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Sample records for recombinant fgf21 protein

  1. Circulating FGF21 proteolytic processing mediated by fibroblast activation protein

    PubMed Central

    Zhen, Eugene Y.; Jin, Zhaoyan; Ackermann, Bradley L.; Thomas, Melissa K.; Gutierrez, Jesus A.

    2015-01-01

    Fibroblast growth factor 21 (FGF21), a hormone implicated in the regulation of glucose homoeostasis, insulin sensitivity, lipid metabolism and body weight, is considered to be a promising therapeutic target for the treatment of metabolic disorders. Despite observations that FGF21 is rapidly proteolysed in circulation rending it potentially inactive, little is known regarding mechanisms by which FGF21 protein levels are regulated. We systematically investigated human FGF21 protein processing using mass spectrometry. In agreement with previous reports, circulating human FGF21 was found to be cleaved primarily after three proline residues at positions 2, 4 and 171. The extent of FGF21 processing was quantified in a small cohort of healthy human volunteers. Relative abundance of FGF21 proteins cleaved after Pro-2, Pro-4 and Pro-171 ranged from 16 to 30%, 10 to 25% and 10 to 34%, respectively. Dipeptidyl peptidase IV (DPP-IV) was found to be the primary protease responsible for N-terminal cleavages after residues Pro-2 and Pro-4. Importantly, fibroblast activation protein (FAP) was implicated as the protease responsible for C-terminal cleavage after Pro-171, rendering the protein inactive. The requirement of FAP for FGF21 proteolysis at the C-terminus was independently demonstrated by in vitro digestion, immunodepletion of FAP in human plasma, administration of an FAP-specific inhibitor and by human FGF21 protein processing patterns in FAP knockout mouse plasma. The discovery that FAP is responsible for FGF21 inactivation extends the FGF21 signalling pathway and may enable novel approaches to augment FGF21 actions for therapeutic applications. PMID:26635356

  2. Recruitment of histone methyltransferase G9a mediates transcriptional repression of Fgf21 gene by E4BP4 protein.

    PubMed

    Tong, Xin; Zhang, Deqiang; Buelow, Katie; Guha, Anirvan; Arthurs, Blake; Brady, Hugh J M; Yin, Lei

    2013-02-22

    The liver responds to fasting-refeeding cycles by reprogramming expression of metabolic genes. Fasting potently induces one of the key hepatic hormones, fibroblast growth factor 21 (FGF21), to promote lipolysis, fatty acid oxidation, and ketogenesis, whereas refeeding suppresses its expression. We previously reported that the basic leucine zipper transcription factor E4BP4 (E4 binding protein 4) represses Fgf21 expression and disrupts its circadian oscillations in cultured hepatocytes. However, the epigenetic mechanism for E4BP4-dependent suppression of Fgf21 has not yet been addressed. Here we present evidence that histone methyltransferase G9a mediates E4BP4-dependent repression of Fgf21 during refeeding by promoting repressive histone modification. We find that Fgf21 expression is up-regulated in E4bp4 knock-out mouse liver. We demonstrate that the G9a-specific inhibitor BIX01294 abolishes suppression of the Fgf21 promoter activity by E4BP4, whereas overexpression of E4bp4 leads to increased levels of dimethylation of histone 3 lysine 9 (H3K9me2) around the Fgf21 promoter region. Furthermore, we also show that E4BP4 interacts with G9a, and knockdown of G9a blocks repression of Fgf21 promoter activity and expression in cells overexpressing E4bp4. A G9a mutant lacking catalytic activity, due to deletion of the SET domain, fails to inhibit the Fgf21 promoter activity. Importantly, acute hepatic knockdown by adenoviral shRNA targeting G9a abolishes Fgf21 repression by refeeding, concomitant with decreased levels of H3K9me2 around the Fgf21 promoter region. In summary, we show that G9a mediates E4BP4-dependent suppression of hepatic Fgf21 by enhancing histone methylation (H3K9me2) of the Fgf21 promoter. PMID:23283977

  3. FGF21 ameliorates nonalcoholic fatty liver disease by inducing autophagy.

    PubMed

    Zhu, Shenglong; Wu, Yunzhou; Ye, Xianlong; Ma, Lei; Qi, Jianying; Yu, Dan; Wei, Yuquan; Lin, Guangxiao; Ren, Guiping; Li, Deshan

    2016-09-01

    The aim of this study is to evaluate the role of fibroblast growth factor 21 (FGF21) in nonalcoholic fatty liver disease (NAFLD) and seek to determine if its therapeutic effect is through induction of autophagy. In this research, Monosodium L-glutamate (MSG)-induced obese mice or normal lean mice were treated with vehicle, Fenofibrate, and recombinant murine FGF21, respectively. After 5 weeks of treatment, metabolic parameters including body weight, blood glucose and lipid levels, hepatic and fat gene expression levels were monitored and analyzed. Also, fat-loaded HepG2 cells were treated with vehicle or recombinant murine FGF21. The expression levels of proteins associated with autophagy were detected by western blot, real-time PCR, and transmission electron microscopy (TEM). Autophagic flux was monitored by laser confocal microscopy and western blot. Results showed that FGF21 significantly reduced body weight (P < 0.01) and serum triglyceride, improved insulin sensitivity, and reversed hepatic steatosis in the MSG model mice. In addition, FGF21 significantly increased the expression of several proteins related to autophagy both in MSG mice and fat-loaded HepG2 cells, such as microtubule associated protein 1 light chain 3, Bcl-2-interacting myosin-like coiled-coil protein-1 (Beclin-1), and autophagy-related gene 5. Furthermore, the evidence of TEM revealed an increased number of autophagosomes and lysosomes in the model cells treated with FGF21. In vitro experimental results also showed that FGF21 remarkably increased autophagic flux. Taken together, FGF21 corrects multiple metabolic parameters on NAFLD in vitro and in vivo by inducing autophagy. PMID:27435856

  4. Metabolic Responses to Dietary Protein Restriction Require an Increase in FGF21 that Is Delayed by the Absence of GCN2.

    PubMed

    Laeger, Thomas; Albarado, Diana C; Burke, Susan J; Trosclair, Lexus; Hedgepeth, John W; Berthoud, Hans-Rudolf; Gettys, Thomas W; Collier, J Jason; Münzberg, Heike; Morrison, Christopher D

    2016-07-19

    FGF21 contributes to the metabolic response to dietary protein restriction, and prior data implicate GCN2 as the amino acid sensor linking protein restriction to FGF21 induction. Here, we demonstrate the persistent and essential role of FGF21 in the metabolic response to protein restriction. We show that Fgf21 KO mice are fully resistant to low protein (LP)-induced changes in food intake, energy expenditure (EE), body weight gain, and metabolic gene expression for 6 months. Gcn2 KO mice recapitulate this phenotype, but LP-induced effects on food intake, EE, and body weight subsequently begin to appear after 14 days on diet. We show that this delayed emergence of LP-induced metabolic effects in Gcn2 KO mice coincides with a delayed but progressive increase of hepatic Fgf21 expression and blood FGF21 concentrations over time. These data indicate that FGF21 is essential for the metabolic response to protein restriction but that GCN2 is only transiently required for LP-induced FGF21. PMID:27396336

  5. Molecular Characterization and Mapping of Fgf21 Gene in a Foodfish Species Asian Seabass

    PubMed Central

    Wang, Le; Xia, Jun Hong; Liu, Xiao Jun; Liu, Peng; Wan, Zi Yi; Yue, Gen Hua

    2014-01-01

    Fgf21 is a newly discovered fibroblast growth factor. It is typically induced by fasting and plays important roles in the regulation of glucose and lipid metabolisms and energy balance in mammals, whereas potential functions of this gene in teleosts are still unknown. We identified the Fgf21 gene and studied its functions in Asian seabass (Lates calcarifer). The cDNA of the Fgf21 encoded a protein with 206 amino acids. Analysis of DNA and amino acid sequences of Fgf21 genes revealed that the sequences and structure of the Fgf21 genes were highly conserved in vertebrates. Real-time PCR revealed that Fgf21 was exclusively expressed in the intestine and kidney, which was different from the expression profiles of mammals. Fgf21 was down-regulated under fasting, whereas it was significantly increased during the LPS challenge. Exogenous recombinant FGF21 significantly suppressed the appetite of Asian seabass. Our data suggest that Fgf21 plays a role in energy regulation and acute phase response in Asian seabass, and may have different functions in fish and mammals. In addition, we identified one SNP in Fgf21. By using this SNP, the gene was mapped on the linkage group 23, where a suggestive QTL for growth was mapped previously. Association mapping identified significant associations between Fgf21 genotypes at the SNP and growth traits. These results not only provide important information of the functions of Fgf21, but also suggest that the SNP in this gene can be used as a marker in selecting fast-growing individuals of Asian seabass. PMID:24587261

  6. Raised FGF-21 and Triglycerides Accompany Increased Energy Intake Driven by Protein Leverage in Lean, Healthy Individuals: A Randomised Trial

    PubMed Central

    Gosby, Alison K.; Lau, Namson S.; Tam, Charmaine S.; Iglesias, Miguel A.; Morrison, Christopher D.; Caterson, Ian D.; Brand-Miller, Jennie; Conigrave, Arthur D.; Raubenheimer, David; Simpson, Stephen J.

    2016-01-01

    A dominant appetite for protein drives increased energy intake in humans when the proportion of protein in the diet is reduced down to approximately 10% of total energy. Compensatory feeding for protein is apparent over a 1–2 d period but the mechanisms driving this regulation are not fully understood. Fibroblast growth factor-21 (FGF-21) has been identified as a candidate protein signal as levels increase in the circulation when dietary protein is low. The aim of this randomised controlled trial was to assess whether changes in percent dietary protein over a 4 d ad libitum experimental period in lean, healthy participants influenced energy intake, metabolic health, circulating FGF-21 and appetite regulating hormones including ghrelin, glucagon like peptide-1 and cholecystokinin. Twenty-two lean, healthy participants were fed ad libitum diets containing 10, 15 and 25% protein, over three, 4 d controlled, in-house experimental periods. Reduced dietary protein intake from 25% to 10% over a period of 4 d was associated with 14% increased energy intake (p = 0.02) as previously reported, and a 6-fold increase in fasting circulating plasma FGF-21 levels (p<0.0001), a 1.5-fold increase in serum triglycerides (p<0.0001), and a 0.9-fold decrease in serum total cholesterol (p = 0.02). Serum HDL cholesterol was reduced with a reduction in dietary protein from 15% to 10% (p = 0.01) over 4 d but not from 25% to 10% (p = 0.1) and the change from baseline was not different between diets. Plasma fasting insulin levels following the 4 d study period were significantly lower following the 25% ad libitum study period compared to the 15% protein period (p = 0.014) but not the 10% protein period (p = 0.2). Variability in interstitial glucose during each study period increased with a decrease in dietary protein from 25% to 15% and 10% (p = 0.001 and p = 0.04, respectively). Ghrelin, glucagon-like peptide-1 and cholecystokinin were unchanged. Increases in energy intake, plasma FGF-21

  7. Photoperiodic regulation of FGF21 production in the Siberian hamster.

    PubMed

    Samms, Ricardo J; Fowler, Maxine J; Cooper, Scott; Emmerson, Paul; Coskun, Tamer; Adams, Andrew C; Kharitonenkov, Alexei; Tsintzas, Kostas; Ebling, Francis J P

    2014-06-01

    This article is part of a Special Issue "Energy Balance". FGF21 is an endocrine member of the fibroblast growth factor superfamily that has been shown to play an important role in the physiological response to nutrient deprivation. Food restriction enhances hepatic FGF21 production, which serves to engage an integrated response to energy deficit. Specifically, elevated FGF21 levels lead to reduced gluconeogenesis and increased hepatic ketogenesis. However, circulating FGF21 concentrations also paradoxically rise in states of metabolic dysfunction such as obesity. Furthermore, multiple peripheral tissues also produce FGF21 in addition to the liver, raising questions as to its endocrine and paracrine roles in the control of energy metabolism. The objectives of this study were to measure plasma FGF21 concentrations in the Siberian hamster, a rodent which undergoes a seasonal cycle of fattening and body weight gain in the long days (LD) of summer, followed by reduction of appetite and fat catabolism in the short days (SD) of winter. Groups of adult male hamsters were raised in long days, and then exposed to SD for up to 12 weeks. Chronic exposure of LD animals to SD led to a significant increase in circulating FGF21 concentrations. This elevation of circulating FGF21 was preceded by an increase in liver FGF21 protein production evident as early as 4 weeks of exposure to SD. FGF21 protein abundance was also increased significantly in interscapular brown adipose tissue, with a positive correlation between plasma levels of FGF21 and BAT protein abundance throughout the experimental period. Epididymal white adipose tissue and skeletal muscle (gastrocnemius) also produced FGF21, but levels did not change in response to a change in photoperiod. In summary, a natural programmed state of fat catabolism was associated with increased FGF21 production in the liver and BAT, consistent with the view that FGF21 has a role in adapting hamsters to the hypophagic winter state.

  8. FGF-21: promising biomarker for detecting ketosis in dairy cows.

    PubMed

    Xu, Chuang; Xu, Qiushi; Chen, Yuanyuan; Yang, Wei; Xia, Cheng; Yu, Hongjiang; Zhu, Kuilin; Shen, Taiyu; Zhang, Ziyang

    2016-03-01

    The objective of this study was to investigate the measurement of serum fibroblast growth factor-21 (FGF-21), a protein mainly synthesized by the liver, as a sensitive biomarker for diagnosis of ketosis in dairy cows. Ninety Holstein-Friesian dairy cows (60 healthy and 30 ketosis cases) were selected and divided into a Ketosis group (K), and a Control group (C). We measured serum FGF-21 and other biochemical parameters by commercial ELISA kits. In a combined population of all 90 cows, we found that serum FGF-21 level was lower (P < 0.001) in cows suffering from ketosis. When the β-hydroxybutyric acid (BHBA) level increased over 1.2 mmol/L, the FGF-21 level tended to decline below 300.85 pg/ml. The area under the receiver operating characteristic curve (AUC-ROC) for serum FGF-21 for diagnosis of fatty liver was 0.952-0.025 [95% confidence interval (CI) 0.904, 1.000] which was higher than the AUC-ROC for glucose (Glc) and other tested parameters. We concluded that FGF-21 could be a diagnostic parameter in the evaluation and auxiliary diagnosis of changes in the energy metabolism state, and serum FGF-21 measurement would have a considerable clinical impact and lead to greater profitability in the dairy industry.

  9. FGF-21: promising biomarker for detecting ketosis in dairy cows.

    PubMed

    Xu, Chuang; Xu, Qiushi; Chen, Yuanyuan; Yang, Wei; Xia, Cheng; Yu, Hongjiang; Zhu, Kuilin; Shen, Taiyu; Zhang, Ziyang

    2016-03-01

    The objective of this study was to investigate the measurement of serum fibroblast growth factor-21 (FGF-21), a protein mainly synthesized by the liver, as a sensitive biomarker for diagnosis of ketosis in dairy cows. Ninety Holstein-Friesian dairy cows (60 healthy and 30 ketosis cases) were selected and divided into a Ketosis group (K), and a Control group (C). We measured serum FGF-21 and other biochemical parameters by commercial ELISA kits. In a combined population of all 90 cows, we found that serum FGF-21 level was lower (P < 0.001) in cows suffering from ketosis. When the β-hydroxybutyric acid (BHBA) level increased over 1.2 mmol/L, the FGF-21 level tended to decline below 300.85 pg/ml. The area under the receiver operating characteristic curve (AUC-ROC) for serum FGF-21 for diagnosis of fatty liver was 0.952-0.025 [95% confidence interval (CI) 0.904, 1.000] which was higher than the AUC-ROC for glucose (Glc) and other tested parameters. We concluded that FGF-21 could be a diagnostic parameter in the evaluation and auxiliary diagnosis of changes in the energy metabolism state, and serum FGF-21 measurement would have a considerable clinical impact and lead to greater profitability in the dairy industry. PMID:26728033

  10. FGF21 ameliorates the neurocontrol of blood pressure in the high fructose-drinking rats.

    PubMed

    He, Jian-Li; Zhao, Miao; Xia, Jing-Jun; Guan, Jian; Liu, Yang; Wang, Lu-Qi; Song, Dong-Xue; Qu, Mei-Yu; Zuo, Meng; Wen, Xin; Yu, Xue; Huo, Rong; Pan, Zhen-Wei; Ban, Tao; Zhang, Yan; Zhu, Jiu-Xin; Shou, Weinian; Qiao, Guo-Fen; Li, Bai-Yan

    2016-01-01

    Fibroblast growth factor-21 (FGF21) is closely related to various metabolic and cardiovascular disorders. However, the direct targets and mechanisms linking FGF21 to blood pressure control and hypertension are still elusive. Here we demonstrated a novel regulatory function of FGF21 in the baroreflex afferent pathway (the nucleus tractus solitarii, NTS; nodose ganglion, NG). As the critical co-receptor of FGF21, β-klotho (klb) significantly expressed on the NTS and NG. Furthermore, we evaluated the beneficial effects of chronic intraperitoneal infusion of recombinant human FGF21 (rhFGF21) on the dysregulated systolic blood pressure, cardiac parameters, baroreflex sensitivity (BRS) and hyperinsulinemia in the high fructose-drinking (HFD) rats. The BRS up-regulation is associated with Akt-eNOS-NO signaling activation in the NTS and NG induced by acute intravenous rhFGF21 administration in HFD and control rats. Moreover, the expressions of FGF21 receptors were aberrantly down-regulated in HFD rats. In addition, the up-regulated peroxisome proliferator-activated receptor-γ and -α (PPAR-γ/-α) in the NTS and NG in HFD rats were markedly reversed by chronic rhFGF21 infusion. Our study extends the work of the FGF21 actions on the neurocontrol of blood pressure regulations through baroreflex afferent pathway in HFD rats. PMID:27387420

  11. FGF21 ameliorates the neurocontrol of blood pressure in the high fructose-drinking rats

    PubMed Central

    He, Jian-Li; Zhao, Miao; Xia, Jing-Jun; Guan, Jian; Liu, Yang; Wang, Lu-Qi; Song, Dong-Xue; Qu, Mei-Yu; Zuo, Meng; Wen, Xin; Yu, Xue; Huo, Rong; Pan, Zhen-Wei; Ban, Tao; Zhang, Yan; Zhu, Jiu-Xin; Shou, Weinian; Qiao, Guo-Fen; Li, Bai-Yan

    2016-01-01

    Fibroblast growth factor-21 (FGF21) is closely related to various metabolic and cardiovascular disorders. However, the direct targets and mechanisms linking FGF21 to blood pressure control and hypertension are still elusive. Here we demonstrated a novel regulatory function of FGF21 in the baroreflex afferent pathway (the nucleus tractus solitarii, NTS; nodose ganglion, NG). As the critical co-receptor of FGF21, β-klotho (klb) significantly expressed on the NTS and NG. Furthermore, we evaluated the beneficial effects of chronic intraperitoneal infusion of recombinant human FGF21 (rhFGF21) on the dysregulated systolic blood pressure, cardiac parameters, baroreflex sensitivity (BRS) and hyperinsulinemia in the high fructose-drinking (HFD) rats. The BRS up-regulation is associated with Akt-eNOS-NO signaling activation in the NTS and NG induced by acute intravenous rhFGF21 administration in HFD and control rats. Moreover, the expressions of FGF21 receptors were aberrantly down-regulated in HFD rats. In addition, the up-regulated peroxisome proliferator-activated receptor-γ and -α (PPAR-γ/-α) in the NTS and NG in HFD rats were markedly reversed by chronic rhFGF21 infusion. Our study extends the work of the FGF21 actions on the neurocontrol of blood pressure regulations through baroreflex afferent pathway in HFD rats. PMID:27387420

  12. Muscle mitochondrial stress adaptation operates independently of endogenous FGF21 action

    PubMed Central

    Ost, Mario; Coleman, Verena; Voigt, Anja; van Schothorst, Evert M.; Keipert, Susanne; van der Stelt, Inge; Ringel, Sebastian; Graja, Antonia; Ambrosi, Thomas; Kipp, Anna P.; Jastroch, Martin; Schulz, Tim J.; Keijer, Jaap; Klaus, Susanne

    2015-01-01

    Objective Fibroblast growth factor 21 (FGF21) was recently discovered as stress-induced myokine during mitochondrial disease and proposed as key metabolic mediator of the integrated stress response (ISR) presumably causing systemic metabolic improvements. Curiously, the precise cell-non-autonomous and cell-autonomous relevance of endogenous FGF21 action remained poorly understood. Methods We made use of the established UCP1 transgenic (TG) mouse, a model of metabolic perturbations made by a specific decrease in muscle mitochondrial efficiency through increased respiratory uncoupling and robust metabolic adaptation and muscle ISR-driven FGF21 induction. In a cross of TG with Fgf21-knockout (FGF21−/−) mice, we determined the functional role of FGF21 as a muscle stress-induced myokine under low and high fat feeding conditions. Results Here we uncovered that FGF21 signaling is dispensable for metabolic improvements evoked by compromised mitochondrial function in skeletal muscle. Strikingly, genetic ablation of FGF21 fully counteracted the cell-non-autonomous metabolic remodeling and browning of subcutaneous white adipose tissue (WAT), together with the reduction of circulating triglycerides and cholesterol. Brown adipose tissue activity was similar in all groups. Remarkably, we found that FGF21 played a negligible role in muscle mitochondrial stress-related improved obesity resistance, glycemic control and hepatic lipid homeostasis. Furthermore, the protective cell-autonomous muscle mitohormesis and metabolic stress adaptation, including an increased muscle proteostasis via mitochondrial unfolded protein response (UPRmt) and amino acid biosynthetic pathways did not require the presence of FGF21. Conclusions Here we demonstrate that although FGF21 drives WAT remodeling, the adaptive pseudo-starvation response under elevated muscle mitochondrial stress conditions operates independently of both WAT browning and FGF21 action. Thus, our findings challenge FGF21 as key

  13. Association between insulin resistance and impairment of FGF21 signal transduction in skeletal muscles.

    PubMed

    Jeon, Ja Young; Choi, Sung-E; Ha, Eun Suk; Kim, Tae Ho; Jung, Jong Gab; Han, Seung Jin; Kim, Hae Jin; Kim, Dae Jung; Kang, Yup; Lee, Kwan-Woo

    2016-07-01

    Fibroblast growth factor (FGF) 21, was identified as a potent metabolic regulator of glucose and lipid metabolism. We investigated whether the levels and signalings of FGF21 changed in the skeletal muscle of type 2 diabetes mellitus (T2DM) patients, participants with impaired glucose tolerance (IGT), human skeletal muscle myotubes (HSMMs) under insulin-resistant conditions, and mice with diet-induced obesity (DIO). A percutaneous biopsy sample of the vastus lateralis muscle of T2DM patients, IGT subjects, and participants with normal glucose tolerance was obtained and the levels and signalings of FGF21 were assessed. We determined whether the expression and signalings of FGF21 in HSMMs altered according to palmitate concentrations and exposure time. Also, we confirmed whether changes of FGF21 signal transduction resulted in the alteration of FGF21 functions. DIO mice were treated intravenously with recombinant FGF21, and the levels and signalings of FGF21 were assessed in their soleus muscles. We checked whether or not FGF21 played a role in the gene transcription related to lipid oxidation. Levels of FGF21 increased, whereas levels of phosphorylated FGF receptor (p-FGFR), phosphorylated FGFR substrates 2α (p-FRS2α), and phosphorylated extracellular signal-regulated kinases (p-ERK) decreased in the skeletal muscle of both T2DM patients and IGT subjects. In vitro, palmitate increased the levels of FGF21 and significantly reduced the levels of β-klotho, p-FGFR, p-FRS2α, and p-ERK1/2 in HSMMs exposed to palmitate. Palmitate also decreased glucose uptake and glycogen contents of FGF21. Consistently, the levels of FGF21 were significantly higher and the levels of β-klotho and p-FGFR were lower in the DIO mice than in normal lean mice. The levels of FGF21 increased but its signal transduction and actions were impaired in skeletal muscles of T2DM patients, IGT subjects, in insulin-resistant HSMMs, and DIO mice.

  14. Fibroblast growth factor (Fgf) 21 is a novel target gene of the aryl hydrocarbon receptor (AhR)

    SciTech Connect

    Cheng, Xingguo; Vispute, Saurabh G.; Liu, Jie; Cheng, Christine; Kharitonenkov, Alexei; Klaassen, Curtis D.

    2014-07-01

    The toxic effects of dioxins, such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), mainly through activation of the aryl hydrocarbon receptor (AhR) are well documented. Fibroblast growth factor (Fgf) 21 plays critical roles in metabolic adaptation to fasting by increasing lipid oxidation and ketogenesis in the liver. The present study was performed to determine whether activation of the AhR induces Fgf21 expression. In mouse liver, TCDD increased Fgf21 mRNA in both dose- and time-dependent manners. In addition, TCDD markedly increased Fgf21 mRNA expression in cultured mouse and human hepatocytes. Moreover, TCDD increased mRNA (in liver) and protein levels (in both liver and serum) of Fgf21 in wild-type mice, but not in AhR-null mice. Chromatin immunoprecipitation assays showed that TCDD increased AhR protein binding to the Fgf21 promoter (− 105/+ 1 base pair). Fgf21-null mice administered 200 μg/kg of TCDD died within 20 days, whereas wild-type mice receiving the same treatment were still alive at one month after administration. This indicates that TCDD-induced Fgf21 expression protects against TCDD toxicity. Diethylhexylphthalate (DEHP) pretreatment attenuated TCDD-induced Fgf21 expression in mouse liver and white adipose tissue, which may explain a previous report that DEHP pretreatment decreases TCDD-induced wasting. In conclusion, Fgf21 appears to be a target gene of AhR-signaling pathway in mouse and human liver. - Highlights: • TCDD induced Fgf21 expression at both mRNA and protein levels. • Fgf21 induction by TCDD is AhR-dependent. • DEHP attenuated TCDD-induced Fgf21 expression.

