch to demonstrate clear links between recombination rate and a number of attributes of reduced DNA content. Recombination rate was negatively correlated with the length of introns, transposable elements, and intergenic spacer and with the rate of short insertions. Importantly, it was positively correlated with gene density, the rate of short deletions, the deletion bias, and the net change in sequence length. All these observations point at a pattern of more condensed genome structure in regions of high recombination. Based on the observed rates of small insertions and deletions and assuming that these rates are representative for the whole genome, we estimate that the genome of the most recent common ancestor of birds and lizards has lost nearly 20% of its DNA content up until the present. Expansion of transposable elements can counteract the effect of deletions in an equilibrium mutation model; however, since the activity of transposable elements has been low in the avian lineage, the deletion bias is likely to have had a significant effect on genome size evolution in dinosaurs and birds, contributing to the maintenance of a small genome. We also demonstrate that most of the observed correlations between recombination rate and genome contraction parameters are seen in the human genome, including for segregating indel polymorphisms. Our data are compatible with a neutral model in which recombination drives vertebrate genome size evolution and gives no direct support for a role of natural selection in this process. PMID:22570634

  2. DNA recombination. Recombination initiation maps of individual human genomes.

    PubMed

    Pratto, Florencia; Brick, Kevin; Khil, Pavel; Smagulova, Fatima; Petukhova, Galina V; Camerini-Otero, R Daniel

    2014-11-14

    DNA double-strand breaks (DSBs) are introduced in meiosis to initiate recombination and generate crossovers, the reciprocal exchanges of genetic material between parental chromosomes. Here, we present high-resolution maps of meiotic DSBs in individual human genomes. Comparing DSB maps between individuals shows that along with DNA binding by PRDM9, additional factors may dictate the efficiency of DSB formation. We find evidence for both GC-biased gene conversion and mutagenesis around meiotic DSB hotspots, while frequent colocalization of DSB hotspots with chromosome rearrangement breakpoints implicates the aberrant repair of meiotic DSBs in genomic disorders. Furthermore, our data indicate that DSB frequency is a major determinant of crossover rate. These maps provide new insights into the regulation of meiotic recombination and the impact of meiotic recombination on genome function.

  3. Gene Delivery into Plant Cells for Recombinant Protein Production

    PubMed Central

    Chen, Qiang

    2015-01-01

    Recombinant proteins are primarily produced from cultures of mammalian, insect, and bacteria cells. In recent years, the development of deconstructed virus-based vectors has allowed plants to become a viable platform for recombinant protein production, with advantages in versatility, speed, cost, scalability, and safety over the current production paradigms. In this paper, we review the recent progress in the methodology of agroinfiltration, a solution to overcome the challenge of transgene delivery into plant cells for large-scale manufacturing of recombinant proteins. General gene delivery methodologies in plants are first summarized, followed by extensive discussion on the application and scalability of each agroinfiltration method. New development of a spray-based agroinfiltration and its application on field-grown plants is highlighted. The discussion of agroinfiltration vectors focuses on their applications for producing complex and heteromultimeric proteins and is updated with the development of bridge vectors. Progress on agroinfiltration in Nicotiana and non-Nicotiana plant hosts is subsequently showcased in context of their applications for producing high-value human biologics and low-cost and high-volume industrial enzymes. These new advancements in agroinfiltration greatly enhance the robustness and scalability of transgene delivery in plants, facilitating the adoption of plant transient expression systems for manufacturing recombinant proteins with a broad range of applications. PMID:26075275

  4. PROGENITORS OF RECOMBINING SUPERNOVA REMNANTS

    SciTech Connect

    Moriya, Takashi J.

    2012-05-01

    Usual supernova remnants have either ionizing plasma or plasma in collisional ionization equilibrium, i.e., the ionization temperature is lower than or equal to the electron temperature. However, the existence of recombining supernova remnants, i.e., supernova remnants with ionization temperature higher than the electron temperature, has been recently confirmed. One suggested way to have recombining plasma in a supernova remnant is to have a dense circumstellar medium at the time of the supernova explosion. If the circumstellar medium is dense enough, collisional ionization equilibrium can be established in the early stage of the evolution of the supernova remnant and subsequent adiabatic cooling, which occurs after the shock wave gets out of the dense circumstellar medium, makes the electron temperature lower than the ionization temperature. We study the circumstellar medium around several supernova progenitors and show which supernova progenitors can have a circumstellar medium dense enough to establish collisional ionization equilibrium soon after the explosion. We find that the circumstellar medium around red supergiants (especially massive ones) and the circumstellar medium dense enough to make Type IIn supernovae can establish collisional ionization equilibrium soon after the explosion and can evolve to become recombining supernova remnants. Wolf-Rayet stars and white dwarfs have the possibility to be recombining supernova remnants but the fraction is expected to be very small. As the occurrence rate of the explosions of red supergiants is much higher than that of Type IIn supernovae, the major progenitors of recombining supernova remnants are likely to be red supergiants.

  5. Systemic delivery of recombinant proteins by genetically modified myoblasts

    SciTech Connect

    Barr, E.; Leiden, J.M. )

    1991-12-06

    The ability to stably deliver recombinant proteins to the systemic circulation would facilitate the treatment of a variety of acquired and inherited diseases. To explore the feasibility of the use of genetically engineered myoblasts as a recombinant protein delivery system, stable transfectants of the murine C2C12 myoblast cell line were produced that synthesize and secrete high levels of human growth hormone (hGH) in vitro. Mice injected with hGH-transfected myoblasts had significant levels of hGH in both muscle and serum that were stable for at least 3 weeks after injection. Histological examination of muscles injected with {beta}-galactosidase-expressing C2C12 myoblasts demonstrated that many of the injected cells had fused to form multinucleated myotubes. Thus, genetically engineered myoblasts can be used for the stable delivery of recombinant proteins into the circulation.

  6. Self-assembly of tunable protein suprastructures from recombinant oleosin

    PubMed Central

    Vargo, Kevin B.; Parthasarathy, Ranganath; Hammer, Daniel A.

    2012-01-01

    Using recombinant amphiphilic proteins to self-assemble suprastructures would allow precise control over surfactant chemistry and the facile incorporation of biological functionality. We used cryo-TEM to confirm self-assembled structures from recombinantly produced mutants of the naturally occurring sunflower protein, oleosin. We studied the phase behavior of protein self-assembly as a function of solution ionic strength and protein hydrophilic fraction, observing nanometric fibers, sheets, and vesicles. Vesicle membrane thickness correlated with increasing hydrophilic fraction for a fixed hydrophobic domain length. The existence of a bilayer membrane was corroborated in giant vesicles through the localized encapsulation of hydrophobic Nile red and hydrophilic calcein. Circular dichroism revealed that changes in nanostructural morphology in this family of mutants was unrelated to changes in secondary structure. Ultimately, we envision the use of recombinant techniques to introduce novel functionality into these materials for biological applications. PMID:22753512

  7. A Multi-Omics Analysis of Recombinant Protein Production in Hek293 Cells

    PubMed Central

    Dietmair, Stefanie; Hodson, Mark P.; Quek, Lake-Ee; Timmins, Nicholas E.; Gray, Peter; Nielsen, Lars K.

    2012-01-01

    Hek293 cells are the predominant hosts for transient expression of recombinant proteins and are used for stable expression of proteins where post-translational modifications performed by CHO cells are inadequate. Nevertheless, there is little information available on the key cellular features underpinning recombinant protein production in Hek293 cells. To improve our understanding of recombinant protein production in Hek293 cells and identify targets for the engineering of an improved host cell line, we have compared a stable, recombinant protein producing Hek293 cell line and its parental cell line using a combination of transcriptomics, metabolomics and fluxomics. Producer cultures consumed less glucose than non-producer cultures while achieving the same growth rate, despite the additional burden of recombinant protein production. Surprisingly, there was no indication that producer cultures compensated for the reduction in glycolytic energy by increasing the efficiency of glucose utilization or increasing glutamine consumption. In contrast, glutamine consumption was lower and the majority of genes involved in oxidative phosphorylation were downregulated in producer cultures. We observed an overall downregulation of a large number of genes associated with broad cellular functions (e.g., cell growth and proliferation) in producer cultures, and therefore speculate that a broad adaptation of the cellular network freed up resources for recombinant protein production while maintaining the same growth rate. Increased abundance of genes associated with endoplasmic reticulum stress indicated a possible bottleneck at the point of protein folding and assembly. PMID:22937046

  8. The Dissociative Recombination of OH(+)

    NASA Technical Reports Server (NTRS)

    Guberman, Steven L.

    1995-01-01

    Theoretical quantum chemical calculations of the cross sections and rates for the dissociative recombination of the upsilon = 0 level of the ground state of OH(+) show that recombination occurs primarily along the 2 (2)Pi diabatic route. The products are 0((1)D) and a hot H atom with 6.1 eV kinetic energy. The coupling to the resonances is very small and the indirect recombination mechanism plays only a minor role. The recommended value for the rate coefficient is (6.3 +/- 0.7) x 10(exp -9)x (T(e)/1300)(exp -0.48) cu.cm/s for 10 less than T(e) less than 1000 K.

  9. DNA recombination: the replication connection.

    PubMed

    Haber, J E

    1999-07-01

    Chromosomal double-strand breaks (DSBs) arise after exposure to ionizing radiation or enzymatic cleavage, but especially during the process of DNA replication itself. Homologous recombination plays a critical role in repair of such DSBs. There has been significant progress in our understanding of two processes that occur in DSB repair: gene conversion and recombination-dependent DNA replication. Recent evidence suggests that gene conversion and break-induced replication are related processes that both begin with the establishment of a replication fork in which both leading- and lagging-strand synthesis occur. There has also been much progress in characterization of the biochemical roles of recombination proteins that are highly conserved from yeast to humans.

  10. Variation in Recombination Rate: Adaptive or Not?

    PubMed

    Ritz, Kathryn R; Noor, Mohamed A F; Singh, Nadia D

    2017-03-27

    Rates of meiotic recombination are widely variable both within and among species. However, the functional significance of this variation remains largely unknown. Is the observed within-species variation in recombination rate adaptive? Recent work has revealed new insight into the scale and scope of population-level variation in recombination rate. These data indicate that the magnitude of within-population variation in recombination is similar among taxa. The apparent similarity of the variance in recombination rate among individuals between distantly related species suggests that the relative costs and benefits of recombination that establish the upper and lower bounds may be similar across species. Here we review the current data on intraspecific variation in recombination rate and discuss the molecular and evolutionary costs and benefits of recombination frequency. We place this variation in the context of adaptation and highlight the need for more empirical studies focused on the adaptive value of variation in recombination rate.

  11. Biochemical and immunological characterization of recombinant allergen Lol p 1.

    PubMed

    Tamborini, E; Faccini, S; Lidholm, J; Svensson, M; Brandazza, A; Longhi, R; Groenlund, H; Sidoli, A; Arosio, P

    1997-11-01

    Pollen from perennial rye grass (Lolium perenne), a major cause of type-I allergy worldwide, contains a complex mixture of allergenic proteins among which Lol p 1 is one of the most important. We describe the expression, purification and characterization of a recombinant Lol p 1 overproduced in Escherichia coli. The recombinant allergen, expressed in high yields and purified in milligram amounts, bound to specific IgE antibodies from human sera, induced histamine release from sensitized human basophils, and elicited rabbit antisera that recognize specifically recombinant Lol p 1 and natural Lol p 1 of pollen extract. Recombinant Lol p 1 was used to develop ImmunoCAP assays for analysis of 150 sera that were Radioallergosorbent test positive to L. perenne pollen. In 130 of them (87%) the assay detected a significant level of IgE antibodies to Lol p 1, reaching on average 37% of the level obtained with a test for IgE to the whole grass pollen extract. To map epitopes on Lol p 1, we produced three deletion mutants [des-(116-240)-Lol p 1, des-(1-88)-Lol p 1 and des-(133-189)-Lol p 1], which were efficiently expressed in bacteria. These all showed a strong reactivity with the specific rabbit IgG antibodies, but lacked most or all the allergenic properties of recombinant Lol p 1. A study of the antigenic structure of Lol p 1 was performed using the three deletion mutants and a set of 17-18-residue overlapping synthetic peptides covering the whole allergen sequence. The results indicate that human IgE and rabbit IgG antibodies bind to distinct regions of Lol p 1, and that at least some important IgE epitopes are mainly conformational. The findings suggest that recombinant allergens constitute useful reagents for further development of serological diagnosis of allergy, and that it should be possible to produce immunogenic fragments of allergenic proteins without allergenic properties.

  12. Selenium incorporation using recombinant techniques

    SciTech Connect

    Walden, Helen

    2010-04-01

    An overview of techniques for recombinant incorporation of selenium and subsequent purification and crystallization of the resulting labelled protein. Using selenomethionine to phase macromolecular structures is common practice in structure determination, along with the use of selenocysteine. Selenium is consequently the most commonly used heavy atom for MAD. In addition to the well established recombinant techniques for the incorporation of selenium in prokaryal expression systems, there have been recent advances in selenium labelling in eukaryal expression, which will be discussed. Tips and things to consider for the purification and crystallization of seleno-labelled proteins are also included.

  13. Recombinant host cells and media for ethanol production

    DOEpatents

    Wood, Brent E; Ingram, Lonnie O; Yomano, Lorraine P; York, Sean W

    2014-02-18

    Disclosed are recombinant host cells suitable for degrading an oligosaccharide that have been optimized for growth and production of high yields of ethanol, and methods of making and using these cells. The invention further provides minimal media comprising urea-like compounds for economical production of ethanol by recombinant microorganisms. Recombinant host cells in accordance with the invention are modified by gene mutation to eliminate genes responsible for the production of unwanted products other than ethanol, thereby increasing the yield of ethanol produced from the oligosaccharides, relative to unmutated parent strains. The new and improved strains of recombinant bacteria are capable of superior ethanol productivity and yield when grown under conditions suitable for fermentation in minimal growth media containing inexpensive reagents. Systems optimized for ethanol production combine a selected optimized minimal medium with a recombinant host cell optimized for use in the selected medium. Preferred systems are suitable for efficient ethanol production by simultaneous saccharification and fermentation (SSF) using lignocellulose as an oligosaccharide source. The invention also provides novel isolated polynucleotide sequences, polypeptide sequences, vectors and antibodies.

  14. Expression and purification of recombinant polyomavirus VP2 protein and its interactions with polyomavirus proteins

    NASA Technical Reports Server (NTRS)

    Cai, X.; Chang, D.; Rottinghaus, S.; Consigli, R. A.; Spooner, B. S. (Principal Investigator)

    1994-01-01

    Recombinant polyomavirus VP2 protein was expressed in Escherichia coli (RK1448), using the recombinant expression system pFPYV2. Recombinant VP2 was purified to near homogeneity by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, electroelution, and Extracti-Gel chromatography. Polyclonal serum to this protein which reacted specifically with recombinant VP2 as well as polyomavirus virion VP2 and VP3 on Western blots (immunoblots) was produced. Purified VP2 was used to establish an in vitro protein-protein interaction assay with polyomavirus structural proteins and purified recombinant VP1. Recombinant VP2 interacted with recombinant VP1, virion VP1, and the four virion histones. Recombinant VP1 coimmunoprecipitated with recombinant VP2 or truncated VP2 (delta C12VP2), which lacked the carboxy-terminal 12 amino acids. These experiments confirmed the interaction between VP1 and VP2 and revealed that the carboxyterminal 12 amino acids of VP2 and VP3 were not necessary for formation of this interaction. In vivo VP1-VP2 interaction study accomplished by cotransfection of COS-7 cells with VP2 and truncated VP1 (delta N11VP1) lacking the nuclear localization signal demonstrated that VP2 was capable of translocating delta N11VP1 into the nucleus. These studies suggest that complexes of VP1 and VP2 may be formed in the cytoplasm and cotransported to the nucleus for virion assembly to occur.

  15. Recombinant bacterial amylopullulanases

    PubMed Central

    Nisha, M; Satyanarayana, T

    2013-01-01

    Pullulanases are endo-acting enzymes capable of hydrolyzing α-1, 6-glycosidic linkages in starch, pullulan, amylopectin, and related oligosaccharides, while amylopullulanases are bifunctional enzymes with an active site capable of cleaving both α-1, 4 and α-1, 6 linkages in starch, amylose and other oligosaccharides, and α-1, 6 linkages in pullulan. The amylopullulanases are classified in GH13 and GH57 family enzymes based on the architecture of catalytic domain and number of conserved sequences. The enzymes with two active sites, one for the hydrolysis of α-1, 4- glycosidic bond and the other for α-1, 6-glycosidic bond, are called α-amylase-pullulanases, while amylopullulanases have only one active site for cleaving both α-1, 4- and α-1, 6-glycosidic bonds. The amylopullulanases produced by bacteria find applications in the starch and baking industries as a catalyst for one step starch liquefaction-saccharification for making various sugar syrups, as antistaling agent in bread and as a detergent additive. PMID:23645215

  16. Recombination in Hepatitis C Virus

    PubMed Central

    González-Candelas, Fernando; López-Labrador, F. Xavier; Bracho, María Alma

    2011-01-01

    Hepatitis C virus (HCV) is a Flavivirus with a positive-sense, single-stranded RNA genome of about 9,600 nucleotides. It is a major cause of liver disease, infecting almost 200 million people all over the world. Similarly to most RNA viruses, HCV displays very high levels of genetic diversity which have been used to differentiate six major genotypes and about 80 subtypes. Although the different genotypes and subtypes share basic biological and pathogenic features they differ in clinical outcomes, response to treatment and epidemiology. The first HCV recombinant strain, in which different genome segments derived from parentals of different genotypes, was described in St. Petersburg (Russia) in 2002. Since then, there have been only a few more than a dozen reports including descriptions of HCV recombinants at all levels: between genotypes, between subtypes of the same genotype and even between strains of the same subtype. Here, we review the literature considering the reasons underlying the difficulties for unequivocally establishing recombination in this virus along with the analytical methods necessary to do it. Finally, we analyze the potential consequences, especially in clinical practice, of HCV recombination in light of the coming new therapeutic approaches against this virus. PMID:22069526

  17. Dissociative Recombination of Complex Ions

    NASA Astrophysics Data System (ADS)

    Mitchell, J. Brian A.

    1999-10-01

    The FALP-MS apparatus at the University of Rennes allows the measurement of rate coefficients for the recombination of molecular ions to be made (at 300K) even though several ions may be present in the afterglow. The recombination of a number of hydrocarbon ions derived from alkane ( Lehfaoui et al. J. Chem. Phys. 106, 5406, 1997.), alkene ( Rebrion-Rowe et al. J. Chem. Phys. 108, 7185, 1998.) and aromatic (Rebrion-Rowe et al. (Submitted to J. Chem. Phys.)) parent molecules has been studied. Despite the wide range of complexity of these compounds, the measured recombination rates are remarkably similar having values in the range of 4-10-7 cm^3.s-1. Plans are being laid for a new version of this apparatus that will allow pre-prepared ions to be injected into the inert buffer gas flow. This will allow reactive ions to be studied as well as halogen containing ions whose recombination rates would normally be masked by electron attachment to their parent gases in a conventional flowing afterglow apparatus. A high temperature modification to the CRESU supersonic flow apparatus (J.L. Le Garrec et al. J. Chem. Phys. 107, 54, 1997.) in our laboratory will allow electron attachment to radicals to be studied by means of the mass spectrometric detection of products, Langmuir probe measurement of the electron density in the flow and Laser Induced Fluorescent identification of the radical species. Such measurements are needed for the modeling of semiconductor processing plasmas.

  18. CRMAGE: CRISPR Optimized MAGE Recombineering

    PubMed Central

    Ronda, Carlotta; Pedersen, Lasse Ebdrup; Sommer, Morten O. A.; Nielsen, Alex Toftgaard

    2016-01-01

    A bottleneck in metabolic engineering and systems biology approaches is the lack of efficient genome engineering technologies. Here, we combine CRISPR/Cas9 and λ Red recombineering based MAGE technology (CRMAGE) to create a highly efficient and fast method for genome engineering of Escherichia coli. Using CRMAGE, the recombineering efficiency was between 96.5% and 99.7% for gene recoding of three genomic targets, compared to between 0.68% and 5.4% using traditional recombineering. For modulation of protein synthesis (small insertion/RBS substitution) the efficiency was increased from 6% to 70%. CRMAGE can be multiplexed and enables introduction of at least two mutations in a single round of recombineering with similar efficiencies. PAM-independent loci were targeted using degenerate codons, thereby making it possible to modify any site in the genome. CRMAGE is based on two plasmids that are assembled by a USER-cloning approach enabling quick and cost efficient gRNA replacement. CRMAGE furthermore utilizes CRISPR/Cas9 for efficient plasmid curing, thereby enabling multiple engineering rounds per day. To facilitate the design process, a web-based tool was developed to predict both the λ Red oligos and the gRNAs. The CRMAGE platform enables highly efficient and fast genome editing and may open up promising prospective for automation of genome-scale engineering. PMID:26797514

  19. Improving recombinant protein purification yield

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Production of adequate amounts of recombinant proteins is essential for antibody production, biochemical activity study, and structural determination during the post-genomic era. It’s technologically challenging and a limiting factor for tung oil research because analytical reagents such as high qua...