  15. Mapping the response of human fibroblast growth factor 21 (FGF21) promoter to serum availability and lipoic acid in HepG2 hepatoma cells.

    PubMed

    Xia, Mengna; Erickson, Anjeza; Yi, Xiaohua; Moreau, Régis

    2016-03-01

    The hormone-like polypeptide, fibroblast growth factor 21 (FGF21), is a major modulator of lipid and glucose metabolism and an exploratory treatment strategy for obesity related metabolic disorders. The costs of recombinant FGF21 and mode of delivery by injection are important constraints to its wide therapeutic use. The stimulation of endogenous FGF21 production through diet is being explored as an alternative approach. To that end, we examined the mechanism(s) by which serum manipulation and lipoic acid (a dietary activator of FGF21) induce FGF21 in human hepatocellular carcinoma HepG2 cells. Serum withdrawal markedly induced FGF21 mRNA levels (88 fold) and FGF21 secreted in the media (19 fold). Lipoic acid induced FGF21 mRNA 7 fold above DMSO-treated control cells and FGF21 secretion 3 fold. These effects were several-fold greater than those of PPARα agonist, Wy14643, which failed to induce FGF21 above and beyond the induction seen with serum withdrawal. The use of transcription inhibitor, actinomycin D, revealed that de novo mRNA synthesis drives FGF21 secretion in response to serum starvation. Four previously unrecognized loci in FGF21 promoter were nucleosome depleted and enriched in acetylated histone H3 revealing their role as transcriptional enhancers and putative transcription factor binding sites. FGF21 did not accumulate to a significant degree in induced HepG2 cells, which secreted FGF21 time dependently in media. We conclude that lipoic acid cell signaling connects with the transcriptional upregulation of FGF21 and it may prove to be a safe and affordable means to stimulate FGF21 production. PMID:26691139

  16. mTORC1 Is a Major Regulatory Node in the FGF21 Signaling Network in Adipocytes.

    PubMed

    Minard, Annabel Y; Tan, Shi-Xiong; Yang, Pengyi; Fazakerley, Daniel J; Domanova, Westa; Parker, Benjamin L; Humphrey, Sean J; Jothi, Raja; Stöckli, Jacqueline; James, David E

    2016-09-27

    FGF21 improves the metabolic profile of obese animals through its actions on adipocytes. To elucidate the signaling network responsible for mediating these effects, we quantified dynamic changes in the adipocyte phosphoproteome following acute exposure to FGF21. FGF21 regulated a network of 821 phosphosites on 542 proteins. A major FGF21-regulated signaling node was mTORC1/S6K. In contrast to insulin, FGF21 activated mTORC1 via MAPK rather than through the canonical PI3K/AKT pathway. Activation of mTORC1/S6K by FGF21 was surprising because this is thought to contribute to deleterious metabolic effects such as obesity and insulin resistance. Rather, mTORC1 mediated many of the beneficial actions of FGF21 in vitro, including UCP1 and FGF21 induction, increased adiponectin secretion, and enhanced glucose uptake without any adverse effects on insulin action. This study provides a global view of FGF21 signaling and suggests that mTORC1 may act to facilitate FGF21-mediated health benefits in vivo. PMID:27681418

  17. Novel Insights into the Cardio-Protective Effects of FGF21 in Lean and Obese Rat Hearts

    PubMed Central

    Chen, Jing; Ramanjaneya, Manjunath; Bari, Muhammad F.; Bhudia, Sunil K.; Hillhouse, Edward W.; Tan, Bee K.; Randeva, Harpal S.

    2014-01-01

    Aims Fibroblast growth factor 21 (FGF21) is a hepatic metabolic regulator with pleotropic actions. Its plasma concentrations are increased in obesity and diabetes; states associated with an increased incidence of cardiovascular disease. We therefore investigated the direct effect of FGF21 on cardio-protection in obese and lean hearts in response to ischemia. Methods and Results FGF21, FGF21-receptor 1 (FGFR1) and beta-Klotho (βKlotho) were expressed in rodent, human hearts and primary rat cardiomyocytes. Cardiac FGF21 was expressed and secreted (real time RT-PCR/western blot and ELISA) in an autocrine-paracrine manner, in response to obesity and hypoxia, involving FGFR1-βKlotho components. Cardiac-FGF21 expression and secretion were increased in response to global ischemia. In contrast βKlotho was reduced in obese hearts. In isolated adult rat cardiomyocytes, FGF21 activated PI3K/Akt (phosphatidylinositol 3-kinase/Akt), ERK1/2(extracellular signal-regulated kinase) and AMPK (AMP-activated protein kinase) pathways. In Langendorff perfused rat [adult male wild-type wistar] hearts, FGF21 administration induced significant cardio-protection and restoration of function following global ischemia. Inhibition of PI3K/Akt, AMPK, ERK1/2 and ROR-α (retinoic-acid receptor alpha) pathway led to significant decrease of FGF21 induced cardio-protection and restoration of cardiac function in response to global ischemia. More importantly, this cardio-protective response induced by FGF21 was reduced in obesity, although the cardiac expression profiles and circulating FGF21 levels were increased. Conclusion In an ex vivo Langendorff system, we show that FGF21 induced cardiac protection and restoration of cardiac function involving autocrine-paracrine pathways, with reduced effect in obesity. Collectively, our findings provide novel insights into FGF21-induced cardiac effects in obesity and ischemia. PMID:24498293

  18. Heme-Regulated eIF2α Kinase Modulates Hepatic FGF21 and Is Activated by PPARβ/δ Deficiency.

    PubMed

    Zarei, Mohammad; Barroso, Emma; Leiva, Rosana; Barniol-Xicota, Marta; Pujol, Eugènia; Escolano, Carmen; Vázquez, Santiago; Palomer, Xavier; Pardo, Virginia; González-Rodríguez, Águeda; Valverde, Ángela M; Quesada-López, Tania; Villarroya, Francesc; Wahli, Walter; Vázquez-Carrera, Manuel

    2016-10-01

    Fibroblast growth factor 21 (FGF21), a peptide hormone with pleiotropic effects on carbohydrate and lipid metabolism, is considered a target for the treatment of diabetes. We investigated the role of peroxisome proliferator-activated receptor (PPAR) β/δ deficiency in hepatic FGF21 regulation. Increased Fgf21 expression was observed in the livers of PPARβ/δ-null mice and in mouse primary hepatocytes when this receptor was knocked down by small interfering RNA (siRNA). Increased Fgf21 was associated with enhanced protein levels in the heme-regulated eukaryotic translation initiation factor 2α (eIF2α) kinase (HRI). This increase caused enhanced levels of phosphorylated eIF2α and activating transcription factor (ATF) 4, which is essential for Fgf21-induced expression. siRNA analysis demonstrated that HRI regulates Fgf21 expression in primary hepatocytes. Enhanced Fgf21 expression attenuated tunicamycin-induced endoplasmic reticulum stress, as demonstrated by using a neutralizing antibody against FGF21. Of note, increased Fgf21 expression in mice fed a high-fat diet or hepatocytes exposed to palmitate was accompanied by reduced PPARβ/δ and activation of the HRI-eIF2α-ATF4 pathway. Moreover, pharmacological activation of HRI increased Fgf21 expression and reduced lipid-induced hepatic steatosis and glucose intolerance, but these effects were not observed in Fgf21-null mice. Overall, these findings suggest that HRI is a potential target for regulating hepatic FGF21 levels.

  19. Hepatic Fgf21 Expression Is Repressed after Simvastatin Treatment in Mice

    PubMed Central

    Ziros, Panos; Zagoriti, Zoi; Lagoumintzis, George; Kyriazopoulou, Venetsana; Iskrenova, Ralitsa P.; Habeos, Evagelia I.; Sykiotis, Gerasimos P.; Chartoumpekis, Dionysios V.; Habeos, Ioannis G

    2016-01-01

    Fibroblast growth factor 21 (Fgf21) is a hormone with emerging beneficial roles in glucose and lipid homeostasis. The interest in Fgf21 as a potential antidiabetic drug and the factors that regulate its production and secretion is growing. Statins are the most widely prescribed drug for the treatment of dyslipidemia. However, the function of statins is not limited to the lowering of cholesterol as they are associated with pleiotropic actions such as antioxidant, anti-inflammatory and cytoprotective effects. The recently described effect of statins on mitochondrial function and the induction of Fgf21 by mitochondrial stress prompted us to investigate the effect of statin treatment on Fgf21 expression in the liver. To this end, C57BL6J male mice and primary mouse hepatocytes were treated with simvastatin, and Fgf21 expression was subsequently assessed by immunoblotting and quantitative real-time PCR. Hepatic Fgf21 protein and mRNA and circulating levels of FGF21significantly decreased in mice that had received simvastatin in their food (0.1% w/w) for 1 week. This effect was also observed with simvastatin doses as low as 0.01% w/w for 1 week or following 2 intraperitoneal injections within a single day. The reduction in Fgf21 mRNA levels was further verified in primary mouse hepatocytes, indicating that the effect of simvastatin is cell autonomous. In conclusion, simvastatin treatment reduced the circulating and hepatic Fgf21 levels and this effect warrants further investigation with reference to its role in metabolism. PMID:27583452

  20. Hepatic Fgf21 Expression Is Repressed after Simvastatin Treatment in Mice.

    PubMed

    Ziros, Panos; Zagoriti, Zoi; Lagoumintzis, George; Kyriazopoulou, Venetsana; Iskrenova, Ralitsa P; Habeos, Evagelia I; Sykiotis, Gerasimos P; Chartoumpekis, Dionysios V; Habeos, Ioannis G

    2016-01-01

    Fibroblast growth factor 21 (Fgf21) is a hormone with emerging beneficial roles in glucose and lipid homeostasis. The interest in Fgf21 as a potential antidiabetic drug and the factors that regulate its production and secretion is growing. Statins are the most widely prescribed drug for the treatment of dyslipidemia. However, the function of statins is not limited to the lowering of cholesterol as they are associated with pleiotropic actions such as antioxidant, anti-inflammatory and cytoprotective effects. The recently described effect of statins on mitochondrial function and the induction of Fgf21 by mitochondrial stress prompted us to investigate the effect of statin treatment on Fgf21 expression in the liver. To this end, C57BL6J male mice and primary mouse hepatocytes were treated with simvastatin, and Fgf21 expression was subsequently assessed by immunoblotting and quantitative real-time PCR. Hepatic Fgf21 protein and mRNA and circulating levels of FGF21significantly decreased in mice that had received simvastatin in their food (0.1% w/w) for 1 week. This effect was also observed with simvastatin doses as low as 0.01% w/w for 1 week or following 2 intraperitoneal injections within a single day. The reduction in Fgf21 mRNA levels was further verified in primary mouse hepatocytes, indicating that the effect of simvastatin is cell autonomous. In conclusion, simvastatin treatment reduced the circulating and hepatic Fgf21 levels and this effect warrants further investigation with reference to its role in metabolism. PMID:27583452

  1. Hepatic Fgf21 Expression Is Repressed after Simvastatin Treatment in Mice.

    PubMed

    Ziros, Panos; Zagoriti, Zoi; Lagoumintzis, George; Kyriazopoulou, Venetsana; Iskrenova, Ralitsa P; Habeos, Evagelia I; Sykiotis, Gerasimos P; Chartoumpekis, Dionysios V; Habeos, Ioannis G

    2016-01-01

    Fibroblast growth factor 21 (Fgf21) is a hormone with emerging beneficial roles in glucose and lipid homeostasis. The interest in Fgf21 as a potential antidiabetic drug and the factors that regulate its production and secretion is growing. Statins are the most widely prescribed drug for the treatment of dyslipidemia. However, the function of statins is not limited to the lowering of cholesterol as they are associated with pleiotropic actions such as antioxidant, anti-inflammatory and cytoprotective effects. The recently described effect of statins on mitochondrial function and the induction of Fgf21 by mitochondrial stress prompted us to investigate the effect of statin treatment on Fgf21 expression in the liver. To this end, C57BL6J male mice and primary mouse hepatocytes were treated with simvastatin, and Fgf21 expression was subsequently assessed by immunoblotting and quantitative real-time PCR. Hepatic Fgf21 protein and mRNA and circulating levels of FGF21significantly decreased in mice that had received simvastatin in their food (0.1% w/w) for 1 week. This effect was also observed with simvastatin doses as low as 0.01% w/w for 1 week or following 2 intraperitoneal injections within a single day. The reduction in Fgf21 mRNA levels was further verified in primary mouse hepatocytes, indicating that the effect of simvastatin is cell autonomous. In conclusion, simvastatin treatment reduced the circulating and hepatic Fgf21 levels and this effect warrants further investigation with reference to its role in metabolism.

  2. Pilot-scale production and characterization of PEGylated human FGF-21 analog.

    PubMed

    Ye, Xianlong; Qi, Jianying; Yu, Dan; Li, Shujie; Wu, Qiang; Wu, Yunzhou; Ren, Guiping; Han, Jun; Li, Deshan

    2016-06-20

    FGF-21 has become a potential drug candidate for the treatment of type 2 diabetes. Previous studies have demonstrated that PEGylation of FGF-21 could significantly increase its in vivo half-life and provide its long-lasting blood glucose-lowering effect. To accelerate the development of PEGylated FGF-21 for clinical application as a long-acting antidiabetes drug, we prepared ahmFGF-21 (FGF-21 mutant) and PEGylated ahmFGF-21 in Escherichia coli Rosetta (DE3) by high cell density fermentation at a 50-L scale and pilot-scale purification. The physical and chemical properties of the purified proteins were analyzed in this study, including purity, molecular weight, isoelectric point, bacterial endotoxin, PEGylated site and second structure. As well as the in vitro glucose uptake activity and in vivo anti-diabetic effect were evaluated. Under the optimal fermentation and purification conditions, the average bacterial yield and expression level of target protein of three batches attained 52.2±4.6g/L and 223.92±5.41mg/L, respectively. The purity of pilot product was above 98% by SDS-PAGE (non-reducing or reducing) and HPLC (SEC or RPC) analysis and the final yield of PEGylated ahmFGF-21 was 87.91±1.49mg/L, which indicated that the pilot-scale production process was relatively stable. N-terminal sequencing and circular dichroism (CD) spectroscopy results showed that modification site of PEGylated ahmFGF-21 was alanine at N-terminal and the second structure of ahmFGF-21 had no obvious changes after PEGylation. Compared with ahmFGF-21, the long-acting hypoglycemic effect of PEGylated ahmFGF-21 prepared in the pilot-scale production was significantly improved in type 2 diabetic db/db mice. Our results demonstrated that the pilot-scale production process of PEGylated ahmFGF-21 was successfully established, which was very important for the clinical application. PMID:27109774

  3. Pilot-scale production and characterization of PEGylated human FGF-21 analog.

    PubMed

    Ye, Xianlong; Qi, Jianying; Yu, Dan; Li, Shujie; Wu, Qiang; Wu, Yunzhou; Ren, Guiping; Han, Jun; Li, Deshan

    2016-06-20

    FGF-21 has become a potential drug candidate for the treatment of type 2 diabetes. Previous studies have demonstrated that PEGylation of FGF-21 could significantly increase its in vivo half-life and provide its long-lasting blood glucose-lowering effect. To accelerate the development of PEGylated FGF-21 for clinical application as a long-acting antidiabetes drug, we prepared ahmFGF-21 (FGF-21 mutant) and PEGylated ahmFGF-21 in Escherichia coli Rosetta (DE3) by high cell density fermentation at a 50-L scale and pilot-scale purification. The physical and chemical properties of the purified proteins were analyzed in this study, including purity, molecular weight, isoelectric point, bacterial endotoxin, PEGylated site and second structure. As well as the in vitro glucose uptake activity and in vivo anti-diabetic effect were evaluated. Under the optimal fermentation and purification conditions, the average bacterial yield and expression level of target protein of three batches attained 52.2±4.6g/L and 223.92±5.41mg/L, respectively. The purity of pilot product was above 98% by SDS-PAGE (non-reducing or reducing) and HPLC (SEC or RPC) analysis and the final yield of PEGylated ahmFGF-21 was 87.91±1.49mg/L, which indicated that the pilot-scale production process was relatively stable. N-terminal sequencing and circular dichroism (CD) spectroscopy results showed that modification site of PEGylated ahmFGF-21 was alanine at N-terminal and the second structure of ahmFGF-21 had no obvious changes after PEGylation. Compared with ahmFGF-21, the long-acting hypoglycemic effect of PEGylated ahmFGF-21 prepared in the pilot-scale production was significantly improved in type 2 diabetic db/db mice. Our results demonstrated that the pilot-scale production process of PEGylated ahmFGF-21 was successfully established, which was very important for the clinical application.

  4. Metformin-induced inhibition of the mitochondrial respiratory chain increases FGF21 expression via ATF4 activation

    SciTech Connect

    Kim, Kook Hwan; Jeong, Yeon Taek; Kim, Seong Hun; Jung, Hye Seung; Park, Kyong Soo; Lee, Hae-Youn; Lee, Myung-Shik

    2013-10-11

    Highlights: •Metformin induces FGF21 expression in an AMPK independent manner. •Metformin enhances FGF21 expression by inhibiting mitochondrial complex I activity. •The PERK-eIF2α-ATF4 axis is required for metformin-induced FGF21 expression. •Metformin activates the ATF4-FGF21 axis in the liver of mouse. •Metformin increases serum FGF21 level in diabetic human subjects. -- Abstract: Fibroblast growth factor 21 (FGF21) is an endocrine hormone that exhibits anti-obesity and anti-diabetes effects. Because metformin is widely used as a glucose-lowering agent in patients with type 2 diabetes (T2D), we investigated whether metformin modulates FGF21 expression in cell lines, and in mice or human subjects. We found that metformin increased the expression and release of FGF21 in a diverse set of cell types, including rat hepatoma FaO, primary mouse hepatocytes, and mouse embryonic fibroblasts (MEFs). Intriguingly, AMP-activated protein kinase (AMPK) was dispensable for the induction of FGF21 by metformin. Mammalian target of rapamycin complex 1 (mTORC1) and peroxisome proliferator-activated receptor α (PPARα), which are additional targets of metformin, were not involved in metformin-induced FGF21 expression. Importantly, inhibition of mitochondrial complex I activity by metformin resulted in FGF21 induction through PKR-like ER kinase (PERK)-eukaryotic translation factor 2α (eIF2α)-activating transcription factor 4 (ATF4). We showed that metformin activated ATF4 and increased FGF21 expression in the livers of mice, which led to increased serum levels of FGF21. We also found that serum FGF21 level was increased in human subjects with T2D after metformin therapy for 6 months. In conclusion, our results indicate that metformin induced expression of FGF21 through an ATF4-dependent mechanism by inhibiting mitochondrial respiration independently of AMPK. Therefore, FGF21 induction by metformin might explain a portion of the beneficial metabolic effects of metformin.

  5. NS5ATP6 modulates intracellular triglyceride content through FGF21 and independently of SIRT1 and SREBP1.

    PubMed

    Li, Zhongshu; Feng, Shenghu; Zhou, Li; Liu, Shunai; Cheng, Jun

    2016-06-17

    The prevalence of nonalcoholic fatty liver disease (NAFLD) is rising strikingly in Western countries and China. The molecular biological mechanism of NAFLD remains unclear, with no effective therapies developed so far. Fibroblast growth factor 21 (FGF21) is a recently discovered hormone, with safe lipid lowering effects. FGF21 analogs are being developed for clinical application. Here we demonstrated that a novel gene, NS5ATP6, modulated intracellular triglyceride (TG) content independently of sirtuin1 (SIRT1) and sterol regulatory element binding protein 1 (SREBP1) in HepG2 cells. Interestingly, NS5ATP6 regulated FGF21 expression both at the mRNA and protein levels. The modulatory effects of NS5ATP6 on intracellular TG content depended upon FGF21. Further studies revealed that NS5ATP6 decreased the promoter activity of FGF21. In addition, NS5ATP6 regulated the expression of miR-577, which directly targeted and regulated FGF21. Therefore, miR-577 might be involved in NS5ATP6 regulation of FGF21 at the post-transcriptional level. In conclusion, NS5ATP6 regulates the intracellular TG level via FGF21, and independently of SIRT1 and SREBP1. PMID:27179781

  6. Fundamentals of FGF19 & FGF21 action in vitro and in vivo.

    PubMed

    Adams, Andrew C; Coskun, Tamer; Rovira, Armando R Irizarry; Schneider, Michael A; Raches, David W; Micanovic, Radmila; Bina, Holly A; Dunbar, James D; Kharitonenkov, Alexei

    2012-01-01

    Fibroblast growth factors 19 (FGF19) and 21 (FGF21) have emerged as key regulators of energy metabolism. Several studies have been conducted to understand the mechanism of FGF19 and FGF21 action, however, the data presented has often been inconsistent and at times contradictory. Here in a single study we compare the mechanisms mediating FGF19/FGF21 actions, and how similarities/differences in actions at the cellular level between these two factors translate to common/divergent physiological outputs. Firstly, we show that in cell culture FGF19/FGF21 are very similar, however, key differences are still observed differentiating the two. In vitro we found that both FGF's activate FGFRs in the context of βKlotho (KLB) expression. Furthermore, both factors alter ERK phosphorylation and glucose uptake with comparable potency. Combination treatment of cells with both factors did not have additive effects and treatment with a competitive inhibitor, the FGF21 delta N17 mutant, also blocked FGF19's effects, suggestive of a shared receptor activation mechanism. The key differences between FGF21/FGF19 were noted at the receptor interaction level, specifically the unique ability of FGF19 to bind/signal directly via FGFR4. To determine if differential effects on energy homeostasis and hepatic mitogenicity exist we treated DIO and ob/ob mice with FGF19/FGF21. We find comparable efficacy of the two proteins to correct body weight and serum glucose in both DIO and ob/ob mice. Nevertheless, FGF21 and FGF19 had distinctly different effects on proliferation in the liver. Interestingly, in vivo blockade of FGF21 signaling in mice using ΔN17 caused profound changes in glycemia indicative of the critical role KLB and FGF21 play in the regulation of glucose homeostasis. Overall, our data demonstrate that while subtle differences exist in vitro the metabolic effects in vivo of FGF19/FGF21 are indistinguishable, supporting a shared mechanism of action for these two hormones in the

  7. Fibroblast growth factor (Fgf) 21 is a novel target gene of the aryl hydrocarbon receptor (AhR).

    PubMed

    Cheng, Xingguo; Vispute, Saurabh G; Liu, Jie; Cheng, Christine; Kharitonenkov, Alexei; Klaassen, Curtis D

    2014-07-01

    The toxic effects of dioxins, such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), mainly through activation of the aryl hydrocarbon receptor (AhR) are well documented. Fibroblast growth factor (Fgf) 21 plays critical roles in metabolic adaptation to fasting by increasing lipid oxidation and ketogenesis in the liver. The present study was performed to determine whether activation of the AhR induces Fgf21 expression. In mouse liver, TCDD increased Fgf21 mRNA in both dose- and time-dependent manners. In addition, TCDD markedly increased Fgf21 mRNA expression in cultured mouse and human hepatocytes. Moreover, TCDD increased mRNA (in liver) and protein levels (in both liver and serum) of Fgf21 in wild-type mice, but not in AhR-null mice. Chromatin immunoprecipitation assays showed that TCDD increased AhR protein binding to the Fgf21 promoter (-105/+1 base pair). Fgf21-null mice administered 200μg/kg of TCDD died within 20days, whereas wild-type mice receiving the same treatment were still alive at one month after administration. This indicates that TCDD-induced Fgf21 expression protects against TCDD toxicity. Diethylhexylphthalate (DEHP) pretreatment attenuated TCDD-induced Fgf21 expression in mouse liver and white adipose tissue, which may explain a previous report that DEHP pretreatment decreases TCDD-induced wasting. In conclusion, Fgf21 appears to be a target gene of AhR-signaling pathway in mouse and human liver.

  8. Fibroblast growth factor (Fgf) 21 is a novel target gene of the aryl hydrocarbon receptor (AhR).

    PubMed

    Cheng, Xingguo; Vispute, Saurabh G; Liu, Jie; Cheng, Christine; Kharitonenkov, Alexei; Klaassen, Curtis D

    2014-07-01

    The toxic effects of dioxins, such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), mainly through activation of the aryl hydrocarbon receptor (AhR) are well documented. Fibroblast growth factor (Fgf) 21 plays critical roles in metabolic adaptation to fasting by increasing lipid oxidation and ketogenesis in the liver. The present study was performed to determine whether activation of the AhR induces Fgf21 expression. In mouse liver, TCDD increased Fgf21 mRNA in both dose- and time-dependent manners. In addition, TCDD markedly increased Fgf21 mRNA expression in cultured mouse and human hepatocytes. Moreover, TCDD increased mRNA (in liver) and protein levels (in both liver and serum) of Fgf21 in wild-type mice, but not in AhR-null mice. Chromatin immunoprecipitation assays showed that TCDD increased AhR protein binding to the Fgf21 promoter (-105/+1 base pair). Fgf21-null mice administered 200μg/kg of TCDD died within 20days, whereas wild-type mice receiving the same treatment were still alive at one month after administration. This indicates that TCDD-induced Fgf21 expression protects against TCDD toxicity. Diethylhexylphthalate (DEHP) pretreatment attenuated TCDD-induced Fgf21 expression in mouse liver and white adipose tissue, which may explain a previous report that DEHP pretreatment decreases TCDD-induced wasting. In conclusion, Fgf21 appears to be a target gene of AhR-signaling pathway in mouse and human liver. PMID:24769090

  9. FGF-21, a novel metabolic regulator, has a robust neuroprotective role and is markedly elevated in neurons by mood stabilizers.