  20. Antigenicity of partial fragments of recombinant Pasteurella multocida toxin.

    PubMed

    Lee, Jeongmin; Woo, Hee-Jong

    2010-12-01

    Pasteurella multocida serogroup D strain, which produces P. multocida toxin (PMT), is a widespread and harmful pathogen of respiratory diseases such as pneumonia and progressive atrophic rhinitis (PAR) in swine. Vaccination has been considered the most desirable and effective approach for controlling the diseases caused by toxigenic P. multocida. To investigate the antigenicity and immunogenicity of partial fragments of recombinant PMT, recombinant proteins of the N-terminal (PMT-A), middle (PMT-B), Cterminal (PMT-C), and middle-C-terminal (PMT2.3) regions of PMT were successfully produced in an Escherichia coli expression system. The molecular masses of PMT-A, PMT-B, PMT-C, and PMT2.3 were ca. 53, 55, 35, and 84 kDa, respectively, purified by nickel-nitrilotriacetic acid (Ni-NTA) affinity column chromatography. All the recombinant proteins except for PMT-A showed immune responses to antisera obtained from a swine showing symptoms of PAR. Moreover, high titers of PMT-specific antibodies were raised from mice immunized with each of the recombinant proteins; however, the immunoreactivities of the antibodies to authentic PMT and heat-inactivated whole bacteria were different, respectively. In the protection study, the highest protection against homologous challenge was shown in the case of PMT2.3; relatively poor protections occurred for the other PMT fragments.

  1. High yield production of extracellular recombinant levansucrase by Bacillus megaterium.

    PubMed

    Korneli, Claudia; Biedendieck, Rebekka; David, Florian; Jahn, Dieter; Wittmann, Christoph

    2013-04-01

    In this study, a high yield production bioprocess with recombinant Bacillus megaterium for the production of the extracellular enzyme levansucrase (SacB) was developed. For basic optimization of culture parameters and nutrients, a recombinant B. megaterium reporter strain that produced green fluorescent protein under control of a vector-based xylose-inducible promoter was used. It enabled efficient microtiter plate-based screening via fluorescence analysis. A pH value of pH 6, 20 % of dissolved oxygen, 37 °C, and elevated levels of biotin (100 μg L(-1)) were found optimal with regard to high protein yield and reduced overflow metabolism. Among the different compounds tested, fructose and glycerol were identified as the preferred source of carbon. Subsequently, the settings were transferred to a B. megaterium strain recombinantly producing levansucrase SacB based on the plasmid-located xylose-inducible expression system. In shake flask culture under the optimized conditions, the novel strain already secreted the target enzyme in high amounts (14 U mL(-1) on fructose and 17.2 U mL(-1) on glycerol). This was further increased in high cell density fed-batch processes up to 55 U mL(-1), reflecting a levansucrase concentration of 0.52 g L(-1). This is 100-fold more than previous efforts for this enzyme in B. megaterium and more than 10-fold higher than reported values of other extracellular protein produced in this microorganism so far. The recombinant strain could also handle raw glycerol from biodiesel industry which provided the same amount and quality of the recombinant protein and suggests future implementation into existing biorefinery concepts.

  2. Recombinant protein production in a variety of Nicotiana hosts: a comparative analysis.

    PubMed

    Conley, Andrew J; Zhu, Hong; Le, Linda C; Jevnikar, Anthony M; Lee, Byong H; Brandle, Jim E; Menassa, Rima

    2011-05-01

    Although many different crop species have been used to produce a wide range of vaccines, antibodies, biopharmaceuticals and industrial enzymes, tobacco has the most established history for the production of recombinant proteins. To further improve the heterologous protein yield of tobacco platforms, transient and stable expression of four recombinant proteins (i.e. human erythropoietin and interleukin-10, an antibody against Pseudomonas aeruginosa, and a hyperthermostable α-amylase) was evaluated in numerous species and cultivars of Nicotiana. Whereas the transient level of recombinant protein accumulation varied significantly amongst the different Nicotiana plant hosts, the variety of Nicotiana had little practical impact on the recombinant protein concentration in stable transgenic plants. In addition, this study examined the growth rate, amount of leaf biomass, total soluble protein levels and the alkaloid content of the various Nicotiana varieties to establish the best plant platform for commercial production of recombinant proteins. Of the 52 Nicotiana varieties evaluated, Nicotiana tabacum (cv. I 64) produced the highest transient concentrations of recombinant proteins, in addition to producing a large amount of biomass and a relatively low quantity of alkaloids, probably making it the most effective plant host for recombinant protein production.

  3. Overview of the purification of recombinant proteins.

    PubMed

    Wingfield, Paul T

    2015-04-01

    When the first version of this unit was written in 1995, protein purification of recombinant proteins was based on a variety of standard chromatographic methods and approaches, many of which were described and mentioned throughout Current Protocols in Protein Science. In the interim, there has been a shift toward an almost universal usage of the affinity or fusion tag. This may not be the case for biotechnology manufacture where affinity tags can complicate producing proteins under regulatory conditions. Regardless of the protein expression system, questions are asked as to which and how many affinity tags to use, where to attach them in the protein, and whether to engineer a self-cleavage system or simply leave them on. We will briefly address some of these issues. Also, although this overview focuses on E.coli, protein expression and purification, other commonly used expression systems are mentioned and, apart from cell-breakage methods, protein purification methods and strategies are essentially the same.

  4. Dielectronic Recombination In Active Galactic Nuclei

    NASA Astrophysics Data System (ADS)

    Lukić, D.; Savin, D. W.; Schnell, M.; Brandau, C.; Schmidt, E.; Schippers, S.; Müller, A.; Lestinsky, M.; Sprenger, F.; Wolf, A.; Altun, Z.; Badnell, N. R.

    2006-05-01

    Recent X-ray satelitte observations of active galactic nuclei point out shortcomings in our understanding of low temperature dielectronic recombination (DR) for iron M- shell ions. In order to resolve this issue and to provide reliable iron M-shell DR data for modeling astrophysical plasmas, we are carrying out a series of laboratory measurements using the heavy-ion Test Storage Ring at the Max- Plank-Institute for Nuclear Physics in Heidelberg, Germany. Storage rings are currently the only laboratory method capable of studying low temperature DR. We use our results to produce experimentally- derived DR rate coefficients. We are also providing our data to atomic theorist to benchmark their DR calculations. Here we will report our recent DR results for selected Fe M-shell ions. At temperatures where these ions are predicted to form in photoionized gas, we find a significant discrepancy between our experimental results and previously recommended DR rate coefficients.

  5. Meiotic Recombination: The Essence of Heredity.

    PubMed

    Hunter, Neil

    2015-10-28

    The study of homologous recombination has its historical roots in meiosis. In this context, recombination occurs as a programmed event that culminates in the formation of crossovers, which are essential for accurate chromosome segregation and create new combinations of parental alleles. Thus, meiotic recombination underlies both the independent assortment of parental chromosomes and genetic linkage. This review highlights the features of meiotic recombination that distinguish it from recombinational repair in somatic cells, and how the molecular processes of meiotic recombination are embedded and interdependent with the chromosome structures that characterize meiotic prophase. A more in-depth review presents our understanding of how crossover and noncrossover pathways of meiotic recombination are differentiated and regulated. The final section of this review summarizes the studies that have defined defective recombination as a leading cause of pregnancy loss and congenital disease in humans.

  6. Super-dissociative recombination of H2+?

    NASA Astrophysics Data System (ADS)

    Mitchell, J. B. A.; Yousif, F. B.; van der Donk, P. J. T.; Morgan, T. J.; Chibisov, M. I.

    1995-11-01

    The dissociative recombination of vibrationally excited H2+ ions to form products in high Rydberg states has been measured. Surprisingly large cross-sections are found for this channel. This seems to be an example of super-dissociative recombination.

  7. Recombinant DNA: History of the Controversy.

    ERIC Educational Resources Information Center

    Vigue, Charles L.; Stanziale, William G.

    1979-01-01

    The hazards associated with recombinant DNA research are presented along with some social implications and the development of recombinant DNA research guidelines by the National Institutes of Health. (SA)

  8. Nondisjunction of chromosome 15: origin and recombination.

    PubMed Central

    Robinson, W P; Bernasconi, F; Mutirangura, A; Ledbetter, D H; Langlois, S; Malcolm, S; Morris, M A; Schinzel, A A

    1993-01-01

    Thirty-two cases of uniparental disomy (UPD), ascertained from Prader-Willi syndrome patients (N = 27) and Angelman syndrome patients (N = 5), are used to investigate the pattern of recombination associated with nondisjunction of chromosome 15. In addition, the meiotic stage of nondisjunction is inferred by using markers mapping near the centromere. Two basic approaches to the analysis of recombination are utilized. Standard methods of centromere mapping are employed to determine the level of recombination in specific pairwise intervals along the chromosome. This method shows a significant reduction in recombination for two of five intervals examined. Second, the observed frequency of each recombinant class (i.e., zero, one, two, three, or more observable crossovers) is compared with expected values. This is useful for testing whether the reduction in recombination can be attributed solely to a proportion of cases with no recombination at all (because of asynapsis), with the remaining groups showing normal recombination (or even excess recombination), or whether recombination is uniformly reduced. Analysis of maternal UPD(15) data shows a slight reduction in the multiple-recombinant classes, with a corresponding increase in both the zero- and one-recombinant classes over expected values. The majority, more than 82%, of the extra chromosomes in maternal UPD(15) cases are due to meiotic I nondisjunction events. In contrast, most paternal UPD(15) cases so far examined appear to have a postzygotic origin of the extra paternal chromosome. PMID:8352279

  9. Theoretical prediction of the impact of Auger recombination on charge collection from an ion track

    NASA Technical Reports Server (NTRS)

    Edmonds, Larry D.

    1991-01-01

    A recombination mechanism that significantly reduces charge collection from very dense ion tracks in silicon devices was postulated by Zoutendyk et al. The theoretical analysis presented here concludes that Auger recombination is such a mechanism and is of marginal importance for higher density tracks produced by 270-MeV krypton, but of major importance for higher density tracks. The analysis shows that recombination loss is profoundly affected by track diffusion. As the track diffuses, the density and recombination rate decrease so fast that the linear density (number of electron-hole pairs per unit length) approaches a non-zero limiting value as t yields infinity. Furthermore, the linear density is very nearly equal to this limiting value in a few picoseconds or less. When Auger recombination accompanies charge transport processes that have much longer time scales, it can be simulated by assigning a reduced linear energy transfer to the ion.

  10. Heterologous Expression, Purification, and Immunological Reactivity of a Recombinant HSP60 from Paracoccidioides brasiliensis

    PubMed Central

    Cunha, Daniela A.; Zancopé-Oliveira, Roseli M.; Sueli, M.; Felipe, S.; Salem-Izacc, Silvia M.; Deepe Jr., George S.; Soares, Célia M. A.

    2002-01-01

    The complete coding cDNA of HSP60 from Paracoccidioides brasiliensis was overexpressed in an Escherichia coli host to produce high levels of recombinant protein. The protein was purified by affinity chromatography. A total of 169 human serum samples were tested for reactivity by Western blot analysis with the purified HSP60 recombinant protein. Immunoblots indicated that the recombinant P. brasiliensis HSP60 was recognized by antibodies in 72 of 75 sera from paracoccidioidomycosis patients. No cross-reactivity was detected with individual sera from patients with aspergillosis, sporotrichosis, cryptococcosis, and tuberculosis. Reactivity to HSP60 was observed in sera from 9.52% of control healthy individuals and 11.5% of patients with histoplasmosis. The high sensitivity and specificity (97.3 and 92.5%, respectively) for HSP60 suggested that the recombinant protein can be used singly or in association with other recombinant antigens to detect antibody responses in P. brasiliensis-infected patients. PMID:11874881

  11. Production of recombinant miraculin using transgenic tomatoes in a closed cultivation system.

    PubMed

    Hirai, Tadayoshi; Fukukawa, Go; Kakuta, Hideo; Fukuda, Naoya; Ezura, Hiroshi

    2010-05-26

    We constructed a cultivation system with a controlled light period, light intensity, temperature, and CO(2) concentration for mass production of the taste-modifying protein miraculin from transgenic tomatoes. The tomato plants exhibited normal growth and produced over 270 g of fresh weight (FW) fruit per plant, with the recombinant miraculin concentration reaching up to 90 microg per g FW of tomatoes. The recombinant miraculin content of transgenic tomatoes was compared to that of plants grown in a netted greenhouse. The recombinant miraculin content of transgenic tomatoes grown in a closed cultivation system was more stable than that of tomatoes grown in a netted greenhouse, suggesting that the closed cultivation system is suitable for the production of recombinant miraculin. We estimate that 45 tFW of tomatoes and 4 kg of recombinant miraculin per 1,000 m(2) of cultivation area can be harvested per year.

  12. [Quantitative specific detection of Staphylococcus aureus based on recombinant lysostaphin and ATP bioluminescence].

    PubMed

    Li, Yuyuan; Mi, Zhiqiang; An, Xiaoping; Zhou, Yusen; Tong, Yigang

    2014-08-01

    Quantitative specific detection of Staphylococcus aureus is based on recombinant lysostaphin and ATP bioluminescence. To produce recombinant lysostaphin, the lysostaphin gene was chemically synthesized and inserted it into prokaryotic expression vector pQE30, and the resulting expression plasmid pQE30-Lys was transformed into E. coli M15 for expressing lysostaphin with IPTG induction. The recombinant protein was purified by Ni(2+)-NTA affinity chromatography. Staphylococcus aureus was detected by the recombinant lysostaphin with ATP bioluminescence, and plate count method. The results of the two methods were compared. The recombinant lysostaphin was successfully expressed, and a method of quantitative specific detection of S. aureus has been established, which showed a significant linear correlation with the colony counting. The detection method developed has good perspective to quantify S. aureus.

  13. Extended recombinant bacterial ghost system.

    PubMed

    Lubitz, W; Witte, A; Eko, F O; Kamal, M; Jechlinger, W; Brand, E; Marchart, J; Haidinger, W; Huter, V; Felnerova, D; Stralis-Alves, N; Lechleitner, S; Melzer, H; Szostak, M P; Resch, S; Mader, H; Kuen, B; Mayr, B; Mayrhofer, P; Geretschläger, R; Haslberger, A; Hensel, A

    1999-08-20

    Controlled expression of cloned PhiX174 gene E in Gram-negative bacteria results in lysis of the bacteria by formation of an E-specific transmembrane tunnel structure built through the cell envelope complex. Bacterial ghosts from a variety of bacteria are used as non-living candidate vaccines. In the recombinant ghost system, foreign proteins are attached on the inside of the inner membrane as fusions with specific anchor sequences. Ghosts have a sealed periplasmic space and the export of proteins into this space vastly extends the capacity of ghosts or recombinant ghosts to function as carriers of foreign antigens. In addition, S-layer proteins forming shell-like self assembly structures can be expressed in candidate vaccine strains prior to E-mediated lysis. Such recombinant S-layer proteins carrying foreign epitopes further extend the possibilities of ghosts as carriers of foreign epitopes. As ghosts have inherent adjuvant properties, they can be used as adjuvants in combination with subunit vaccines. Subunits or other ligands can also be coupled to matrixes like dextran which are used to fill the internal lumen of ghosts. Oral, aerogenic or parenteral immunization of experimental animals with recombinant ghosts induced specific humoral and cellular immune responses against bacterial and target components including protective mucosal immunity. The most relevant advantage of recombinant bacterial ghosts as immunogens is that no inactivation procedures that denature relevant immunogenic determinants are employed in this production. This fact explains the superior quality of ghosts when compared to other inactivated vaccines. The endotoxic component of the outer membrane does not limit the use of ghosts as vaccine candidates but triggers the release of several potent immunoregulatory cytokines. As carriers, there is no limitation in the size of foreign antigens that can be inserted in the membrane and the capacity of all spaces including the membranes, peri

  14. Adaptive evolution of a lactose-consuming Saccharomyces cerevisiae recombinant.

    PubMed

    Guimarães, Pedro M R; François, Jean; Parrou, Jean Luc; Teixeira, José A; Domingues, Lucília

    2008-03-01

    The construction of Saccharomyces cerevisiae strains that ferment lactose has biotechnological interest, particularly for cheese whey fermentation. A flocculent lactose-consuming S. cerevisiae recombinant expressing the LAC12 (lactose permease) and LAC4 (beta-galactosidase) genes of Kluyveromyces lactis was constructed previously but showed poor efficiency in lactose fermentation. This strain was therefore subjected to an evolutionary engineering process (serial transfer and dilution in lactose medium), which yielded an evolved recombinant strain that consumed lactose twofold faster, producing 30% more ethanol than the original recombinant. We identified two molecular events that targeted the LAC construct in the evolved strain: a 1,593-bp deletion in the intergenic region (promoter) between LAC4 and LAC12 and a decrease of the plasmid copy number by about 10-fold compared to that in the original recombinant. The results suggest that the intact promoter was unable to mediate the induction of the transcription of LAC4 and LAC12 by lactose in the original recombinant and that the deletion established the transcriptional induction of both genes in the evolved strain. We propose that the tuning of the expression of the heterologous LAC genes in the evolved recombinant was accomplished by the interplay between the decreased copy number of both genes and the different levels of transcriptional induction for LAC4 and LAC12 resulting from the changed promoter structure. Nevertheless, our results do not exclude other possible mutations that may have contributed to the improved lactose fermentation phenotype. This study illustrates the usefulness of simple evolutionary engineering approaches in strain improvement. The evolved strain efficiently fermented threefold-concentrated cheese whey, providing an attractive alternative for the fermentation of lactose-based media.

  15. Mechanisms of fever induced by recombinant human interferon.

    PubMed Central

    Dinarello, C A; Bernheim, H A; Duff, G W; Le, H V; Nagabhushan, T L; Hamilton, N C; Coceani, F

    1984-01-01

    Since the early trials using human interferon (hIFN) derived from blood leukocytes or cell lines, fever has been a prominent component of IFN therapy. Human protein impurities might account for the fever to cell-derived hIFN, but recombinant hIFN, free of extraneous human proteins, has produced fever in nearly all recipients during clinical trials. Our present studies were carried out to determine the mechanisms of fever due to recombinant hIFN currently being used in humans. Because recombinant hIFN is produced in Escherichia coli, in these experiments we considered contaminating endotoxin as the cause of fever. Polymyxin B, which blocks endotoxin, had no effect on the pyrogenicity of hIFN in rabbits. In addition, hIFN injected into an endotoxin-resistant strain of mice produced fever. The pyrogenicity of hIFN does not appear to involve production of leukocytic pyrogen (LP), since no circulating LP was detected in rabbits during IFN fever. Furthermore, human mononuclear cells incubated with hIFN in vitro at 10(4)-10(6) U/ml did not release LP. However, hIFN stimulated prostaglandin E2 (PGE2) release from rabbit hypothalamic tissue in vitro. Intracerebroventricular injection of hIFN into the awake cat also produced fever and a rise in PGE2 levels in the cerebrospinal fluid; both effects were reversed by treatment with indomethacin. We conclude that the fever of recombinant hIFN is not due to endotoxin but that hIFN is intrinsically pyrogenic by inducing PGE2 in the hypothalamus. PMID:6590569

  16. Recombination Catalysts for Hypersonic Fuels

    NASA Technical Reports Server (NTRS)

    Chinitz, W.

    1998-01-01

    The goal of commercially-viable access to space will require technologies that reduce propulsion system weight and complexity, while extracting maximum energy from the products of combustion. This work is directed toward developing effective nozzle recombination catalysts for the supersonic and hypersonic aeropropulsion engines used to provide such access to space. Effective nozzle recombination will significantly reduce rk=le length (hence, propulsion system weight) and reduce fuel requirements, further decreasing the vehicle's gross lift-off weight. Two such catalysts have been identified in this work, barium and antimony compounds, by developing chemical kinetic reaction mechanisms for these materials and determining the engine performance enhancement for a typical flight trajectory. Significant performance improvements are indicated, using only 2% (mole or mass) of these compounds in the combustor product gas.

  17. Selection of Recombinant Human Antibodies.

    PubMed

    Tomszak, Florian; Weber, Susanne; Zantow, Jonas; Schirrmann, Thomas; Hust, Michael; Frenzel, André

    2016-01-01

    Since the development of therapeutic antibodies the demand of recombinant human antibodies is steadily increasing. Traditionally, therapeutic antibodies were generated by immunization of rat or mice, the generation of hybridoma clones, cloning of the antibody genes and subsequent humanization and engineering of the lead candidates. In the last few years, techniques were developed that use transgenic animals with a human antibody gene repertoire. Here, modern recombinant DNA technologies can be combined with well established immunization and hybridoma technologies to generate already affinity maturated human antibodies. An alternative are in vitro technologies which enabled the generation of fully human antibodies from antibody gene libraries that even exceed the human antibody repertoire. Specific antibodies can be isolated from these libraries in a very short time and therefore reduce the development time of an antibody drug at a very early stage.In this review, we describe different technologies that are currently used for the in vitro and in vivo generation of human antibodies.