    PubMed

    Leng, Y; Wang, Z; Tsai, L-K; Leeds, P; Fessler, E B; Wang, J; Chuang, D-M

    2015-02-01

    Fibroblast growth factor-21 (FGF-21) is a new member of the FGF super-family and an important endogenous regulator of glucose and lipid metabolism. It has been proposed as a therapeutic target for diabetes and obesity. Its function in the central nervous system (CNS) remains unknown. Previous studies from our laboratory demonstrated that aging primary neurons are more vulnerable to glutamate-induced excitotoxicity, and that co-treatment with the mood stabilizers lithium and valproic acid (VPA) induces synergistic neuroprotective effects. This study sought to identify molecule(s) involved in these synergistic effects. We found that FGF-21 mRNA was selectively and markedly elevated by co-treatment with lithium and VPA in primary rat brain neurons. FGF-21 protein levels were also robustly increased in neuronal lysates and culture medium following lithium-VPA co-treatment. Combining glycogen synthase kinase-3 (GSK-3) inhibitors with VPA or histone deacetylase (HDAC) inhibitors with lithium synergistically increased FGF-21 mRNA levels, supporting that synergistic effects of lithium and VPA are mediated via GSK-3 and HDAC inhibition, respectively. Exogenous FGF-21 protein completely protected aging neurons from glutamate challenge. This neuroprotection was associated with enhanced Akt-1 activation and GSK-3 inhibition. Lithium-VPA co-treatment markedly prolonged lithium-induced Akt-1 activation and augmented GSK-3 inhibition. Akt-1 knockdown markedly decreased FGF-21 mRNA levels and reduced the neuroprotection induced by FGF-21 or lithium-VPA co-treatment. In addition, FGF-21 knockdown reduced lithium-VPA co-treatment-induced Akt-1 activation and neuroprotection against excitotoxicity. Together, our novel results suggest that FGF-21 is a key mediator of the effects of these mood stabilizers and a potential new therapeutic target for CNS disorders.

  10. FGF-21, a novel metabolic regulator, has a robust neuroprotective role and is dramatically elevated in neurons by mood stabilizers

    PubMed Central

    Leng, Yan; Wang, Zhifei; Tsai, Li-Kai; Leeds, Peter; Fessler, Emily Bame; Wang, Junyu; Chuang, De-Maw

    2013-01-01

    Fibroblast growth factor-21 (FGF-21) is a new member of the FGF super-family and an important endogenous regulator of glucose and lipid metabolism. It has been proposed as a therapeutic target for diabetes and obesity. Its function in the central nervous system (CNS) remains unknown. Previous studies from our laboratory demonstrated that aging primary neurons are more vulnerable to glutamate-induced excitotoxicity, and that co-treatment with the mood stabilizers lithium and valproic acid (VPA) induces synergistic neuroprotective effects. This study sought to identify molecule(s) involved in these synergistic effects. We found that FGF-21 mRNA was selectively and dramatically elevated by co-treatment with lithium and VPA in primary rat brain neurons. FGF-21 protein levels were also robustly increased in neuronal lysates and culture medium following lithium-VPA co-treatment. Combining glycogen synthase kinase-3 (GSK-3) inhibitors with VPA or histone deacetylase (HDAC) inhibitors with lithium synergistically increased FGF-21 mRNA levels, supporting that synergistic effects of lithium and VPA are mediated via GSK-3 and HDAC inhibition, respectively. Exogenous FGF-21 protein completely protected aging neurons from glutamate challenge. This neuroprotection was associated with enhanced Akt-1 activation and GSK-3 inhibition. Lithium-VPA co-treatment dramatically prolonged lithium-induced Akt-1 activation and augmented GSK-3 inhibition. Akt-1 knockdown markedly decreased FGF-21 mRNA levels, and reduced the neuroprotection induced by FGF-21 or lithium-VPA co-treatment. In addition, FGF-21 knockdown reduced lithium-VPA co-treatment-induced Akt-1 activation and neuroprotection against excitotoxicity. Together, our novel results suggest that FGF-21 is a key mediator of the effects of these mood stabilizers, and a potential new therapeutic target for CNS disorders. PMID:24468826

  11. Single ingestion of soy β-conglycinin induces increased postprandial circulating FGF21 levels exerting beneficial health effects

    PubMed Central

    Hashidume, Tsutomu; Kato, Asuka; Tanaka, Tomohiro; Miyoshi, Shoko; Itoh, Nobuyuki; Nakata, Rieko; Inoue, Hiroyasu; Oikawa, Akira; Nakai, Yuji; Shimizu, Makoto; Inoue, Jun; Sato, Ryuichiro

    2016-01-01

    Soy protein β-conglycinin has serum lipid-lowering and anti-obesity effects. We showed that single ingestion of β-conglycinin after fasting alters gene expression in mouse liver. A sharp increase in fibroblast growth factor 21 (FGF21) gene expression, which is depressed by normal feeding, resulted in increased postprandial circulating FGF21 levels along with a significant decrease in adipose tissue weights. Most increases in gene expressions, including FGF21, were targets for the activating transcription factor 4 (ATF4), but not for peroxisome proliferator-activated receptor α. Overexpression of a dominant-negative form of ATF4 significantly reduced β-conglycinin-induced increases in hepatic FGF21 gene expression. In FGF21-deficient mice, β-conglycinin effects were partially abolished. Methionine supplementation to the diet or primary hepatocyte culture medium demonstrated its importance for activating liver or hepatocyte ATF4-FGF21 signaling. Thus, dietary β-conglycinin intake can impact hepatic and systemic metabolism by increasing the postprandial circulating FGF21 levels. PMID:27312476

  12. Single ingestion of soy β-conglycinin induces increased postprandial circulating FGF21 levels exerting beneficial health effects.

    PubMed

    Hashidume, Tsutomu; Kato, Asuka; Tanaka, Tomohiro; Miyoshi, Shoko; Itoh, Nobuyuki; Nakata, Rieko; Inoue, Hiroyasu; Oikawa, Akira; Nakai, Yuji; Shimizu, Makoto; Inoue, Jun; Sato, Ryuichiro

    2016-01-01

    Soy protein β-conglycinin has serum lipid-lowering and anti-obesity effects. We showed that single ingestion of β-conglycinin after fasting alters gene expression in mouse liver. A sharp increase in fibroblast growth factor 21 (FGF21) gene expression, which is depressed by normal feeding, resulted in increased postprandial circulating FGF21 levels along with a significant decrease in adipose tissue weights. Most increases in gene expressions, including FGF21, were targets for the activating transcription factor 4 (ATF4), but not for peroxisome proliferator-activated receptor α. Overexpression of a dominant-negative form of ATF4 significantly reduced β-conglycinin-induced increases in hepatic FGF21 gene expression. In FGF21-deficient mice, β-conglycinin effects were partially abolished. Methionine supplementation to the diet or primary hepatocyte culture medium demonstrated its importance for activating liver or hepatocyte ATF4-FGF21 signaling. Thus, dietary β-conglycinin intake can impact hepatic and systemic metabolism by increasing the postprandial circulating FGF21 levels. PMID:27312476

  13. Circulating CTRP1 Levels in Type 2 Diabetes and Their Association with FGF21

    PubMed Central

    Han, Sora; Kim, Jong Dai; Lee, Sunyi; Jeong, Ae Lee; Park, Jeong Su; Yong, Hyo Jeong; Boldbaatar, Ariundavaa; Ka, Hye In; Rhee, Eun-Jung; Lee, Won-Young; Yang, Young

    2016-01-01

    The goal of this study was to investigate whether circulating C1q/TNF-α-related protein 1 (CTRP1) levels are associated with diabetes. In addition, relationships between CTRP1 and other diabetes-related cytokines were elucidated, including adiponectin and fibroblast growth factor 21 (FGF21). A total of 178 subjects (78 men and 100 women) aged 29–70 years (mean age, 46.1 years) were randomly selected. The sera from a normal glucose tolerance group (n = 68) and a prediabetes/type 2 diabetes group (n = 110) were collected; then, circulating levels of CTRP1, adiponectin, and FGF21 were determined via enzyme-linked immunosorbent assay in all sera. Subjects with either prediabetes or diabetes exhibited higher circulating CTRP1 levels than healthy subjects. Sera analysis revealed that CTRP1 was positively correlated with age, body mass index, fasting blood glucose, and circulating FGF21 levels. However, CTRP1 was negatively correlated with total cholesterol and total circulating adiponectin levels in univariate analysis. In addition, multivariate analysis found that CTRP1 was independently associated with age, fasting blood glucose, and circulating FGF21 levels. CTRP1 was correlated with homeostasis model assessment-β (HOMA-β), but no correlation was observed with HOMA-insulin resistance. In conclusion, circulating CTRP1 levels are increased in subjects with type 2 diabetes and are positively associated with circulating FGF21 levels. PMID:27313611

  14. Physiological and Pharmacological Roles of FGF21 in Cardiovascular Diseases.

    PubMed

    Cheng, Peng; Zhang, Fangfang; Yu, Lechu; Lin, Xiufei; He, Luqing; Li, Xiaokun; Lu, Xuemian; Yan, Xiaoqing; Tan, Yi; Zhang, Chi

    2016-01-01

    Cardiovascular disease (CVD) is one of the most severe diseases in clinics. Fibroblast growth factor 21 (FGF21) is regarded as an important metabolic regulator playing a therapeutic role in diabetes and its complications. The heart is a key target as well as a source of FGF21 which is involved in heart development and also induces beneficial effects in CVDs. Our review is to clarify the roles of FGF21 in CVDs. Strong evidence showed that the development of CVDs including atherosclerosis, coronary heart disease, myocardial ischemia, cardiac hypertrophy, and diabetic cardiomyopathy is associated with serum FGF21 levels increase which was regarded as a compensatory response to induced cardiac protection. Furthermore, administration of FGF21 suppressed the above CVDs. Mechanistic studies revealed that FGF21 induced cardiac protection likely by preventing cardiac lipotoxicity and the associated oxidative stress, inflammation, and apoptosis. Normally, FGF21 induced therapeutic effects against CVDs via activation of the above kinases-mediated pathways by directly binding to the FGF receptors of the heart in the presence of β-klotho. However, recently, growing evidence showed that FGF21 induced beneficial effects on peripheral organs through an indirect way mediated by adiponectin. Therefore whether adiponectin is also involved in FGF21-induced cardiac protection still needs further investigation. PMID:27247947

  15. Physiological and Pharmacological Roles of FGF21 in Cardiovascular Diseases

    PubMed Central

    Cheng, Peng; Zhang, Fangfang; Yu, Lechu; Lin, Xiufei; He, Luqing; Li, Xiaokun; Lu, Xuemian; Yan, Xiaoqing; Tan, Yi; Zhang, Chi

    2016-01-01

    Cardiovascular disease (CVD) is one of the most severe diseases in clinics. Fibroblast growth factor 21 (FGF21) is regarded as an important metabolic regulator playing a therapeutic role in diabetes and its complications. The heart is a key target as well as a source of FGF21 which is involved in heart development and also induces beneficial effects in CVDs. Our review is to clarify the roles of FGF21 in CVDs. Strong evidence showed that the development of CVDs including atherosclerosis, coronary heart disease, myocardial ischemia, cardiac hypertrophy, and diabetic cardiomyopathy is associated with serum FGF21 levels increase which was regarded as a compensatory response to induced cardiac protection. Furthermore, administration of FGF21 suppressed the above CVDs. Mechanistic studies revealed that FGF21 induced cardiac protection likely by preventing cardiac lipotoxicity and the associated oxidative stress, inflammation, and apoptosis. Normally, FGF21 induced therapeutic effects against CVDs via activation of the above kinases-mediated pathways by directly binding to the FGF receptors of the heart in the presence of β-klotho. However, recently, growing evidence showed that FGF21 induced beneficial effects on peripheral organs through an indirect way mediated by adiponectin. Therefore whether adiponectin is also involved in FGF21-induced cardiac protection still needs further investigation. PMID:27247947

  16. Recombinant murine fibroblast growth factor 21 ameliorates obesity-related inflammation in monosodium glutamate-induced obesity rats.

    PubMed

    Wang, Wen-Fei; Li, Si-Ming; Ren, Gui-Ping; Zheng, Wei; Lu, Yu-Jia; Yu, Yin-Hang; Xu, Wen-Juan; Li, Tian-He; Zhou, Li-Hong; Liu, Yan; Li, De-Shan

    2015-05-01

    The aim of this study is to investigate the role of FGF21 in obesity-related inflammation in livers of monosodium glutamate (MSG)-induced obesity rats. The MSG rats were injected with recombinant murine fibroblast growth factor 21(FGF21) or equal volumes of vehicle. Metabolic parameters including body weight, Lee's index, food intake, visceral fat and liver weight, intraperitoneal glucose tolerance, glucose, and lipid levels were dynamically measured at specific time points. Liver function and routine blood test were also analyzed. Further, systemic inflammatory cytokines such as glucose transporter 1 (GLUT-1), leptin, TNF-α, and IL-6 mRNAs were determined by real-time PCR. FGF21 independently decreased body weight and whole-body fat mass without reducing food intake in the MSG rats. FGF21 reduced blood glucose level, Lee's index, visceral fat, and liver weight, and improved glucose tolerance, lipid metabolic spectrum, and hepatic steatosis in the MSG-obesity rats. Liver function parameters including AST, ALT, ALP, TP, T.Bili, and D.Bili levels significantly reduced in the FGF21-treated obesity rats compared to the controls. Further, FGF21 ameliorated the total and differential white blood cell (WBC) count, serum C-reactive protein (CRP), IL-6, and TNF-α levels in adipose tissues of the obesity rats, suggesting inflammation amelioration in the in the obesity rats by FGF21. FGF21 improves multiple metabolic disorders and ameliorates obesity-related inflammation in the MSG-induced obesity rats.

  17. FGF21 improves cognition by restored synaptic plasticity, dendritic spine density, brain mitochondrial function and cell apoptosis in obese-insulin resistant male rats.

    PubMed

    Sa-Nguanmoo, Piangkwan; Tanajak, Pongpan; Kerdphoo, Sasiwan; Satjaritanun, Pattarapong; Wang, Xiaojie; Liang, Guang; Li, Xiaokun; Jiang, Chao; Pratchayasakul, Wasana; Chattipakorn, Nipon; Chattipakorn, Siriporn C

    2016-09-01

    Fibroblast growth factor 21 (FGF21) is an endocrine hormone which exerts beneficial effects on metabolic regulation in obese and diabetic models. However, the effect of FGF21 on cognition in obese-insulin resistant rats has not been investigated. We hypothesized that FGF21 prevented cognitive decline in obese-insulin resistant rats by improving hippocampal synaptic plasticity, dendritic spine density, brain mitochondrial function and brain FGF21 signaling as well as decreasing brain cell apoptosis. Eighteen male Wistar rats were divided into two groups, and received either a normal diet (ND) (n=6) or a high fat diet (HFD) (n=12) for 12weeks. At week 13, the HFD-fed rats were subdivided into two subgroups (n=6/subgroup) to receive either vehicle or recombinant human FGF21 (0.1mg/kg/day) for four weeks. ND-fed rats were given vehicle for four weeks. At the end of the treatment, cognitive function, metabolic parameters, pro-inflammatory markers, brain mitochondrial function, cell apoptosis, hippocampal synaptic plasticity, dendritic spine density and brain FGF21 signaling were determined. The results showed that vehicle-treated HFD-fed rats developed obese-insulin resistance and cognitive decline with impaired hippocampal synaptic plasticity, decreased dendritic spine density, brain mitochondrial dysfunction and increased brain cell apoptosis. Impaired brain FGF 21 signaling was found in these obese-insulin resistant rats. FGF21-treated obese-insulin resistant rats had improved peripheral insulin sensitivity, increased hippocampal synaptic plasticity, increased dendritic spine density, restored brain mitochondrial function, attenuated brain cells apoptosis and increased brain FGF21 signaling, leading to a prevention of cognitive decline. These findings suggest that FGF21 treatment exerts neuroprotection in obese-insulin resistant rats. PMID:27566237

  18. Metabolic hormone FGF21 is induced in ground squirrels during hibernation but its overexpression is not sufficient to cause torpor.

    PubMed

    Nelson, Bethany T; Ding, Xunshan; Boney-Montoya, Jamie; Gerard, Robert D; Kliewer, Steven A; Andrews, Matthew T

    2013-01-01

    Hibernation is a natural adaptation that allows certain mammals to survive physiological extremes that are lethal to humans. Near freezing body temperatures, heart rates of 3-10 beats per minute, absence of food consumption, and depressed metabolism are characteristic of hibernation torpor bouts that are periodically interrupted by brief interbout arousals (IBAs). The molecular basis of torpor induction is unknown, however starved mice overexpressing the metabolic hormone fibroblast growth factor 21 (FGF21) promote fat utilization, reduce body temperature, and readily enter torpor-all hallmarks of mammalian hibernation. In this study we cloned FGF21 from the naturally hibernating thirteen-lined ground squirrel (Ictidomys tridecemlineatus) and found that levels of FGF21 mRNA in liver and FGF21 protein in serum are elevated during hibernation torpor bouts and significantly elevated during IBAs compared to summer active animals. The effects of artificially elevating circulating FGF21 concentrations 50 to 100-fold via adenoviral-mediated overexpression were examined at three different times of the year. This is the first time that a transgenic approach has been used in a natural hibernator to examine mechanistic aspects of hibernation. Surgically implanted transmitters measured various metrics of the hibernation phenotype over a 7-day period including changes in motor activity, heart rate and core body temperature. In April fed-state animals, FGF21 overexpression decreased blood insulin and free fatty acid concentrations, effects similar to those seen in obese mice. However, elevated FGF21 concentrations did not cause torpor in these fed-state animals nor did they cause torpor or affect metabolic parameters in fasted-state animals in March/April, August or October. We conclude that FGF21 is strongly regulated during torpor and IBA but that its overexpression is not sufficient to cause torpor in naturally hibernating ground squirrels.

  19. Decreased beige adipocyte number and mitochondrial respiration coincide with reduced FGF21 gene expression in Sprague Dawley rats fed prenatal low protein and postnatal high fat diets

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We have shown that protein malnutrition during fetal growth followed by postnatal high-fat diets results in a rapid increase in subcutaneous adipose tissue mass in the offspring contributing to development of obesity and insulin resistance. Recent studies have shown that the absence of a key transcr...

  20. 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...

  1. Loss of FGF21 in diabetic mouse during hepatocellular carcinogenetic transformation

    PubMed Central

    Zhang, Quan; Li, Yan; Liang, Tingting; Lu, Xuemian; Liu, Xingkai; Zhang, Chi; Jiang, Xin; Martin, Robert C; Cheng, Mingliang; Cai, Lu

    2015-01-01

    Diabetes associated metabolic syndrome has been shown to be an independent risk factor for the development of hepatocellular carcinoma (HCC). Cirrhosis, in fact, was not always a prerequisite of HCC development and this might particularly apply to the metabolic abnormality associated HCC. This study was to investigate diabetes associated HCC and the potential role of FGF21 during carcinogenetic transformation of HCC. Dimethylnitrosamine (DEN) was used to induce HCC in the diabetic OVE26 mice. Pronounced damage characterized by steatohepatitis was found in the liver of diabetic mice. Steatohepatitis accompanied by constant cell proliferation and tumor cell growth were also found in the hepatic tissues of diabetic OVE26 mice when DEN being administrated. FGF21 protein level increased in liver tissues at an early stage along with steatohepatitis in diabetic OVE26 mice, but decreased in liver tissues later when HCC was developed. In addition, decreased FGF21 protein level was associated with cancerous hyper-proliferation and aberrant p53 and TGF-β/Smad signaling during HCC development. Loss of FGF21 may play an important role in HCC carcinogenetic transformation during metabolic liver injury in diabetic animals. The present finding calls attention to the need to control metabolic disorders associated with diabetes and may further develop a protective strategy against HCC. PMID:26175944

  2. Differentiated embryo chondrocyte 1 (DEC1) is a novel negative regulator of hepatic fibroblast growth factor 21 (FGF21) in aging mice.

    PubMed

    Fujita, Yu; Makishima, Makoto; Bhawal, Ujjal K

    2016-01-15

    Human differentiated embryo chondrocyte expressed gene 1 (DEC1) is frequently used as a marker of senescence in vivo. Fibroblast growth factor 21 (FGF21), a novel endocrine-like member of the FGF superfamily, is highly expressed in the liver, and FGF21-transgenic mice have extended lifespans. Thus, we hypothesized that FGF21 may play a role in the DEC1-mediated aging process. In this study, DEC1 knockout (KO) mice were used to characterize the mechanism by which FGF21 protects mice from aging. Aging is strongly diminished in DEC1 KO mice, which is reflected by decreased lipid levels and oxidative stress, leading to an amelioration of liver function and structure. The expression of FGF21 decreased with aging in wild-type (WT) mice, whereas ATF4, Phospho-ERK and Phospho-p38 expression was maintained and was accompanied by a compensatory rise of FGF21 mRNA and protein expression in DEC1 KO mice. Over-expression of DEC1 markedly abolished the hepatic expression of FGF21, and siRNA-mediated inhibition of endogenous DEC1 increased the expression of FGF21. DEC1 further diminished the expression of ATF4 in HepG2 cells over-expressing DEC1. The induction of FGF21 and ATF4 at the mRNA and protein levels during the course of aging supports the view that DEC1 KO mice are able to restore the age-related imbalance of metabolism. Collectively, the data obtained in this study suggest that DEC1 is a novel negative regulator of hepatic FGF21 expression.

  3. FGF21 Is Associated with Acanthosis Nigricans in Obese Patients

    PubMed Central

    Huang, Yueye; Yang, Jie; Li, Yan; Chen, Jiaqi; Song, Kexiu; Wang, Xingchun; Bu, Le; Cheng, Xiaoyun; Wang, Jiying; Qu, Shen

    2016-01-01

    Objective. We aimed to investigate the relationship between FGF21 and obesity-related acanthosis nigricans (AN). Methods. 40 obese patients without AN (OB group), 40 obese patients with AN (AN group), and 40 healthy volunteers (control group, CON) were included in this study. Weight, BMI, lipid profile, FFA, UA, and CRP were measured in all participants. Oral glucose tolerance tests (OGTT) were performed and serum glucose and plasma insulin were measured. Serum FGF21 was measured by ELISA. Results. Compared with OB group, AN group had higher levels of fasting insulin and homeostasis model of assessment for insulin resistance (HOMA-IR) (P < 0.05), but lower serum levels of blood glucose. The difference of FGF21 among three groups was significant and AN group showed the highest serum level of FGF21 (P < 0.05). Serum FGF21 was most positively correlated with fasting insulin and HOMA-IR. Multiple logistic analysis showed that FGF21 was the independent risk factor for AN (OR 4.550; 95% CI 1.054–19.635; P = 0.042). Conclusion. AN patients had more serious hyperinsulinemia but better serum levels of blood glucose than OB. Increased FGF21 is associated with AN in obese patients and may be considered as compensatory response to the decreased insulin sensitivity. PMID:27190511

  4. TM-25659-Induced Activation of FGF21 Level Decreases Insulin Resistance and Inflammation in Skeletal Muscle via GCN2 Pathways

    PubMed Central

    Jung, Jong Gab; Yi, Sang-A; Choi, Sung-E; Kang, Yup; Kim, Tae Ho; Jeon, Ja Young; Bae, Myung Ae; Ahn, Jin Hee; Jeong, Hana; Hwang, Eun Sook; Lee, Kwan-Woo

    2015-01-01

    The TAZ activator 2-butyl-5-methyl-6-(pyridine-3-yl)-3-[2′-(1H-tetrazole-5-yl)-biphenyl-4-ylmethyl]-3H-imidazo[4,5-b]pyridine] (TM-25659) inhibits adipocyte differentiation by interacting with peroxisome proliferator-activated receptor gamma. TM-25659 was previously shown to decrease weight gain in a high fat (HF) diet-induced obesity (DIO) mouse model. However, the fundamental mechanisms underlying the effects of TM-25659 remain unknown. Therefore, we investigated the effects of TM-25659 on skeletal muscle functions in C2 myotubes and C57BL/6J mice. We studied the molecular mechanisms underlying the contribution of TM-25659 to palmitate (PA)-induced insulin resistance in C2 myotubes. TM-25659 improved PA-induced insulin resistance and inflammation in C2 myotubes. In addition, TM-25659 increased FGF21 mRNA expression, protein levels, and FGF21 secretion in C2 myotubes via activation of GCN2 pathways (GCN2-phosphoeIF2α-ATF4 and FGF21). This beneficial effect of TM-25659 was diminished by FGF21 siRNA. C57BL/6J mice were fed a HF diet for 30 weeks. The HF-diet group was randomly divided into two groups for the next 14 days: the HF-diet and HF-diet + TM-25659 groups. The HF diet + TM-25659-treated mice showed improvements in their fasting blood glucose levels, insulin sensitivity, insulin-stimulated Akt phosphorylation, and inflammation, but neither body weight nor food intake was affected. The HF diet + TM-25659-treated mice also exhibited increased expression of both FGF21 mRNA and protein. These data indicate that TM-25659 may be beneficial for treating insulin resistance by inducing FGF21 in models of PA-induced insulin resistance and HF diet-induced insulin resistance. PMID:26537193

  5. Ampelopsin Improves Insulin Resistance by Activating PPARγ and Subsequently Up-Regulating FGF21-AMPK Signaling Pathway

    PubMed Central

    Qin, Yu; Liu, Lei; Wan, Jing; Zou, Lingyun; Zhang, Qianyong; Zhu, Jundong; Mi, Mantian

    2016-01-01

    Ampelopsin (APL), a major bioactive constituent of Ampelopsis grossedentata, exerts a number of biological effects. Here, we explored the anti-diabetic activity of APL and elucidate the underlying mechanism of this action. In palmitate-induced insulin resistance of L6 myotubes, APL treatment markedly up- regulated phosphorylated insulin receptor substrate-1 and protein kinase B, along with a corresponding increase of glucose uptake capacity. APL treatment also increased expressions of fibroblast growth factor (FGF21) and phosphorylated adenosine 5’-monophosphate -activated protein kinase (p-AMPK), however inhibiting AMPK by Compound C or AMPK siRNA, or blockage of FGF21 by FGF21 siRNA, obviously weakened APL -induced increases of FGF21 and p-AMPK as well as glucose uptake capacity in palmitate -pretreated L6 myotubes. Furthermore, APL could activate PPAR γ resulting in increases of glucose uptake capacity and expressions of FGF21 and p-AMPK in palmitate -pretreated L6 myotubes, whereas all those effects were obviously abolished by addition of GW9662, a specific inhibitor of peroxisome proliferator- activated receptor –γ (PPARγ), and PPARγsiRNA. Using molecular modeling and the luciferase reporter assays, we observed that APL could dock with the catalytic domain of PPARγ and dose-dependently up-regulate PPARγ activity. In summary, APL maybe a potential agonist of PPARγ and promotes insulin sensitization by activating PPARγ and subsequently regulating FGF21- AMPK signaling pathway. These results provide new insights into the protective health effects of APL, especially for the treatment of Type 2 diabetes mellitus. PMID:27391974

  6. FGF21 represses cerebrovascular aging via improving mitochondrial biogenesis and inhibiting p53 signaling pathway in an AMPK-dependent manner.

    PubMed

    Wang, Xiao-Mei; Xiao, Hang; Liu, Ling-Lin; Cheng, Dang; Li, Xue-Jun; Si, Liang-Yi

    2016-08-15

    Cerebrovascular aging has a high relationship with stroke and neurodegenerative disease. In the present study, we evaluated the influence of fibroblast growth factor 21 (FGF21) on angiotensin (Ang II)-mediated cerebrovascular aging in human brain vascular smooth muscle cells (hBVSMCs). Ang II induced remarkable aging-phenotypes in hBVSMCs, including enhanced SA-β-gal staining and NBS1 protein expression. First, we used immunoblotting assay to confirm protein expression of FGF21 receptor (FGFR1) and the co-receptor β-Klotho in cultured hBVSMCs. Second, we found that FGF21 treatment partly prevented the aging-related changes induced by Ang II. FGF21 inhibited Ang II-enhanced ROS production/superoxide anion levels, rescued the Ang II-reduced Complex IV and citrate synthase activities, and suppressed the Ang II-induced meprin protein expression. Third, we showed that FGF21 not only inhibited the Ang II-induced p53 activation, but also blocked the action of Ang II on Siah-1-TRF signaling pathway which is upstream factors for p53 activation. At last, either chemical inhibition of AMPK signaling pathway by a specific antagonist Compound C or knockdown of AMPKα1/2 isoform using siRNA, successfully abolished the anti-aging action of FGF21 in hBVSMCs. These results indicate that FGF21 protects against Ang II-induced cerebrovascular aging via improving mitochondrial biogenesis and inhibiting p53 activation in an AMPK-dependent manner, and highlight the therapeutic value of FGF21 in cerebrovascular aging-related diseases such as stroke and neurodegenerative disease. PMID:27364911

  7. Impaired mitochondrial fat oxidation induces FGF21 in muscle

    PubMed Central

    Vandanmagsar, Bolormaa; Warfel, Jaycob D.; Wicks, Shawna E.; Ghosh, Sujoy; Salbaum, J. Michael; Burk, David; Dubuisson, Olga S.; Mendoza, Tamra M.; Zhang, Jingying; Noland, Robert C.; Mynatt, Randall L.

    2016-01-01

    SUMMARY Fatty acids are the primary fuel source for skeletal muscle during most of our daily activities and impaired fatty acid oxidation (FAO) is associated with insulin resistance. We have developed a mouse model of impaired FAO by deleting carnitine palmitoyltransferase-1b specifically in skeletal muscle (Cpt1bm−/−). Cpt1bm−/− mice have increased glucose utilization and are resistant to diet induced obesity. Here we show that inhibition of mitochondrial FAO induces FGF21 expression specifically in skeletal muscle. The induction of FGF21 in Cpt1b-deficient muscle is dependent on AMPK and Akt1 signaling but independent on the stress signaling pathways. FGF21 appears to act in a paracrine manner to increase glucose uptake under low insulin conditions, but does not contribute to the resistance to diet induced obesity. PMID:27184848

  8. Exercise Increases Serum Fibroblast Growth Factor 21 (FGF21) Levels

    PubMed Central

    Cuevas-Ramos, Daniel; Almeda-Valdés, Paloma; Meza-Arana, Clara Elena; Brito-Córdova, Griselda; Gómez-Pérez, Francisco J.; Mehta, Roopa; Oseguera-Moguel, Jorge; Aguilar-Salinas, Carlos A.

    2012-01-01

    Background Fibroblast growth factor 21 (FGF21) increases glucose uptake. It is unknown if FGF21 serum levels are affected by exercise. Methodology/Principal Findings This was a comparative longitudinal study. Anthropometric and biochemical evaluation were carried out before and after a bout of exercise and repeated after two weeks of daily supervised exercise. The study sample was composed of 60 sedentary young healthy women. The mean age was 24±3.7 years old, and the mean BMI was 21.4±7.0 kg/m2. The anthropometric characteristics did not change after two weeks of exercise. FGF21 levels significantly increased after two weeks of exercise (276.8 ng/l (142.8–568.6) vs. (460.8 (298.2–742.1), p<0.0001)). The delta (final–basal) log of serum FGF21, adjusted for BMI, showed a significant positive correlation with basal glucose (r = 0.23, p = 0.04), mean maximal heart rate (MHR) (r = 0.54, p<0.0001), mean METs (r = 0.40, p = 0.002), delta plasma epinephrine (r = 0.53, p<0.0001) and delta plasma FFAs (r = 0.35, p = 0.006). A stepwise linear regression model showed that glucose, MHR, METs, FFAs, and epinephrine, were factors independently associated with the increment in FGF21 after the exercise program (F = 4.32; r2 = 0.64, p<0.0001). Conclusions Serum FGF21 levels significantly increased after two weeks of physical activity. This increment correlated positively with clinical parameters related to the adrenergic and lipolytic response to exercise. Trial Registration ClinicalTrials.gov NCT01512368 PMID:22701542

  9. FGF21 and the late adaptive response to starvation in humans.

    PubMed

    Fazeli, Pouneh K; Lun, Mingyue; Kim, Soo M; Bredella, Miriam A; Wright, Spenser; Zhang, Yang; Lee, Hang; Catana, Ciprian; Klibanski, Anne; Patwari, Parth; Steinhauser, Matthew L

    2015-11-03

    In mice, FGF21 is rapidly induced by fasting, mediates critical aspects of the adaptive starvation response, and displays a number of positive metabolic properties when administered pharmacologically. In humans, however, fasting does not consistently increase FGF21, suggesting a possible evolutionary divergence in FGF21 function. Moreover, many key aspects of FGF21 function in mice have been identified in the context of transgenic overexpression or administration of supraphysiologic doses, rather than in a physiologic setting. Here, we explored the dynamics and function of FGF21 in human volunteers during a 10-day fast. Unlike mice, which show an increase in circulating FGF21 after only 6 hours, human subjects did not have a notable surge in FGF21 until 7 to 10 days of fasting. Moreover, we determined that FGF21 induction was associated with decreased thermogenesis and adiponectin, an observation that directly contrasts with previous reports based on supraphysiologic dosing. Additionally, FGF21 levels increased after ketone induction, demonstrating that endogenous FGF21 does not drive starvation-mediated ketogenesis in humans. Instead, a longitudinal analysis of biologically relevant variables identified serum transaminases--markers of tissue breakdown--as predictors of FGF21. These data establish FGF21 as a fasting-induced hormone in humans and indicate that FGF21 contributes to the late stages of adaptive starvation, when it may regulate the utilization of fuel derived from tissue breakdown.

  10. FGF21 and the late adaptive response to starvation in humans

    PubMed Central

    Fazeli, Pouneh K.; Lun, Mingyue; Kim, Soo M.; Bredella, Miriam A.; Wright, Spenser; Zhang, Yang; Lee, Hang; Catana, Ciprian; Klibanski, Anne; Patwari, Parth; Steinhauser, Matthew L.

    2015-01-01

    In mice, FGF21 is rapidly induced by fasting, mediates critical aspects of the adaptive starvation response, and displays a number of positive metabolic properties when administered pharmacologically. In humans, however, fasting does not consistently increase FGF21, suggesting a possible evolutionary divergence in FGF21 function. Moreover, many key aspects of FGF21 function in mice have been identified in the context of transgenic overexpression or administration of supraphysiologic doses, rather than in a physiologic setting. Here, we explored the dynamics and function of FGF21 in human volunteers during a 10-day fast. Unlike mice, which show an increase in circulating FGF21 after only 6 hours, human subjects did not have a notable surge in FGF21 until 7 to 10 days of fasting. Moreover, we determined that FGF21 induction was associated with decreased thermogenesis and adiponectin, an observation that directly contrasts with previous reports based on supraphysiologic dosing. Additionally, FGF21 levels increased after ketone induction, demonstrating that endogenous FGF21 does not drive starvation-mediated ketogenesis in humans. Instead, a longitudinal analysis of biologically relevant variables identified serum transaminases — markers of tissue breakdown — as predictors of FGF21. These data establish FGF21 as a fasting-induced hormone in humans and indicate that FGF21 contributes to the late stages of adaptive starvation, when it may regulate the utilization of fuel derived from tissue breakdown. PMID:26529252

  11. Research Perspectives on the Regulation and Physiological Functions of FGF21 and its Association with NAFLD

    PubMed Central

    Inagaki, Takeshi

    2015-01-01

    Fibroblast growth factor 21 (FGF21) is a metabolic hormone primarily secreted from the liver and functions in multiple tissues. Various transcription factors induce FGF21 expression in the liver, which indicates that FGF21 is a mediator of multiple environmental cues. FGF21 alters metabolism under starvation conditions, protects the body from energy depletion, and extends life span. Pharmacological administration of FGF21 alleviates dyslipidemia and induces weight loss in obese animals. In addition to the well-studied functions of FG21, several lines of recent evidence indicate a possible link between FGF21 and non-alcoholic fatty liver disease (NAFLD). High serum levels of FGF21 are associated with NAFLD and its risk factors, such as endoplasmic reticulum stress and chronic inflammation. In addition, FGF21 alleviates the major risk factors of NAFLD, including obesity, dyslipidemia, and insulin insensitivity. Thus, FGF21 is a potential drug candidate for diseases, such as NAFLD, dyslipidemia, and type 2 diabetes. In this review, the research perspectives of FGF21 and therapeutic potencies of FGF21 as a modulator of NAFLD are summarized. PMID:26441837

  12. Fenofibrate increases cardiac autophagy via FGF21/SIRT1 and prevents fibrosis and inflammation in the hearts of Type 1 diabetic mice.

    PubMed

    Zhang, Jingjing; Cheng, Yanli; Gu, Junlian; Wang, Shudong; Zhou, Shanshan; Wang, Yuehui; Tan, Yi; Feng, Wenke; Fu, Yaowen; Mellen, Nicholas; Cheng, Rui; Ma, Jianxing; Zhang, Chi; Li, Zhanquan; Cai, Lu

    2016-04-01

    Fenofibrate (FF), as a peroxisome-proliferator-activated receptor α (PPARα) agonist, has been used clinically for decades to lower lipid levels. In the present study, we examined whether FF can be repurposed to prevent the pathogenesi of the heart in Type 1 diabetes and to describe the underlying mechanism of its action. Streptozotocin (STZ)-induced diabetic mice and their age-matched control mice were treated with vehicle or FF by gavage every other day for 3 or 6 months. FF prevented diabetes-induced cardiac dysfunction (e.g. decreased ejection fraction and hypertrophy), inflammation and remodelling. FF also increased cardiac expression of fibroblast growth factor 21 (FGF21) and sirtuin 1 (Sirt1) in non-diabetic and diabetic conditions. Deletion of FGF21 gene (FGF21-KO) worsened diabetes-induced pathogenic effects in the heart. FF treatment prevented heart deterioration in the wild-type diabetic mice, but could not do so in the FGF21-KO diabetic mice although the systemic lipid profile was lowered in both wild-type and FGF21-KO diabetic mice. Mechanistically, FF treatment prevented diabetes-impaired autophagy, reflected by increased microtubule-associated protein 1A/1B-light chain 3, in the wild-type diabetic mice but not in the FGF21-KO diabetic mice. Studies with H9C2 cells in vitro demonstrated that exposure to high glucose (HG) significantly increased inflammatory response, oxidative stress and pro-fibrotic response and also significantly inhibited autophagy. These effects of HG were prevented by FF treatment. Inhibition of either autophagy by 3-methyladenine (3MA) or Sirt1 by sirtinol (SI) abolished FF's prevention of HG-induced effects. These results suggested that FF could prevent Type 1 diabetes-induced pathological and functional abnormalities of the heart by increasing FGF21 that may up-regulate Sirt1-mediated autophagy. PMID:26795437

  13. PPAR{alpha} is a key regulator of hepatic FGF21

    SciTech Connect

    Lundasen, Thomas; Hunt, Mary C.; Nilsson, Lisa-Mari; Sanyal, Sabyasachi; Angelin, Bo; Alexson, Stefan E.H.; Rudling, Mats . E-mail: mats.rudling@cnt.ki.se

    2007-08-24

    The metabolic regulator fibroblast growth factor 21 (FGF21) has antidiabetic properties in animal models of diabetes and obesity. Using quantitative RT-PCR, we here show that the hepatic gene expression of FGF21 is regulated by the peroxisome proliferator-activated receptor alpha (PPAR{alpha}). Fasting or treatment of mice with the PPAR{alpha} agonist Wy-14,643 induced FGF21 mRNA by 10-fold and 8-fold, respectively. In contrast, FGF21 mRNA was low in PPAR{alpha} deficient mice, and fasting or treatment with Wy-14,643 did not induce FGF21. Obese ob/ob mice, known to have increased PPAR{alpha} levels, displayed 12-fold increased hepatic FGF21 mRNA levels. The potential importance of PPAR{alpha} for FGF21 expression also in human liver was shown by Wy-14,643 induction of FGF21 mRNA in human primary hepatocytes, and PPAR{alpha} response elements were identified in both the human and mouse FGF21 promoters. Further studies on the mechanisms of regulation of FGF21 by PPAR{alpha} in humans will be of great interest.

  14. FGF21 attenuates pathological myocardial remodeling following myocardial infarction through the adiponectin-dependent mechanism.

    PubMed

    Joki, Yusuke; Ohashi, Koji; Yuasa, Daisuke; Shibata, Rei; Ito, Masanori; Matsuo, Kazuhiro; Kambara, Takahiro; Uemura, Yusuke; Hayakawa, Satoko; Hiramatsu-Ito, Mizuho; Kanemura, Noriyoshi; Ogawa, Hayato; Daida, Hiroyuki; Murohara, Toyoaki; Ouchi, Noriyuki

    2015-03-27

    Ischemic heart disease is one of the leading causes of death. Fibroblast growth factor 21 (FGF21) is a circulating factor with an anti-diabetic property. Skeletal muscle is an important source of FGF21 production. Here, we investigated whether skeletal muscle-derived FGF21 modulates cardiac remodeling in a murine model of myocardial infarction. Myocardial infarction was produced in C57BL/6J wild-type (WT) mice by the permanent ligation of the left anterior descending coronary artery (LAD). Adenoviral vectors expressing FGF21 (Ad-FGF21) or control β-galactosidase were intramuscularly injected into mice at 3 days before permanent LAD ligation. Intramuscular injection of Ad-FGF21 increased plasma FGF21 levels in WT mice compared with control. Treatment of WT mice with Ad-FGF21 led to improvement of left ventricular systolic dysfunction and dilatation at 2 weeks after LAD ligation. Ad-FGF21 administration to WT mice also led to enhancement of capillary density in the infarct border zone, and reduction of myocyte apoptosis in the remote zone, which were accompanied by decreased expression of pro-inflammatory cytokines. Furthermore, treatment of WT mice with Ad-FGF21 increased plasma levels of adiponectin, which is a cardioprotective adipokine. The beneficial effects of Ad-FGF21 on cardiac dysfunction and inflammatory response after myocardial infarction were diminished in adiponectin-knockout mice. These data suggest that muscle-derived FGF21 ameliorates adverse cardiac remodeling after myocardial infarction, at least in part, through an adiponectin-dependent mechanism.

  15. FGF21 mediates the lipid metabolism response to amino acid starvation

    PubMed Central

    De Sousa-Coelho, Ana Luísa; Relat, Joana; Hondares, Elayne; Pérez-Martí, Albert; Ribas, Francesc; Villarroya, Francesc; Marrero, Pedro F.; Haro, Diego

    2013-01-01

    Lipogenic gene expression in liver is repressed in mice upon leucine deprivation. The hormone fibroblast growth factor 21 (FGF21), which is critical to the adaptive metabolic response to starvation, is also induced under amino acid deprivation. Upon leucine deprivation, we found that FGF21 is needed to repress expression of lipogenic genes in liver and white adipose tissue, and stimulate phosphorylation of hormone-sensitive lipase in white adipose tissue. The increased expression of Ucp1 in brown adipose tissue under these circumstances is also impaired in FGF21-deficient mice. Our results demonstrate the important role of FGF21 in the regulation of lipid metabolism during amino acid starvation. PMID:23661803

  16. Glucocorticoids Regulate the Metabolic Hormone FGF21 in a Feed-Forward Loop

    PubMed Central

    Patel, Rucha; Bookout, Angie L.; Magomedova, Lilia; Owen, Bryn M.; Consiglio, Giulia P.; Shimizu, Makoto; Zhang, Yuan; Mangelsdorf, David J.; Kliewer, Steven A.

    2015-01-01

    Hormones such as fibroblast growth factor 21 (FGF21) and glucocorticoids (GCs) play crucial roles in coordinating the adaptive starvation response. Here we examine the interplay between these hormones. It was previously shown that FGF21 induces corticosterone levels in mice by acting on the brain. We now show that this induces the expression of genes required for GC synthesis in the adrenal gland. FGF21 also increases corticosterone secretion from the adrenal in response to ACTH. We further show that the relationship between FGF21 and GCs is bidirectional. GCs induce Fgf21 expression in the liver by acting on the GC receptor (GR). The GR binds in a ligand-dependent manner to a noncanonical GR response element located approximately 4.4 kb upstream of the Fgf21 transcription start site. The GR cooperates with the nuclear fatty acid receptor, peroxisome proliferator-activated receptor-α, to stimulate Fgf21 transcription. GR and peroxisome proliferator-activated receptor-α ligands have additive effects on Fgf21 expression both in vivo and in primary cultures of mouse hepatocytes. We conclude that FGF21 and GCs regulate each other's production in a feed-forward loop and suggest that this provides a mechanism for bypassing negative feedback on the hypothalamic-pituitary-adrenal axis to allow sustained gluconeogenesis during starvation. PMID:25495872

  17. Treatment of CIA Mice with FGF21 Down-regulates TH17-IL-17 Axis.

    PubMed

    Li, Si-ming; Yu, Yin-hang; Li, Lu; Wang, Wen-fei; Li, De-shan

    2016-02-01

    Recently, FGF21 was reported to play an important role in anti-inflammation. The aim of the study is to explore the mechanism for FGF21 alleviating inflammation of CIA. CIA mice were injected with FGF21 once a day for 28 days after first booster immunization. The results showed that FGF21 alleviates arthritis severity and decreases serum anti-CII antibodies levels in CIA mice. Compared with CIA model, the number of the splenic TH17 cells was significantly decreased in FGF21-treated mice. FGF21 treatment reduced the mRNA expression of IL-17, TNF-α, IL-1β, IL-6, IL-8, and MMP3 and increased level of IL-10 in the spleen tissue. The expression of STAT3 and phosphorylated STAT3 was suppressed in FGF21-treated group. The mRNA expression of RORγt and IL-23 also decreased. In conclusion, these findings suggest that the beneficial effects of FGF21 on CIA mice were achieved by down-regulating Th17-IL-17 axis through STAT3/RORγt pathway. Modulating of Th17-mediated inflammatory response may be one of the mechanisms for FGF21 attenuating inflammation in CIA.

  18. GCN2 and FGF21 are likely mediators of the protection from cancer, autoimmunity, obesity, and diabetes afforded by vegan diets.

    PubMed

    McCarty, Mark F

    2014-09-01

    Third World quasi-vegan cultures have been characterized by low risks for "Western" cancers, autoimmune disorders, obesity, and diabetes. The relatively low essential amino acid contents of many vegan diets may play a role in this regard. It is proposed that such diets modestly activate the kinase GCN2 - a physiological detector of essential amino acid paucity - within the liver, resulting in up-regulated production of fibroblast growth factor 21 (FGF21). FGF21, by opposing the stimulatory effect of growth hormone on hepatic IGF-I production, may be responsible for the down-regulation of plasma IGF-I observed in vegans consuming diets of modest protein content. Decreased IGF-I bioactivity throughout life can be expected to have a favorable impact on cancer risk, as observed in rodents that are calorie restricted or genetically defective in IGF-I activity. Increased FGF21 in vegans might also contribute to their characteristic leanness and low LDL cholesterol by promoting hepatic lipid oxidation while inhibiting lipogenesis. Direct trophic effects of FGF21 on pancreatic beta-cells may help to explain the low risk for diabetes observed in vegans, and the utility of vegan diets in diabetes management. And up-regulation of GCN2 in immune cells, by boosting T regulatory activity, might play some role in the reduced risk for autoimmunity reported in some quasi-vegan cultures. The fact that bone density tends to be no greater in vegans than omnivores, despite consumption of a more "alkaline" diet, might be partially attributable to the fact that FGF21 opposes osteoblastogenesis and decreases IGF-I. If these speculations have merit, it should be possible to demonstrate that adoption of a vegan diet of modest protein content increases plasma FGF21 levels.

  19. GCN2 and FGF21 are likely mediators of the protection from cancer, autoimmunity, obesity, and diabetes afforded by vegan diets.

    PubMed

    McCarty, Mark F

    2014-09-01

    Third World quasi-vegan cultures have been characterized by low risks for "Western" cancers, autoimmune disorders, obesity, and diabetes. The relatively low essential amino acid contents of many vegan diets may play a role in this regard. It is proposed that such diets modestly activate the kinase GCN2 - a physiological detector of essential amino acid paucity - within the liver, resulting in up-regulated production of fibroblast growth factor 21 (FGF21). FGF21, by opposing the stimulatory effect of growth hormone on hepatic IGF-I production, may be responsible for the down-regulation of plasma IGF-I observed in vegans consuming diets of modest protein content. Decreased IGF-I bioactivity throughout life can be expected to have a favorable impact on cancer risk, as observed in rodents that are calorie restricted or genetically defective in IGF-I activity. Increased FGF21 in vegans might also contribute to their characteristic leanness and low LDL cholesterol by promoting hepatic lipid oxidation while inhibiting lipogenesis. Direct trophic effects of FGF21 on pancreatic beta-cells may help to explain the low risk for diabetes observed in vegans, and the utility of vegan diets in diabetes management. And up-regulation of GCN2 in immune cells, by boosting T regulatory activity, might play some role in the reduced risk for autoimmunity reported in some quasi-vegan cultures. The fact that bone density tends to be no greater in vegans than omnivores, despite consumption of a more "alkaline" diet, might be partially attributable to the fact that FGF21 opposes osteoblastogenesis and decreases IGF-I. If these speculations have merit, it should be possible to demonstrate that adoption of a vegan diet of modest protein content increases plasma FGF21 levels. PMID:25015767

  20. 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.

  1. Decreased beige adipocyte number and mitochondrial respiration coincide with increased histone methyl transferase (G9a) and reduced FGF21 gene expression in Sprague Dawley rats fed prenatal low protein and postnatal high fat

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We have shown that protein malnutrition during fetal growth followed by postnatal high-fat diets results in a rapid increase in subcutaneous adipose tissue mass in the offspring contributing to development of obesity and insulin resistance. Recent studies have shown that the absence of a key transcr...

  2. [Effect of FGF-21 on learning and memory ability and antioxidant capacity in brain tissue of D-galactose-induced aging mice].