  18. Recombinant protein polymers in biomaterials.

    PubMed

    Kim, Wookhyun

    2013-01-01

    Naturally occurring protein-based materials have been found that function as critical components in biomechanical response, fibers and adhesives. A relatively small but growing number of recombinant protein-based materials that mimic the desired features of their natural sources, such as collagens, elastins and silks, are considered as an alternative to conventional synthetic polymers. Advances in genetic engineering have facilitated the synthesis of repetitive protein polymers with precise control of molecular weights which are designed by using synthetic genes encoding tandem repeats of oligopeptide originating from a modular domain of natural proteins. Many repeat sequences as protein polymer building blocks adopt a well-defined secondary structure and undergo self-assembly to result in physically cross-linked networks or with chemical cross-linking so that further form three-dimensional architectures similar to natural counterparts. In this review, recombinant protein polymers currently developed will be presented that have emerged as promising class of next generation biomaterials.

  19. Recombinant vector and eukaryotic host transformed thereby

    SciTech Connect

    Sugden, W.M.

    1987-08-11

    A recombinant plasmid is described comprising: a segment from a first plasmid which is not a lymphotrophic herpes virus segment and which facilitates the replication of the recombinant plasmid in a prokaryotic host; a segment from a lymphotrophic herpes virus which is linked to the first plasmid segment such that is a capable of assisting in maintaining the recombinant plasmid as a plasmid if the recombinant plasmid is inserted into a eukaryotic host that has been transformed by the lymphotrophic herpes virus; and a foreign eukaryotic gene component linked as part of the recombinant plasmid.

  20. Novel applications of recombinant erythropoietin.

    PubMed

    Sharples, Edward J; Thiemermann, Christoph; Yaqoob, Magdi M

    2006-04-01

    Recombinant erythropoietin (EPO) was introduced into clinical practice after the identification of EPO as the major haemopoietic growth factor determining survival and maturation of erythroid precursors. Advances in our understanding of the novel sites of action of EPO in the vasculature, brain, heart and kidney have opened new avenues of therapeutic potential for EPO, and have led to an increased understanding of the biological roles of EPO and its mechanisms of cell protection.

  1. Selenium incorporation using recombinant techniques.

    PubMed

    Walden, Helen

    2010-04-01

    Using selenomethionine to phase macromolecular structures is common practice in structure determination, along with the use of selenocysteine. Selenium is consequently the most commonly used heavy atom for MAD. In addition to the well established recombinant techniques for the incorporation of selenium in prokaryal expression systems, there have been recent advances in selenium labelling in eukaryal expression, which will be discussed. Tips and things to consider for the purification and crystallization of seleno-labelled proteins are also included.

  2. Contribution of near threshold states to dielectronic recombination in recombining plasma with Li-like Al ions

    NASA Astrophysics Data System (ADS)

    Stancalie, V.

    2012-05-01

    The dielectronic recombination of Li-like into Be-like aluminum ions, in laser-produced plasmas with Li-like ions, is re-visited with respect to the contribution of near-threshold states. Results are shown for recombining laser-produced plasma regime. The relativistic Dirac R-matrix calculation is performed to output resonance energy levels and rates. The target energies, and orbitals, are calculated with the extended average level multi-configurational Dirac-Fock method in the general-purpose relativistic atomic structure package (GRASP), while for determining the plasma population densities distribution over the excited Rydberg states, the Atomic Data Analysis System (ADAS) package programs is used.

  3. Interchromosomal recombination in Zea mays.

    PubMed Central

    Hu, W; Timmermans, M C; Messing, J

    1998-01-01

    A new allele of the 27-kD zein locus in maize has been generated by interchromosomal recombination between chromosomes of two different inbred lines. A continuous patch of at least 11,817 bp of inbred W64A, containing the previously characterized Ra allele of the 27-kD zein gene, has been inserted into the genome of A188 by a single crossover. While both junction sequences are conserved, sequences of the two homologs between these junctions differ considerably. W64A contains the 7313-bp-long retrotransposon, Zeon-1. A188 contains a second copy of the 27-kD zein gene and a 2-kb repetitive element. Therefore, recombination results in a 7.3-kb insertion and a 14-kb deletion compared to the original S+A188 allele. If nonpairing sequences are looped out, 206 single base changes, frequently clustered, are present. The structure of this allele may explain how a recently discovered example of somatic recombination occurred in an A188/W64A hybrid. This would indicate that despite these sequence differences, pairing between these alleles could occur early during plant development. Therefore, such a somatically derived chimeric chromosome can also be heritable and give rise to new alleles. PMID:9799274

  4. Nondisjunction of chromosome 15: Origin and recombination

    SciTech Connect

    Robinson, W.P.; Bernasconi, F.; Schinzel, A.A.; Mutirangura, A.; Ledbetter, D.H. ); Langlois, S. ); Morris, M.A.; Malcolm, S.

    1993-09-01

    Thirty-two cases of uniparental disomy (UPD), ascertained from Prader-Willi syndrome patients (N=27) and Angelman syndrome patients (N-5), are used to investigate the pattern of recombination associated with nondisjunction of chromosome 15. In addition, the meiotic stage of nondisjunction is inferred by using markers mapping near the centromere. Two basic approaches to the analysis of recombination in specific pairwise intervals along the chromosome. This method shows a significant reduction in recombination for two of five intervals examined. Second, the observed frequency of each recombinant class (i.e., zero, one, two, three, or more observable crossovers) is compared with expected values. This is useful for testing whether the reduction in recombination can be attributed solely to a proportion of cases with no recombination at all (because of asynapsis), with the remaining groups showing normal recombination (or even excess recombination), or whether recombination is uniformly reduced. Analysis of maternal UPD(15) data shows a slight reduction in the multiple-recombinant classes, with a corresponding increase in both the zero- and one-recombinant classes over expected values. The majority, more than 82%, of the extra chromosomes in maternal UPD(15) cases are due to meiotic I nondisjunction events. In contrast, more paternal UPD(15) cases so far examined appear to have a postzygotic origin of the extra paternal chromosome. 33 refs., 1 fig., 7 tabs.

  5. Factors influencing recombination frequency and distribution in a human meiotic crossover hotspot.

    PubMed

    Jeffreys, Alec J; Neumann, Rita

    2005-08-01

    Little is known about the factors that influence the frequency and distribution of meiotic recombination events within human crossover hotspots. We now describe the detailed analysis of sperm recombination in the NID1 hotspot. Like the neighbouring MS32 hotspot, the NID1 hotspot is associated with a minisatellite, suggesting that hotspots predispose DNA to tandem repetition. Unlike MS32, crossover resolution breakpoints in NID1 avoid the minisatellite, producing a cold spot within the hotspot. This avoidance may be related to the palindromic nature of the minisatellite interfering with the generation and/or processing of recombination intermediates. The NID1 hotspot also contains a single nucleotide polymorphism (SNP) close to the centre, which appears to directly influence the frequency of crossover initiation. Quantitative gene conversion assays show that this SNP affects the frequency of gene conversion and crossover to a very similar extent, providing evidence that conversions and crossovers are triggered by the same recombination initiating events. The recombination-suppressing allele is over-transmitted to recombinant progeny, and provides the most dramatic example to date of recombination-mediated meiotic drive, of a magnitude sufficient to virtually guarantee that the recombination suppressor will eventually replace the more active allele in human populations.

  6. The advances and perspectives of recombinant protein production in the silk gland of silkworm Bombyx mori.

    PubMed

    Xu, Hanfu

    2014-10-01

    The silk gland of silkworm Bombyx mori, is one of the most important organs that has been fully studied and utilized so far. It contributes finest silk fibers to humankind. The silk gland has excellent ability of synthesizing silk proteins and is a kind tool to produce some useful recombinant proteins, which can be widely used in the biological, biotechnical and pharmaceutical application fields. It's a very active area to express recombinant proteins using the silk gland as a bioreactor, and great progress has been achieved recently. This review recapitulates the progress of producing recombinant proteins and silk-based biomaterials in the silk gland of silkworm in addition to the construction of expression systems. Current challenges and future trends in the production of valuable recombinant proteins using transgenic silkworms are also discussed.

  7. In situ recovery of lycopene during biosynthesis with recombinant Escherichia coli.

    PubMed

    Yoon, Ko-Woon; Doo, Eun-Hee; Kim, Seon-Won; Park, Jin-Byung

    2008-06-30

    Lycopene is produced by recombinant Escherichia coli expressing genes to encode for the lycopene biosynthesis. However, the productivity of lycopene seemed to be limited by many factors including product toxicity. In the present study, we have investigated physiology of recombinant E. coli during biosynthesis and in situ recovery of lycopene based on an organic/aqueous two-phase system. Lycopene, the 40-carbon molecule product, was little extracted from recombinant E. coli cells to octane or decane phase. However, partial digestion of cell walls with lysozyme promoted extraction of lycopene into the organic phases. Engineering of an organic/aqueous two-phase system allowed recombinant E. coli cells to produce ca. 40% larger amount of lycopene compared to that in a conventional aqueous single-phase system. Optimization of the in situ product recovery process will lead to further increase of product concentration and productivity.

  8. Mechanistic features of recombination in HIV.

    PubMed

    Galetto, Román; Negroni, Matteo

    2005-01-01

    The importance of recombination in retroviral evolution has been acknowledged for several decades. Consequently, after the identification of HIV as the etiological agent of AIDS, it was suspected that recombination could also play a central role in the evolution of this virus. However, only recently, extensive epidemiologic studies of HIV infections worldwide have provided an estimate for the occurrence of recombination in vivo, unveiling recombination frequencies that dwarf those initially expected. Nowadays, recombination is regarded as an integral part of the infectious cycle of this retrovirus, which impacts on diagnosis and treatment of infections, especially when genetically distant viruses have been at the origin of the recombinant forms. Retroviral recombination is observed when two genetically divergent genomic RNA molecules are present in the same viral particle, and arises during the reverse transcription step. This review focuses on the mechanisms that have been proposed to account for the occurrence of recombination in retroviruses, from the strand displacement model, according to which recombination occurs during second DNA strand synthesis; to the description of the factors responsible for copy-choice recombination during first DNA strand synthesis, such as the presence of breaks, pause sites, or secondary structures in the genomic RNA. Most of these models have been supported by experimental data obtained from in vitro reconstituted systems or from cell infection studies using academic model sequences. The situation in vivo is expected to be more complex, since several factors come into play when recombination involves relatively distant isolates, as in the case of inter-subtype recombination. At present, it is clear that further studies are needed in order to evaluate whether a prevailing mechanism exists for in vivo recombination, and these studies will also be essential for understanding how the underlying mechanisms of recombination contribute

  9. Generation of pure spin currents via Auger recombination in quantum wells with Rashba splitting

    SciTech Connect

    Afanasiev, A. N. Greshnov, A. A. Greshnov, A. A.

    2015-10-15

    We propose a nonoptical mechanism for generating spin current via Auger recombination in semiconductor quantum wells (QWs) with spin–orbit splitting associated with structural QW asymmetry. It is shown that Auger recombination in narrow-bandgap semiconductors makes it possible to produce spin currents that exceed those that are obtained in the case of intraband as well as interband optical excitation. Analysis shows that the interference term in the expression for the Auger-recombination rate is responsible for the generation of spin currents.

  10. Strand invasion promoted by recombination protein of coliphage

    NASA Astrophysics Data System (ADS)

    Rybalchenko, Nataliya; Golub, Efim I.; Bi, Baoyuan; Radding, Charles M.

    2004-12-01

    Studies of phage in vivo have indicated that its own recombination enzymes, protein and exonuclease, are capable of catalyzing two dissimilar pathways of homologous recombination that are widely distributed in nature: single-strand annealing and strand invasion. The former is an enzymatic splicing of overlapping ends of broken homologous DNA molecules, whereas the latter is characterized by the formation of a three-stranded synaptic intermediate and subsequent strand exchange. Previous studies in vitro have shown that protein has annealing activity, and that exonuclease, acting on branched substrates, can produce a perfect splice that requires only ligation for completion. The present study shows that protein can initiate strand invasion in vitro, as evidenced both by the formation of displacement loops (D-loops) in superhelical DNA and by strand exchange between colinear single-stranded and double-stranded molecules. Thus, protein can catalyze steps that are central to both strand annealing and strand invasion pathways of recombination. These observations add protein to a set of diverse proteins that appear to promote recognition of homology by a unitary mechanism governed by the intrinsic dynamic properties of base pairs in DNA. genetic recombination | phage λ

  11. Role of Recombinant DNA Technology to Improve Life

    PubMed Central

    Khan, Suliman; Ullah, Muhammad Wajid; Siddique, Rabeea; Nabi, Ghulam; Manan, Sehrish; Yousaf, Muhammad

    2016-01-01

    In the past century, the recombinant DNA technology was just an imagination that desirable characteristics can be improved in the living bodies by controlling the expressions of target genes. However, in recent era, this field has demonstrated unique impacts in bringing advancement in human life. By virtue of this technology, crucial proteins required for health problems and dietary purposes can be produced safely, affordably, and sufficiently. This technology has multidisciplinary applications and potential to deal with important aspects of life, for instance, improving health, enhancing food resources, and resistance to divergent adverse environmental effects. Particularly in agriculture, the genetically modified plants have augmented resistance to harmful agents, enhanced product yield, and shown increased adaptability for better survival. Moreover, recombinant pharmaceuticals are now being used confidently and rapidly attaining commercial approvals. Techniques of recombinant DNA technology, gene therapy, and genetic modifications are also widely used for the purpose of bioremediation and treating serious diseases. Due to tremendous advancement and broad range of application in the field of recombinant DNA technology, this review article mainly focuses on its importance and the possible applications in daily life. PMID:28053975

  12. Production of recombinant proteins in microalgae at pilot greenhouse scale.

    PubMed

    Gimpel, Javier A; Hyun, James S; Schoepp, Nathan G; Mayfield, Stephen P

    2015-02-01

    Recombinant protein production in microalgae chloroplasts can provide correctly folded proteins in significant quantities and potentially inexpensive costs compared to other heterologous protein production platforms. The best results have been achieved by using the psbA promoter and 5' untranslated region (UTR) to drive the expression of heterologous genes in a psbA-deficient, non-photosynthetic, algal host. Unfortunately, using such a strategy makes the system unviable for large scale cultivation using natural sunlight for photosynthetic growth. In this study we characterized eight different combinations of 5' regulatory regions and psbA coding sequences for their ability to restore photosynthesis in a psbA-deficient Chlamydomonas reinhardtii, while maintaining robust accumulation of a commercially viable recombinant protein driven by the psbA promoter/5'UTR. The recombinant protein corresponded to bovine Milk Amyloid A (MAA), which is present in milk colostrum and could be used to prevent infectious diarrhea in mammals. This approach allowed us to identify photosynthetic strains that achieved constitutive production of MAA when grown photosynthetically in 100 L bags in a greenhouse. Under these conditions, the maximum MAA expression achieved was 1.86% of total protein, which corresponded to 3.28 mg/L of culture medium. Within our knowledge, this is the first report of a recombinant protein being produced this way in microalgae.

  13. Role of Recombinant DNA Technology to Improve Life.

    PubMed

    Khan, Suliman; Ullah, Muhammad Wajid; Siddique, Rabeea; Nabi, Ghulam; Manan, Sehrish; Yousaf, Muhammad; Hou, Hongwei

    2016-01-01

    In the past century, the recombinant DNA technology was just an imagination that desirable characteristics can be improved in the living bodies by controlling the expressions of target genes. However, in recent era, this field has demonstrated unique impacts in bringing advancement in human life. By virtue of this technology, crucial proteins required for health problems and dietary purposes can be produced safely, affordably, and sufficiently. This technology has multidisciplinary applications and potential to deal with important aspects of life, for instance, improving health, enhancing food resources, and resistance to divergent adverse environmental effects. Particularly in agriculture, the genetically modified plants have augmented resistance to harmful agents, enhanced product yield, and shown increased adaptability for better survival. Moreover, recombinant pharmaceuticals are now being used confidently and rapidly attaining commercial approvals. Techniques of recombinant DNA technology, gene therapy, and genetic modifications are also widely used for the purpose of bioremediation and treating serious diseases. Due to tremendous advancement and broad range of application in the field of recombinant DNA technology, this review article mainly focuses on its importance and the possible applications in daily life.

  14. Clonal reproduction with androgenesis and somatic recombination: the case of the ant Cardiocondyla kagutsuchi.

    PubMed

    Okita, Ichiro; Tsuchida, Koji

    2016-04-01

    In haplodiploid insects such as ants, male sexuals develop from unfertilised haploid eggs, while female sexuals and workers develop from fertilized diploid eggs. However, some ant species do not exchange their gene pool between sexes; both male and female sexuals are clonally produced, while workers are sexually produced. To date, three ant species, Wasmannia auropunctata, Vollenhovia emeryi, and Paratrechina longicornis, have been reported to reproduce using such reproductive systems. In this study, we reveal that in one lineage of the ant Cardiocondyla kagutsuchi, male and female sexuals are also clonally produced. In contrast to the abovementioned three species, the workers were not only sexually produced but had recombinant sequences in their nuclear internal transcribed spacer regions, although the recombinant sequences were not detected in male or female sexuals. These results suggest that the lineage likely possesses a mechanism to compensate for the reduction in genetic variation due to clonal reproduction with somatic recombination that occurs within the workers.

  15. Clonal reproduction with androgenesis and somatic recombination: the case of the ant Cardiocondyla kagutsuchi

    NASA Astrophysics Data System (ADS)

    Okita, Ichiro; Tsuchida, Koji

    2016-04-01

    In haplodiploid insects such as ants, male sexuals develop from unfertilised haploid eggs, while female sexuals and workers develop from fertilized diploid eggs. However, some ant species do not exchange their gene pool between sexes; both male and female sexuals are clonally produced, while workers are sexually produced. To date, three ant species, Wasmannia auropunctata, Vollenhovia emeryi, and Paratrechina longicornis, have been reported to reproduce using such reproductive systems. In this study, we reveal that in one lineage of the ant Cardiocondyla kagutsuchi, male and female sexuals are also clonally produced. In contrast to the abovementioned three species, the workers were not only sexually produced but had recombinant sequences in their nuclear internal transcribed spacer regions, although the recombinant sequences were not detected in male or female sexuals. These results suggest that the lineage likely possesses a mechanism to compensate for the reduction in genetic variation due to clonal reproduction with somatic recombination that occurs within the workers.

  16. Gene Disruption by Homologous Recombination in the Xylella fastidiosa Citrus Variegated Chlorosis Strain

    PubMed Central

    Gaurivaud, Patrice; Souza, Leonardo C. A.; Virgílio, Andrea C. D.; Mariano, Anelise G.; Palma, Renê R.; Monteiro, Patrícia B.

    2002-01-01

    Mutagenesis by homologous recombination was evaluated in Xylella fastidiosa by using the bga gene, coding for β-galactosidase, as a model. Integration of replicative plasmids by homologous recombination between the cloned truncated copy of bga and the endogenous gene was produced by one or two crossover events leading to β-galactosidase mutants. A promoterless chloramphenicol acetyltransferase gene was used to monitor the expression of the target gene and to select a cvaB mutant. PMID:12200328

  17. Developing recombinant antibodies for biomarker detection

    SciTech Connect

    Baird, Cheryl L.; Fischer, Christopher J.; Pefaur, Noah B.; Miller, Keith D.; Kagen, Jacob; Srivastava, Sudhir; Rodland, Karin D.

    2010-10-01

    Monoclonal antibodies (mAbs) have an essential role in biomarker validation and diagnostic assays. A barrier to pursuing these applications is the reliance on immunization and hybridomas to produce mAbs, which is time-consuming and may not yield the desired mAb. We recommend a process flow for affinity reagent production that utilizes combinatorial protein display systems (eg, yeast surface display or phage display) rather than hybridomas. These systems link a selectable phenotype-binding conferred by an antibody fragment-with a means for recovering the encoding gene. Recombinant libraries obtained from immunizations can produce high-affinity antibodies (<10 nM) more quickly than other methods. Non-immune libraries provide an alternate route when immunizations are not possible, or when suitable mAbs are not recovered from an immune library. Directed molecular evolution (DME) is an integral part of optimizing mAbs obtained from combinatorial protein display, but can also be used on hybridoma-derived mAbs. Variants can easily be obtained and screened to increase the affinity of the parent mAb (affinity maturation). We discuss examples where DME has been used to tailor affinity reagents to specific applications. Combinatorial protein display also provides an accessible method for identifying antibody pairs, which are necessary for sandwich-type diagnostic assays.

  18. Hydrogen from Water in a Novel Recombinant Oxygen-Tolerant Cyanobacterial System (Presentation)

    SciTech Connect

    Xu, Q.; Smith, H. O.; Maness, P.-C.

    2007-05-01

    The objective of this report is to develop an O{sub 2}-tolerant cyanobacterial system for continuous light-driven H{sub 2} production from water. The overall goal is to produce a cyanobacterial recombinant to produce H{sub 2} continuously.