    PubMed

    Yu, Yin-Hang; Ren, Gui-Ping; Liu, Yao-Nan; Qu, Su-Su; Bai, Fu-Liang; Zhang, Tong; Wang, Wen-Fei; Tian, Gui-You; Ye, Xian-Long; Li, De-Shan

    2014-07-01

    This study aims to investigate the effects of fibroblast growth factor 21 (FGF-21) on learning and memory abilities and antioxidant capacity of D-galactose-induced aging mice. Kunming mice (37.1 +/- 0.62) g were randomly divided into normal control group, model group and FGF-21 high, medium and low dose groups (n = 8). Each group was injected in cervical part subcutaneously with D-galactose 180 mg x kg(-1) x d(-1) once a day for 8 weeks. At the same time, FGF-21-treated mice were administered with FGF-21 by giving subcutaneous injection in cervical part at the daily doses of 5, 2 and 1 mg x kg(-1) x d(-1). The normal control group was given with normal saline by subcutaneous injection in cervical part. At seventh week of the experiment, the learning and memory abilities of mice were determined by water maze and jumping stand tests. At the end of the experiment, the mice were sacrificed and the cells damage of hippocampus was observed by HE staining in each group. Reactive oxygen species (ROS), malondialdehyde (MDA), superoxide dismutase (SOD), glutathione peroxidase (GPx), catalase (CAT) and total antioxidant capacity (T-AOC) in the brain of mice were determined. The results showed that different doses of FGF-21 could reduce the time reaching the end (P < 0.01 or P < 0.05) and the number of touching blind side (P < 0.01 or P < 0.05) in the water maze comparing with the model group. It could also prolong the latency time (P < 0.05) and decrease the number of errors (P < 0.01 or P < 0.05) in the step down test. The result of HE staining showed that FGF-21 could significantly reduce brain cell damage in the hippocampus. The ROS and MDA levels of three different doses FGF-21 treatment group reduced significantly than that of the model group [(5.58 +/- 1.07), (7.78 +/- 1.92), (9.03 +/- 1.77) vs (12.75 +/- 2.02) pmol (DCF) x min(-1) x mg(-1), P < 0.01 or P < 0.05], [(2.92 +/- 0.71), (4.21 +/- 0.81), (4.41 +/- 0.97) vs (5.62 +/- 0.63) nmol x mg(-1) (protein), P < 0

  3. CREBH-FGF21 axis improves hepatic steatosis by suppressing adipose tissue lipolysis

    PubMed Central

    Park, Jong-Gil; Xu, Xu; Cho, Sungyun; Hur, Kyu Yeon; Lee, Myung-Shik; Kersten, Sander; Lee, Ann-Hwee

    2016-01-01

    Adipose tissue lipolysis produces glycerol and nonesterified fatty acids (NEFA) that serve as energy sources during nutrient scarcity. Adipose tissue lipolysis is tightly regulated and excessive lipolysis causes hepatic steatosis, as NEFA released from adipose tissue constitutes a major source of TG in the liver of patients with nonalcoholic fatty liver diseases. Here we show that the liver-enriched transcription factor CREBH is activated by TG accumulation and induces FGF21, which suppresses adipose tissue lipolysis, ameliorating hepatic steatosis. CREBH-deficient mice developed severe hepatic steatosis due to increased adipose tissue lipolysis, when fasted or fed a high-fat low-carbohydrate ketogenic diet. FGF21 production was impaired in CREBH-deficient mice, and adenoviral overexpression of FGF21 suppressed adipose tissue lipolysis and improved hepatic steatosis in these mice. Thus, our results uncover a negative feedback loop in which CREBH regulates NEFA flux from adipose tissue to the liver via FGF21. PMID:27301791

  4. Prolongevity hormone FGF21 protects against immune senescence by delaying age-related thymic involution.

    PubMed

    Youm, Yun-Hee; Horvath, Tamas L; Mangelsdorf, David J; Kliewer, Steven A; Dixit, Vishwa Deep

    2016-01-26

    Age-related thymic degeneration is associated with loss of naïve T cells, restriction of peripheral T-cell diversity, and reduced healthspan due to lower immune competence. The mechanistic basis of age-related thymic demise is unclear, but prior evidence suggests that caloric restriction (CR) can slow thymic aging by maintaining thymic epithelial cell integrity and reducing the generation of intrathymic lipid. Here we show that the prolongevity ketogenic hormone fibroblast growth factor 21 (FGF21), a member of the endocrine FGF subfamily, is expressed in thymic stromal cells along with FGF receptors and its obligate coreceptor, βKlotho. We found that FGF21 expression in thymus declines with age and is induced by CR. Genetic gain of FGF21 function in mice protects against age-related thymic involution with an increase in earliest thymocyte progenitors and cortical thymic epithelial cells. Importantly, FGF21 overexpression reduced intrathymic lipid, increased perithymic brown adipose tissue, and elevated thymic T-cell export and naïve T-cell frequencies in old mice. Conversely, loss of FGF21 function in middle-aged mice accelerated thymic aging, increased lethality, and delayed T-cell reconstitution postirradiation and hematopoietic stem cell transplantation (HSCT). Collectively, FGF21 integrates metabolic and immune systems to prevent thymic injury and may aid in the reestablishment of a diverse T-cell repertoire in cancer patients following HSCT. PMID:26755598

  5. Circulating Fibroblast Growth Factor 21 (Fgf21) as Diagnostic and Prognostic Biomarker in Renal Cancer

    PubMed Central

    Knott, ME; Minatta, JN; Roulet, L; Gueglio, G; Pasik, L; Ranuncolo, SM; Nuñez, M; Puricelli, L; De Lorenzo, MS

    2016-01-01

    Background The finding of new biomarkers is needed to have a better sub-classification of primary renal tumors (RCC) as well as more reliable predictors of outcome and therapy response. In this study, we evaluated the role of circulating FGF21, an endocrine factor, as a diagnostic and prognostic biomarker for ccRCC. Materials and Methods Serum samples from healthy controls (HC), clear cell and chromophobe RCC cancer patients were obtained from the serum biobank “Biobanco Público de Muestras Séricas Oncológicas” (BPMSO) of the “Instituto de Oncología “Ángel H. Roffo”. Serum FGF21 and leptin were measured by ELISA while other metabolic markers were measured following routinely clinical procedures. Results One of our major findings was that FGF21 levels were significantly increased in ccRCC patients compared with HC. Moreover, we showed an association between the increased serum FGF21 levels and the shorter disease free survival in a cohort of 98 ccRCC patients, after adjustment for other predictors of outcome. Conclusion Our results suggest that higher FGF21 serum level is an independent prognostic biomarker, associated with worse free-disease survival. PMID:27358750

  6. Fibroblast Growth Factor 21 (FGF21) Protects against High Fat Diet Induced Inflammation and Islet Hyperplasia in Pancreas

    PubMed Central

    Singhal, Garima; Fisher, ffolliott Martin; Chee, Melissa J.; Tan, Tze Guan; El Ouaamari, Abdelfattah; Adams, Andrew C.; Najarian, Robert; Kulkarni, Rohit N.; Benoist, Christophe; Flier, Jeffrey S.; Maratos-Flier, Eleftheria

    2016-01-01

    Fibroblast growth factor 21 (FGF21) is an important endocrine metabolic regulator expressed in multiple tissues including liver and adipose tissue. Although highest levels of expression are in pancreas, little is known about the function of FGF21 in this tissue. In order to understand the physiology of FGF21 in the pancreas, we analyzed its expression and regulation in both acinar and islet tissues. We found that acinar tissue express 20-fold higher levels than that observed in islets. We also observed that pancreatic FGF21 is nutritionally regulated; a marked reduction in FGF21 expression was noted with fasting while obesity is associated with 3–4 fold higher expression. Acinar and islet cells are targets of FGF21, which when systemically administered, leads to phosphorylation of the downstream target ERK 1/2 in about half of acinar cells and a small subset of islet cells. Chronic, systemic FGF21 infusion down-regulates its own expression in the pancreas. Mice lacking FGF21 develop significant islet hyperplasia and periductal lymphocytic inflammation when fed with a high fat obesogenic diet. Inflammatory infiltrates consist of TCRb+ Thy1+ T lymphocytes with increased levels of Foxp3+ regulatory T cells. Increased levels of inflammatory cells were coupled with elevated expression of cytokines such as TNFα, IFNγ and IL1β. We conclude that FGF21 acts to limit islet hyperplasia and may also prevent pancreatic inflammation. PMID:26872145

  7. Tissue-specific Actions of the Metabolic Hormones FGF15/19 and FGF21

    PubMed Central

    Owen, Bryn M.; Mangelsdorf, David J.; Kliewer, Steven A.

    2014-01-01

    Fibroblast growth factors (FGFs) 15/19 and 21 belong to a subfamily of FGFs that function as hormones. Produced in response to specific nutritional cues, they act on overlapping sets of cell surface receptors composed of classic FGF receptors in complex with βKlotho, and regulate metabolism and related processes during periods of fluctuating energy availability. Pharmacologically, both FGF15/19 and FGF21 cause weight loss and improve both insulin sensitivity and lipid parameters, in rodent and primate models of metabolic disease. Recently, FGF21 was shown to have similar effects in obese patients with type 2 diabetes. Here, we discuss emerging concepts in FGF15/19 and FGF21 tissue specific actions and critically assess their putative role as candidate targets for treating metabolic disease. PMID:25476453

  8. The PPARα - FGF21 hormone axis contributes to metabolic regulation by the hepatic JNK signaling pathway

    PubMed Central

    Vernia, Santiago; Cavanagh-Kyros, Julie; Garcia-Haro, Luisa; Sabio, Guadalupe; Barrett, Tamera; Jung, Dae Young; Kim, Jason K.; Xu, Jia; Shulha, Hennady P.; Garber, Manuel; Gao, Guangping; Davis, Roger J.

    2014-01-01

    The cJun NH2-terminal kinase (JNK) stress signaling pathway is implicated in the metabolic response to the consumption of a high fat diet, including the development of obesity and insulin resistance. These metabolic adaptations involve altered liver function. Here we demonstrate that hepatic JNK potently represses the nuclear hormone receptor peroxisome proliferator-activated receptor α (PPARα). JNK therefore causes decreased expression of PPARα target genes that increase fatty acid oxidation / ketogenesis and promote the development of insulin resistance. We show that the PPARα target gene fibroblast growth factor 21 (Fgf21) plays a key role in this response because disruption of the hepatic PPARα - FGF21 hormone axis suppresses the metabolic effects of JNK-deficiency. This analysis identifies the hepatokine FGF21 as a critical mediator of JNK signaling in the liver. PMID:25043817

  9. 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. PMID:23276922

  10. Time-imposed daily restricted feeding induces rhythmic expression of Fgf21 in white adipose tissue of mice.

    PubMed

    Oishi, Katsutaka; Konishi, Morichika; Murata, Yusuke; Itoh, Nobuyuki

    2011-08-26

    Fibroblast growth factor 21 (FGF21) is a key metabolic regulator that is induced by fasting and starvation, and its expression is thought to be regulated by the circadian clock in the liver. To evaluate the functional role of FGF21 in the circadian regulation of physiology and behavior, we examined the temporal expression profiles of Fgf21 and circadian clock genes in addition to behavioral activity rhythms under adlibitum feeding (ALF) and time-imposed restricted feeding (RF) in mice. Four hours of daily restricted feeding during the daytime induced over an 80-fold increase in feeding-dependent rhythmic Fgf21 mRNA expression in epididymal white adipose tissue (eWAT), although the expression levels were continuously increased 10-fold in the liver of wild-type (WT) mice. Refeeding subsequent to transient fasting revealed that refeeding but not fasting remarkably induces Fgf21 expression in eWAT, although fasting-induced hepatic Fgf21 expression is completely reversed by refeeding. The free-running period of locomotor activity rhythm under ALF and the food anticipatory activity (FAA) under RF remained intact in Fgf21 knockout (KO) mice, suggesting that FGF21 is dispensable for both the central clock in the suprachiasmatic nucleus (SCN) and the food-entrainable oscillator that governs the FAA. Temporal expression profiles of circadian genes such as mPer2 and BMAL1 were essentially identical in both tissues between WT and Fgf21 KO mice under RF. The physiological role of the refeeding-induced adipose Fgf21 expression remains to be elucidated.

  11. FGF21 Promotes Endothelial Cell Angiogenesis through a Dynamin-2 and Rab5 Dependent Pathway

    PubMed Central

    Yaqoob, Usman; Jagavelu, Kumaravelu; Shergill, Uday; de Assuncao, Thiago; Cao, Sheng; Shah, Vijay H.

    2014-01-01

    Binding of angiogenic molecules with cognate receptor tyrosine kinases (RTK) is required for angiogenesis however the precise link between RTK binding, endocytosis, and signaling requires further investigation. Here, we use FGFR1 as a model to test the effects of the large GTPase and endocytosis regulatory molecule dynamin-2 on angiogenic signaling in context of distinct FGF ligands. In vitro, overexpression of dominant negative dynamin-2 (DynK44A) attenuates FGFR1 activation of Erk and tubulogenesis by FGF2. Furthermore, we identify FGF21, a non-classical, FGF ligand implicated in diverse human pathologies as an angiogenic molecule acting through FGFR1 and β-Klotho coreceptor. Disruption of FGFR1 activation of ERK by FGF21 is achieved by perturbation of the function of both dynamin-2 and Rab5 GTPase. In vivo, mice harboring endothelial selective overexpression of DynK44A, show impaired angiogenesis in response to FGF21. In conclusion, dynamin dependent endocytosis of FGFR1 is required for in vitro and in vivo angiogenesis in response to FGF2 and the non-classical FGF ligand, FGF21. These studies extend our understanding of the relationships between RTK binding, internalization, endosomal targeting, and angiogenic signaling. PMID:24848261

  12. Hepatic mTORC1 controls locomotor activity, body temperature, and lipid metabolism through FGF21.

    PubMed

    Cornu, Marion; Oppliger, Wolfgang; Albert, Verena; Robitaille, Aaron M; Trapani, Francesca; Quagliata, Luca; Fuhrer, Tobias; Sauer, Uwe; Terracciano, Luigi; Hall, Michael N

    2014-08-12

    The liver is a key metabolic organ that controls whole-body physiology in response to nutrient availability. Mammalian target of rapamycin (mTOR) is a nutrient-activated kinase and central controller of growth and metabolism that is negatively regulated by the tumor suppressor tuberous sclerosis complex 1 (TSC1). To investigate the role of hepatic mTOR complex 1 (mTORC1) in whole-body physiology, we generated liver-specific Tsc1 (L-Tsc1 KO) knockout mice. L-Tsc1 KO mice displayed reduced locomotor activity, body temperature, and hepatic triglyceride content in a rapamycin-sensitive manner. Ectopic activation of mTORC1 also caused depletion of hepatic and plasma glutamine, leading to peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α)-dependent fibroblast growth factor 21 (FGF21) expression in the liver. Injection of glutamine or knockdown of PGC-1α or FGF21 in the liver suppressed the behavioral and metabolic defects due to mTORC1 activation. Thus, mTORC1 in the liver controls whole-body physiology through PGC-1α and FGF21. Finally, mTORC1 signaling correlated with FGF21 expression in human liver tumors, suggesting that treatment of glutamine-addicted cancers with mTOR inhibitors might have beneficial effects at both the tumor and whole-body level.

  13. Hormone Resistance in Diabetes and Obesity: Insulin, Leptin, and FGF21

    PubMed Central

    Flier, Jeffrey S.

    2012-01-01

    This an edited transcript of the Lee E. Farr Lecture given by Dr. Jeffrey Flier on May 8, 2012, at the culmination of the annual Student Research Day at the Yale School of Medicine. In this presentation, Dr. Flier discusses his and his wife’s research on insulin, leptin, and FGF21 in the context of his reflections upon his life’s work and his advice for young investigators. PMID:23012588

  14. FGF21 signalling pathway and metabolic traits – genetic association analysis

    PubMed Central

    Kaess, Bernhard M; Barnes, Timothy A; Stark, Klaus; Charchar, Fadi J; Waterworth, Dawn; Song, Kijoung; Wang, William Y S; Vollenweider, Peter; Waeber, Gerard; Mooser, Vincent; Zukowska-Szczechowska, Ewa; Samani, Nilesh J; Hengstenberg, Christian; Tomaszewski, Maciej

    2010-01-01

    Fibroblast growth factor 21 (FGF21) is a novel master regulator of metabolic profile. The biological actions of FGF21 are elicited upon its klotho beta (KLB)-facilitated binding to FGF receptor 1 (FGFR1), FGFR2 and FGFR3. We hypothesised that common polymorphisms in the FGF21 signalling pathway may be associated with metabolic risk. At the screening stage, we examined associations between 63 common single-nucleotide polymorphisms (SNPs) in five genes of this pathway (FGF21, KLB, FGFR1, FGFR2, FGFR3) and four metabolic phenotypes (LDL cholesterol – LDL-C, HDL-cholesterol – HDL-C, triglycerides and body mass index) in 629 individuals from Silesian Hypertension Study (SHS). Replication analyses were performed in 5478 unrelated individuals of the Swiss CoLaus cohort (imputed genotypes) and in 3030 directly genotyped individuals of the German Myocardial Infarction Family Study (GerMIFS). Of 54 SNPs that met quality control criteria after genotyping in SHS, 4 (rs4733946 and rs7012413 in FGFR1; rs2071616 in FGFR2 and rs7670903 in KLB) showed suggestive association with LDL-C (P=0.0006, P=0.0013, P=0.0055, P=0.011, respectively) and 1 (rs2608819 in KLB) was associated with body mass index (P=0.011); all with false discovery rate q<0.5. Of these, only one FGFR2 polymorphism (rs2071616) showed replicated association with LDL-C in both CoLaus (P=0.009) and men from GerMIFS (P=0.017). The direction of allelic effect of rs2071616 upon LDL-C was consistent in all examined populations. These data show that common genetic variations in FGFR2 may be associated with LDL-C in subjects of white European ancestry. PMID:20717167

  15. Long-lasting hypoglycemic effect of modified FGF-21 analog with polyethylene glycol in type 1 diabetic mice and its systematic toxicity.

    PubMed

    Ye, Xianlong; Qi, Jianying; Wu, Qiang; Yu, Dan; Li, Shujie; Wu, Yunzhou; Li, Deshan

    2016-06-15

    Fibroblast growth factor-21 (FGF-21) is a novel metabolic regulator and has the potential to become a powerful therapy to treat diabetes mellitus. However, we found that the clinical application of wild type FGF-21 was influenced by its low intrinsic bio-stability and poor hypoglycemic potency. In this study, The N-terminus of FGF-21 analog (mFGF-21) was PEGylated in a site-specific manner by 20kD methoxy poly-ethylene glycol-propionaldehyde (mPEG-ALD). PEGylated mFGF-21 was isolated by Capto Q anion exchange chromatography. The properties of PEGylated mFGF-21 including the in vitro bio-stability and biological activity were evaluated. As well as the anti-diabetic effect of PEGylated mFGF-21 were studied in streptozotocin (STZ)-induced type 1 diabetic mice. Results demonstrated that PEGylated mFGF-21 had a similar capacity of stimulating glucose uptake in HepG2 cells with mFGF-21 and PEGylation of mFGF-21 significantly enhanced the anti-protease ability and the long acting anti-diabetic effect in type 1 diabetic mice. Furthermore, the preliminary safety of PEGylated mFGF-21 following subcutaneously injection was assessed using healthy mice by measuring the body weight, histopathology and clinical biochemical parameters, and the results showed no subacute toxicity to major organs or tissues and no significant changes in physiological and biochemical parameters in healthy mice. Taken together, under the premise of remaining the in vitro biological activity of mFGF-21, PEGylation significantly improves the long lasting hypoglycemic effect of mFGF-21 in type 1 diabetic mice. Our valuation shows that PEGylated mFGF-21 is a potential drug for the effective treatment of type 1 diabetes. PMID:27089817

  16. Long-lasting hypoglycemic effect of modified FGF-21 analog with polyethylene glycol in type 1 diabetic mice and its systematic toxicity.

    PubMed

    Ye, Xianlong; Qi, Jianying; Wu, Qiang; Yu, Dan; Li, Shujie; Wu, Yunzhou; Li, Deshan

    2016-06-15

    Fibroblast growth factor-21 (FGF-21) is a novel metabolic regulator and has the potential to become a powerful therapy to treat diabetes mellitus. However, we found that the clinical application of wild type FGF-21 was influenced by its low intrinsic bio-stability and poor hypoglycemic potency. In this study, The N-terminus of FGF-21 analog (mFGF-21) was PEGylated in a site-specific manner by 20kD methoxy poly-ethylene glycol-propionaldehyde (mPEG-ALD). PEGylated mFGF-21 was isolated by Capto Q anion exchange chromatography. The properties of PEGylated mFGF-21 including the in vitro bio-stability and biological activity were evaluated. As well as the anti-diabetic effect of PEGylated mFGF-21 were studied in streptozotocin (STZ)-induced type 1 diabetic mice. Results demonstrated that PEGylated mFGF-21 had a similar capacity of stimulating glucose uptake in HepG2 cells with mFGF-21 and PEGylation of mFGF-21 significantly enhanced the anti-protease ability and the long acting anti-diabetic effect in type 1 diabetic mice. Furthermore, the preliminary safety of PEGylated mFGF-21 following subcutaneously injection was assessed using healthy mice by measuring the body weight, histopathology and clinical biochemical parameters, and the results showed no subacute toxicity to major organs or tissues and no significant changes in physiological and biochemical parameters in healthy mice. Taken together, under the premise of remaining the in vitro biological activity of mFGF-21, PEGylation significantly improves the long lasting hypoglycemic effect of mFGF-21 in type 1 diabetic mice. Our valuation shows that PEGylated mFGF-21 is a potential drug for the effective treatment of type 1 diabetes.

  17. Nrf2 Represses FGF21 During Long-Term High-Fat Diet–Induced Obesity in Mice

    PubMed Central

    Chartoumpekis, Dionysios V.; Ziros, Panos G.; Psyrogiannis, Agathoklis I.; Papavassiliou, Athanasios G.; Kyriazopoulou, Venetsana E.; Sykiotis, Gerasimos P.; Habeos, Ioannis G.

    2011-01-01

    OBJECTIVE Obesity is characterized by chronic oxidative stress. Fibroblast growth factor 21 (FGF21) has recently been identified as a novel hormone that regulates metabolism. NFE2-related factor 2 (Nrf2) is a transcription factor that orchestrates the expression of a battery of antioxidant and detoxification genes under both basal and stress conditions. The current study investigated the role of Nrf2 in a mouse model of long-term high-fat diet (HFD)-induced obesity and characterized its crosstalk to FGF21 in this process. RESEARCH DESIGN AND METHODS Wild-type (WT) and Nrf2 knockout (Nrf2-KO) mice were fed an HFD for 180 days. During this period, food consumption and body weights were measured. Glucose metabolism was assessed by an intraperitoneal glucose tolerance test and intraperitoneal insulin tolerance test. Total RNA was prepared from liver and adipose tissue and was used for quantitative real-time RT-PCR. Fasting plasma was collected and analyzed for blood chemistries. The ST-2 cell line was used for transfection studies. RESULTS Nrf2-KO mice were partially protected from HFD-induced obesity and developed a less insulin-resistant phenotype. Importantly, Nrf2-KO mice had higher plasma FGF21 levels and higher FGF21 mRNA levels in liver and white adipose tissue than WT mice. Thus, the altered metabolic phenotype of Nrf2-KO mice under HFD was associated with higher expression and abundance of FGF21. Consistently, the overexpression of Nrf2 in ST-2 cells resulted in decreased FGF21 mRNA levels as well as in suppressed activity of a FGF21 promoter luciferase reporter. CONCLUSIONS The identification of Nrf2 as a novel regulator of FGF21 expands our understanding of the crosstalk between metabolism and stress defense. PMID:21852674

  18. Plasma FGF21 concentrations, adipose fibroblast growth factor receptor-1 and β-klotho expression decrease with fasting in northern elephant seals.

    PubMed

    Suzuki, Miwa; Lee, Andrew Y; Vázquez-Medina, José Pablo; Viscarra, Jose A; Crocker, Daniel E; Ortiz, Rudy M

    2015-05-15

    Fibroblast growth factor (FGF)-21 is secreted from the liver, pancreas, and adipose in response to prolonged fasting/starvation to facilitate lipid and glucose metabolism. Northern elephant seals naturally fast for several months, maintaining a relatively elevated metabolic rate to satisfy their energetic requirements. Thus, to better understand the impact of prolonged food deprivation on FGF21-associated changes, we analyzed the expression of FGF21, FGF receptor-1 (FGFR1), β-klotho (KLB; a co-activator of FGFR) in adipose, and plasma FGF21, glucose and 3-hydroxybutyrate in fasted elephant seal pups. Expression of FGFR1 and KLB mRNA decreased 98% and 43%, respectively, with fasting duration. While the 80% decrease in mean adipose FGF21 mRNA expression with fasting did not reach statistical significance, it paralleled the 39% decrease in plasma FGF21 concentrations suggesting that FGF21 is suppressed with fasting in elephant seals. Data demonstrate an atypical response of FGF21 to prolonged fasting in a mammal suggesting that FGF21-mediated mechanisms have evolved differentially in elephant seals. Furthermore, the typical fasting-induced, FGF21-mediated actions such as the inhibition of lipolysis in adipose may not be required in elephant seals as part of a naturally adapted mechanism to support their unique metabolic demands during prolonged fasting.

  19. Plasma FGF21 Concentrations, Adipose Fibroblast Growth Factor Receptor-1 and β-Klotho Expression Decrease with Fasting in Northern Elephant Seals

    PubMed Central

    Suzuki, Miwa; Lee, Andrew; Vázquez-Medina, Jose Pablo; Viscarra, Jose A.; Crocker, Daniel E.; Ortiz, Rudy M.

    2015-01-01

    Fibroblast growth factor (FGF)-21 is secreted from the liver, pancreas, and adipose in response to prolonged fasting/starvation to facilitate lipid and glucose metabolism. Northern elephant seals naturally fast for several months, maintaining a relatively elevated metabolic rate to satisfy their energetic requirements. Thus, to better understand the impact of prolonged food deprivation on FGF21-associated changes, we analyzed the expression of FGF21, FGF receptor-1 (FGFR1), β-klotho (KLB; a co-activator of FGFR) in adipose, and plasma FGF21, glucose and 3-hydroxybutyrate in fasted elephant seal pups. Expression of FGFR1 and KLB mRNA decreased 98% and 43%, respectively, with fasting duration. While the 80% decrease in mean adipose FGF21 mRNA expression with fasting did not reach statistical significance, it paralleled the 39% decrease in plasma FGF21 concentrations suggesting that FGF21 is suppressed with fasting in elephant seals. Data demonstrate an atypical response of FGF21 to prolonged fasting in a mammal suggesting that FGF21-mediated mechanisms have evolved differentially in elephant seals. Furthermore, the typical fasting-induced, FGF21-mediated actions such as the inhibition of lipolysis in adipose may not be required in elephant seals as part of a naturally adapted mechanism to support their unique metabolic demands during prolonged fasting. PMID:25857751

  20. Plasma FGF21 concentrations, adipose fibroblast growth factor receptor-1 and β-klotho expression decrease with fasting in northern elephant seals.

    PubMed

    Suzuki, Miwa; Lee, Andrew Y; Vázquez-Medina, José Pablo; Viscarra, Jose A; Crocker, Daniel E; Ortiz, Rudy M

    2015-05-15

    Fibroblast growth factor (FGF)-21 is secreted from the liver, pancreas, and adipose in response to prolonged fasting/starvation to facilitate lipid and glucose metabolism. Northern elephant seals naturally fast for several months, maintaining a relatively elevated metabolic rate to satisfy their energetic requirements. Thus, to better understand the impact of prolonged food deprivation on FGF21-associated changes, we analyzed the expression of FGF21, FGF receptor-1 (FGFR1), β-klotho (KLB; a co-activator of FGFR) in adipose, and plasma FGF21, glucose and 3-hydroxybutyrate in fasted elephant seal pups. Expression of FGFR1 and KLB mRNA decreased 98% and 43%, respectively, with fasting duration. While the 80% decrease in mean adipose FGF21 mRNA expression with fasting did not reach statistical significance, it paralleled the 39% decrease in plasma FGF21 concentrations suggesting that FGF21 is suppressed with fasting in elephant seals. Data demonstrate an atypical response of FGF21 to prolonged fasting in a mammal suggesting that FGF21-mediated mechanisms have evolved differentially in elephant seals. Furthermore, the typical fasting-induced, FGF21-mediated actions such as the inhibition of lipolysis in adipose may not be required in elephant seals as part of a naturally adapted mechanism to support their unique metabolic demands during prolonged fasting. PMID:25857751

  1. Effects of lifestyle modification and metformin on irisin and FGF21 among HIV-infected subjects with the metabolic syndrome

    PubMed Central

    Srinivasa, Suman; Wong, Kimberly; Fitch, Kathleen V.; Wei, Jeffrey; Petrow, Eva; Cypess, Aaron M.; Torriani, Martin; Grinspoon, Steven K.