  19. Infectious vaccinia virus recombinants that express hepatitis B virus surface antigen

    NASA Astrophysics Data System (ADS)

    Smith, Geoffrey L.; Mackett, Michael; Moss, Bernard

    1983-04-01

    Potential live vaccines against hepatitis B virus have been produced. The coding sequence for hepatitis B virus surface antigen (HBsAg) has been inserted into the vaccinia virus genome under control of vaccinia virus early promoters. Cells infected with these vaccinia virus recombinants synthesize and excrete HBsAg and vaccinated rabbits rapidly produce antibodies to HBsAg.

  20. Analysis of Recombinant Proteins in Transgenic Rice Seeds: Identity, Localization, Tolerance to Digestion, and Plant Stress Response.

    PubMed

    Wakasa, Yuhya; Takaiwa, Fumio

    2016-01-01

    Rice seeds are an ideal production platform for high-value recombinant proteins in terms of economy, scalability, safety, and stability. Strategies for the expression of large amounts of recombinant proteins in rice seeds have been established in the past decade and transgenic rice seeds that accumulate recombinant products such as bioactive peptides and proteins, which promote the health and quality of life of humans, have been generated in many laboratories worldwide. One of the most important advantages is the potential for direct oral delivery of transgenic rice seeds without the need for recombinant protein purification (downstream processing), which has been attributed to the high expression levels of recombinant products. Transgenic rice will be beneficial as a delivery system for pharmaceuticals and nutraceuticals in the future. This chapter introduces the strategy for producing recombinant protein in the edible part (endosperm) of the rice grain and describes methods for the analysis of transgenic rice seeds in detail.

  1. Frequency and genetic characterization of V(DD)J recombinants in the human peripheral blood antibody repertoire.

    PubMed

    Briney, Bryan S; Willis, Jordan R; Hicar, Mark D; Thomas, James W; Crowe, James E

    2012-09-01

    Antibody heavy-chain recombination that results in the incorporation of multiple diversity (D) genes, although uncommon, contributes substantially to the diversity of the human antibody repertoire. Such recombination allows the generation of heavy chain complementarity determining region 3 (HCDR3) regions of extreme length and enables junctional regions that, because of the nucleotide bias of N-addition regions, are difficult to produce through normal V(D)J recombination. Although this non-classical recombination process has been observed infrequently, comprehensive analysis of the frequency and genetic characteristics of such events in the human peripheral blood antibody repertoire has not been possible because of the rarity of such recombinants and the limitations of traditional sequencing technologies. Here, through the use of high-throughput sequencing of the normal human peripheral blood antibody repertoire, we analysed the frequency and genetic characteristics of V(DD)J recombinants. We found that these recombinations were present in approximately 1 in 800 circulating B cells, and that the frequency was severely reduced in memory cell subsets. We also found that V(DD)J recombination can occur across the spectrum of diversity genes, indicating that virtually all recombination signal sequences that flank diversity genes are amenable to V(DD)J recombination. Finally, we observed a repertoire bias in the diversity gene repertoire at the upstream (5') position, and discovered that this bias was primarily attributable to the order of diversity genes in the genomic locus.

  2. Two-photon transitions in primordial hydrogen recombination

    NASA Astrophysics Data System (ADS)

    Hirata, Christopher M.

    2008-07-01

    The subject of cosmological hydrogen recombination has received much attention recently because of its importance to predictions for and cosmological constraints from cosmic microwave background observations. While the central role of the two-photon decay 2s→1s has been recognized for many decades, high-precision calculations require us to consider two-photon decays from the higher states ns, nd→1s (n≥3). Simple attempts to include these processes in recombination calculations with an effective two-photon decay coefficient analogous to the 2s decay coefficient Λ2s=8.22s-1 have suffered from physical problems associated with the existence of kinematically allowed sequences of one-photon decays, e.g. 3d→2p→1s, that technically also produce two photons. These correspond to resonances in the two-photon spectrum that are optically thick to two-photon absorption, necessitating a radiative transfer calculation. We derive the appropriate equations, develop a numerical code to solve them, and verify the results by finding agreement with analytic approximations to the radiative transfer equation. The related processes of Raman scattering and two-photon recombination are included using similar machinery. Our results show that early in recombination the two-photon decays act to speed up recombination, reducing the free electron abundance by 1.3% relative to the standard calculation at z=1300. However, we find that some photons between Lyα and Lyβ are produced, mainly by 3d→1s two-photon decay and 2s→1s Raman scattering. At later times, these photons redshift down to Lyα, excite hydrogen atoms, and act to slow recombination. Thus, the free electron abundance is increased by 1.3% relative to the standard calculation at z=900. Our calculation involves a very different physical argument than the recent studies of Wong and Scott and Chluba and Sunyaev, and produces a much larger effect on the ionization history. The implied correction to the cosmic microwave

  3. Current Trends of HIV Recombination Worldwide

    PubMed Central

    Lau, Katherine A.; Wong, Justin J.L.

    2013-01-01

    One of the major characteristics of HIV-1 is its high genetic variability and extensive heterogeneity. This characteristic is due to its molecular traits, which in turn allows it to vary, recombine, and diversify at a high frequency. As such, it generates complex molecular forms, termed recombinants, which evade the human immune system and so survive. There is no sequence constraint to the recombination pattern as it appears to occur at inter-group (between groups M and O), as well as interand intra-subtype within group M. Rapid emergence and active global transmission of HIV-1 recombinants, known as circulating recombinant forms (CRFs) and unique recombinant forms (URFs), requires urgent attention. To date, 55 CRFs have been reported around the world. The first CRF01_AE originated from Central Africa but spread widely in Asia. The most recent CRF; CRF55_01B is a recombinant form of CRF01_AE and subtype B, although its origin is yet to be publicly disclosed. HIV-1 recombination is an ongoing event and plays an indispensable role in HIV epidemics in different regions. Africa, Asia and South America are identified as recombination hot-spots. They are affected by continual emergence and cocirculation of newly emerging CRFs and URFs, which are now responsible for almost 20% of HIV-1 infections worldwide. Better understanding of recombinants is necessary to determine their biological and molecular attributes. PMID:24470968

  4. Method of producing a cellulase-containing cell-free fermentate produced from microorganism ATCC 55702

    DOEpatents

    Dees, H. Craig

    1998-01-01

    Bacteria which produce large amounts of cellulose-containing cell-free fermentate have been identified. The original bacterium (ATCC 55703) was genetically altered using nitrosoguanidine (MNNG) treatment to produce the enhanced cellulase producing bacterium (ATCC 55702), which was identified through replicate plating. ATCC 55702 has improved characteristics and qualities for the degradation of cellulosic waste materials for fuel production, food processing, textile processing, and other industrial applications. ATCC 55702 is an improved bacterial host for genetic manipulations using recombinant DNA techniques, and is less likely to destroy genetic manipulations using standard mutagenesis techniques.

  5. Method of producing a cellulase-containing cell-free fermentate produced from microorganism ATCC 55702

    DOEpatents

    Dees, H.C.

    1998-05-26

    Bacteria which produce large amounts of cellulose-containing cell-free fermentate have been identified. The original bacterium (ATCC 55703) was genetically altered using nitrosoguanidine (MNNG) treatment to produce the enhanced cellulase producing bacterium (ATCC 55702), which was identified through replicate plating. ATCC 55702 has improved characteristics and qualities for the degradation of cellulosic waste materials for fuel production, food processing, textile processing, and other industrial applications. ATCC 55702 is an improved bacterial host for genetic manipulations using recombinant DNA techniques, and is less likely to destroy genetic manipulations using standard mutagenesis techniques. 5 figs.

  6. Recombinant erythropoietin in clinical practice

    PubMed Central

    Ng, T; Marx, G; Littlewood, T; Macdougall, I

    2003-01-01

    The introduction of recombinant human erythropoietin (RHuEPO) has revolutionised the treatment of patients with anaemia of chronic renal disease. Clinical studies have demonstrated that RHuEPO is also useful in various non-uraemic conditions including haematological and oncological disorders, prematurity, HIV infection, and perioperative therapies. Besides highlighting both the historical and functional aspects of RHuEPO, this review discusses the applications of RHuEPO in clinical practice and the potential problems of RHuEPO treatment. PMID:12897214

  7. Recombinant canine single chain insulin analogues: insulin receptor binding capacity and ability to stimulate glucose uptake.

    PubMed

    Adams, Jamie P; Holder, Angela L; Catchpole, Brian

    2014-12-01

    Virtually all diabetic dogs require exogenous insulin therapy to control their hyperglycaemia. In the UK, the only licensed insulin product currently available is a purified porcine insulin preparation. Recombinant insulin is somewhat problematic in terms of its manufacture, since the gene product (preproinsulin) undergoes substantial post-translational modification in pancreatic β cells before it becomes biologically active. The aim of the present study was to develop recombinant canine single chain insulin (SCI) analogues that could be produced in a prokaryotic expression system and which would require minimal processing. Three recombinant SCI constructs were developed in a prokaryotic expression vector, by replacing the insulin C-peptide sequence with one encoding a synthetic peptide (GGGPGKR), or with one of two insulin-like growth factor (IGF)-2 C-peptide coding sequences (human: SRVSRRSR; canine: SRVTRRSSR). Recombinant proteins were expressed in the periplasmic fraction of Escherichia coli and assessed for their ability to bind to the insulin and IGF-1 receptors, and to stimulate glucose uptake in 3T3-L1 adipocytes. All three recombinant SCI analogues demonstrated preferential binding to the insulin receptor compared to the IGF-1 receptor, with increased binding compared to recombinant canine proinsulin. The recombinant SCI analogues stimulated glucose uptake in 3T3-L1 adipocytes compared to negligible uptake using recombinant canine proinsulin, with the canine insulin/cIGF-2 chimaeric SCI analogue demonstrating the greatest effect. Thus, biologically-active recombinant canine SCI analogues can be produced relatively easily in bacteria, which could potentially be used for treatment of diabetic dogs.

  8. Regulation of recombination and genomic maintenance.

    PubMed

    Heyer, Wolf-Dietrich

    2015-08-03

    Recombination is a central process to stably maintain and transmit a genome through somatic cell divisions and to new generations. Hence, recombination needs to be coordinated with other events occurring on the DNA template, such as DNA replication, transcription, and the specialized chromosomal functions at centromeres and telomeres. Moreover, regulation with respect to the cell-cycle stage is required as much as spatiotemporal coordination within the nuclear volume. These regulatory mechanisms impinge on the DNA substrate through modifications of the chromatin and directly on recombination proteins through a myriad of posttranslational modifications (PTMs) and additional mechanisms. Although recombination is primarily appreciated to maintain genomic stability, the process also contributes to gross chromosomal arrangements and copy-number changes. Hence, the recombination process itself requires quality control to ensure high fidelity and avoid genomic instability. Evidently, recombination and its regulatory processes have significant impact on human disease, specifically cancer and, possibly, neurodegenerative diseases.

  9. Recombinant shrimp (Litopenaeus vannamei) trypsinogen production in Pichia pastoris.

    PubMed

    Guerrero-Olazarán, Martha; Escamilla-Treviño, Luis L; Castillo-Galván, Mauricio; Gallegos-López, Juan A; Viader-Salvadó, José M

    2009-01-01

    Shrimp (Litopenaeus vannamei) trypsinogen has never been isolated from its natural source. To assess the production of L. vannamei trypsinogen, we engineered Pichia pastoris strains and evaluated two culture approaches with three induction culture media, to produce recombinant shrimp trypsinogen for the first time. The trypsinogen II cDNA was fused to the signal sequence of the Saccharomyces cerevisiae alpha mating factor, placed under the control of the P. pastoris AOX1 promoter, and integrated into the genome of P. pastoris host strain GS115. Using standard culture conditions for heterologous gene induction of a GS115 strain in shake flasks, recombinant shrimp trypsinogen was not detected by SDS-PAGE and Western blot analysis. Growth kinetics revealed a toxicity of recombinant shrimp trypsinogen or its activated form over the cell host. Thus, a different culture approach was tested for the induction step, involving the use of high cell density cultures, a higher frequency of methanol feeding (every 12 h), and a buffered minimal methanol medium supplemented with sorbitol or alanine; alanine supplemented medium was found to be more efficient. After 96 h of induction with alanine supplemented medium, a 29-kDa band from the cell-free culture medium was clearly observed by SDS-PAGE, and confirmed by Western blot to be shrimp trypsinogen, at a concentration of 14 microg/mL. Our results demonstrate that high density cell cultures with alanine in the induction medium allow the production of recombinant shrimp trypsinogen using the P. pastoris expression system, because of improved cell viability and greater stability of the recombinant trypsinogen.

  10. Transient Expression of Tetrameric Recombinant Human Butyrylcholinesterase in Nicotiana benthamiana

    PubMed Central

    Alkanaimsh, Salem; Karuppanan, Kalimuthu; Guerrero, Andrés; Tu, Aye M.; Hashimoto, Bryce; Hwang, Min Sook; Phu, My L.; Arzola, Lucas; Lebrilla, Carlito B.; Dandekar, Abhaya M.; Falk, Bryce W.; Nandi, Somen; Rodriguez, Raymond L.; McDonald, Karen A.

    2016-01-01

    To optimize the expression, extraction and purification of plant-derived tetrameric recombinant human butyrylcholinesterase (prBChE), we describe the development and use of plant viral amplicon-based gene expression system; Tobacco Mosaic Virus (TMV) RNA-based overexpression vector (TRBO) to express enzymatically active FLAG-tagged plant made recombinant butyrylcholinesterase (rBChE) in Nicotiana benthamiana leaves using transient agroinfiltration. Two gene expression cassettes were designed to express the recombinant protein in either the ER or to the apoplastic compartment. Leaf homogenization was used to isolate ER-retained recombinant butyrylcholinesterase (prBChE-ER) while apoplast-targeted rBChE was isolated by either leaf homogenization (prBChE) or vacuum-extraction of apoplastic wash fluid (prBChE-AWF). rBChE from apoplast wash fluid had a higher specific activity but lower enzyme yield than leaf homogenate. To optimize the isolation and purification of total recombinant protein from leaf homogenates, an acidic extraction buffer was used. The acidic extraction buffer yielded >95% enzymatically active tetrameric rBChE as verified by Coomassie stained and native gel electrophoresis. Furthermore, when compared to human butyrylcholinesterase, the prBChE was found to be similar in terms of tetramerization and enzyme kinetics. The N-linked glycan profile of purified prBChE-ER was found to be mostly high mannose structures while the N-linked glycans on prBChE-AWF were primarily complex. The glycan profile of the prBChE leaf homogenates showed a mixture of high mannose, complex and paucimannose type N-glycans. These findings demonstrate the ability of plants to produce rBChE that is enzymatically active and whose oligomeric state is comparable to mammalian butyrylcholinesterase. The process of plant made rBChE tetramerization and strategies for improving its pharmacokinetics properties are also discussed. PMID:27379103

  11. Production of recombinant proteins in suspension-cultured plant cells.

    PubMed

    Plasson, Carole; Michel, Rémy; Lienard, David; Saint-Jore-Dupas, Claude; Sourrouille, Christophe; de March, Ghislaine Grenier; Gomord, Véronique

    2009-01-01

    Plants have emerged in the past decade as a suitable alternative to the current production systems for recombinant pharmaceutical proteins and, today their potential for low-cost production of high quality, much safer and biologically active mammalian proteins is largely documented. Among various plant expression systems being explored, genetically modified suspension-cultured plant cells offer a promising system for production of biopharmaceuticals. Indeed, when compared to other plant-based production platforms that have been explored, suspension-cultured plant cells have the advantage of being totally devoid of problems associated with the vagaries of weather, pest, soil and gene flow in the environment. Because of short growth cycles, the timescale needed for the production of recombinant proteins in plant cell culture can be counted in days or weeks after transformation compared to months needed for the production in transgenic plants. Moreover, recovery and purification of recombinant proteins from plant biomass is an expensive and technically challenging business that may amount to 80-94% of the final product cost. One additional advantage of plant cell culture is that the recombinant protein fused with a signal sequence can be expressed and secreted into the culture medium, and therefore recovered and purified in the absence of large quantities of contaminating proteins. Consequently, the downstream processing of proteins extracted from plant cell culture medium is less expensive, which may/does balance the higher costs of fermentation. When needed for clinical use, recombinant proteins are easily produced in suspension-cultured plant cells under certified, controllable and sterile conditions that offer improved safety and provide advantages for good manufacturing practices and regulatory compliance. In this chapter, we present basic protocols for rapid generation of transgenic suspension-cultured cells of Nicotiana tabacum, Oriza sativa and Arabidopis

  12. Rapid purification of recombinant histones.

    PubMed

    Klinker, Henrike; Haas, Caroline; Harrer, Nadine; Becker, Peter B; Mueller-Planitz, Felix

    2014-01-01

    The development of methods to assemble nucleosomes from recombinant histones decades ago has transformed chromatin research. Nevertheless, nucleosome reconstitution remains time consuming to this day, not least because the four individual histones must be purified first. Here, we present a streamlined purification protocol of recombinant histones from bacteria. We termed this method "rapid histone purification" (RHP) as it circumvents isolation of inclusion bodies and thereby cuts out the most time-consuming step of traditional purification protocols. Instead of inclusion body isolation, whole cell extracts are prepared under strongly denaturing conditions that directly solubilize inclusion bodies. By ion exchange chromatography, the histones are purified from the extracts. The protocol has been successfully applied to all four canonical Drosophila and human histones. RHP histones and histones that were purified from isolated inclusion bodies had similar purities. The different purification strategies also did not impact the quality of octamers reconstituted from these histones. We expect that the RHP protocol can be readily applied to the purification of canonical histones from other species as well as the numerous histone variants.

  13. Theory of recombination in directed molecular evolution

    NASA Astrophysics Data System (ADS)

    Peng, Weiqun; Hwa, Terence; Levine, Herbert; Kessler, David A.

    2003-03-01

    Recombination is a fundamental process driving the evolution of biological organisms in nature. It is also a very efficient method being used in in vitro directed evolution of molecules. Here we propose a simple model for the directed evolution of protein-binding DNA sequences subject to recombination, substitution, and competitive selection. This turns out to be a rare model of involving recombination which is analytically tractable. We characterize the dynamical and steady-state behaviors of this model and verify them numerically. We discuss the manner in which recombination drastically speeds up the evolutionary process.

  14. Recombinant cells that highly express chromosomally-integrated heterologous gene

    DOEpatents

    Ingram, Lonnie O.; Ohta, Kazuyoshi; Wood, Brent E.

    2007-03-20

    Recombinant host cells are obtained that comprise (A) a heterologous, polypeptide-encoding polynucleotide segment, stably integrated into a chromosome, which is under transcriptional control of an endogenous promoter and (B) a mutation that effects increased expression of the heterologous segment, resulting in enhanced production by the host cells of each polypeptide encoded by that segment, relative to production of each polypeptide by the host cells in the absence of the mutation. The increased expression thus achieved is retained in the absence of conditions that select for cells displaying such increased expression. When the integrated segment comprises, for example, ethanol-production genes from an efficient ethanol producer like Zymomonas mobilis, recombinant Escherichia coli and other enteric bacterial cells within the present invention are capable of converting a wide range of biomass-derived sugars efficiently to ethanol.

  15. Recombinant cells that highly express chromosomally-integrated heterologous genes

    DOEpatents

    Ingram, L.O.; Ohta, Kazuyoshi; Wood, B.E.

    1998-10-13

    Recombinant host cells are obtained that comprise (A) a heterologous, polypeptide-encoding polynucleotide segment, stably integrated into a chromosome, which is under transcriptional control of an endogenous promoter and (B) a mutation that effects increased expression of the heterologous segment, resulting in enhanced production by the host cells of each polypeptide encoded by that segment, relative to production of each polypeptide by the host cells in the absence of the mutation. The increased expression thus achieved is retained in the absence of conditions that select for cells displaying such increased expression. When the integrated segment comprises, for example, ethanol-production genes from an efficient ethanol producer like Zymomonas mobilis, recombinant Escherichia coli and other enteric bacterial cells within the present invention are capable of converting a wide range of biomass-derived sugars efficiently to ethanol. 13 figs.

  16. Recombinant cells that highly express chromosomally-integrated heterologous genes

    DOEpatents

    Ingram, Lonnie O.; Ohta, Kazuyoshi; Wood, Brent E.

    2000-08-22

    Recombinant host cells are obtained that comprise (A) a heterologous, polypeptide-encoding polynucleotide segment, stably integrated into a chromosome, which is under transcriptional control of an endogenous promoter and (B) a mutation that effects increased expression of the heterologous segment, resulting in enhanced production by the host cells of each polypeptide encoded by that segment, relative to production of each polypeptide by the host cells in the absence of the mutation. The increased expression thus achieved is retained in the absence of conditions that select for cells displaying such increased expression. When the integrated segment comprises, for example, ethanol-production genes from an efficient ethanol producer like Zymomonas mobilis, recombinant Escherichia coli and other enteric bacterial cells within the present invention are capable of converting a wide range of biomass-derived sugars efficiently to ethanol.

  17. Recombinant cells that highly express chromosomally-integrated heterologous genes

    DOEpatents

    Ingram, Lonnie O.; Ohta, Kazuyoshi; Wood, Brent E.

    1998-01-01

    Recombinant host cells are obtained that comprise (A) a heterologous, polypeptide-encoding polynucleotide segment, stably integrated into a chromosome, which is under transcriptional control of an endogenous promoter and (B) a mutation that effects increased expression of the heterologous segment, resulting in enhanced production by the host cells of each polypeptide encoded by that segment, relative to production of each polypeptide by the host cells in the absence of the mutation. The increased expression thus achieved is retained in the absence of conditions that select for cells displaying such increased expression. When the integrated segment comprises, for example, ethanol-production genes from an efficient ethanol producer like Zymomonas mobilis, recombinant Escherichia coli and other enteric bacterial cells within the present invention are capable of converting a wide range of biomass-derived sugars efficiently to ethanol.

  18. Genetically Engineered Poxviruses for Recombinant Gene Expression, Vaccination, and Safety

    NASA Astrophysics Data System (ADS)

    Moss, Bernard

    1996-10-01

    Vaccinia virus, no longer required for immunization against smallpox, now serves as a unique vector for expressing genes within the cytoplasm of mammalian cells. As a research tool, recombinant vaccinia viruses are used to synthesize and analyze the structure--function relationships of proteins, determine the targets of humoral and cell-mediated immunity, and investigate the types of immune response needed for protection against specific infectious diseases and cancer. The vaccine potential of recombinant vaccinia virus has been realized in the form of an effective oral wild-life rabies vaccine, although no product for humans has been licensed. A genetically altered vaccinia virus that is unable to replicate in mammalian cells and produces diminished cytopathic effects retains the capacity for high-level gene expression and immunogenicity while promising exceptional safety for laboratory workers and potential vaccine recipients.

  19. Production of recombinant chemokines and validation of refolding

    PubMed Central

    Veldkamp, Christopher T.; Koplinski, Chad A.; Jensen, Davin R.; Peterson, Francis C.; Smits, Kaitlin M.; Smith, Brittney L.; Johnson, Scott K.; Lettieri, Christina; Buchholz, Wallace G.; Solheim, Joyce C.; Volkman, Brian F.

    2016-01-01

    The diverse roles of chemokines in normal immune function and many human diseases have motivated numerous investigations into the structure and function of this family of proteins. Recombinant chemokines are often used to study how chemokines coordinate the trafficking of immune cells in various biological contexts. A reliable source of biologically active protein is vital for any in vitro or in vivo functional analysis. In this chapter, we describe a general method for the production of recombinant chemokines and robust techniques for efficient refolding that ensure consistently high biological activity. Considerations for initiating development of protocols consistent with Current Good Manufacturing Practices (cGMPs) to produce biologically active chemokines suitable for use in clinical trials are also discussed. PMID:26921961

  20. [Advances of consolidated bioprocessing based on recombinant strategy].

    PubMed

    Zheng, Zongbao; Zhao, Meina; Chen, Tao; Zhao, Xueming

    2013-10-01

    Lignocellulosic biomass represents an abundant, low-cost and renewable source of potentially fermentable sugars. It is acandidate besides petroleum as feedstock for fuel and chemical production. Recent researches on utilizing lignocellulosicsas feedstock boost development of numerous-promising processes for a variety of fuels and chemicals, such as biodiesel, biohydrogen and ethanol. However, high cost in depolymerization is a primary obstacle preventing the use of lignocellulosic biomass as feedstock. Consolidated bioprocessing (CBP), refers to the bioprocess without any exogenous cellulolyotic enzymes added, converting the lignocellulosic material into biochemicals directly, which could potentially avoid the cost of the dedicated enzyme generation step by incorporating enzyme-generating, biomass-degrading and bioproduct-producing capabilities into a single organism through genetic engineering. There are two CBP strategies, native strategy and recombinant strategy. We mainly introduce the recombinant strategy, including its principle, the two responding styles, the contributions of synthetic biology and metabolic engineering and the future challenges.

  1. Development and application of a TaqMan single nucleotide polymorphism genotyping assay to study infectious laryngotracheitis virus recombination in the natural host.

    PubMed

    Loncoman, Carlos A; Hartley, Carol A; Coppo, Mauricio J C; Vaz, Paola K; Diaz-Méndez, Andrés; Browning, Glenn F; Lee, Sang-Won; Devlin, Joanne M

    2017-01-01

    To date, recombination between different strains of the avian alphaherpesvirus infectious laryngotracheitis virus (ILTV) has only been detected in field samples using full genome sequencing and sequence analysis. These previous studies have revealed that natural recombination is widespread in ILTV and have demonstrated that recombination between two attenuated ILTV vaccine strains generated highly virulent viruses that produced widespread disease within poultry flocks in Australia. In order to better understand ILTV recombination, this study developed a TaqMan single nucleotide polymorphism (SNP) genotyping assay to detect recombination between two field strains of ILTV (CSW-1 and V1-99 ILTV) under experimental conditions. Following in vivo co-inoculation of these two ILTV strains in specific pathogen free (SPF) chickens, recovered viruses were plaque purified and subjected to the SNP genotyping assay. This assay revealed ILTV recombinants in all co-inoculated chickens. In total 64/87 (74%) of the recovered viruses were recombinants and 23 different recombination patterns were detected, with some of them occurring more frequently than others. The results from this study demonstrate that the TaqMan SNP genotyping assay is a useful tool to study recombination in ILTV and also show that recombination occurs frequently during experimental co-infection with ILTV in SPF chickens. This tool, when used to assess ILTV recombination in the natural host, has the potential to greatly contribute to our understanding of alphaherpesvirus recombination.

  2. Development and application of a TaqMan single nucleotide polymorphism genotyping assay to study infectious laryngotracheitis virus recombination in the natural host

    PubMed Central

    Hartley, Carol A.; Coppo, Mauricio J. C.; Vaz, Paola K.; Diaz-Méndez, Andrés; Browning, Glenn F.; Lee, Sang-won; Devlin, Joanne M.

    2017-01-01

    To date, recombination between different strains of the avian alphaherpesvirus infectious laryngotracheitis virus (ILTV) has only been detected in field samples using full genome sequencing and sequence analysis. These previous studies have revealed that natural recombination is widespread in ILTV and have demonstrated that recombination between two attenuated ILTV vaccine strains generated highly virulent viruses that produced widespread disease within poultry flocks in Australia. In order to better understand ILTV recombination, this study developed a TaqMan single nucleotide polymorphism (SNP) genotyping assay to detect recombination between two field strains of ILTV (CSW-1 and V1-99 ILTV) under experimental conditions. Following in vivo co-inoculation of these two ILTV strains in specific pathogen free (SPF) chickens, recovered viruses were plaque purified and subjected to the SNP genotyping assay. This assay revealed ILTV recombinants in all co-inoculated chickens. In total 64/87 (74%) of the recovered viruses were recombinants and 23 different recombination patterns were detected, with some of them occurring more frequently than others. The results from this study demonstrate that the TaqMan SNP genotyping assay is a useful tool to study recombination in ILTV and also show that recombination occurs frequently during experimental co-infection with ILTV in SPF chickens. This tool, when used to assess ILTV recombination in the natural host, has the potential to greatly contribute to our understanding of alphaherpesvirus recombination. PMID:28350819

  3. Reduced genetic distance and high replication levels increase the RNA recombination rate of hepatitis delta virus.

    PubMed

    Lin, Chia-Chi; Yang, Zhi-Wei; Iang, Shan-Bei; Chao, Mei

    2015-01-02

    Hepatitis delta virus (HDV) replication is carried out by host RNA polymerases. Since homologous inter-genotypic RNA recombination is known to occur in HDV, possibly via a replication-dependent process, we hypothesized that the degree of sequence homology and the replication level should be related to the recombination frequency in cells co-expressing two HDV sequences. To confirm this, we separately co-transfected cells with three different pairs of HDV genomic RNAs and analyzed the obtained recombinants by RT-PCR followed by restriction fragment length polymorphism and sequencing analyses. The sequence divergence between the clones ranged from 24% to less than 0.1%, and the difference in replication levels was as high as 100-fold. As expected, significant differences were observed in the recombination frequencies, which ranged from 0.5% to 47.5%. Furthermore, varying the relative amounts of parental RNA altered the dominant recombinant species produced, suggesting that template switching occurs frequently during the synthesis of genomic HDV RNA. Taken together, these data suggest that during the host RNA polymerase-driven RNA recombination of HDV, both inter- and intra-genotypic recombination events are important in shaping the genetic diversity of HDV.

  4. The effect of gamma radiation on recombination frequency in Caenorhabditis elegans.

    PubMed

    Kim, J S; Rose, A M

    1987-06-01

    We have studied the effect of gamma radiation on recombination frequency for intervals across the cluster of linkage group I in Caenorhabditis elegans. Recombination frequency increased approximately twofold across the dpy-5-unc-13 interval after treatment with 2000 rads (1 rad = 10 mGy) of cobalt 60 gamma radiation. Several factors affecting the magnitude of the increase have been characterized. Recombination frequency increased more with higher doses of radiation. However, the increase in recombination frequency with increasing dose was accompanied by a reduced average number of progeny from radiation-treated individuals. The amount of the increase was affected by meiotic stage, age at the time of treatment (premeiotic), and radiation dose. The increase in recombination was detectable in the B brood and remained elevated for the remainder of egg production. X-chromosome nondisjunction was also increased by radiation treatment. A high frequency of the recombinant progeny produced with radiation treatment were sterile unlike their nonrecombinant siblings. When parameters affecting recombination frequency are held constant during treatment, chromosomal regions of high gene density on the meiotic map increased more (fourfold) than an adjacent region of low gene density (no increase). The greatest increase was across the dpy-14-unc-13 interval near the center of the gene cluster. These results may suggest that the physical length of DNA per map unit is greater within the cluster than outside.

  5. The effect of the unfolded protein response on the production of recombinant proteins in plants.

    PubMed

    Thomas, David Rhys; Walmsley, Amanda Maree

    2015-02-01

    Recombinant proteins are currently produced through a wide variety of host systems, including yeast, E. coli, insect and mammalian cells. One of the most recent systems developed uses plant cells. While considerable advances have been made in the yields and fidelity of plant-made recombinant proteins, many of these gains have arisen from the development of recombinant factors. This includes elements such as highly effective promoters and untranslated regions, deconstructed viral vectors, silencing inhibitors, and improved DNA delivery techniques. However, unlike other host systems, much of the work on recombinant protein production in plants uses wild-type hosts that have not been modified to facilitate recombinant protein expression. As such, there are still endogenous mechanisms functioning to maintain the health of the cell. The result is that these pathways, such as the unfolded protein response, can actively work to reduce recombinant protein production to maintain the integrity of the cell. This review examines how issues arising from the unfolded protein response have been addressed in other systems, and how these methods may be transferable to plant systems. We further identify several areas of host plant biology that present attractive targets for modification to facilitate recombinant protein production.

  6. Observation of Plasma Recombination with the Negative Ions in Detached Plasma

    NASA Astrophysics Data System (ADS)

    Tonegawa, Akira; Shirota, Isao; Yoshida, Kenichi; Sugimoto, Tatunori; Kawamura, Kazutaka; Watanabe, Tsuguhiro; Ohyabu, Nobuyoshi; Takayama, Kazuo

    2000-10-01

    Detached divertors regimes are characterized by a low temperature (few eV) and high density plasma near the divertor plates. Recently, a new recombination process associated with excited hydrogen molecule, that is, molecular activated recombination (MAR), is expected to lead to an enhancement of the reduction of ion particle flux, and to modify the structure of detached recombining plasmas. In particular, negative ions play a key role in detached divertors regimes in charge exchange recombination of MAR. We present the experimental investigation of effects of the negative ions on detached plasma with MAR in the linear divertor plasma simulator, TPDSHEET-IV (Test Plasma produced by Directed current for SHEET plasma) device. The hydrogen plasma were generated with the hydrogen gas flow of 100 sccm at the discharge current of 50 A and the magnetic field of 0.7 kG. The negative ion density of hydrogen atom was measure by a probe-assisted laser photodetachment method. The reduction of the heat load to the target plate was clearly observed in hydrogen plasma with the hydrogen gas puff. At the same time, negative ions of hydrogen atom are localized in the region of cold electrons (2 eV) of the circumference of the sheet plasma. The charge exchange recombination rate of MAR is about 4 times of magnitude larger than three body recombination rate coefficients at this temperature. These experimental results suggest that the plasma recombination process comes from the negative ion of hydrogen atom.

  7. Stimulation of intrachromosomal homologous recombination in human cells by electroporation with site-specific endonucleases.

    PubMed Central

    Brenneman, M; Gimble, F S; Wilson, J H

    1996-01-01

    In somatic mammalian cells, homologous recombination is a rare event. To study the effects of chromosomal breaks on frequency of homologous recombination, site-specific endonucleases were introduced into human cells by electroporation. Cell lines with a partial duplication within the HPRT (hypoxanthine phosphoribosyltransferase) gene were created through gene targeting. Homologous intrachromosomal recombination between the repeated regions of the gene can reconstruct a functioning, wild-type gene. Treatment of these cells with the restriction endonuclease Xba I, which has a recognition site within the repeated region of HPRT homology, increased the frequency or homologous recombination bv more than 10-fold. Recombination frequency was similarly increased by treatment with the rare-cutting yeast endonuclease PI-Sce I when a cleavage site was placed within the repeated region of HPRT. In contrast, four restriction enzymes that cut at positions either outside of the repeated regions or between them produced no change in recombination frequency. The results suggest that homologous recombination between intrachromosomal repeats can be specifically initiated by a double-strand break occurring within regions of homology, consistent with the predictions of a model. PMID:8622983

  8. Recombinant spider silk genetically functionalized with affinity domains.

    PubMed

    Jansson, Ronnie; Thatikonda, Naresh; Lindberg, Diana; Rising, Anna; Johansson, Jan; Nygren, Per-Åke; Hedhammar, My

    2014-05-12

    Functionalization of biocompatible materials for presentation of active protein domains is an area of growing interest. Herein, we describe a strategy for functionalization of recombinant spider silk via gene fusion to affinity domains of broad biotechnological use. Four affinity domains of different origin and structure; the IgG-binding domains Z and C2, the albumin-binding domain ABD, and the biotin-binding domain M4, were all successfully produced as soluble silk fusion proteins under nondenaturing purification conditions. Silk films and fibers produced from the fusion proteins were demonstrated to be chemically and thermally stable. Still, the bioactive domains are concluded to be folded and accessible, since their respective targets could be selectively captured from complex samples, including rabbit serum and human plasma. Interestingly, materials produced from mixtures of two different silk fusion proteins displayed combined binding properties, suggesting that tailor-made materials with desired stoichiometry and surface distributions of several binding domains can be produced. Further, use of the IgG binding ability as a general mean for presentation of desired biomolecules could be demonstrated for a human vascular endothelial growth factor (hVEGF) model system, via a first capture of anti-VEGF IgG to silk containing the Z-domain, followed by incubation with hVEGF. Taken together, this study demonstrates the potential of recombinant silk, genetically functionalized with affinity domains, for construction of biomaterials capable of presentation of almost any desired biomolecule.

  9. Viral RNA-directed RNA polymerases use diverse mechanisms to promote recombination between RNA molecules.

    PubMed

    Chetverin, Alexander B; Kopein, Damir S; Chetverina, Helena V; Demidenko, Alexander A; Ugarov, Victor I

    2005-03-11

    An earlier developed purified cell-free system was used to explore the potential of two RNA-directed RNA polymerases (RdRps), Qbeta phage replicase and the poliovirus 3Dpol protein, to promote RNA recombination through a primer extension mechanism. The substrates of recombination were fragments of complementary strands of a Qbeta phage-derived RNA, such that if aligned at complementary 3'-termini and extended using one another as a template, they would produce replicable molecules detectable as RNA colonies grown in a Qbeta replicase-containing agarose. The results show that while 3Dpol efficiently extends the aligned fragments to produce the expected homologous recombinant sequences, only nonhomologous recombinants are generated by Qbeta replicase at a much lower yield and through a mechanism not involving the extension of RNA primers. It follows that the mechanisms of RNA recombination by poliovirus and Qbeta RdRps are quite different. The data favor an RNA transesterification reaction catalyzed by a conformation acquired by Qbeta replicase during RNA synthesis and provide a likely explanation for the very low frequency of homologous recombination in Qbeta phage.

  10. Statistical approaches to maximize recombinant protein expression in Escherichia coli: a general review.

    PubMed

    Papaneophytou, Christos P; Kontopidis, George

    2014-02-01

    The supply of many valuable proteins that have potential clinical or industrial use is often limited by their low natural availability. With the modern advances in genomics, proteomics and bioinformatics, the number of proteins being produced using recombinant techniques is exponentially increasing and seems to guarantee an unlimited supply of recombinant proteins. The demand of recombinant proteins has increased as more applications in several fields become a commercial reality. Escherichia coli (E. coli) is the most widely used expression system for the production of recombinant proteins for structural and functional studies. However, producing soluble proteins in E. coli is still a major bottleneck for structural biology projects. One of the most challenging steps in any structural biology project is predicting which protein or protein fragment will express solubly and purify for crystallographic studies. The production of soluble and active proteins is influenced by several factors including expression host, fusion tag, induction temperature and time. Statistical designed experiments are gaining success in the production of recombinant protein because they provide information on variable interactions that escape the "one-factor-at-a-time" method. Here, we review the most important factors affecting the production of recombinant proteins in a soluble form. Moreover, we provide information about how the statistical design experiments can increase protein yield and purity as well as find conditions for crystal growth.

  11. Recombinant Zymomonas for pentose fermentation

    DOEpatents

    Picataggio, S.K.; Zhang, M.; Eddy, C.K.; Deanda, K.A.; Finkelstein, M.

    1996-05-07

    The invention relates to microorganisms which normally do not ferment a pentose sugar and which are genetically altered to ferment this pentose to produce ethanol. A representative example is Zymomonas mobilis which has been transformed with E. coli xylose isomerase, xylulokinase, transaldolase and transketolase genes. Expression of the added genes are under the control of Zymomonas mobilis promoters. This newly created microorganism is useful for fermenting pentoses and glucose, produced by hydrolysis of hemicellulose and cellulose, to produce ethanol. 2 figs.

  12. Recombinant zymomonas for pentose fermentation

    DOEpatents

    Picataggio, Stephen K.; Zhang, Min; Eddy, Christina K.; Deanda, Kristine A.; Finkelstein, Mark

    1996-01-01

    The invention relates to microorganisms which normally do not ferment a pentose sugar and which are genetically altered to ferment this pentose to produce ethanol. A representative example is Zymomonas mobilis which has been transformed with E. coli xylose isomerase, xylulokinase, transaldolase and transketolase genes. Expression of the added genes are under the control of Zymomonas mobilis promoters. This newly created microorganism is useful for fermenting pentoses and glucose, produced by hydrolysis of hemicellulose and cellulose, to produce ethanol.

  13. Genetic Recombination in Bacteriophage T4: Single-Burst Analysis of Cosegregants and Evidence in Favor of a Splice/Patch Coupling Model

    PubMed Central

    Shcherbakov, V. P.; Plugina, L. A.; Nesheva, M. A.

    1992-01-01

    To reveal the structure of penultimate DNA intermediates in T4 bacteriophage recombination, resolution of which produces free recombinant molecules, a single-burst analysis of the recombinant progeny was made in multifactor crosses, enabling one to determine quantitatively the different recombinants generated by one or two exchanges within the same chromosome segment. It was found that double and single exchanges are highly correlated in T4 recombination. These results were interpreted as evidence for simultaneous formation of a splice/patch pair as the primary recombination products. A recombination model called here the ``splice/patch coupling model'' is presented according to which resolution of a single DNA intermediate results in two linear heterozygous molecules containing a patch and a splice, respectively, in homologous positions. PMID:1516814

  14. Theoretical prediction of the impact of Auger recombination on charge collection from an ion track

    NASA Technical Reports Server (NTRS)

    Edmonds, Larry D.

    1991-01-01

    The theoretical analysis presented indicates that Auger recombination can reduce charge collection from very dense ion tracks in silicon devices. It is of marginal importance for tracks produced by 270-MeV krypton, and therefore it is of major importance for ions exhibiting a significantly larger loss. The analysis shows that recombination loss is profoundly affected by track diffusion. As the track diffuses, the density and recombination rate decrease so fast that the linear density (number of electron-hole pairs per unit length) approaches a nonzero limiting value as t approaches infinity. Furthermore, the linear density is very nearly equal to this limiting value in a few picoseconds or less. When Auger recombination accompanies charge transport processes that have much longer time scales, it can be simulated by assigning a reduced linear energy transfer to the ion.