    2015-01-01

    Summary Objective Few studies have investigated irisin and FGF21 to elucidate the role of these hormones to regulate ‘beiging’ in HIV-infected patients. Design Fifty HIV-infected subjects with the metabolic syndrome were previously recruited and randomized to receive lifestyle modification (LSM) and/or metformin over 12 months. In the current study, we assessed FGF21 and irisin at baseline and after intervention. In addition, we assessed circulating FGF21 and irisin in relationship to brown adipose tissue (BAT) gene expression in dorsocervical subcutaneous fat biopsies from 13 HIV-infected subjects. Results At baseline, prior to intervention, HIV-infected subjects demonstrated increased log FGF21 (2·13 ± 0·06 vs 1·98 ± 0·05 pg/ml, P = 0·05) and log irisin (0·33 ± 0·02 vs 0·17 ± 0·04 μg/ml, P = 0·003) compared with healthy controls well matched based on waist circumference. After 12 months, HIV-infected subjects randomized to LSM demonstrated a relative reduction in FGF21 compared with those not randomized to LSM (−10 [−35,22] vs 40 [0,94] %change, P = 0·01). Changes in FGF21 were inversely associated with improved parameters of energy homoeostasis, including increased REE (ρ = −0·34, P = 0·046) and max VO2 (ρ = −0·38, P = 0·02), and reduced RQ (ρ = 0·40, P = 0·02) among all HIV-infected subjects. Increased UCP-1 (r = 0·75, P = 0·003), DIO2 (r = 0·58, P = 0·04) and CideA (r = 0·73, P = 0·01) gene expression in dorsocervical fat was significantly associated with FGF21 in HIV-infected subjects. Conclusion HIV-infected subjects with metabolic complications demonstrate increases in FGF21 in relationship to BAT gene expression. Relative reductions in FGF21 in those receiving long-term LSM relate to overall improvements in energy expenditure parameters. In contrast, irisin levels are elevated in HIV-infected subjects, but are not influenced by LSM nor associated with BAT gene expression. PMID:25130061

  2. Metformin Prevents Fatty Liver and Improves Balance of White/Brown Adipose in an Obesity Mouse Model by Inducing FGF21

    PubMed Central

    Kim, Eun Kyung; Lee, Seung Hoon; Jhun, Joo Yeon; Byun, Jae Kyeong; Jeong, Jeong Hee; Lee, Seon-Young; Kim, Jae Kyung; Choi, Jong Young; Cho, Mi-La

    2016-01-01

    Obesity and its associated metabolic disorders are related to the onset of fatty liver and the balance of white adipose tissue (WAT) and brown adipose tissue (BAT). We hypothesized that metformin, an effective pharmacological treatment for type 2 diabetes, would inhibit white adipogenesis, fatty liver, and metabolic dysfunction. Metformin was treated daily for 14 weeks in a high-fat dieting C57BL/6J mice. Serum biomarkers were analyzed and protein level was assessed using confocal staining or flow cytometry. The development of lipid drops in the liver cells and white adipocyte was measured using hematoxylin and eosin or Oil Red O stains. Gene expressions were analyzed with quantitative real-time PCR. Metformin treatment decreased the body weight and improved the metabolic profile of obese mice. In obese mice, metformin also induced the expression of BAT-related markers and increased fibroblast growth factor (FGF) 21 expression in the liver and in white adipocyte. Metformin suppressed white adipocyte differentiation via induction of FGF21. Metformin improves Treg/Th17 balance in CD4+ T cells in mice with high-fat diet-induced obesity. Metformin also improves glucose metabolism and metabolic disorder. Interleukin-17 deficiency also decreases inflammation in mice. Therefore, metformin may be therapeutically useful for the treatment of obesity and metabolic dysfunction. PMID:27057099

  3. FGF21 as a mediator of adaptive responses to stress and metabolic benefits of anti-diabetic drugs.

    PubMed

    Kim, Kook Hwan; Lee, Myung-Shik

    2015-07-01

    Most hormones secreted from specific organs of the body in response to diverse stimuli contribute to the homeostasis of the whole organism. Fibroblast growth factor 21 (FGF21), a hormone induced by a variety of environmental or metabolic stimuli, plays a crucial role in the adaptive response to these stressful conditions. In addition to its role as a stress hormone, FGF21 appears to function as a mediator of the therapeutic effects of currently available drugs and those under development for treatment of metabolic diseases. In this review, we highlight molecular mechanisms and the functional importance of FGF21 induction in response to diverse stress conditions such as changes of nutritional status, cold exposure, and exercise. In addition, we describe recent findings regarding the role of FGF21 in the pathogenesis and treatment of diabetes associated with obesity, liver diseases, pancreatitis, muscle atrophy, atherosclerosis, cardiac hypertrophy, and diabetic nephropathy. Finally, we discuss the current understanding of the actions of FGF21 as a crucial regulator mediating beneficial metabolic effects of therapeutic agents such as metformin, glucagon/glucagon-like peptide 1 analogues, thiazolidinedione, sirtuin 1 activators, and lipoic acid. PMID:26116622

  4. iNKT Cells Induce FGF21 for Thermogenesis and Are Required for Maximal Weight Loss in GLP1 Therapy.

    PubMed

    Lynch, Lydia; Hogan, Andrew E; Duquette, Danielle; Lester, Chantel; Banks, Alexander; LeClair, Katherine; Cohen, David E; Ghosh, Abhisek; Lu, Bing; Corrigan, Michelle; Stevanovic, Darko; Maratos-Flier, Eleftheria; Drucker, Daniel J; O'Shea, Donal; Brenner, Michael

    2016-09-13

    Adipose-resident invariant natural killer T (iNKT) cells are key players in metabolic regulation. iNKT cells are innate lipid sensors, and their activation, using their prototypic ligand α-galactosylceramide (αGalCer), induces weight loss and restores glycemic control in obesity. Here, iNKT activation induced fibroblast growth factor 21 (FGF21) production and thermogenic browning of white fat. Complete metabolic analysis revealed that iNKT cell activation induced increased body temperature, V02, VC02, and fatty acid oxidation, without affecting food intake or activity. FGF21 induction played a major role in iNKT cell-induced weight loss, as FGF21 null mice lost significantly less weight after αGalCer treatment. The glucagon-like peptide 1 (GLP-1) receptor agonist, liraglutide, also activated iNKT cells in humans and mice. In iNKT-deficient mice, liraglutide promoted satiety but failed to induce FGF21, resulting in less weight loss. These findings reveal an iNKT cell-FGF21 axis that defines a new immune-mediated pathway that could be targeted for glycemic control and weight regulation. PMID:27593966

  5. [Recombinant protein production in Escherichia coli].

    PubMed

    Nuc, Przemysław; Nuc, Katarzyna

    2006-01-01

    Growing needs for efficient recombinant production pose new challenges; starting from cell growth optimization under overexpression conditions, improving vectors, gene and protein sequence to suit them to protein biosynthesis machinery of the host, through extending the knowledge of protein folding, fusion protein construction, and coexpression systems, to improvements in protein purification and renaturation technologies. Hitherto Escherichia coli is the most defined and the cheapest protein biosynthesis system. With its wealth of available mutants tested is the best suited to economically test new gene constructs and to scale up the recombinant protein production.

  6. 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

  7. Dietary Betaine Supplementation Increases Fgf21 Levels to Improve Glucose Homeostasis and Reduce Hepatic Lipid Accumulation in Mice.

    PubMed

    Ejaz, Asma; Martinez-Guino, Laura; Goldfine, Allison B; Ribas-Aulinas, Francesc; De Nigris, Valeria; Ribó, Sílvia; Gonzalez-Franquesa, Alba; Garcia-Roves, Pablo M; Li, Elizabeth; Dreyfuss, Jonathan M; Gall, Walt; Kim, Jason K; Bottiglieri, Teodoro; Villarroya, Francesc; Gerszten, Robert E; Patti, Mary-Elizabeth; Lerin, Carles

    2016-04-01

    Identifying markers of human insulin resistance may permit development of new approaches for treatment and prevention of type 2 diabetes. To this end, we analyzed the fasting plasma metabolome in metabolically characterized human volunteers across a spectrum of insulin resistance. We demonstrate that plasma betaine levels are reduced in insulin-resistant humans and correlate closely with insulin sensitivity. Moreover, betaine administration to mice with diet-induced obesity prevents the development of impaired glucose homeostasis, reduces hepatic lipid accumulation, increases white adipose oxidative capacity, and enhances whole-body energy expenditure. In parallel with these beneficial metabolic effects, betaine supplementation robustly increased hepatic and circulating fibroblast growth factor (Fgf)21 levels. Betaine administration failed to improve glucose homeostasis and liver fat content in Fgf21(-/-) mice, demonstrating that Fgf21 is necessary for betaine's beneficial effects. Together, these data indicate that dietary betaine increases Fgf21 levels to improve metabolic health in mice and suggest that betaine supplementation merits further investigation as a supplement for treatment or prevention of type 2 diabetes in humans. PMID:26858359

  8. Protein building blocks preserved by recombination.

    PubMed

    Voigt, Christopher A; Martinez, Carlos; Wang, Zhen-Gang; Mayo, Stephen L; Arnold, Frances H

    2002-07-01

    Borrowing concepts from the schema theory of genetic algorithms, we have developed a computational algorithm to identify the fragments of proteins, or schemas, that can be recombined without disturbing the integrity of the three-dimensional structure. When recombination leaves these schemas undisturbed, the hybrid proteins are more likely to be folded and functional. Crossovers found by screening libraries of several randomly shuffled proteins for functional hybrids strongly correlate with those predicted by this approach. Experimental results from the construction of hybrids of two beta-lactamases that share 40% amino acid identity demonstrate a threshold in the amount of schema disruption that the hybrid protein can tolerate. To the extent that introns function to promote recombination within proteins, natural selection would serve to bias their locations to schema boundaries. PMID:12042875

  9. Recombinant protein blends: silk beyond natural design.

    PubMed

    Dinjaski, Nina; Kaplan, David L

    2016-06-01

    Recombinant DNA technology and new material concepts are shaping future directions in biomaterial science for the design and production of the next-generation biomaterial platforms. Aside from conventionally used synthetic polymers, numerous natural biopolymers (e.g., silk, elastin, collagen, gelatin, alginate, cellulose, keratin, chitin, polyhydroxyalkanoates) have been investigated for properties and manipulation via bioengineering. Genetic engineering provides a path to increase structural and functional complexity of these biopolymers, and thereby expand the catalog of available biomaterials beyond that which exists in nature. In addition, the integration of experimental approaches with computational modeling to analyze sequence-structure-function relationships is starting to have an impact in the field by establishing predictive frameworks for determining material properties. Herein, we review advances in recombinant DNA-mediated protein production and functionalization approaches, with a focus on hybrids or combinations of proteins; recombinant protein blends or 'recombinamers'. We highlight the potential biomedical applications of fibrous protein recombinamers, such as Silk-Elastin Like Polypeptides (SELPs) and Silk-Bacterial Collagens (SBCs). We also discuss the possibility for the rationale design of fibrous proteins to build smart, stimuli-responsive biomaterials for diverse applications. We underline current limitations with production systems for these proteins and discuss the main trends in systems/synthetic biology that may improve recombinant fibrous protein design and production. PMID:26686863

  10. Fibroblast growth factor 21 (FGF21) inhibits macrophage-mediated inflammation by activating Nrf2 and suppressing the NF-κB signaling pathway.

    PubMed

    Yu, Yinhang; He, Jinjiao; Li, Siming; Song, Liying; Guo, Xiaochen; Yao, Wenbing; Zou, Dehua; Gao, Xinyu; Liu, Yunye; Bai, Fuliang; Ren, Guiping; Li, Deshan

    2016-09-01

    Our previous report has shown that FGF21 has anti-inflammatory properties in a collagen-induced arthritis (CIA) model. In this study, the underlying molecular mechanisms of action were also investigated using RAW 264.7 cells, a murine monocyte-macrophage. RAW 264.7 cells were pre-incubated with various concentrations (2000, 500, 100ng/ml) of FGF21 and stimulated with LPS to induce oxidative stress and inflammation. The result of flow cytometry showed that β-Klotho, FGF21 specific receptor, was expressed in murine splenic macrophages and RAW 264.7. In vitro, FGF21 reduced the expression of TNF-α, IL-1β, IL-6 and IFN-γ and increased the level of IL-10 in a dose-dependent manner in LPS-stimulated RAW 264.7 macrophages. FGF21 also suppressed profound elevation of ROS production and oxidative stress, as evidenced by an increase of the MDA level and depletion of the intracellular GSH level, and restored the activities of antioxidant enzymes SOD and GSH-Px in LPS-stimulated RAW 264.7 macrophages. Moreover, FGF21 inhibited LPS-induced nuclear factor-κB (NF-κB) activation, including degradation of I-κB and nuclear translocation of p65. In addition, the result of Western blot and real-time PCR showed that FGF21 induced heme oxygenase-1 (HO-1) expression and increased the nuclear transcription factor-E2-related factor 2 (Nrf2) levels in a dose-dependent manner in LPS-stimulated RAW 264.7 macrophages. In conclusion, the results suggest that macrophages are the targets for the anti-inflammatory effects of FGF21, and FGF21 exerted an anti-inflammatory effect mainly via enhancing Nrf2-mediated anti-oxidant capacity and suppressing NF-κB signaling pathway.

  11. Fibroblast growth factor 21 (FGF21) ameliorates collagen-induced arthritis through modulating oxidative stress and suppressing nuclear factor-kappa B pathway.

    PubMed

    Yu, Yinhang; Li, Siming; Liu, Yaonan; Tian, Guiyou; Yuan, Qingyan; Bai, Fuliang; Wang, Wenfei; Zhang, Zhiyi; Ren, Guiping; Zhang, Yu; Li, Deshan

    2015-03-01

    It has been demonstrated that circulating FGF21 levels are elevated in the serum and synovial fluid of patients with rheumatoid arthritis (RA). The aim of this study is to investigate efficacy of FGF21 for treatment of RA and the molecular mechanisms of the therapeutic effect on collagen-induced arthritis (CIA). Mice with CIA were subcutaneously administered with FGF21 (5, 2 or 1mg·kg(-1)·d(-1)), IL-1β antibody (5mg·kg(-1)·d(-1)), IL-17A antibody (5mg·kg(-1)·d(-1)) and dexamethasone (DEX) (1mg·kg(-1)·d(-1)), respectively. The effects of treatment were determined by arthritis severity score, histological damage and cytokine production. The activation of NF-κB was analyzed by Western blotting. We also detected the levels of oxidative stress parameters. Our results showed that FGF21 had beneficial effects on clinical symptom and histological lesion of CIA mice. Similar to antibody and DEX, FGF21 treatment alleviated the severity of arthritis by reducing humoral and cellular immune responses and down-regulating the expression of pro-inflammatory cytokines. FGF21 treatment also reduced the expression of TNF-α, IL-1β, IL-6, IFN-γ and MMP-3 and increased level of IL-10 in the spleen tissue or the plasma of CIA mice in a dose-dependent manner. Furthermore, FGF21 inhibited IκBα degradation and NF-κB p65 nuclear translocation and induced significant changes of oxidative stress parameters (MDA, SOD, CAT, GSH-PX and GSH) in the plasma. FGF21 exerts therapeutic efficacy for RA through antioxidant reaction and inhibiting NF-κB inflammatory pathway. This study provides evidence that FGF21 may be a promising therapeutic agent for RA patients. PMID:25601498

  12. Fibroblast growth factor 21 (FGF21) inhibits macrophage-mediated inflammation by activating Nrf2 and suppressing the NF-κB signaling pathway.

    PubMed

    Yu, Yinhang; He, Jinjiao; Li, Siming; Song, Liying; Guo, Xiaochen; Yao, Wenbing; Zou, Dehua; Gao, Xinyu; Liu, Yunye; Bai, Fuliang; Ren, Guiping; Li, Deshan

    2016-09-01

    Our previous report has shown that FGF21 has anti-inflammatory properties in a collagen-induced arthritis (CIA) model. In this study, the underlying molecular mechanisms of action were also investigated using RAW 264.7 cells, a murine monocyte-macrophage. RAW 264.7 cells were pre-incubated with various concentrations (2000, 500, 100ng/ml) of FGF21 and stimulated with LPS to induce oxidative stress and inflammation. The result of flow cytometry showed that β-Klotho, FGF21 specific receptor, was expressed in murine splenic macrophages and RAW 264.7. In vitro, FGF21 reduced the expression of TNF-α, IL-1β, IL-6 and IFN-γ and increased the level of IL-10 in a dose-dependent manner in LPS-stimulated RAW 264.7 macrophages. FGF21 also suppressed profound elevation of ROS production and oxidative stress, as evidenced by an increase of the MDA level and depletion of the intracellular GSH level, and restored the activities of antioxidant enzymes SOD and GSH-Px in LPS-stimulated RAW 264.7 macrophages. Moreover, FGF21 inhibited LPS-induced nuclear factor-κB (NF-κB) activation, including degradation of I-κB and nuclear translocation of p65. In addition, the result of Western blot and real-time PCR showed that FGF21 induced heme oxygenase-1 (HO-1) expression and increased the nuclear transcription factor-E2-related factor 2 (Nrf2) levels in a dose-dependent manner in LPS-stimulated RAW 264.7 macrophages. In conclusion, the results suggest that macrophages are the targets for the anti-inflammatory effects of FGF21, and FGF21 exerted an anti-inflammatory effect mainly via enhancing Nrf2-mediated anti-oxidant capacity and suppressing NF-κB signaling pathway. PMID:27276443

  13. Brown Adipose Tissue and Browning Agents: Irisin and FGF21 in the Development of Obesity in Children and Adolescents.

    PubMed

    Pyrżak, B; Demkow, U; Kucharska, A M

    2015-01-01

    In the pediatric population, especially in early infancy, the activity of brown adipose tissue (BAT) is the highest. Further in life BAT is more active in individuals with a lower body mass index and one can expect that BAT is protective against childhood obesity. The development of BAT throughout the whole life can be regulated by genetic, endocrine, and environmental factors. Three distinct adipose depots have been identified: white, brown, and beige adipocytes. The process by which BAT can become beige is still unclear and is an area of intensive research. The "browning agents" increase energy expenditure through the production of heat. Numerous factors known as "browning agents" have currently been described. In humans, recent studies justify a notion of a role of novel myokines: irisin and fibroblast growth factor 21 (FGF21) in the metabolism and development of obesity. This review describes a possible role of irisin and FGF21 in the pathogenesis of obesity in children.

  14. High-fat diet and FGF21 cooperatively promote aerobic thermogenesis in mtDNA mutator mice.

    PubMed

    Wall, Christopher E; Whyte, Jamie; Suh, Jae M; Fan, Weiwei; Collins, Brett; Liddle, Christopher; Yu, Ruth T; Atkins, Annette R; Naviaux, Jane C; Li, Kefeng; Bright, Andrew Taylor; Alaynick, William A; Downes, Michael; Naviaux, Robert K; Evans, Ronald M

    2015-07-14

    Mitochondria are highly adaptable organelles that can facilitate communication between tissues to meet the energetic demands of the organism. However, the mechanisms by which mitochondria can nonautonomously relay stress signals remain poorly understood. Here we report that mitochondrial mutations in the young, preprogeroid polymerase gamma mutator (POLG) mouse produce a metabolic state of starvation. As a result, these mice exhibit signs of metabolic imbalance including thermogenic defects in brown adipose tissue (BAT). An unexpected benefit of this adaptive response is the complete resistance to diet-induced obesity when POLG mice are placed on a high-fat diet (HFD). Paradoxically, HFD further increases oxygen consumption in part by inducing thermogenesis and mitochondrial biogenesis in BAT along with enhanced expression of fibroblast growth factor 21 (FGF21). Collectively, these findings identify a mechanistic link between FGF21, a long-known marker of mitochondrial disease, and systemic metabolic adaptation in response to mitochondrial stress.

  15. High-fat diet and FGF21 cooperatively promote aerobic thermogenesis in mtDNA mutator mice

    PubMed Central

    Wall, Christopher E.; Whyte, Jamie; Suh, Jae M.; Fan, Weiwei; Collins, Brett; Liddle, Christopher; Yu, Ruth T.; Atkins, Annette R.; Naviaux, Jane C.; Li, Kefeng; Bright, Andrew Taylor; Alaynick, William A.; Downes, Michael; Naviaux, Robert K.; Evans, Ronald M.

    2015-01-01

    Mitochondria are highly adaptable organelles that can facilitate communication between tissues to meet the energetic demands of the organism. However, the mechanisms by which mitochondria can nonautonomously relay stress signals remain poorly understood. Here we report that mitochondrial mutations in the young, preprogeroid polymerase gamma mutator (POLG) mouse produce a metabolic state of starvation. As a result, these mice exhibit signs of metabolic imbalance including thermogenic defects in brown adipose tissue (BAT). An unexpected benefit of this adaptive response is the complete resistance to diet-induced obesity when POLG mice are placed on a high-fat diet (HFD). Paradoxically, HFD further increases oxygen consumption in part by inducing thermogenesis and mitochondrial biogenesis in BAT along with enhanced expression of fibroblast growth factor 21 (FGF21). Collectively, these findings identify a mechanistic link between FGF21, a long-known marker of mitochondrial disease, and systemic metabolic adaptation in response to mitochondrial stress. PMID:26124126

  16. 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).

  17. Protein Crystal Recombinant Human Insulin

    NASA Technical Reports Server (NTRS)

    1994-01-01

    The comparison of protein crystal, Recombiant Human Insulin; space-grown (left) and earth-grown (right). On STS-60, Spacehab II indicated that space-grown crystals are larger and of greater optical clarity than their earth-grown counterparts. Recombiant Human Insulin facilitates the incorporation of glucose into cells. In diabetics, there is either a decrease in or complete lack of insulin, thereby leading to several harmful complications. Principal Investigator is Larry DeLucas.

  18. Extracellular secretion of recombinant proteins

    SciTech Connect

    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.

  19. Heavy labeling of recombinant proteins.

    PubMed

    Rodriguez, Eric

    2007-01-01

    Because of the cost of isotopic chemicals and heterologous proteins to produce, an economical 15N/13C isotopic labeling method is critically needed. Four protocols have been tested for the expression of Ovine interferon-tau in Pichia pastoris. 13C-glucose in place of 13C-glycerol as well as the need for 15N/13C-sources were evaluated during the growth phase. Sequential addition of 15NH4Cl and 13C-methanol were also evaluated at different ratio. Our results demonstrate that 15N/13C isotopes are not required throughout the initial growth period but are necessary at low concentration a few hours prior to the methanol induction period. We have evaluated the cost of the use of isotopes 15NH4Cl, 13C-glucose and 13C-methanol in our optimised P4 protocol conditions. The cost was one-third that of the standard method using 15NH4Cl and 13C-glucose throughout the entire growth period and was even lower using 13C-glycerol.

  20. Astragalus polysaccharides affect insulin resistance by regulating the hepatic SIRT1-PGC-1α/PPARα-FGF21 signaling pathway in male Sprague Dawley rats undergoing catch-up growth.

    PubMed

    Gu, Chengying; Zeng, Yipeng; Tang, Zhaosheng; Wang, Chaoxun; He, Yanju; Feng, Xinge; Zhou, Ligang

    2015-11-01

    The present study investigated the effects of Astragalus polysaccharides (APS) on insulin resistance by modulation of hepatic sirtuin 1 (SIRT1)‑peroxisome proliferator‑activated receptor (PPAR)‑γ coactivator (PGC)‑1α/PPARα‑fibroblast growth factor (FGF)21, and glucose and lipid metabolism. Thirty male Sprague Dawley rats were divided into three groups: A normal control group, a catch‑up growth group and an APS‑treated (APS-G) group. The latter two groups underwent food restriction for 4 weeks, prior to being provided with a high fat diet, which was available ad libitum. The APS‑G group was orally treated with APS for 8 weeks, whereas the other groups were administered saline. Body weight was measured and an oral glucose tolerance test (OGTT) was conducted after 8 weeks. The plasma glucose and insulin levels obtained from the OGTT were assayed, and hepatic morphology was observed by light and transmission electron microscopy. In addition, the mRNA expression levels of PGC‑1α/PPARα, and the protein expression levels of SIRT1, FGF21 and nuclear factor‑κB were quantified in the liver and serum. APS treatment suppressed abnormal glycolipid metabolism and insulin resistance following 8 weeks of catch‑up growth by improving hepatic SIRT1‑PPARα‑FGF21 intracellular signaling and reducing chronic inflammation, and by partially attenuating hepatic steatosis. The suppressive effects of APS on liver acetylation and glycolipid metabolism‑associated molecules contributed to the observed suppression of insulin resistance. However, the mechanism underlying the effects of APS on insulin resistance requires further research in order to be elucidated. Rapid and long‑term treatment with APS may provide a novel, safe and effective therapeutic strategy for type 2 diabetes. PMID:26323321

  1. Recombinant protein scaffolds for tissue engineering.

    PubMed

    Werkmeister, Jerome A; Ramshaw, John A M

    2012-02-01

    New biological materials for tissue engineering are now being developed using common genetic engineering capabilities to clone and express a variety of genetic elements that allow cost-effective purification and scaffold fabrication from these recombinant proteins, peptides or from chimeric combinations of these. The field is limitless as long as the gene sequences are known. The utility is dependent on the ease, product yield and adaptability of these protein products to the biomedical field. The development of recombinant proteins as scaffolds, while still an emerging technology with respect to commercial products, is scientifically superior to current use of natural materials or synthetic polymer scaffolds, in terms of designing specific structures with desired degrees of biological complexities and motifs. In the field of tissue engineering, next generation scaffolds will be the key to directing appropriate tissue regeneration. The initial period of biodegradable synthetic scaffolds that provided shape and mechanical integrity, but no biological information, is phasing out. The era of protein scaffolds offers distinct advantages, particularly with the combination of powerful tools of molecular biology. These include, for example, the production of human proteins of uniform quality that are free of infectious agents and the ability to make suitable quantities of proteins that are found in low quantity or are hard to isolate from tissue. For the particular needs of tissue engineering scaffolds, fibrous proteins like collagens, elastin, silks and combinations of these offer further advantages of natural well-defined structural scaffolds as well as endless possibilities of controlling functionality by genetic manipulation.