  15. Biomimetic production of silk-like recombinant squid sucker ring teeth proteins.

    PubMed

    Ding, Dawei; Guerette, Paul A; Hoon, Shawn; Kong, Kiat Whye; Cornvik, Tobias; Nilsson, Martina; Kumar, Akshita; Lescar, Julien; Miserez, Ali

    2014-09-08

    The sucker ring teeth (SRT) of Humboldt squid exhibit mechanical properties that rival those of robust engineered synthetic polymers. Remarkably, these properties are achieved without a mineral phase or covalent cross-links. Instead, SRT are exclusively made of silk-like proteins called "suckerins", which assemble into nanoconfined β-sheet reinforced supramolecular networks. In this study, three streamlined strategies for full-length recombinant suckerin protein production and purification were developed. Recombinant suckerin exhibited high solubility and colloidal stability in aqueous-based solvents. In addition, the colloidal suspensions exhibited a concentration-dependent conformational switch, from random coil to β-sheet enriched structures. Our results demonstrate that recombinant suckerin can be produced in a facile manner in E. coli and processed from mild aqueous solutions into materials enriched in β-sheets. We suggest that recombinant suckerin-based materials offer potential for a range of biomedical and engineering applications.

  16. Recombinant Phage Elicits Protective Immune Response against Systemic S. globosa Infection in Mouse Model

    PubMed Central

    Chen, Feng; Jiang, Rihua; Wang, Yicun; Zhu, Mingji; Zhang, Xu; Dong, Shuai; Shi, Hongxi; Wang, Li

    2017-01-01

    Sporothrix globosa is a type of fungus that typically infects immunocompromised patients. Its prevention continues to pose a challenge. A 70-KDa glycoprotein (Gp70) of Sporothrix has been previously reported to protect host against infection from this fungus. Here, we displayed an epitope peptide (kpvqhalltplgldr) of Gp70 on the major coat protein (pIII), and investigated its efficiency as a vaccine for preventing S. globosa infection. The recombinant phage and the heat-killed S. globosa were used to immunize mice separately. In this study, we evaluated the humoral and cellular immune responses in the mice and demonstrated that recombinant phage could induce mice to produce a stronger immune response and generate antibodies to inhibit S. globosa infection. Furthermore, immunization with recombinant phage could increase the survival rate of S. globosa infection in mice. All these results together indicated that recombinant phages displaying kpvqhalltplgldr are a potential vaccine candidate against S. globosa infection. PMID:28165018

  17. DNA induces conformational changes in a recombinant human minichromosome maintenance complex.

    PubMed

    Hesketh, Emma L; Parker-Manuel, Richard P; Chaban, Yuriy; Satti, Rabab; Coverley, Dawn; Orlova, Elena V; Chong, James P J

    2015-03-20

    ATP-dependent DNA unwinding activity has been demonstrated for recombinant archaeal homohexameric minichromosome maintenance (MCM) complexes and their yeast heterohexameric counterparts, but in higher eukaryotes such as Drosophila, MCM-associated DNA helicase activity has been observed only in the context of a co-purified Cdc45-MCM-GINS complex. Here, we describe the production of the recombinant human MCM (hMCM) complex in Escherichia coli. This protein displays ATP hydrolysis activity and is capable of unwinding duplex DNA. Using single-particle asymmetric EM reconstruction, we demonstrate that recombinant hMCM forms a hexamer that undergoes a conformational change when bound to DNA. Recombinant hMCM produced without post-translational modifications is functional in vitro and provides an important tool for biochemical reconstitution of the human replicative helicase.

  18. Recombination-cascade X-ray spectra of highly charged helium-like ions

    NASA Technical Reports Server (NTRS)

    Pradhan, A. K.

    1985-01-01

    It is shown that the relative intensity distribution among the X-ray spectral lines of helium-like ions from the n = 2 states produced through recombination processes such as radiative and charge transfer recombination may be given by considering in detail the radiative cascades following recombination. Model calculations are presented with predicted line ratios for Ar XVII and Fe XXV in recombination-dominated noncoronal plasmas. In particular, compared to coronal intensities, the singlet resonance line (w) should be much weaker relative to the triplet intercombination (x, y) and forbidden (z) lines, yielding large values for the ratio G = (x + y + z)/w. Accurate configuration interaction type wave functions are employed to calculate the eigenenergies, transition probabilities, and cascade coefficients. Certain relevant tokamak and astrophysical observations are discussed.

  19. Free recombination within Helicobacter pylori

    PubMed Central

    Suerbaum, Sebastian; Smith, John Maynard; Bapumia, Khairun; Morelli, Giovanna; Smith, Noel H.; Kunstmann, Erdmute; Dyrek, Isabelle; Achtman, Mark

    1998-01-01

    Sequences of three gene fragments (flaA, flaB, and vacA) from Helicobacter pylori strains isolated from patients in Germany, Canada, and South Africa were analyzed for diversity and for linkage equilibrium by using the Homoplasy Test and compatibility matrices. Horizontal genetic exchange in H. pylori is so frequent that different loci and polymorphisms within each locus are all at linkage equilibrium. These results indicate that H. pylori is panmictic. Comparisons with sequences from Escherichia coli, Neisseria meningitidis, and Drosophila melanogaster showed that recombination in H. pylori was much more frequent than in other species. In contrast, when multiple family members infected with H. pylori were investigated, some strains were indistinguishable at all three loci. Thus, H. pylori is clonal over short time periods after natural transmission. PMID:9770535

  20. Deleterious background selection with recombination

    SciTech Connect

    Hudson, R.R.; Kaplan, N.L.

    1995-12-01

    An analytic expression for the expected nucleotide diversity is obtained for a neutral locus in a region with deleterious mutation and recombination. Our analytic results are used to predict levels of variation for the entire third chromosome of Drosophila melanogaster. The predictions are consistent with the low levels of variation that have been observed at loci near the centromeres of the third chromosome of D. melanogaster. However, the low levels of variation observed near the tips of this chromosome are not predicted using currently available estimates of the deleterious mutation rate and of selection coefficients. If considerably smaller selection coefficients are assumed, the low observed levels of variation at the tips of the third chromosome are consistent with the background selection model. 33 refs., 4 figs., 1 tab.

  1. Dielectronic recombination of tungsten ions

    NASA Astrophysics Data System (ADS)

    Li, Bowen; O'Sullivan, Gerry; Dong, Chenzhong; Chen, Ximeng

    2016-08-01

    Ab initio calculations of dielectronic recombination rate coefficients of Ne-, Pd- and Ag-like tungsten have been performed. Energy levels, radiative transition probabilities and autoionization rates were calculated using the Flexible Atomic Code. The contributions from different channels to the total rate coefficients are discussed. The present calculated rate coefficients are compared with other calculations where available. Excellent agreement has been found for Ne-like W while a large discrepancy was found for Pd-like W, which implies that more ab initio calculations and experimental measurements are badly needed. Further calculations demonstrated that the influence of configuration interaction is small while nonresonant radiative stabilizing (NRS) contribution to doubly excited non-autoionizing states are vital. The data obtained are expected to be useful for modeling plasmas for fusion applications, especially for the ITER community, which makes experimental verification even more essential.

  2. Overview of the Purification of Recombinant Proteins

    PubMed Central

    Wingfield, Paul T.

    2015-01-01

    When the first version of this unit was written in 1995 protein purification of recombinant proteins was based on a variety of standard chromatographic methods and approaches many of which were described and mentioned in this unit and elsewhere in the book. In the interim there has been a shift towards an almost universal usage of the affinity or fusion tag. This may not be the case for biotechnology manufacture where affinity tags can complicate producing proteins under regulatory conditions. Regardless of the protein expression system, questions are asked as to which and how many affinity tags to use, where to attach them in the protein and whether to engineer a self cleavage system or simply leave them on. We will briefly address some of these issues. Also although this overview focuses on E.coli, protein expression and purification from the other commonly used expression systems are mentioned and apart from cell breakage methods, the protein purification methods and strategies are essentially the same. PMID:25829302

  3. Rad54, the Motor of Homologous Recombination

    PubMed Central

    Mazin, Alexander V.; Mazina, Olga M.; Bugreev, Dmitry V.; Rossi, Matthew J.

    2009-01-01

    Homologous recombination (HR) performs crucial functions including DNA repair, segregation of homologous chromosomes, propagation of genetic diversity, and maintenance of telomeres. HR is responsible for the repair of DNA double-strand breaks and DNA interstrand cross-links. The process of HR is initiated at the site of DNA breaks and gaps and involves a search for homologous sequences promoted by Rad51 and auxiliary proteins followed by the subsequent invasion of broken DNA ends into the homologous duplex DNA that then serves as a template for repair. The invasion produces a cross-stranded structure, known as the Holliday junction. Here, we describe the properties of Rad54, an important and versatile HR protein that is evolutionarily conserved in eukaryotes. Rad54 is a motor protein that translocates along dsDNA and performs several important functions in HR. The current review focuses on the recently identified Rad54 activities which contribute to the late phase of HR, especially the branch migration of Holliday junctions. PMID:20089461

  4. Method for producing electrodes using microscale or nanoscale materials obtained from hydrogendriven metallurgical reactions

    DOEpatents

    Reilly, James J.; Adzic, Gordana D.; Johnson, John R.; Vogt, Thomas; McBreen, James

    2003-09-02

    A method is provided for producing electrodes using microscale and nanoscale metal materials formed from hydrogen driven metallurgical processes; such a the HD (hydriding, dehydriding) process, the HDDR (hydriding, dehydriding, disproportionation, and recombination) process, and variants thereof.

  5. Recombining without Hotspots: A Comprehensive Evolutionary Portrait of Recombination in Two Closely Related Species of Drosophila

    PubMed Central

    Smukowski Heil, Caiti S.; Ellison, Chris; Dubin, Matthew; Noor, Mohamed A.F.

    2015-01-01

    Meiotic recombination rate varies across the genome within and between individuals, populations, and species in virtually all taxa studied. In almost every species, this variation takes the form of discrete recombination hotspots, determined in some mammals by a protein called PRDM9. Hotspots and their determinants have a profound effect on the genomic landscape, and share certain features that extend across the tree of life. Drosophila, in contrast, are anomalous in their absence of hotspots, PRDM9, and other species-specific differences in the determination of recombination. To better understand the evolution of meiosis and general patterns of recombination across diverse taxa, we present a truly comprehensive portrait of recombination across time, combining recently published cross-based contemporary recombination estimates from each of two sister species with newly obtained linkage-disequilibrium-based historic estimates of recombination from both of these species. Using Drosophila pseudoobscura and Drosophila miranda as a model system, we compare recombination rate between species at multiple scales, and we suggest that Drosophila replicate the pattern seen in human–chimpanzee in which recombination rate is conserved at broad scales. We also find evidence of a species-wide recombination modifier(s), resulting in both a present and historic genome-wide elevation of recombination rates in D. miranda, and identify broad scale effects on recombination from the presence of an inversion. Finally, we reveal an unprecedented view of the distribution of recombination in D. pseudoobscura, illustrating patterns of linked selection and where recombination is taking place. Overall, by combining these estimation approaches, we highlight key similarities and differences in recombination between Drosophila and other organisms. PMID:26430062

  6. Recombining without Hotspots: A Comprehensive Evolutionary Portrait of Recombination in Two Closely Related Species of Drosophila.

    PubMed

    Smukowski Heil, Caiti S; Ellison, Chris; Dubin, Matthew; Noor, Mohamed A F

    2015-10-01

    Meiotic recombination rate varies across the genome within and between individuals, populations, and species in virtually all taxa studied. In almost every species, this variation takes the form of discrete recombination hotspots, determined in some mammals by a protein called PRDM9. Hotspots and their determinants have a profound effect on the genomic landscape, and share certain features that extend across the tree of life. Drosophila, in contrast, are anomalous in their absence of hotspots, PRDM9, and other species-specific differences in the determination of recombination. To better understand the evolution of meiosis and general patterns of recombination across diverse taxa, we present a truly comprehensive portrait of recombination across time, combining recently published cross-based contemporary recombination estimates from each of two sister species with newly obtained linkage-disequilibrium-based historic estimates of recombination from both of these species. Using Drosophila pseudoobscura and Drosophila miranda as a model system, we compare recombination rate between species at multiple scales, and we suggest that Drosophila replicate the pattern seen in human-chimpanzee in which recombination rate is conserved at broad scales. We also find evidence of a species-wide recombination modifier(s), resulting in both a present and historic genome-wide elevation of recombination rates in D. miranda, and identify broad scale effects on recombination from the presence of an inversion. Finally, we reveal an unprecedented view of the distribution of recombination in D. pseudoobscura, illustrating patterns of linked selection and where recombination is taking place. Overall, by combining these estimation approaches, we highlight key similarities and differences in recombination between Drosophila and other organisms.

  7. The dissociative recombination of NO+: the influence of the vibrational excitation state

    NASA Astrophysics Data System (ADS)

    Mostefaoui, T.; Laubé, S.; Gautier, G.; Rebrion-Rowe, C.; Rowe, B. R.; Mitchell, J. B. A.

    1999-11-01

    The dissociative recombination rate coefficient for NO+ has been measured at 300 K using a flowing afterglow Langmuir probe-mass spectrometer apparatus. A value of (4±1) × 10-7 cm3 s-1 has been found for ground electronic and vibrational state ions produced from the parent NO gas. Ions produced from a N2:O2 mixture which are in a wide range of vibrationally excited states display a smaller recombination rate of (1.6±0.5) × 10-7 cm3 s-1.

  8. Vaccinia Virus Recombinants: Expression of VSV Genes and Protective Immunization of Mice and Cattle

    NASA Astrophysics Data System (ADS)

    Mackett, M.; Yilma, T.; Rose, J. K.; Moss, B.

    1985-01-01

    Vesicular stomatitis virus (VSV) causes a contagious disease of horses, cattle, and pigs. When DNA copies of messenger RNA's for the G or N proteins of VSV were linked to a vaccinia virus promoter and inserted into the vaccinia genome, the recombinants retained infectivity and synthesized VSV polypeptides. After intradermal vaccination with live recombinant virus expressing the G protein, mice produced VSV-neutralizing antibodies and were protected against lethal encephalitis upon intravenous challenge with VSV. In cattle, the degree of protection against intradermalingually injected VSV was correlated with the level of neutralizing antibody produced following vaccination.

  9. Oligonucleotide recombination in gram negative bacteria

    Technology Transfer Automated Retrieval System (TEKTRAN)

    This report describes several key aspects of a novel form of RecA-independent homologous recombination. We found that synthetic single stranded DNA oligonucleotides (oligos) introduced into bacteria by transformation can site-specifically recombine with bacterial chromosomes in the absence of any a...

  10. Oligo Recombination in Gram Negative Bacteria

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Homologous recombination is important for bacterial survival because it simultaneously provides genomic stability as well as genomic plasticity. Of the mechanistic pathways for homologous recombination, those mediated by RecA are the most thoroughly characterized and are understood to be structural...

  11. Dielectronic recombination lines of C{sup +}

    SciTech Connect

    Sochi, Taha Storey, Peter J.

    2013-11-15

    The present paper presents atomic data generated to investigate the recombination lines of C II in the spectra of planetary nebulae. These data include energies of bound and autoionizing states, oscillator strengths and radiative transition probabilities, autoionization probabilities, and recombination coefficients. The R-matrix method of electron scattering theory was used to describe the C{sup 2+} plus electron system.

  12. Recombinant DNA encoding a desulfurization biocatalyst

    DOEpatents

    Rambosek, J.; Piddington, C.S.; Kovacevich, B.R.; Young, K.D.; Denome, S.A.

    1994-10-18

    This invention relates to a recombinant DNA molecule containing a gene or genes which encode a biocatalyst capable of desulfurizing a fossil fuel which contains organic sulfur molecules. For example, the present invention encompasses a recombinant DNA molecule containing a gene or genes of a strain of Rhodococcus rhodochrous. 13 figs.

  13. Recombinant DNA encoding a desulfurization biocatalyst

    DOEpatents

    Rambosek, John; Piddington, Chris S.; Kovacevich, Brian R.; Young, Kevin D.; Denome, Sylvia A.

    1994-01-01

    This invention relates to a recombinant DNA molecule containing a gene or genes which encode a biocatalyst capable of desulfurizing a fossil fuel which contains organic sulfur molecules. For example, the present invention encompasses a recombinant DNA molecule containing a gene or genes of a strain of Rhodococcus rhodochrous.

  14. Dissociative recombination: Results from storage rings

    SciTech Connect

    Larsson, Mats

    2005-05-27

    The focus of this article is on the measurement of product branching ratios in the dissociative recombination of polyatomic molecular ions with electrons by means of ion storage rings. Recombination of ions of interest in astrophysics, plasma-assisted combustion, thermonuclear fusion, protein fragmentation, and atmospheric physics are reviewed and discussed.

  15. Dissociative recombination of highly symmetric polyatomic ions.

    PubMed

    Douguet, Nicolas; Orel, Ann E; Greene, Chris H; Kokoouline, Viatcheslav

    2012-01-13

    A general first-principles theory of dissociative recombination is developed for highly symmetric molecular ions and applied to H(3)O(+) and CH(3)(+), which play an important role in astrophysical, combustion, and laboratory plasma environments. The theoretical cross sections obtained for the dissociative recombination of the two ions are in good agreement with existing experimental data from storage ring experiments.

  16. Radiofrequency recombination lines from the interstellar medium

    NASA Technical Reports Server (NTRS)

    Dupree, A. K.

    1971-01-01

    Observations of recombination lines form normal H II regions, extended H II regions, nonthermal sources, and the H I medium are discussed. Detection of recombination lines from elements other than hydrogen may provide a means of identifying fossil Stromgren spheres at high temperature.

  17. V(D)J recombination deficiencies.

    PubMed

    de Villartay, Jean-Pierre

    2009-01-01

    V(D)J recombination not only comprises the molecular mechanism that insures diversity of the immune system but also constitutes a critical checkpoint in the developmental program of B- and T-lymphocytes. The analysis of human patients with Severe Combined Immune Deficiency (SCID) has contributed to the understanding of the biochemistry of the V(D)J recombination reaction. The molecular study V(D)J recombination settings in humans, mice and in cellular mutants has allowed to unravel the process of Non Homologous End Joining (NHEJ), one of the key pathway that insure proper repair of DNA double strand breaks (dsb), whether they occur during V(D)J recombination or secondary to other DNA injuries. Two NHEJ factors, Artemis and Cernunnos, were indeed discovered through the study of human V(D)J recombination defective human SCID patients.

  18. Stabilized pigment and method for producing the same

    NASA Technical Reports Server (NTRS)

    Morrison, Stanley Roy (Inventor); Freund, Thomas (Inventor)

    1976-01-01

    A chemical species, present in two oxidation states which differ from one another by one equivalent, is added to pigment materials to serve as a recombination center for alternately capturing electrons and holes produced by the pigment materials when they are subjected to ultraviolet light exposure.

  19. Measurement of the recombination of photoproduced PAH ions

    NASA Astrophysics Data System (ADS)

    Novotny, O.; Sivaraman, B.; Rebrion-Rowe, C.; Travers, D.; Mitchell, J. B. A.; Rowe, B. R.

    2005-01-01

    A new technique, Flowing Afterglow with Photo Ions - FLAPI, has been developed for measuring the rate coefficient for the recombination of complex ions with electrons. The method is based on the FALP-MS apparatus at the Université de Rennes I. A helium plasma is generated by a microwave discharge in a He buffer gas and downstream a small amount of argon gas is injected to get rid of helium metastables. A very small amount of neutral PAH molecules is added to the afterglow plasma by evaporation from a plate coated with the PAH to be studied. PAH ions are then produced by photoionization of the parent molecule using a pulsed UV laser (157 nm). The laser beam is oriented along the flow-tube and so a constant spatial concentration of photoions is obtained. The electron concentration along the flow-tube is measured by means of a movable Langmuir probe. The decay of the ion concentration in time is measured at a fixed position using a quadrupole mass spectrometer which is triggered by the laser pulse. Anthracene ion recombination has been studied using this technique and we have obtained the preliminary recombination rate coefficient (1.1 +/- 0.5) × 10-6 cm3 s-1.

  20. Radiative and interfacial recombination in CdTe heterostructures

    SciTech Connect

    Swartz, C. H. Edirisooriya, M.; LeBlanc, E. G.; Noriega, O. C.; Jayathilaka, P. A. R. D.; Ogedengbe, O. S.; Hancock, B. L.; Holtz, M.; Myers, T. H.; Zaunbrecher, K. N.

    2014-12-01

    Double heterostructures (DH) were produced consisting of a CdTe film between two wide band gap barriers of CdMgTe alloy. A combined method was developed to quantify radiative and non-radiative recombination rates by examining the dependence of photoluminescence (PL) on both excitation intensity and time. The measured PL characteristics, and the interface state density extracted by modeling, indicate that the radiative efficiency of CdMgTe/CdTe DHs is comparable to that of AlGaAs/GaAs DHs, with interface state densities in the low 10{sup 10 }cm{sup −2} and carrier lifetimes as long as 240 ns. The radiative recombination coefficient of CdTe is found to be near 10{sup −10} cm{sup 3}s{sup −1}. CdTe film growth on bulk CdTe substrates resulted in a homoepitaxial interface layer with a high non-radiative recombination rate.

  1. Phenol removal from refinery wastewater by mutant recombinant horseradish peroxidase.

    PubMed

    Asad, Sedigheh; Dabirmanesh, Bahareh; Khajeh, Khosro

    2014-01-01

    Application of mutated recombinant horseradish peroxidase (HRP) for phenol removal from refinery effluents is reported. Recombinant HRP produced in Escherichia coli suffers from the disadvantage of lacking glycosylation, which affects its catalytic efficiency and stability toward inactivating parameters such as increased temperature and enhanced amounts of hydrogen peroxide. In the present study, the previously reported variant (in which Asn268 was substituted with Asp, N268D) with improved stability characteristics and catalytic efficiency was used to remove phenol from a petroleum refinery effluent. The presence and removal of phenol was studied by high-performance liquid chromatography; the precipitated oxidized phenol was also observed and removed from the sample by centrifugation. Results showed that the N268D variant can remove 61%, 67%, and 81% of phenol from effluent in 1, 2, and 16 H, respectively. By exploiting the N268D mutant, removal of 50% phenol could be achieved in 42 Min, which was more than 22 times less than the treatment time required by native recombinant enzyme.

  2. Identifying the Important HIV-1 Recombination Breakpoints

    PubMed Central

    Fan, Jun; Simon-Loriere, Etienne; Arts, Eric J.; Negroni, Matteo; Robertson, David L.

    2008-01-01

    Recombinant HIV-1 genomes contribute significantly to the diversity of variants within the HIV/AIDS pandemic. It is assumed that some of these mosaic genomes may have novel properties that have led to their prevalence, particularly in the case of the circulating recombinant forms (CRFs). In regions of the HIV-1 genome where recombination has a tendency to convey a selective advantage to the virus, we predict that the distribution of breakpoints—the identifiable boundaries that delimit the mosaic structure—will deviate from the underlying null distribution. To test this hypothesis, we generate a probabilistic model of HIV-1 copy-choice recombination and compare the predicted breakpoint distribution to the distribution from the HIV/AIDS pandemic. Across much of the HIV-1 genome, we find that the observed frequencies of inter-subtype recombination are predicted accurately by our model. This observation strongly indicates that in these regions a probabilistic model, dependent on local sequence identity, is sufficient to explain breakpoint locations. In regions where there is a significant over- (either side of the env gene) or under- (short regions within gag, pol, and most of env) representation of breakpoints, we infer natural selection to be influencing the recombination pattern. The paucity of recombination breakpoints within most of the envelope gene indicates that recombinants generated in this region are less likely to be successful. The breakpoints at a higher frequency than predicted by our model are approximately at either side of env, indicating increased selection for these recombinants as a consequence of this region, or at least part of it, having a tendency to be recombined as an entire unit. Our findings thus provide the first clear indication of the existence of a specific portion of the genome that deviates from a probabilistic null model for recombination. This suggests that, despite the wide diversity of recombinant forms seen in the viral

  3. Pentose fermentation by recombinant Zymomonas

    DOEpatents

    Picataggio, S.K.; Zhang, M.; Eddy, C.K.; Deanda, K.A.; Finkelstein, M.; Mohagheghi, A.; Newman, M.M.; McMillan, J.D.

    1998-01-27

    The invention relates to microorganisms which normally do not ferment pentose sugar and which are genetically altered to ferment pentose sugar to produce ethanol, and fermentation processes utilizing the same. Examples include Zymomonas mobilis which has been transformed with combinations of E. coli genes for xylose isomerase, xylulokinase, transaldolase, transketolase, L-arabinose isomerase, L-ribulokinase, and L-ribulose 5-phosphate 4-epimerase. Expression of the added genes are under the control of Zymomonas mobilis promoters. These newly created microorganisms are useful for fermenting pentoses and glucose, produced by hydrolysis of hemicellulose and cellulose, to produce ethanol. 7 figs.

  4. Recombinant Zymomonas for pentose fermentation

    DOEpatents

    Picataggio, S.K.; Min Zhang; Eddy, C.K.; Deanda, K.A.

    1998-03-10

    The invention relates to microorganisms which normally do not ferment pentose sugar and which are genetically altered to ferment pentose sugar to produce ethanol, and fermentation processes utilizing the same. Examples include Zymomonas mobilis which has been transformed with combinations of E. coli genes for xylose isomerase, xylulokinase, transaldolase, transketolase, L-arabinose isomerase, L-ribulokinase, and L-ribulose-5-phosphate 4-epimerase. Expression of the added genes are under the control of Zymomonas mobilis promoters. These newly created microorganisms are useful for fermenting pentoses and glucose, produced by hydrolysis of hemicellulose and cellulose, to produce ethanol. 7 figs.

  5. Pentose fermentation by recombinant zymomonas

    DOEpatents

    Picataggio, Stephen K.; Zhang, Min; Eddy, Christina K.; Deanda, Kristine A.; Finkelstein, Mark; Mohagheghi, Ali; Newman, Mildred M.; McMillan, James D.

    1998-01-01

    The invention relates to microorganisms which normally do not ferment pentose sugar and which are genetically altered to ferment pentose sugar to produce ethanol, and fermentation processes utilizing the same. Examples include Zymomonas mobilis which has been transformed with combinations of E. coli genes for xylose isomerase, xylulokinase, transaldolase, transketolase, L-arabinose isomerase, L-ribulokinase, and L-ribulose 5-phosphate 4-epimerase. Expression of the added genes are under the control of Zymomonas mobilis promoters. These newly created microorganisms are useful for fermenting pentoses and glucose, produced by hydrolysis of hemicellulose and cellulose, to produce ethanol.

  6. Recombinant Zymomonas for pentose fermentation

    DOEpatents

    Picataggio, Stephen K.; Zhang, Min; Eddy, Christina K.; Deanda, Kristine A.

    1998-01-01

    The invention relates to microorganisms which normally do not ferment pentose sugar and which are genetically altered to ferment pentose sugar to produce ethanol, and fermentation processes utilizing the same. Examples include Zymomonas mobilis which has been transformed with combinations of E. coli genes for xylose isomerase, xylulokinase, transaldolase, transketolase, L-arabinose isomerase, L-ribulokinase, and L-ribulose-5-phosphate 4-epimerase. Expression of the added genes are under the control of Zymomonas mobilis promoters. These newly created microorganisms are useful for fermenting pentoses and glucose, produced by hydrolysis of hemicellulose and cellulose, to produce ethanol.

  7. Staphylococcus simulans Recombinant Lysostaphin: Production, Purification, and Determination of Antistaphylococcal Activity.

    PubMed

    Boksha, I S; Lavrova, N V; Grishin, A V; Demidenko, A V; Lyashchuk, A M; Galushkina, Z M; Ovchinnikov, R S; Umyarov, A M; Avetisian, L R; Chernukha, M Iu; Shaginian, I A; Lunin, V G; Karyagina, A S

    2016-05-01

    Staphylococcus simulans lysostaphin is an endopeptidase lysing staphylococcus cell walls by cleaving pentaglycine cross-bridges in their peptidoglycan. A synthetic gene encoding S. simulans lysostaphin was cloned in Escherichia coli cells, and producer strains were designed. The level of produced biologically active lysostaphin comprised 6-30% of total E. coli cell protein (depending on E. coli M15 or BL21 producer) under batch cultivation conditions. New methods were developed for purification of lysostaphin without affinity domains and for testing its enzymatic activity. As judged by PAGE, the purified recombinant lysostaphin is of >97% purity. The produced lysostaphin lysed cells of Staphylococcus aureus and Staphylococcus haemolyticus clinical isolates. In vitro activity and general biochemical properties of purified recombinant lysostaphin produced by M15 or BL21 E. coli strains were identical to those of recombinant lysostaphin supplied by Sigma-Aldrich (USA) and used as reference in other known studies. The prepared recombinant lysostaphin represents a potential product for development of enzymatic preparation for medicine and veterinary due to the simple purification scheme enabling production of the enzyme of high purity and antistaphylococcal activity.

  8. Products of Dissociative Recombination in the Ionosphere

    NASA Technical Reports Server (NTRS)

    Cosby, Philip

    1996-01-01

    SRI International undertook a novel experimental measurement of the product states formed by dissociative recombination (DR) of O2(+), NO(+), and N2(+) as a function of both electron energy and reactant ion vibrational level. For these measurements we used a recently developed experimental technique for measuring dissociation product distributions that allows both the branching ratios to be accurately determined and the electronic and rovibrational state composition of the reactant ions to be specified. DR is the dominant electron loss mechanism in all regions of the ionosphere. In this process, electron attachment to the molecular ion produces an unstable neutral molecule that rapidly dissociates. For a molecular ion such as O2(+), the dissociation recombination reaction is (1) O2(+) + e yields O + O + W. The atomic products of this reaction, in this case two oxygen atoms, can be produced in a variety of excited states and with a variety of kinetic energies, as represented by W in Eq. (1). These atoms are not only active in the neutral chemistry of the ionosphere, but are also especially important because their optical emissions are often used to infer in situ concentrations of the parent molecular ion and ambient electron densities. Many laboratory measurements have been made of DR reaction rates under a wide range of electron temperatures, but very little is known about the actual distributions among the final states of the atomic products. This lack of knowledge seriously limits the validity and effectiveness of efforts to model both natural and man-made ionospheric disturbances. Bates recently identified major deficiencies in the currently accepted branching ratios for O2(+) as they relate to blue and green line emission measurements in the nocturnal F-region. During our two-year effort, we partially satisfied our ambitious goals. We constructed and operated a variable pressure, electron-impact ion source and a high pressure, hollow-cathode discharge ion

  9. Enhanced Proteolytic Processing of Recombinant Human Coagulation Factor VIII B-Domain Variants by Recombinant Furins.

    PubMed

    Demasi, Marcos A; de S Molina, Erika; Bowman-Colin, Christian; Lojudice, Fernando H; Muras, Angelita; Sogayar, Mari C

    2016-06-01

    Recombinant human factor VIII (rFVIII) is used in replacement therapy for hemophilia A. Current research efforts are focused on bioengineering rFVIII molecules to improve its secretion efficiency and stability, limiting factors for its efficient production. However, high expression yield in mammalian cells of these rFVIII variants is generally associated with limited proteolytic processing. Non-processed single-chain polypeptides constitute non-natural FVIII molecule configurations with unpredictable toxicity and/or antigenicity. Our main objective was to demonstrate the feasibility of promoting full-proteolytic processing of an rFVIII variant retaining a portion of the B-domain, converting it into the smallest natural activatable form of rFVIII, while keeping its main advantage, i.e., improved secretion efficiency. We generated and employed a CHO-DG44 cell clone producing an rFVIII variant retaining a portion of the B-domain and the FVIII native cleavage site between Arg(1648) and Glu(1649). By bioengineering CHO-DG44 cells to express stably the recombinant human endoproteases PACE, PACE-SOL, PCSK5, PCSK6, or PCKS7, we were able to achieve complete intra- or extracellular proteolytic processing of this rFVIII variant. Additionally, our quantitative data indicated that removal of the B-domain segment by intracellular proteolytic processing does not interfere with this rFVIII variant secretion efficiency. This work also provides the first direct evidence of (1) intracellular cleavage at the Arg(1648) FVIII processing site promoted by wild-type PACE and PCSK7 and (2) proteolytic processing at the Arg(1648) FVIII processing site by PCSK6.

  10. Optimal expression condition of recombinant RAP.

    PubMed

    Zhang, Jie; Zhang, Hong; Bi, Hao; Liu, Zhiguo; Guo, Jianli; Qu, Shen

    2007-02-01

    In order to construct the expression recombinant of human receptor associated protein (RAP), optimize its expression condition and obtain the recombinant protein after expression with high efficiency, two prokaryotic expression vectors-pT7-PL and pET-28a(+) were used to construct the expression recombinant containing RAP cDNA, and the expression efficiency of two kinds of expression E. coli of BL21 strains was compared. The effect of different induction conditions on the expression of recombinant RAP was observed. After recombinant protein was purified with Ni(+) -nitrilotriacetic acid (Ni(+) -NTA) affinity chromatogram, its binding ability with microphage was observed. The results showed that two recombinant plasmids both obtained high expression of RAP. The expression levels of RAP in plasmid pT7-PL-RAP in BL21 (DE3, plysS) strain were significantly higher than in BL21 (DE3) strain. The expression of pT7-PL-RAP in the presence of chloramphenicol was higher than in the absence of chloramphenicol, and most of the inducible expressed RAP was soluble. The RAP which was purified by Ni(+) -NTA resin could strongly bind with the RAW264.7 cells rich in low density lipoprotein receptor (LDLR) family receptors. It was concluded that the expression condition of recombinant RAP was optimized and functional RAP was obtained, which offered a good foundation for the further production of RAP as research tool.

  11. Homologous recombination in plants is organ specific.

    PubMed

    Boyko, Alexander; Filkowski, Jody; Hudson, Darryl; Kovalchuk, Igor

    2006-03-20

    In this paper we analysed the genome stability of various Arabidopsis thaliana plant organs using a transgenic recombination system. The system was based on two copies of non-functional GUS (lines #651 and #11) or LUC (line #15D8) reporter genes serving as a recombination substrate. Both reporter assays showed that recombination in flowers or stems were rare events. Most of the recombination sectors were found in leaves and roots, with leaves having over 2-fold greater number of the recombination events per single cell genome as compared to roots. The recombination events per single genome were 9.7-fold more frequent on the lateral half of the leaves than on the medial halves. This correlated with a 2.5-fold higher metabolic activity in the energy source (lateral) versus energy sink (medial) of leaves. Higher metabolic activity was paralleled by a higher anthocyanin production in lateral halves. The level of double strand break (DSB) occurrence was also different among plant organs; the highest level was observed in roots and the lowest in leaves. High level of DSBs strongly positively correlated with the activity of the key repair enzymes, AtKU70 and AtRAD51. The ratio of AtRAD51 to AtKU70 expression was the highest in leaves, supporting the more active involvement of homologous recombination pathway in the repair of DSBs in this organ. Western blot analysis confirmed the real time PCR expression data for AtKU70 gene.

  12. First-principles study of Frenkel pair recombination in tungsten

    NASA Astrophysics Data System (ADS)

    Qin, Shi-Yao; Jin, Shuo; Li, Yu-Hao; Zhou, Hong-Bo; Zhang, Ying; Lu, Guang-Hong

    2017-02-01

    The recombination of one Frenkel pair in tungsten has been investigated through first-principles simulation. Two different recombination types have been identified: instantaneous and thermally activated. The small recombination barriers for thermally activated recombination cases indicate that recombination can occur easily with a slightly increased temperature. For both of the two recombination types, recombination occurs through the self-interstitial atom moving towards the vacancy. The recombination process can be direct or through replacement sequences, depending on the vertical distance between the vacancy and the <1 1 1> line of self-interstitial atom pair.

  13. Extracellular secretion of recombinant proteins

    DOEpatents

    Linger, Jeffrey G.; Darzins, Aldis

    2014-07-22

    Nucleic acids encoding secretion signals, expression vectors containing the nucleic acids, and host cells containing the expression vectors are disclosed. Also disclosed are polypeptides that contain the secretion signals and methods of producing polypeptides, including methods of directing the extracellular secretion of the polypeptides. Exemplary embodiments include cellulase proteins fused to secretion signals, methods to produce and isolate these polypeptides, and methods to degrade lignocellulosic biomass.

  14. Intraspecific variation of recombination rate in maize

    PubMed Central

    2013-01-01

    Background In sexually reproducing organisms, meiotic crossovers ensure the proper segregation of chromosomes and contribute to genetic diversity by shuffling allelic combinations. Such genetic reassortment is exploited in breeding to combine favorable alleles, and in genetic research to identify genetic factors underlying traits of interest via linkage or association-based approaches. Crossover numbers and distributions along chromosomes vary between species, but little is known about their intraspecies variation. Results Here, we report on the variation of recombination rates between 22 European maize inbred lines that belong to the Dent and Flint gene pools. We genotype 23 doubled-haploid populations derived from crosses between these lines with a 50 k-SNP array and construct high-density genetic maps, showing good correspondence with the maize B73 genome sequence assembly. By aligning each genetic map to the B73 sequence, we obtain the recombination rates along chromosomes specific to each population. We identify significant differences in recombination rates at the genome-wide, chromosome, and intrachromosomal levels between populations, as well as significant variation for genome-wide recombination rates among maize lines. Crossover interference analysis using a two-pathway modeling framework reveals a negative association between recombination rate and interference strength. Conclusions To our knowledge, the present work provides the most comprehensive study on intraspecific variation of recombination rates and crossover interference strength in eukaryotes. Differences found in recombination rates will allow for selection of high or low recombining lines in crossing programs. Our methodology should pave the way for precise identification of genes controlling recombination rates in maize and other organisms. PMID:24050704

  15. Production of recombinant immunotherapeutics for anticancer treatment: the role of bioengineering.

    PubMed

    Pranchevicius, Maria-Cristina S; Vieira, Thiessa R

    2013-01-01

    Cancer is one of the most important health problems because many cases are difficult to prevent. Cancer still has unknown mechanisms of pathogenesis, and its capacity to produce temporary or permanent damage, besides death, is very high. Although many anticancer therapies are available, finding a cure for cancer continues to be a difficult task. Thus, many efforts have been made to develop more effective treatments, such as immunotherapy based on a new class of tumor-specific products that are produced using recombinant DNA technology. These recombinant products are used with the main objectives of killing the tumor and stimulating immune cells to respond to the cancer cells. The principal recombinant products in anticancer therapy are immunostimulants, vaccines, antibodies, immunotoxins and fusion proteins. This review focuses on the general aspects of these genetically engineered products, their clinical performance, current advances and future prospects for this type of anticancer therapy.

  16. Evidence of harvesting electricity by exciton recombination in an n-n type solar cell.

    PubMed

    Song, Qun Liang; Yang, Hong Bin; Gan, Ye; Gong, Cheng; Ming Li, Chang

    2010-04-07

    Free electrons and holes bounded by weak interactions in organic molecules must be generated from excitons to produce photocurrent in organic solar cells. Free charge carriers, in either small molecule- or polymer-based solar cells, are generated so far by dissociation of excitons at the donor-acceptor interface through injecting electrons (holes) from a donor (acceptor) into an acceptor (donor) while leaving holes (electrons) in the donor (acceptor). Here we report a new way, intermolecular exciton recombination, to generate free carriers from organic semiconductors. Unlike the exciton dissociation between donor and acceptor, the recombination of electrons from perfluorinated hexadecafluorophthalo-cyaninatozinc (F16ZnPc) with holes from fullerene (C(60)) frees their counterpart carriers. A new organic solar cell based on this intermolecular exciton recombination at the interface is fabricated to clearly demonstrate this new way to produce free carriers and then harvest electricity from sunlight.

  17. Spacecraft thermal energy accommodation from atomic recombination

    NASA Technical Reports Server (NTRS)

    Carleton, Karen L.; Marinelli, William J.

    1991-01-01

    Measurements of atomic recombination probabilities important in determining energy release to reusable spacecraft thermal protection surfaces during reentry are presented. An experimental apparatus constructed to examine recombination of atomic oxygen from thermal protection and reference materials at reentry temperatures is described. The materials are examined under ultrahigh vacuum conditions to develop and maintain well characterized surface conditions that are free of contamination. When compared with stagnation point heat transfer measurements performed in arc jet facilities, these measurements indicate that a significant fraction of the excess energy available from atom recombination is removed from the surface as metastable O2.

  18. Recombination of N4(+) ions with electrons

    NASA Technical Reports Server (NTRS)

    Cao, Y. S.; Johnsen, R.

    1991-01-01

    Using a modified high-pressure-afterglow/mass spectrometer apparatus similar to that described by Lee and Johnsen (1989), spectroscopic observations of afterglow helium plasmas, with N2 as a minor additive, were carried out in order to verify the mechanism suggested by Bates (1991) for dissociative recombination of electrons with N4(+) ions. It was found that dissociative recombination of electrons with N4(+) ions results in the formation of N2 molecules in the C 3Pi(u) (v = 0,1) state, with the recombination rate coefficient of (2.6 +/- 0.3) x 10 exp -6 cu cm/sec at 300 K.

  19. Dissociative Recombination without a Curve Crossing

    NASA Technical Reports Server (NTRS)

    Guberman, Steven L.

    1994-01-01

    Ab initio calculations show that a curve crossing is not always needed for a high dissociative- recombination cross section. For HeH(+), in which no neutral states cross the ion potential curve, dissociative recombination is driven by the nuclear kinetic-energy operator on adiabatic potential curves. The kinetic-energy derivative operator allows for capture into repulsive curves that are outside of the classical turning points for the nuclear motion. The dominant dissociative route is the C (2)Sigma(+) state leading to H(n = 2) atoms. An analogous mechanism is proposed for the dissociative recombination of H3(+).

  20. Disease diagnosis by recombinant DNA methods

    SciTech Connect

    Caskey, C.T.

    1987-06-05

    Recombinant DNA procedures have now been applied to the problem of the identification of molecular defects in man that account for heritable diseases, somatic mutations associated with neoplasia, and acquired infectious disease. Thus, recombinant DNA technology has rapidly expanded the ability to diagnose disease. Substantial advances in the simplification of procedures for diagnostic purposes have been made, and the informed physician has gained in diagnostic accuracy as a consequence of these developments. The wide application of recombinant DNA diagnostics will depend on simplicity, speed of results, and cost containment. 66 references, 7 figures.