  2. Recombinant protein materials for bioengineering and nanomedicine.

    PubMed

    Corchero, José Luis; Vázquez, Esther; García-Fruitós, Elena; Ferrer-Miralles, Neus; Villaverde, Antonio

    2014-12-01

    Proteins are essential macromolecules supporting life. Being efficient catalyzers and offering specific cross-molecular contacts, proteins are largely exploited in biotechnology and biomedicine as therapeutics, in industrial catalysis or as molecular reagents. Recombinant enzymes, hormones, immunogens and antibodies are produced aiming to different applications, on the basis of their ability to interact with or modify substrates or biological targets. In nature, proteins also perform task-specific architectonic roles, and they can organize in supramolecular complexes with intriguing physical properties such as elasticity and adhesiveness, and with regulatable stiffness, flexibility and mechanical strength. Proteins have recently gained interest as materials for bioengineering and nanomedicine as they can combine these features with functionality, biocompatibility and degradability in unusually versatile composites. We revise here the fundamental properties of the diverse categories of emerging protein materials resulting from biological synthesis and how they can be genetically re-designed to engineer the interplay between mechanical and biological properties in a medically oriented exploitable way.

  3. Serum FGF-21 levels are associated with worsened radial trabecular bone microarchitecture and decreased radial bone strength in women with anorexia nervosa

    PubMed Central

    Fazeli, Pouneh K.; Faje, Alexander T.; Cross, Ela J.; Lee, Hang; Rosen, Clifford J.; Bouxsein, Mary L.; Klibanski, Anne

    2015-01-01

    BACKGROUND Anorexia nervosa (AN) is a psychiatric disorder characterized by self-induced starvation and low body weight. Women with AN have impaired bone formation, low bone mass and an increased risk of fracture. FGF-21 is a hormone secreted by the liver in starvation and FGF-21 transgenic mice have significant bone loss due to an uncoupling of bone resorption and bone formation. We hypothesized that FGF-21 may contribute to the low bone mass state of AN. SUBJECTS AND METHODS We studied 46 women: 20 with AN (median age [interquartile range]: 27.5 [25, 30.75] years) and 26 normal-weight controls (NWC) of similar age (25 [24, 28.5] years). We investigated associations between serum FGF-21 and 1) aBMD measured by dual energy X-ray absorptiometry, 2) parameters of bone microarchitecture in the distal radius and tibia measured by high-resolution peripheral quantitative CT and 3) bone strength, estimated by microfinite element analysis. RESULTS FGF-21 levels were similar in AN and NWC (AN: 33.1 [18.1, 117.0] pg/ml vs NWC: 57.4 [23.8, 107.1] pg/ml; p=0.54). There was a significant inverse association between log FGF-21 and trabecular number in the radius in both AN (R= -0.57, p<0.01) and NWC (R= -0.53, p<0.01) and a significant positive association between log FGF-21 and trabecular separation in the radius in AN (R=0.50, p<0.03) and NWC (R=0.52, p<0.01). Estimates of radial bone strength were inversely associated with log FGF-21 in AN (R= -0.50, p<0.03 for both stiffness and failure load). There were no associations between FGF-21 and aBMD, cortical parameters or tibial parameters in the AN or NWC groups. CONCLUSIONS FGF-21 may be an important determinant of trabecular skeletal homeostasis in AN. PMID:25868802

  4. Green biofactories: recombinant protein production in plants.

    PubMed

    Ahmad, Adil; Pereira, Eridan O; Conley, Andrew J; Richman, Alex S; Menassa, Rima

    2010-11-01

    Until recently, low accumulation levels have been the major bottleneck for plant-made recombinant protein production. However, several breakthroughs have been described in the past few years allowing for very high accumulation levels, mainly through chloroplast transformation and transient expression, coupled with subcellular targeting and protein fusions. Another important factor influencing our ability to use plants for the production of recombinant proteins is the availability of quick and simple purification strategies. Recent developments using oleosin, zein, ELP and hydrophobin fusion tags have shown promise as efficient and cost-effective methods for non-chromatographic separation. Furthermore, plant glycosylation is a major barrier to the parenteral administration of plant-made biopharmaceuticals because of potential immunogenicity concerns. A major effort has been invested in humanizing plant glycosylation, and several groups have been able to reduce or eliminate immunogenic glycans while introducing mammalian-specific glycans. Finally, biosafety issues and public perception are essential for the acceptance of plants as bioreactors for the production of proteins. Over recent years, it has become clear that food and feed plants carry an inherent risk of contaminating our food supply, and thus much effort has focused on the use of non-food plants. Presently, Nicotiana benthamiana has emerged as the preferred host for transient expression, while tobacco is most frequently used for chloroplast transformation. In this review, we focus on the main issues hindering the economical production of recombinant proteins in plants, describing the current efforts for addressing these limitations, and we include an extensive list of recent patents generated with the intention of solving these limitations. PMID:21171961

  5. Alterations in Hepatic FGF21, Co-Regulated Genes, and Upstream Metabolic Genes in Response to Nutrition, Ketosis and Inflammation in Peripartal Holstein Cows.

    PubMed

    Akbar, Haji; Batistel, Fernanda; Drackley, James K; Loor, Juan J

    2015-01-01

    In rodents, fibroblast growth factor 21 (FGF21) has emerged as a key metabolic regulator produced by liver. To gather preliminary data on the potential importance of FGF1, co-regulated genes, and upstream metabolic genes, we examined the hepatic mRNA expression in response to nutrition and inflammation in dairy cows. In experiment 1, induction of ketosis through feed restriction on d 5 postpartum upregulated FGF21, its co-receptor KLB, and PPARA but only elicited a numerical increase in serum FGF21 concentration. In experiment 2, cows in control (CON) or receiving 50 g/d of L-carnitine (C50) from -14 through 21 d had increased FGF21, PPARA, and NFIL3 on d 10 compared with d 2 postpartum. In contrast, compared with CON and C50, 100 g/d L-carnitine (C100) resulted in lower FGF21, KLB, ANGPTL4, and ARNTL expression on d 10. In experiment 3, cows were fed during the dry period either a higher-energy (OVE; 1.62 Mcal/kg DM) or lower-energy (CON; 1.34 Mcal/kg DM) diet and received 0 (OVE:N, CON:N) or 200 μg of LPS (OVE:Y, CON:Y) into the mammary gland at d 7 postpartum. For FGF21 mRNA expression in CON, the LPS challenge (CON:Y) prevented a decrease in expression between d 7 and 14 postpartum such that cows in CON:N had a 4-fold lower expression on d 14 compared with d 7. The inflammatory stimulus induced by LPS in CON:Y resulted in upregulation of PPARA on d 14 to a similar level as cows in OVE:N. In OVE:Y, expression of PPARA was lower than CON:N on d 7 and remained unchanged on d 14. On d 7, LPS led to a 4-fold greater serum FGF21 only in OVE but not in CON cows. In fact, OVE:Y reached the same serum FGF21 concentration as CON:N, suggesting a carryover effect of dietary energy level on signaling mechanisms within liver. Overall, results indicate that nutrition, ketosis, and inflammation during the peripartal period can alter hepatic FGF21, co-regulated genes, and upstream metabolic genes to various extents. The functional outcome of these changes merits further study

  6. Alterations in Hepatic FGF21, Co-Regulated Genes, and Upstream Metabolic Genes in Response to Nutrition, Ketosis and Inflammation in Peripartal Holstein Cows

    PubMed Central

    Akbar, Haji; Batistel, Fernanda; Drackley, James K.; Loor, Juan J.

    2015-01-01

    In rodents, fibroblast growth factor 21 (FGF21) has emerged as a key metabolic regulator produced by liver. To gather preliminary data on the potential importance of FGF1, co-regulated genes, and upstream metabolic genes, we examined the hepatic mRNA expression in response to nutrition and inflammation in dairy cows. In experiment 1, induction of ketosis through feed restriction on d 5 postpartum upregulated FGF21, its co-receptor KLB, and PPARA but only elicited a numerical increase in serum FGF21 concentration. In experiment 2, cows in control (CON) or receiving 50 g/d of L-carnitine (C50) from -14 through 21 d had increased FGF21, PPARA, and NFIL3 on d 10 compared with d 2 postpartum. In contrast, compared with CON and C50, 100 g/d L-carnitine (C100) resulted in lower FGF21, KLB, ANGPTL4, and ARNTL expression on d 10. In experiment 3, cows were fed during the dry period either a higher-energy (OVE; 1.62 Mcal/kg DM) or lower-energy (CON; 1.34 Mcal/kg DM) diet and received 0 (OVE:N, CON:N) or 200 μg of LPS (OVE:Y, CON:Y) into the mammary gland at d 7 postpartum. For FGF21 mRNA expression in CON, the LPS challenge (CON:Y) prevented a decrease in expression between d 7 and 14 postpartum such that cows in CON:N had a 4-fold lower expression on d 14 compared with d 7. The inflammatory stimulus induced by LPS in CON:Y resulted in upregulation of PPARA on d 14 to a similar level as cows in OVE:N. In OVE:Y, expression of PPARA was lower than CON:N on d 7 and remained unchanged on d 14. On d 7, LPS led to a 4-fold greater serum FGF21 only in OVE but not in CON cows. In fact, OVE:Y reached the same serum FGF21 concentration as CON:N, suggesting a carryover effect of dietary energy level on signaling mechanisms within liver. Overall, results indicate that nutrition, ketosis, and inflammation during the peripartal period can alter hepatic FGF21, co-regulated genes, and upstream metabolic genes to various extents. The functional outcome of these changes merits further study

  7. 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

  8. Patents in therapeutic recombinant protein production using mammalian cells.

    PubMed

    Picanco-Castro, Virginia; de Freitas, Marcela Cristina Correa; Bomfim, Aline de Sousa; de Sousa Russo, Elisa Maria

    2014-01-01

    The industrial production of recombinant proteins preferentially requires the generation of stable cell lines expressing proteins in a quick, relatively facile, and a reproducible manner. Different methods are used to insert exogenous DNA into the host cell, and choosing the appropriate producing cell is of paramount importance for the efficient production and quality of the recombinant protein. This review addresses the advances in recombinant protein production in mammalian cell lines, according to key patents from the last 30 years.

  9. Production of recombinant proteins by yeast cells.

    PubMed

    Celik, Eda; Calık, Pınar

    2012-01-01

    Yeasts are widely used in production of recombinant proteins of medical or industrial interest. For each individual product, the most suitable expression system has to be identified and optimized, both on the genetic and fermentative level, by taking into account the properties of the product, the organism and the expression cassette. There is a wide range of important yeast expression hosts including the species Saccharomyces cerevisiae, Pichia pastoris, Hansenula polymorpha, Kluyveromyces lactis, Schizosaccharomyces pombe, Yarrowia lipolytica and Arxula adeninivorans, with various characteristics such as being thermo-tolerant or halo-tolerant, rapidly reaching high cell densities or utilizing unusual carbon sources. Several strains were also engineered to have further advantages, such as humanized glycosylation pathways or lack of proteases. Additionally, with a large variety of vectors, promoters and selection markers to choose from, combined with the accumulated knowledge on industrial-scale fermentation techniques and the current advances in the post-genomic technology, it is possible to design more cost-effective expression systems in order to meet the increasing demand for recombinant proteins and glycoproteins. In this review, the present status of the main and most promising yeast expression systems is discussed. PMID:21964262

  10. Recombinant production of spider silk proteins.

    PubMed

    Heidebrecht, Aniela; Scheibel, Thomas

    2013-01-01

    Natural spider silk fibers combine extraordinary properties such as stability and flexibility which results in a toughness superseding that of all other fiber materials. As the spider's aggressive territorial behavior renders their farming not feasible, the biotechnological production of spider silk proteins (spidroins) is essential in order to investigate and employ them for applications. In order to accomplish this task, two approaches have been tested: firstly, the expression of partial cDNAs, and secondly, the expression of synthetic genes in several host organisms, including bacteria, yeast, plants, insect cells, mammalian cells, and transgenic animals. The experienced problems include genetic instability, limitations of the translational and transcriptional machinery, and low solubility of the produced proteins. Here, an overview of attempts to recombinantly produce spidroins will be given, and advantages and disadvantages of the different approaches and host organisms will be discussed. PMID:23415154

  11. Recombinant HT.sub.m4 gene, protein and assays

    DOEpatents

    Lim, Bing; Adra, Chaker N.; Lelias, Jean-Michel

    1996-01-01

    The invention relates to a recombinant DNA molecule which encodes a HT.sub.m4 protein, a transformed host cell which has been stably transfected with a DNA molecule which encodes a HT.sub.m4 protein and a recombinant HT.sub.m4 protein. The invention also relates to a method for detecting the presence of a hereditary atopy.

  12. 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.

  13. Protective effect of FGF21 on type 1 diabetes-induced testicular apoptotic cell death probably via both mitochondrial- and endoplasmic reticulum stress-dependent pathways in the mouse model.

    PubMed

    Jiang, Xin; Zhang, Chi; Xin, Ying; Huang, Zhifeng; Tan, Yi; Huang, Yadong; Wang, Yonggang; Feng, Wenke; Li, Xiaokun; Li, Wei; Qu, Yaqin; Cai, Lu

    2013-05-10

    Fibroblast growth factor 21 (FGF21) is a novel member identified and was reported to express predominantly in pancreas, liver and adipose tissue, and relatively less in other organs, such as the testis. However, the role of FGF21 in the testis has never been addressed. The present study examined FGF21 expression at mRNA level by real-time RT-PCR assay in the testis of fasting and non-fasting mice or mice with type 1 diabetes that was induced with streptozotocin. We also examined the effect of Fgf21 gene deletion or supplementation of the exogenous FGF21 on the testicular apoptotic cell death spontaneously or induced by type 1 diabetes in FGF21 knockout (FGF21-KO) mice. Deletion of Fgf21 gene does not affect testicular cell proliferation, but significantly increases the spontaneous incidence of testicular TUNEL positive cells with increases in the Bax/Bcl2 expression ratio and apoptosis-inducing factor (AIF) expression. Diabetes induced significant increases in testicular TUNEL positive cells, Bax/Bcl2 expression ratio, AIF expression, CHOP and cleaved caspase-12 expression, and oxidative damage, but did not change the expression of cleaved caspase-3 and caspase-8. Deletion of Fgf21 gene also significantly enhances diabetes-induced TUNEL positive cells along with the increased expression of Bax/Bcl2 ratio, AIF, CHOP, cleaved caspase-12, and oxidative damage, which was significantly prevented by the supplementation of exogenous FGF21. These results suggest that Fgf21 gene may involve in maintaining normal spermatogenesis and also protect the germ cells from diabetes-induced apoptotic cell death probably via the prevention of diabetes-induced oxidative damage. PMID:23499715

  14. Elastomeric Recombinant Protein-based Biomaterials

    PubMed Central

    Annabi, Nasim; Mithieux, Suzanne M.; Camci-Unal, Gulden; Dokmeci, Mehmet R.; Weiss, Anthony S.; Khademhosseini, Ali

    2013-01-01

    Elastomeric protein-based biomaterials, produced from elastin derivatives, are widely investigated as promising tissue engineering scaffolds due to their remarkable properties including substantial extensibility, long-term stability, self-assembly, high resilience upon stretching, low energy loss, and excellent biological activity. These elastomers are processed from different sources of soluble elastin such as animal-derived soluble elastin, recombinant human tropoelastin, and elastin-like polypeptides into various forms including three dimensional (3D) porous hydrogels, elastomeric films, and fibrous electrospun scaffolds. Elastin-based biomaterials have shown great potential for the engineering of elastic tissues such as skin, lung and vasculature. In this review, the synthesis and properties of various elastin-based elastomers with their applications in tissue engineering are described. PMID:23935392

  15. Recombinant protein vaccines produced in insect cells.

    PubMed

    Cox, Manon M J

    2012-02-27

    The baculovirus-insect cell expression system is a well known tool for the production of complex proteins. The technology is also used for commercial manufacture of various veterinary and human vaccines. This review paper provides an overview of how this technology can be applied to produce a multitude of vaccine candidates. The key advantage of this recombinant protein manufacturing platform is that a universal "plug and play" process may be used for producing a broad range of protein-based prophylactic and therapeutic vaccines for both human and veterinary use while offering the potential for low manufacturing costs. Large scale mammalian cell culture facilities previously established for the manufacturing of monoclonal antibodies that have now become obsolete due to yield improvement could be deployed for the manufacturing of these vaccines. Alternatively, manufacturing capacity could be established in geographic regions that do not have any vaccine production capability. Dependent on health care priorities, different vaccines could be manufactured while maintaining the ability to rapidly convert to producing pandemic influenza vaccine when the need arises. PMID:22265860

  16. PF-05231023, a long-acting FGF21 analogue, decreases body weight by reduction of food intake in non-human primates.

    PubMed

    Thompson, W Clayton; Zhou, Yingjiang; Talukdar, Saswata; Musante, Cynthia J

    2016-08-01

    PF-05231023, a long-acting FGF21 analogue, is a promising potential pharmacotherapy for the treatment of obesity and associated comorbidities. Previous studies have shown the potential of FGF21 and FGF21-like compounds to decrease body weight in mice, non-human primates, and humans; the precise mechanisms of action remain unclear. In particular, there have been conflicting reports on the degree to which FGF21-induced weight loss in non-human primates is attributable to a decrease in food intake versus an increase in energy expenditure. Here, we present a semi-mechanistic mathematical model of energy balance and body composition developed from similar work in mice. This model links PF-05231023 administration and washout to changes in food intake, which in turn drives changes in body weight. The model is calibrated to and compared with recently published data from cynomolgus macaques treated with PF-05231023, demonstrating its accuracy in describing pharmacotherapy-induced weight loss in these animals. The results are consistent with the hypothesis that PF-05231023 decreases body weight in cynomolgus macaques solely by a reduction in food intake, with no direct effect on energy expenditure. PMID:27405817

  17. A Long-Acting FGF21 Molecule, PF-05231023, Decreases Body Weight and Improves Lipid Profile in Non-human Primates and Type 2 Diabetic Subjects.

    PubMed

    Talukdar, Saswata; Zhou, Yingjiang; Li, Dongmei; Rossulek, Michelle; Dong, Jennifer; Somayaji, Veena; Weng, Yan; Clark, Ronald; Lanba, Adhiraj; Owen, Bryn M; Brenner, Martin B; Trimmer, Jeffrey K; Gropp, Kathryn E; Chabot, Jeffrey R; Erion, Derek M; Rolph, Timothy P; Goodwin, Bryan; Calle, Roberto A

    2016-03-01

    FGF21 plays a central role in energy, lipid, and glucose homeostasis. To characterize the pharmacologic effects of FGF21, we administered a long-acting FGF21 analog, PF-05231023, to obese cynomolgus monkeys. PF-05231023 caused a marked decrease in food intake that led to reduced body weight. To assess the effects of PF-05231023 in humans, we conducted a placebo-controlled, multiple ascending-dose study in overweight/obese subjects with type 2 diabetes. PF-05231023 treatment resulted in a significant decrease in body weight, improved plasma lipoprotein profile, and increased adiponectin levels. Importantly, there were no significant effects of PF-05231023 on glycemic control. PF-05231023 treatment led to dose-dependent changes in multiple markers of bone formation and resorption and elevated insulin-like growth factor 1. The favorable effects of PF-05231023 on body weight support further evaluation of this molecule for the treatment of obesity. Longer studies are needed to assess potential direct effects of FGF21 on bone in humans. PMID:26959184

  18. 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

  19. Data Mining for Expressivity of Recombinant Protein Expression

    NASA Astrophysics Data System (ADS)

    Kira, Satoshi; Isoai, Atsushi; Yamamura, Masayuki

    We analyzed the expressivity of recombinant proteins by using data mining methods. The expression technique of recombinant protein is a key step towards elucidating the functions of genes discovered through genomic sequence projects. We have studied the productive efficiency of recombinant proteins in fission yeast, Schizosaccharomyces pombe (S.pombe), by mining the expression results. We gathered 57 proteins whose expression levels were known roughly in the host. Correlation analysis, principal component analysis and decision tree analysis were applied to these expression data. Analysis featuring codon usage and amino acid composition clarified that the amino acid composition affected to the expression levels of a recombinant protein strongly than the effect of codon usage. Furthermore, analysis of amino acid composition showed that protein solubility and the metabolism cost of amino acids correlated with a protein expressivity. Codon usage was often interesting in the field of recombinant expressions. However, our analysis found the weak correlation codon features with expressivities. These results indicated that ready-made indices of codon bias were irrelevant ones for modeling the expressivities of recombinant proteins. Our data driven approach was an easy and powerful method to improve recombinant protein expression, and this approach should be concentrated attention with the huge amount of expression data accumulating through the post-genome era.

  20. Recombinant pinoresinol/lariciresinol reductase, recombinant dirigent protein, and methods of use

    DOEpatents

    Lewis, Norman G.; Davin, Laurence B.; Dinkova-Kostova, Albena T.; Fujita, Masayuki; Gang, David R.; Sarkanen, Simo; Ford, Joshua D.

    2001-04-03

    Dirigent proteins and pinoresinol/lariciresinol reductases have been isolated, together with cDNAs encoding dirigent proteins and pinoresinol/lariciresinol reductases. Accordingly, isolated DNA sequences are provided which code for the expression of dirigent proteins and pinoresinol/lariciresinol reductases. In other aspects, replicable recombinant cloning vehicles are provided which code for dirigent proteins or pinoresinol/lariciresinol reductases or for a base sequence sufficiently complementary to at least a portion of dirigent protein or pinoresinol/lariciresinol reductase DNA or RNA to enable hybridization therewith. In yet other aspects, modified host cells are provided that have been transformed, transfected, infected and/or injected with a recombinant cloning vehicle and/or DNA sequence encoding dirigent protein or pinoresinol/lariciresinol reductase. Thus, systems and methods are provided for the recombinant expression of dirigent proteins and/or pinoresinol/lariciresinol reductases.

  1. Metal binding proteins, recombinant host cells and methods

    DOEpatents

    Summers, Anne O.; Caguiat, Jonathan J.

    2004-06-15

    The present disclosure provides artificial heavy metal binding proteins termed chelons by the inventors. These chelons bind cadmium and/or mercuric ions with relatively high affinity. Also disclosed are coding sequences, recombinant DNA molecules and recombinant host cells comprising those recombinant DNA molecules for expression of the chelon proteins. In the recombinant host cells or transgenic plants, the chelons can be used to bind heavy metals taken up from contaminated soil, groundwater or irrigation water and to concentrate and sequester those ions. Recombinant enteric bacteria can be used within the gastrointestinal tracts of animals or humans exposed to toxic metal ions such as mercury and/or cadmium, where the chelon recombinantly expressed in chosen in accordance with the ion to be rededicated. Alternatively, the chelons can be immobilized to solid supports to bind and concentrate heavy metals from a contaminated aqueous medium including biological fluids.

  2. Recombinant HT{sub m4} gene, protein and assays

    DOEpatents

    Lim, B.; Adra, C.N.; Lelias, J.M.

    1996-09-03

    The invention relates to a recombinant DNA molecule which encodes a HT{sub m4} protein, a transformed host cell which has been stably transfected with a DNA molecule which encodes a HT{sub m4} protein and a recombinant HT{sub m4} protein. The invention also relates to a method for detecting the presence of a hereditary atopy. 2 figs.

  3. Rescuing Recombinant Proteins by Sequestration Into the P22 VLP

    PubMed Central

    Patterson, Dustin P.; LaFrance, Benjamin; Douglas, Trevor

    2013-01-01

    Here we report the use of a self-assembling protein cage to sequester and solubilize recombinant proteins which are usually trafficked to insoluble inclusion bodies. Our results suggest that protein cages can be used as novel vehicles to rescue and produce soluble proteins that are otherwise difficult to obtain using conventional methods. PMID:24079011

  4. Production and secretion of recombinant proteins in Dictyostelium discoideum.

    PubMed

    Dittrich, W; Williams, K L; Slade, M B

    1994-06-01

    We have expressed useful amounts of three recombinant proteins in a new eukaryotic host/vector system. The cellular slime mold Dictyostelium discoideum efficiently secreted two recombinant products, a soluble form of the normally cell surface associated D. discoideum glycoprotein (PsA) and the heterologous protein glutathione-S-transferase (GST) from Schistosoma japonicum, while the enzyme beta-glucuronidase (GUS) from Escherichia coli was cell associated. Up to 20mg/l of recombinant PsA and 1mg/l of GST were obtained after purification from a standard, peptone based growth medium. The secretion signal peptide was correctly cleaved from the recombinant GST- and PsA-proteins and the expression of recombinant PsA was shown to be stable for at least one hundred generations in the absence of selection. PMID:7764951

  5. Challenges and opportunities in the purification of recombinant tagged proteins.

    PubMed

    Pina, Ana Sofia; Lowe, Christopher R; Roque, Ana Cecília A

    2014-01-01

    The purification of recombinant proteins by affinity chromatography is one of the most efficient strategies due to the high recovery yields and purity achieved. However, this is dependent on the availability of specific affinity adsorbents for each particular target protein. The diversity of proteins to be purified augments the complexity and number of specific affinity adsorbents needed, and therefore generic platforms for the purification of recombinant proteins are appealing strategies. This justifies why genetically encoded affinity tags became so popular for recombinant protein purification, as these systems only require specific ligands for the capture of the fusion protein through a pre-defined affinity tag tail. There is a wide range of available affinity pairs "tag-ligand" combining biological or structural affinity ligands with the respective binding tags. This review gives a general overview of the well-established "tag-ligand" systems available for fusion protein purification and also explores current unconventional strategies under development. PMID:24334194

  6. 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 ...