  1. Bacterial genome remodeling through bacteriophage recombination.

    PubMed

    Menouni, Rachid; Hutinet, Geoffrey; Petit, Marie-Agnès; Ansaldi, Mireille

    2015-01-01

    Bacteriophages co-exist and co-evolve with their hosts in natural environments. Virulent phages lyse infected cells through lytic cycles, whereas temperate phages often remain dormant and can undergo lysogenic or lytic cycles. In their lysogenic state, prophages are actually part of the host genome and replicate passively in rhythm with host division. However, prophages are far from being passive residents: they can modify or bring new properties to their host. In this review, we focus on two important phage-encoded recombination mechanisms, i.e. site-specific recombination and homologous recombination, and how they remodel bacterial genomes.

  2. Advances in recombinant antibody manufacturing.

    PubMed

    Kunert, Renate; Reinhart, David

    2016-04-01

    Since the first use of Chinese hamster ovary (CHO) cells for recombinant protein expression, production processes have steadily improved through numerous advances. In this review, we have highlighted several key milestones that have contributed to the success of CHO cells from the beginning of their use for monoclonal antibody (mAb) expression until today. The main factors influencing the yield of a production process are the time to accumulate a desired amount of biomass, the process duration, and the specific productivity. By comparing maximum cell densities and specific growth rates of various expression systems, we have emphasized the limiting parameters of different cellular systems and comprehensively described scientific approaches and techniques to improve host cell lines. Besides the quantitative evaluation of current systems, the quality-determining properties of a host cell line, namely post-translational modifications, were analyzed and compared to naturally occurring polyclonal immunoglobulin fractions from human plasma. In summary, numerous different expression systems for mAbs are available and also under scientific investigation. However, CHO cells are the most frequently investigated cell lines and remain the workhorse for mAb production until today.

  3. An inducible expression system for high-level expression of recombinant proteins in slow growing mycobacteria.

    PubMed

    Leotta, Lisa; Spratt, Joanne M; Kong, Carlyn U; Triccas, James A

    2015-09-01

    A novel protein expression vector utilising the inducible hspX promoter of Mycobacterium tuberculosis was constructed and evaluated in this study. High-level induction of three mycobacterial antigens, comprising up to 9% of bacterial sonicate, was demonstrated in recombinant Mycobacterium bovis BCG when grown under low-oxygen tension, which serves to enhance hspX promoter activity. Recombinant proteins were efficiently purified from bacterial lysates in a soluble form by virtue of a C-terminal 6-histidine tag. Purification of the immunodominant M. tuberculosis Ag85B antigen using this system resulted in a recombinant protein that stimulated significant IFN-γ release from Ag85B-reactive T cells generated after vaccination of mice with an Ag85B-expressing vaccine. Further, the M. tuberculosis L-alanine dehydrogenase (Ald) protein purified from recombinant BCG displayed strong enzymatic activity in recombinant form. This study demonstrated that high levels of native-like recombinant mycobacterial proteins can be produced in mycobacterial hosts, and this may aid the analysis of mycobacterial protein function and the development of new treatments.

  4. Protection of gerbils from amebic liver abscess by immunization with a recombinant Entamoeba histolytica antigen.

    PubMed Central

    Zhang, T; Cieslak, P R; Stanley, S L

    1994-01-01

    Amebiasis, infection by the intestinal protozoan parasite Entamoeba histolytica, is a leading parasitic cause of death. As a step in the development of a recombinant antigen vaccine to prevent E. histolytica infection, we looked at the ability of a recombinant version of the serine-rich E. histolytica protein (SREHP) to elicit a protective immune response against invasive amebic disease. Gerbils, a standard model for amebic liver abscess, were immunized with either a recombinant SREHP/maltose-binding protein (MBP) fusion, recombinant MBP alone, or phosphate-buffered saline (PBS), all combined with complete Freund's adjuvant. In the first trial (group 1), gerbils received a primary and two booster immunizations intraperitoneally; in the second trial (group 2), gerbils were immunized by a single intradermal injection. SREHP/MBP-immunized gerbils in both groups produced antibody to native SHEHP and developed delayed-type hypersensitivity responses to recombinant SREHP. All gerbils were challenged by an intrahepatic injection with 5 x 10(4) virulent E. histolytica HM1-IMSS trophozoites. Complete protection from amebic liver abscess was seen in 64% of the SHEHP/MBP-immunized gerbils in group 1 and in 100% of the SREHP/MBP-immunized gerbils in group 2. There was no protection observed in MBP- or PBS-immunized gerbils in either group. Our results indicate that the SREHP molecule has potential as a vaccine to prevent amebic infection and demonstrate that successful vaccination of animals with recombinant E. histolytica antigen vaccines is possible. Images PMID:8132322

  5. Ar^+ recombination with negative ions in a flowing afterglow: A new approach.

    NASA Astrophysics Data System (ADS)

    Miller, Thomas M.; Viggiano, A. A.

    2006-05-01

    Ion-ion recombination (mutual neutralization) has been previously studied in a flowing-afterglow Langmuir-probe apparatus, using the probe to measure the positive-ion and negative-ion densities as a function of distance (time) along the flow tube axis.^1 A different approach has been taken in the present work, applicable to Ar^+ (or Kr^+ and Xe^+) recombination reactions. A flowing electron-Ar^+ afterglow plasma is first established, and the ambipolar diffusion frequency is measured. Then, an electron attaching gas is added to the afterglow, and the electron attachment rate constant and product ion branching fractions are measured in the usual manner.^2 Finally, the reactant gas concentration is reduced enough that the attachment reaction has not gone to completion by the end of the flow tube. Modeling of the diffusion, attachment, and recombination processes allows us to determine rate constants for each negative ion type recombining with Ar^+. For example, Ar^+ neutralized by Cl^-, Cl2^-, and CCl2O^- produced in attachment to oxalyl chloride,^2 shows that Cl2^- recombines at about half the rate constant of CCl2O^-, and Cl^- + Ar^+ recombination is negligible. ^1D. Smith and N. G. Adams, in Physics of Ion-Ion and Electron-Ion Collisions, Ed. F. Brouillard and J. W. McGowan (Plenum, New York, 1983). ^2J. M. Van Doren, T. M. Miller, and A. A. Viggiano, J. Chem. Phys. (submitted).

  6. Recombinant infectious hematopoietic necrosis viruses induce protection for rainbow trout Oncorhynchus mykiss.

    PubMed

    Romero, Alejandro; Figueras, Antonio; Thoulouze, Maria-Isabel; Bremont, Michael; Novoa, Beatriz

    2008-07-07

    Infectious hematopoietic necrosis virus (IHNV) and viral hemorrhagic septicaemia virus (VHSV) are rhabdoviruses that infect salmonids, producing serious economic losses. Two recombinant IHN viruses were generated by reverse genetics. For one (rIHNV GFP) the IHNV NV gene was replaced with the green fluorescent protein (GFP) gene. In the other (rIHNV-Gvhsv GFP) the G gene was also exchanged for that of VHSV. No mortalities, external signs or histological lesions were observed in experimental infections conducted with the recombinant viruses. Neither the rIHNV GFP nor rIHNV-Gvhsv GFP was detected by RT-PCR in any of the examined tissues from experimentally infected fish. In order to assess their potential as vaccines against the wild type viruses, rainbow trout were vaccinated with the recombinant viruses by intraperitoneal injection and challenged 30 d later with virulent IHNV or VHSV. The GFP viruses provided protection against both wild type viruses. None of the recombinant viruses induced antibody production, and the expression of interferon (IFNalpha4) and interferon induced genes such as Mx protein and ISG-15 was not different to that of controls. The rIHNV-Gvhsv GFP did not inhibit cellular apoptosis as it was observed in an IHNV inoculated fish cell line. These studies suggest that the recombinant rIHNV-Gvhsv GFP is a promising candidate as a live recombinant vaccine and also provides a good model to further study viral pathogenicity and the molecular basis of protection against these viral infections.

  7. Effect of Auger Recombination on Lasing in Heterostructured Quantum Dots with Engineered Core/Shell Interfaces.

    PubMed

    Park, Young-Shin; Bae, Wan Ki; Baker, Thomas; Lim, Jaehoon; Klimov, Victor I

    2015-11-11

    Nanocrystal quantum dots (QDs) are attractive materials for applications as laser media because of their bright, size-tunable emission and the flexibility afforded by colloidal synthesis. Nonradiative Auger recombination, however, hampers optical amplification in QDs by rapidly depleting the population of gain-active multiexciton states. In order to elucidate the role of Auger recombination in QD lasing and isolate its influence from other factors that might affect optical gain, we study two types of CdSe/CdS core/shell QDs with the same core radii and the same total sizes but different properties of the core/shell interface ("sharp" vs "smooth"). These samples exhibit distinctly different biexciton Auger lifetimes but are otherwise virtually identical. The suppression of Auger recombination in the sample with a smooth (alloyed) interface results in a notable improvement in the optical gain performance manifested in the reduction of the threshold for amplified spontaneous emission and the ability to produce dual-color lasing involving both the band-edge (1S) and the higher-energy (1P) electronic states. We develop a model, which explicitly accounts for the multiexciton nature of optical gain in QDs, and use it to analyze the competition between stimulated emission from multiexcitons and their decay via Auger recombination. These studies re-emphasize the importance of Auger recombination control for the realization of real-life QD-based lasing technologies and offer practical strategies for suppression of Auger recombination via "interface engineering" in core/shell structures.

  8. Cloning and Expression of Soluble Recombinant HIV-1 CRF35 Protease-HP Thioredoxin Fusion Protein

    PubMed Central

    Azarnezhad, Asaad; Sharifi, Zohreh; Seyedabadi, Rahmatollah; Hosseini, Arshad; Johari, Behrooz; Sobhani Fard, Mahsa

    2016-01-01

    Background: As a drug target and an antigenic agent, HIV-1 protease (HIV-1 PR) is at the center of attention for designing anti-AIDS inhibitors and diagnostic tests. In previous studies, the production of the recombinant protease has been faced with several difficulties; therefore, the aims of this study were the easy production, purification of the soluble form of protease in E. coli and investigation of its immunoreactivity. Methods: Protease coding region was isolated from the serum of an infected individual, amplified by RT-PCR and cloned into PTZ57R using TA-cloning. Protease coding frame was isolated by PCR and cloned in pET102/D. TOPO expression vector and cloned protease was expressed in Escherichia coli (E. coli) BL21. Produced recombinant protein was purified by affinity Ni-NTA column and protein concentration was checked by BCA protein assay kit. Subsequently, immunoreactivity of recombinant protease (rPR) was assayed by Western blotting and ELISA. Results: Cloning of the HIV protease by TOPO cloning system in pET102/D.TOPO was confirmed with PCR and sequencing. The concentration range of purified recombinant protein was 85 to 100 μg/ml. Immunogenicity of rPR was confirmed by Western blotting and ELISA. Conclusion: Soluble production of recombinant HIV-1 protease (HIV-1 rPR) was performed successfully. This recombinant protein disclosed 86% specificity and 90% sensitivity in immunoassay tests. PMID:27920885

  9. Synthesis and characterization of biologically active recombinant elk and horse FSH.

    PubMed

    Fachal, María Victoria; Furlan, Mike; Clark, Rena; Card, Claire E; Chedrese, P Jorge

    2010-02-01

    The objective of this investigation was to clone and express the elk and horse common alpha-subunit and FSH beta-subunit cDNAs, and to produce recombinant FSH from both species in vitro. The RNAs extracted from elk and horse pituitary glands were reverse-transcribed and amplified by polymerase chain reaction. The cDNAs corresponding to both subunits of elk and horse were cloned into the expression vector pBudCE4.1 and transfected into CRL-9096 cells. Expression of both genes was determined in the transfected cells by Northern and Western blot analysis. Recombinant elk and horse FSH secreted in culture media were characterized by an in vitro bioassay and RIA. When the recombinant products were assessed as activity over mass of FSH measured by RIA, the horse product was 5.6 times more potent than the elk product. The recombinant products injected to immature female Wistar rats stimulated ovarian growth. The results suggest that the products obtained correspond to recombinant versions of the native elk and horse FSH. The availability of these recombinant products may aid in the development of more predictable and efficient techniques of ovarian stimulation in cervids, equids, and other species as well.

  10. Virulence and Molecular Characterization of Experimental Isolates of the Stripe Rust Pathogen (Puccinia striiformis) Indicate Somatic Recombination.

    PubMed

    Lei, Yu; Wang, Meinan; Wan, Anmin; Xia, Chongjing; See, Deven R; Zhang, Min; Chen, Xianming

    2017-03-01

    Puccinia striiformis causes stripe rust on wheat, barley, and grasses. Natural population studies have indicated that somatic recombination plays a possible role in P. striiformis variation. To determine whether somatic recombination can occur, susceptible wheat or barley plants were inoculated with mixed urediniospores of paired isolates of P. striiformis. Progeny isolates were selected by passing through a series of inoculations of wheat or barley genotypes. Potential recombinant isolates were compared with the parental isolates on the set of 18 wheat or 12 barley genotypes that are used to differentiate races of P. striiformis f. sp. tritici (the wheat stripe rust pathogen) and P. striiformis f. sp. hordei (the barley stripe rust pathogen), respectively, for virulence changes. They were also tested with 51 simple-sequence repeat and 90 single-nucleotide polymorphism markers for genotype changes. From 68 possible recombinant isolates obtained from nine combinations of isolates based on virulence tests, 66 were proven to be recombinant isolates by molecular markers. Various types of recombinants were determined, including lost virulence from both virulent parental isolates, gained virulence from both avirulent isolates, combined virulences from both parents, and inherited virulence from one parent and avirulence from another. Marker data indicate that most of the recombinants were produced through chromosome reassortment and crossover after the hybridization of two parental isolates. The results demonstrate that somatic recombination is a mechanism by which new variants can be generated in P. striiformis.

  11. Recombinant Human Papillomavirus (HPV) Quadrivalent Vaccine

    Cancer.gov

    This page contains brief information about recombinant human papillomavirus (HPV) quadrivalent vaccine and a collection of links to more information about the use of this vaccine, research results, and ongoing clinical trials.

  12. Recombinant Human Papillomavirus (HPV) Bivalent Vaccine

    Cancer.gov

    This page contains brief information about recombinant human papillomavirus (HPV) bivalent vaccine and a collection of links to more information about the use of this vaccine, research results, and ongoing clinical trials.

  13. Recombinant Human Papillomavirus (HPV) Nonavalent Vaccine

    Cancer.gov

    This page contains brief information about recombinant human papillomavirus (HPV) nonavalent vaccine and a collection of links to more information about the use of this vaccine, research results, and ongoing clinical trials.

  14. Epigenetic control of meiotic recombination in plants.

    PubMed

    Yelina, Natasha; Diaz, Patrick; Lambing, Christophe; Henderson, Ian R

    2015-03-01

    Meiotic recombination is a deeply conserved process within eukaryotes that has a profound effect on patterns of natural genetic variation. During meiosis homologous chromosomes pair and undergo DNA double strand breaks generated by the Spo11 endonuclease. These breaks can be repaired as crossovers that result in reciprocal exchange between chromosomes. The frequency of recombination along chromosomes is highly variable, for example, crossovers are rarely observed in heterochromatin and the centromeric regions. Recent work in plants has shown that crossover hotspots occur in gene promoters and are associated with specific chromatin modifications, including H2A.Z. Meiotic chromosomes are also organized in loop-base arrays connected to an underlying chromosome axis, which likely interacts with chromatin to organize patterns of recombination. Therefore, epigenetic information exerts a major influence on patterns of meiotic recombination along chromosomes, genetic variation within populations and evolution of plant genomes.

  15. Chemical recombination in an expansion tube

    NASA Technical Reports Server (NTRS)

    Bakos, Robert J.; Morgan, Richard G.

    1994-01-01

    The note describes the theoretical basis of chemical recombination in an expansion tube which simulates energy, Reynolds number, and stream chemistry at near-orbital velocities. Expansion tubes can satisfy ground-based hypersonic propulsion and aerothermal testing requirements.

  16. The Kinetics of Nitrogen Atom Recombination

    ERIC Educational Resources Information Center

    Brown, G. Ronald; Winkler, C. A.

    1977-01-01

    Describes a study of the kinetics of the recombination of nitrogen atoms in which concentration-time relations are determined directly by utilizing visual observations of emissions to make gas phase titrations of N atoms with NO. (MLH)

  17. Strategies for RNA-Guided DNA Recombination

    NASA Astrophysics Data System (ADS)

    Angeleska, Angela; Jonoska, Nataša; Saito, Masahico; Landweber, Laura F.

    We present a model for homologous DNA recombination events guided by double-stranded RNA (dsRNA) templates, and apply this model to DNA rearrangements in some groups of ciliates, such as Stylonychia or Oxytricha. In these organisms, differentiation of a somatic macronucleus from a germline micronucleus involves extensive gene rearrangement, which can be modeled as topological braiding of the DNA, with the template-guided alignment proceeding through DNA branch migration. We show that a graph structure, which we refer to as an assembly graph, containing only 1- and 4-valent vertices can provide a physical representation of the DNA at the time of recombination. With this representation, 4-valent vertices correspond to the alignment of the recombination sites, and we model the actual recombination event as smoothing of these vertices.

  18. The homologous recombination system of Ustilago maydis.

    PubMed

    Holloman, William K; Schirawski, Jan; Holliday, Robin

    2008-08-01

    Homologous recombination is a high fidelity, template-dependent process that is used in repair of damaged DNA, recovery of broken replication forks, and disjunction of homologous chromosomes in meiosis. Much of what is known about recombination genes and mechanisms comes from studies on baker's yeast. Ustilago maydis, a basidiomycete fungus, is distant evolutionarily from baker's yeast and so offers the possibility of gaining insight into recombination from an alternative perspective. Here we have surveyed the genome of U. maydis to determine the composition of its homologous recombination system. Compared to baker's yeast, there are fundamental differences in the function as well as in the repertoire of dedicated components. These include the use of a BRCA2 homolog and its modifier Dss1 rather than Rad52 as a mediator of Rad51, the presence of only a single Rad51 paralog, and the absence of Dmc1 and auxiliary meiotic proteins.

  19. The homologous recombination system of Ustilago maydis

    PubMed Central

    Holloman, William K.; Schirawski, Jan; Holliday, Robin

    2008-01-01

    Homologous recombination is a high fidelity, template-dependent process that is used in repair of damaged DNA, recovery of broken replication forks, and disjunction of homologous chromosomes in meiosis. Much of what is known about recombination genes and mechanisms comes from studies on baker's yeast. Ustilago maydis, a basidiomycete fungus, is distant evolutionarily from baker's yeast and so offers the possibility of gaining insight into recombination from an alternative perspective. Here we have surveyed the genome of Ustilago maydis to determine the composition of its homologous recombination system. Compared to baker's yeast, there are fundamental differences in the function as well as in the repertoire of dedicated components. These include the use of a BRCA2 homolog and its modifier Dss1 rather than Rad52 as a mediator of Rad51, the presence of only a single Rad51 paralog, and the absence of Dmc1 and auxiliary meiotic proteins. PMID:18502156

  20. Investigating the dynamics of recombinant protein secretion from a microalgal host.

    PubMed

    Lauersen, Kyle J; Huber, Isabel; Wichmann, Julian; Baier, Thomas; Leiter, Andreas; Gaukel, Volker; Kartushin, Viktor; Rattenholl, Anke; Steinweg, Christian; von Riesen, Lena; Posten, Clemens; Gudermann, Frank; Lütkemeyer, Dirk; Mussgnug, Jan H; Kruse, Olaf

    2015-12-10

    Production of recombinant proteins with microalgae represents an alternative platform over plant- or bacterial-based expression systems for certain target proteins. Secretion of recombinant proteins allows accumulation of the target product physically separate from the valuable algal biomass. To date, there has been little investigation into the dynamics of recombinant protein secretion from microalgal hosts-the culture parameters that encourage secreted product accumulation and stability, while encouraging biomass production. In this work, the efficiency of recombinant protein production was optimized by adjusting cultivation parameters for a strain of Chlamydomonas reinhardtii previously engineered to secrete a functional recombinant Lolium perenne ice binding protein (LpIBP), which has applications as a frozen food texturing and cryopreservation additive, into its culture medium. Three media and several cultivation styles were investigated for effects on secreted LpIBP titres and culture growth. A combination of acetate and carbon dioxide feeding with illumination resulted in the highest overall biomass and recombinant protein titres up to 10mgL(-1) in the culture medium. Pure photoautotrophic production was possible using two media types, with recombinant protein accumulation in all cultivations correlating to culture cell density. Two different cultivation systems were used for scale-up to 10L cultivations, one of which produced yields of secreted recombinant protein up to 12mgL(-1) within six cultivation days. Functional ice recrystallization inhibition (IRI) of the LpIBP from total concentrated extracellular protein extracts was demonstrated in a sucrose solution used as a simplified ice cream model. IRI lasted up to 7 days, demonstrating the potential of secreted products from microalgae for use as food additives.