  7. QA-RecombineIt: a server for quality assessment and recombination of protein models

    PubMed Central

    Pawlowski, Marcin; Bogdanowicz, Albert; Bujnicki, Janusz M.

    2013-01-01

    QA-RecombineIt provides a web interface to assess the quality of protein 3D structure models and to improve the accuracy of models by merging fragments of multiple input models. QA-RecombineIt has been developed for protein modelers who are working on difficult problems, have a set of different homology models and/or de novo models (from methods such as I-TASSER or ROSETTA) and would like to obtain one consensus model that incorporates the best parts into one structure that is internally coherent. An advanced mode is also available, in which one can modify the operation of the fragment recombination algorithm by manually identifying individual fragments or entire models to recombine. Our method produces up to 100 models that are expected to be on the average more accurate than the starting models. Therefore, our server may be useful for crystallographic protein structure determination, where protein models are used for Molecular Replacement to solve the phase problem. To address the latter possibility, a special feature was added to the QA-RecombineIt server. The QA-RecombineIt server can be freely accessed at http://iimcb.genesilico.pl/qarecombineit/. PMID:23700309

  8. Structural characterization of recombinant therapeutic proteins by circular dichroism.

    PubMed

    Bertucci, Carlo; Pistolozzi, Marco; De Simone, Angela

    2011-10-01

    Most of the protein therapeutics are now produced by recombinant DNA technology. The advantages of recombinant proteins are related to their higher specificity and to their safety as exposure to animal or human diseases. However, several problems are still present in development of recombinant proteins as therapeutics, such as low bioavailability, short serum half-life, and immune response. Their successful application hinges on the protein stereochemical stability, and on the folding and the tendency to aggregate induced by purification steps and storage. All these aspects determine the failure of many potential protein therapies, and limitations in the development of the formulation. The application of multiple analytical techniques is important in order to obtain a detailed product profile and to understand how manufacturing can influence product structure and activity. Surely the protein conformation is a key aspect to be assessed, because a specific conformation is often essential for the biological function of the protein. Thus, there is a growing need to perform structural studies under the conditions in which the proteins operate, and to monitor the structural changes of the protein. Circular dichroism has been increasingly recognised as a valuable and reliable technique to get this information. In particular, examples will be here reported on the use of circular dichroism spectroscopy in the structural characterization of free and formulated recombinant proteins, looking at the prediction of the secondary structure, propensity to conformational changes, stability, and tendency to aggregate.

  9. Recombinant protein expression in Escherichia coli: advances and challenges

    PubMed Central

    Rosano, Germán L.; Ceccarelli, Eduardo A.

    2014-01-01

    Escherichia coli is one of the organisms of choice for the production of recombinant proteins. Its use as a cell factory is well-established and it has become the most popular expression platform. For this reason, there are many molecular tools and protocols at hand for the high-level production of heterologous proteins, such as a vast catalog of expression plasmids, a great number of engineered strains and many cultivation strategies. We review the different approaches for the synthesis of recombinant proteins in E. coli and discuss recent progress in this ever-growing field. PMID:24860555

  10. Making recombinant proteins in animals--different systems, different applications.

    PubMed

    Dyck, Michael K; Lacroix, Dan; Pothier, François; Sirard, Marc-André

    2003-09-01

    Transgenic animal bioreactors represent a powerful tool to address the growing need for therapeutic recombinant proteins. The ability of transgenic animals to produce complex, biologically active recombinant proteins in an efficient and economic manner has stimulated a great deal of interest in this area. As a result, genetically modified animals of several species, expressing foreign proteins in various tissues, are currently being developed. However, the generation of transgenic animals is a cumbersome process and remains problematic in the application of this technology. The advantages and disadvantages of different transgenic systems in relation to other bioreactor systems are discussed.

  11. Optimizing transient recombinant protein expression in mammalian cells.

    PubMed

    Hopkins, Ralph F; Wall, Vanessa E; Esposito, Dominic

    2012-01-01

    Transient gene expression (TGE) in mammalian cells has become a routine process for expressing recombinant proteins in cell lines such as human embryonic kidney 293 and Chinese hamster ovary cells. The rapidly increasing need for recombinant proteins requires further improvements in TGE technology. While a great deal of focus has been directed toward optimizing the secretion of antibodies and other naturally secreted targets, much less work has been done on ways to improve cytoplasmic expression in mammalian cells. The benefits to protein production in mammalian cells, particularly for eukaryotic proteins, should be very significant - glycosylation and other posttranslational modifications will likely be native or near-native, solubility and protein folding would likely improve overexpression in heterologous hosts, and expression of proteins in their proper intracellular compartments is much more likely to occur. Improvements in this area have been slow, however, due to limited development of the cell culture processes needed for low-cost, higher-throughput expression in mammalian cells, and the relatively low diversity of DNA vectors for protein production in these systems. Here, we describe how the use of recombinational cloning, coupled with improvements in transfection protocols which increase speed and lower cost, can be combined to make mammalian cells much more amenable for routine recombinant protein expression. PMID:21987258

  12. 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. PMID:21924345

  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. 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. PMID:25116083

  15. Molecular hydrogen improves obesity and diabetes by inducing hepatic FGF21 and stimulating energy metabolism in db/db mice.

    PubMed

    Kamimura, Naomi; Nishimaki, Kiyomi; Ohsawa, Ikuroh; Ohta, Shigeo

    2011-07-01

    Recent extensive studies have revealed that molecular hydrogen (H(2)) has great potential for improving oxidative stress-related diseases by inhaling H(2) gas, injecting saline with dissolved H(2), or drinking water with dissolved H(2) (H(2)-water); however, little is known about the dynamic movement of H(2) in a body. First, we show that hepatic glycogen accumulates H(2) after oral administration of H(2)-water, explaining why consumption of even a small amount of H(2) over a short span time efficiently improves various disease models. This finding was supported by an in vitro experiment in which glycogen solution maintained H(2). Next, we examined the benefit of ad libitum drinking H(2)-water to type 2 diabetes using db/db obesity model mice lacking the functional leptin receptor. Drinking H(2)-water reduced hepatic oxidative stress, and significantly alleviated fatty liver in db/db mice as well as high fat-diet-induced fatty liver in wild-type mice. Long-term drinking H(2)-water significantly controlled fat and body weights, despite no increase in consumption of diet and water. Moreover, drinking H(2)-water decreased levels of plasma glucose, insulin, and triglyceride, the effect of which on hyperglycemia was similar to diet restriction. To examine how drinking H(2)-water improves obesity and metabolic parameters at the molecular level, we examined gene-expression profiles, and found enhanced expression of a hepatic hormone, fibroblast growth factor 21 (FGF21), which functions to enhance fatty acid and glucose expenditure. Indeed, H(2) stimulated energy metabolism as measured by oxygen consumption. The present results suggest the potential benefit of H(2) in improving obesity, diabetes, and metabolic syndrome.

  16. 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.

  17. Expression, purification, and immobilization of recombinant tamavidin 2 fusion proteins.

    PubMed

    Takakura, Yoshimitsu; Oka, Naomi; Tsunashima, Masako

    2014-01-01

    Tamavidin 2 is a fungal avidin-like protein that binds biotin with high affinity. Unlike avidin or streptavidin, tamavidin 2 in soluble form is produced at high levels in Escherichia coli. In this chapter, we describe a method for immobilization and purification of recombinant proteins with the use of tamavidin 2 as an affinity tag. The protein fused to tamavidin 2 is tightly immobilized and simultaneously purified on biotinylated magnetic microbeads without loss of activity. PMID:24943317

  18. Chimeragenesis of distantly-related proteins by noncontiguous recombination.

    PubMed

    Smith, Matthew A; Romero, Philip A; Wu, Timothy; Brustad, Eric M; Arnold, Frances H

    2013-02-01

    We introduce a method for identifying elements of a protein structure that can be shuffled to make chimeric proteins from two or more homologous parents. Formulating recombination as a graph-partitioning problem allows us to identify noncontiguous segments of the sequence that should be inherited together in the progeny proteins. We demonstrate this noncontiguous recombination approach by constructing a chimera of β-glucosidases from two different kingdoms of life. Although the protein's alpha-beta barrel fold has no obvious subdomains for recombination, noncontiguous SCHEMA recombination generated a functional chimera that takes approximately half its structure from each parent. The X-ray crystal structure shows that the structural blocks that make up the chimera maintain the backbone conformations found in their respective parental structures. Although the chimera has lower β-glucosidase activity than the parent enzymes, the activity was easily recovered by directed evolution. This simple method, which does not rely on detailed atomic models, can be used to design chimeras that take structural, and functional, elements from distantly-related proteins. PMID:23225662

  19. 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

  20. Gene delivery into plant cells for recombinant protein production.

    PubMed

    Chen, Qiang; Lai, Huafang

    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

  1. 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.

  2. Tailoring recombinant protein quality by rational media design.

    PubMed

    Brühlmann, David; Jordan, Martin; Hemberger, Jürgen; Sauer, Markus; Stettler, Matthieu; Broly, Hervé

    2015-01-01

    Clinical efficacy and safety of recombinant proteins are closely associated with their structural characteristics. The major quality attributes comprise glycosylation, charge variants (oxidation, deamidation, and C- & N-terminal modifications), aggregates, low-molecular-weight species (LMW), and misincorporation of amino acids in the protein backbone. Cell culture media design has a great potential to modulate these quality attributes due to the vital role of medium in mammalian cell culture. The purpose of this review is to provide an overview of the way both classical cell culture medium components and novel supplements affect the quality attributes of recombinant therapeutic proteins expressed in mammalian hosts, allowing rational and high-throughput optimization of mammalian cell culture media. A selection of specific and/or potent inhibitors and activators of oligosaccharide processing as well as components affecting multiple quality attributes are presented. Extensive research efforts in this field show the feasibility of quality engineering through media design, allowing to significantly modulate the protein function.

  3. 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.

  4. Recovery and purification of plant-made recombinant proteins.

    PubMed

    Wilken, Lisa R; Nikolov, Zivko L

    2012-01-01

    Plants are becoming commercially acceptable for recombinant protein production for human therapeutics, vaccine antigens, industrial enzymes, and nutraceuticals. Recently, significant advances in expression, protein glycosylation, and gene-to-product development time have been achieved. Safety and regulatory concerns for open-field production systems have also been addressed by using contained systems to grow transgenic plants. However, using contained systems eliminates several advantages of open-field production, such as inexpensive upstream production and scale-up costs. Upstream technological achievements have not been matched by downstream processing advancements. In the past 10 years, the most research progress was achieved in the areas of extraction and pretreatment. Extraction conditions have been optimized for numerous proteins on a case-by-case basis leading to the development of platform-dependent approaches. Pretreatment advances were made after realizing that plant extracts and homogenates have unique compositions that require distinct conditioning prior to purification. However, scientists have relied on purification methods developed for other protein production hosts with modest investments in developing novel plant purification tools. Recently, non-chromatographic purification methods, such as aqueous two-phase partitioning and membrane filtration, have been evaluated as low-cost purification alternatives to packed-bed adsorption. This paper reviews seed, leafy, and bioreactor-based platforms, highlights strategies for the primary recovery and purification of recombinant proteins, and compares process economics between systems. Lastly, the future direction and research needs for developing economically competitive recombinant proteins with commercial potential are discussed.

  5. Influenza vaccines: from whole virus preparations to recombinant protein technology.

    PubMed

    Huber, Victor C

    2014-01-01

    Vaccination against influenza represents our most effective form of prevention. Historical approaches toward vaccine creation and production have yielded highly effective vaccines that are safe and immunogenic. Despite their effectiveness, these historical approaches do not allow for the incorporation of changes into the vaccine in a timely manner. In 2013, a recombinant protein-based vaccine that induces immunity toward the influenza virus hemagglutinin was approved for use in the USA. This vaccine represents the first approved vaccine formulation that does not require an influenza virus intermediate for production. This review presents a brief history of influenza vaccines, with insight into the potential future application of vaccines generated using recombinant technology.

  6. Recombinant protein production from stable mammalian cell lines and pools.

    PubMed

    Hacker, David L; Balasubramanian, Sowmya

    2016-06-01

    We highlight recent developments for the production of recombinant proteins from suspension-adapted mammalian cell lines. We discuss the generation of stable cell lines using transposons and lentivirus vectors (non-targeted transgene integration) and site-specific recombinases (targeted transgene integration). Each of these methods results in the generation of cell lines with protein yields that are generally superior to those achievable through classical plasmid transfection that depends on the integration of the transfected DNA by non-homologous DNA end-joining. This is the main reason why these techniques can also be used for the generation of stable cell pools, heterogenous populations of recombinant cells generated by gene delivery and genetic selection without resorting to single cell cloning. This allows the time line from gene transfer to protein production to be reduced.

  7. Recombinant protein production from stable mammalian cell lines and pools.

    PubMed

    Hacker, David L; Balasubramanian, Sowmya

    2016-06-01

    We highlight recent developments for the production of recombinant proteins from suspension-adapted mammalian cell lines. We discuss the generation of stable cell lines using transposons and lentivirus vectors (non-targeted transgene integration) and site-specific recombinases (targeted transgene integration). Each of these methods results in the generation of cell lines with protein yields that are generally superior to those achievable through classical plasmid transfection that depends on the integration of the transfected DNA by non-homologous DNA end-joining. This is the main reason why these techniques can also be used for the generation of stable cell pools, heterogenous populations of recombinant cells generated by gene delivery and genetic selection without resorting to single cell cloning. This allows the time line from gene transfer to protein production to be reduced. PMID:27322762

  8. Destabilase-lysozyme of medicinal leech. Multifunctionality of recombinant protein.

    PubMed

    Zavalova, L L; Lazarev, V N; Levitsky, S A; Yudina, T G; Baskova, I P

    2010-09-01

    Preparation and purification of a recombinant protein are described along with characteristics of its specific (for ε-(γ-Glu)-Lys and D-dimer substrates) and nonspecific (for L-γ-Glu-pNA) isopeptidase activities; the absence of peptidase function for α-(α-Glu)-Lys substrate is noted. It is shown that the protein exhibits muramidase (cell walls of Micrococcus lysodeikticus) and specific glycosidase activities. The latter was determined towards the fluorogenic substrate 4-methylumbelliferyl-tetra-N-acetyl-β-chitotetraoxide. Antimicrobial activity of recombinant destabilase-lysozyme protein (recDest-Lys) and its 11-membered amphipathic peptide was revealed towards cells of the strict anaerobic Archaean Methanosarcina barkeri, whose cell walls contain no murein. Possible mechanisms of the effect of recDest-Lys on these cells are discussed.

  9. Metabolic engineering of Escherichia coli to improve recombinant protein production.

    PubMed

    Liu, Min; Feng, Xinjun; Ding, Yamei; Zhao, Guang; Liu, Huizhou; Xian, Mo

    2015-12-01

    Escherichia coli is one of the most widely used strains for recombinant protein production. However, obstacles also exist in both academic researches and industrial applications, such as the metabolic burden, the carbon source waste, and the cells' physiological deterioration. This article reviews recent approaches for improving recombinant protein production in metabolic engineering, including workhorse selection, stress factor application, and carbon flux regulation. Selecting a suitable host is the first key point for recombinant protein production. In general, it all depends on characteristics of the strains and the target proteins. It will be triggered cells physiological deterioration when the medium is significantly different from the cell's natural environment. Coexpression of stress factors can help proteins to fold into their native conformation. Carbon flux regulation is a direct approach for redirecting more carbon flux toward the desirable pathways and products. However, some undesirable consequences are usually found in metabolic engineering, such as glucose transport inhibition, cell growth retardation, and useless metabolite accumulation. More efficient regulators and platform cell factories should be explored to meet a variety of production demands.

  10. Advantage of recombinant borrelial proteins for serodiagnosis of neuroborreliosis.

    PubMed

    Kaiser, R; Rauer, S

    1999-01-01

    Two enzyme immunoassay (EIA) systems were compared for their ability to detect Borrelia burgdorferi sensu lato specific IgG and IgM antibodies and to differentiate between symptomatic (83 patients with neuroborreliosis) and asymptomatic seropositive subjects (80 healthy controls). Antibody concentrations were determined by EIA; the antigens used were either a sonicate of B. burgdorferi or three recombinant borrelial proteins: the 14-kDa flagellin fragment, the outer surface protein C (22 kDa) and the high molecular mass protein p83 (83 kDa). In the sonicate, EIA, IgG or IgM antibodies to B. burgdorferi, or both, were detected in all patients with neuroborreliosis and in all controls. Pre-absorption of sera with Treponema phagedenis sonicate diminished the sensitivity of detection of borrelial specific IgG (IgG or IgM or both) antibodies in patients with neuroborreliosis from 80 to 57% (100 to 82%) and in the controls from 100 to 32% (100 to 37%). While being specific for B. burgdorferi, the recombinant EIAs proved to be significantly more sensitive than the sonicate EIA: IgG or IgM, or both antibodies against any of the recombinant antigens were detected in 92% of patients with neuroborreliosis and in 24% of controls. The increase in sensitivity in patients with neuroborreliosis was mostly due to the higher detection rate of IgM antibodies in the recombinant EIA (77% versus 48% in the sonicate EIA), while IgG antibodies were demonstrated with similar frequencies in both EIA systems (57% versus 60%). It was concluded that the recombinant EIAs are superior to the sonicate EIA with pre-absorption of cross-reactive antibodies in the confirmation of an acute borrelial infection and in the differentiation between symptomatic and asymptomatic infections.

  11. A Bacillus megaterium System for the Production of Recombinant Proteins and Protein Complexes.

    PubMed

    Biedendieck, Rebekka

    2016-01-01

    For many years the Gram-positive bacterium Bacillus megaterium has been used for the production and secretion of recombinant proteins. For this purpose it was systematically optimized. Plasmids with different inducible promoter systems, with different compatible origins, with small tags for protein purification and with various specific signals for protein secretion were combined with genetically improved host strains. Finally, the development of appropriate cultivation conditions for the production strains established this organism as a bacterial cell factory even for large proteins. Along with the overproduction of individual proteins the organism is now also used for the simultaneous coproduction of up to 14 recombinant proteins, multiple subsequently interacting or forming protein complexes. Some of these recombinant strains are successfully used for bioconversion or the biosynthesis of valuable components including vitamins. The titers in the g per liter scale for the intra- and extracellular recombinant protein production prove the high potential of B. megaterium for industrial applications. It is currently further enhanced for the production of recombinant proteins and multi-subunit protein complexes using directed genetic engineering approaches based on transcriptome, proteome, metabolome and fluxome data. PMID:27165321

  12. A Bacillus megaterium System for the Production of Recombinant Proteins and Protein Complexes.

    PubMed

    Biedendieck, Rebekka

    2016-01-01

    For many years the Gram-positive bacterium Bacillus megaterium has been used for the production and secretion of recombinant proteins. For this purpose it was systematically optimized. Plasmids with different inducible promoter systems, with different compatible origins, with small tags for protein purification and with various specific signals for protein secretion were combined with genetically improved host strains. Finally, the development of appropriate cultivation conditions for the production strains established this organism as a bacterial cell factory even for large proteins. Along with the overproduction of individual proteins the organism is now also used for the simultaneous coproduction of up to 14 recombinant proteins, multiple subsequently interacting or forming protein complexes. Some of these recombinant strains are successfully used for bioconversion or the biosynthesis of valuable components including vitamins. The titers in the g per liter scale for the intra- and extracellular recombinant protein production prove the high potential of B. megaterium for industrial applications. It is currently further enhanced for the production of recombinant proteins and multi-subunit protein complexes using directed genetic engineering approaches based on transcriptome, proteome, metabolome and fluxome data.

  13. A general method of protein purification for recombinant unstructured non-acidic proteins.

    PubMed

    Campos, Francisco; Guillén, Gabriel; Reyes, José L; Covarrubias, Alejandra A

    2011-11-01

    Typical late embryogenesis abundant (LEA) proteins accumulate in response to water deficit imposed by the environment or by plant developmental programs. Because of their physicochemical properties, they can be considered as hydrophilins and as a paradigm of intrinsically unstructured proteins (IUPs) in plants. To study their biophysical and biochemical characteristics large quantities of highly purified protein are required. In this work, we report a fast and simple purification method for non-acidic recombinant LEA proteins that does not need the addition of tags and that preserves their in vitro protective activity. The method is based on the enrichment of the protein of interest by boiling the bacterial protein extract, followed by a differential precipitation with trichloroacetic acid (TCA). Using this procedure we have obtained highly pure recombinant LEA proteins of groups 1, 3, and 4 and one recombinant bacterial hydrophilin. This protocol will facilitate the purification of this type of IUPs, and could be particularly useful in proteomic projects/analyses.

  14. Soluble recombinant protein production in Pseudoalteromonas haloplanktis TAC125.

    PubMed

    Giuliani, Maria; Parrilli, Ermenegilda; Sannino, Filomena; Apuzzo, Gennaro; Marino, Gennaro; Tutino, Maria Luisa

    2015-01-01

    Solubility/activity issues are often experienced when immunoglobulin fragments are produced in conventional microbial cell factories. Although several experimental approaches have been followed to solve, or at least minimize, the accumulation of the recombinant proteins into insoluble aggregates, sometimes the only alternative strategy is changing the protein production platform. In this chapter we describe the use of Antarctic bacterium Pseudoalteromonas haloplanktis TAC125 as host of choice for the production of the heavy-chain antibody fragment VHHD6.1. Combining the use of a regulated psychrophilic gene expression system with an optimized fermentation process in defined growth medium, we obtained the recombinant VHHD6.1 in fully soluble form and correctly translocated into host periplasmic space.

  15. Systems Biology of Recombinant Protein Production in Bacillus megaterium

    NASA Astrophysics Data System (ADS)

    Biedendieck, Rebekka; Bunk, Boyke; Fürch, Tobias; Franco-Lara, Ezequiel; Jahn, Martina; Jahn, Dieter

    Over the last two decades the Gram-positive bacterium Bacillus megaterium was systematically developed to a useful alternative protein production host. Multiple vector systems for high yield intra- and extracellular protein production were constructed. Strong inducible promoters were combined with DNA sequences for optimised ribosome binding sites, various leader peptides for protein export and N- as well as C-terminal affinity tags for affinity chromatographic purification of the desired protein. High cell density cultivation and recombinant protein production were successfully tested. For further system biology based control and optimisation of the production process the genomes of two B. megaterium strains were completely elucidated, DNA arrays designed, proteome, fluxome and metabolome analyses performed and all data integrated using the bioinformatics platform MEGABAC. Now, solid theoretical and experimental bases for primary modeling attempts of the production process are available.

  16. Systems biology of recombinant protein production using Bacillus megaterium.

    PubMed

    Biedendieck, Rebekka; Borgmeier, Claudia; Bunk, Boyke; Stammen, Simon; Scherling, Christian; Meinhardt, Friedhelm; Wittmann, Christoph; Jahn, Dieter

    2011-01-01

    The Gram-negative bacterium Escherichia coli is the most widely used production host for recombinant proteins in both academia and industry. The Gram-positive bacterium Bacillus megaterium represents an increasingly used alternative for high yield intra- and extracellular protein synthesis. During the past two decades, multiple tools including gene expression plasmids and production strains have been developed. Introduction of free replicating and integrative plasmids into B. megaterium is possible via protoplasts transformation or transconjugation. Using His(6)- and StrepII affinity tags, the intra- or extracellular produced proteins can easily be purified in one-step procedures. Different gene expression systems based on the xylose controlled promoter P(xylA) and various phage RNA polymerase (T7, SP6, K1E) driven systems enable B. megaterium to produce up to 1.25g of recombinant protein per liter. Biomass concentrations of up to 80g/l can be achieved by high cell density cultivations in bioreactors. Gene knockouts and gene replacements in B. megaterium are possible via an optimized gene disruption system. For a safe application in industry, sporulation and protease-deficient as well as UV-sensitive mutants are available. With the help of the recently published B. megaterium genome sequence, it is possible to characterize bottle necks in the protein production process via systems biology approaches based on transcriptome, proteome, metabolome, and fluxome data. The bioinformatical platform (Megabac, http://www.megabac.tu-bs.de) integrates obtained theoretical and experimental data. PMID:21943898

  17. Functional insights into recombinant TROSPA protein from Ixodes ricinus.

    PubMed

    Figlerowicz, Marek; Urbanowicz, Anna; Lewandowski, Dominik; Jodynis-Liebert, Jadwiga; Sadowski, Czeslaw

    2013-01-01

    Lyme disease (also called borreliosis) is a prevalent chronic disease transmitted by ticks and caused by Borrelia burgdorferi s. l. spirochete. At least one tick protein, namely TROSPA from I. scapularis, commonly occurring in the USA, was shown to be required for colonization of the vector by bacteria. Located in the tick gut, TROSPA interacts with the spirochete outer surface protein A (OspA) and initiates the tick colonization. Ixodes ricinus is a primary vector involved in B. burgdorferi s. l. transmission in most European countries. In this study, we characterized the capacities of recombinant TROSPA protein from I. ricinus to interact with OspA from different Borrelia species and to induce an immune response in animals. We also showed that the N-terminal part of TROSPA (a putative transmembrane domain) is not involved in the interaction with OspA and that reduction of the total negative charge on the TROSPA protein impaired TROSPA-OspA binding. In general, the data presented in this paper indicate that recombinant TROSPA protein retains the capacity to form a complex with OspA and induces a significant level of IgG in orally immunized rats. Thus, I. ricinus TROSPA may be considered a good candidate component for an animal vaccine against Borrelia. PMID:24204685

  18. Staphylokinase as a Plasminogen Activator Component in Recombinant Fusion Proteins

    PubMed Central

    Szarka, S. J.; Sihota, E. G.; Habibi, H. R.; Wong, S.-L.

    1999-01-01

    The plasminogen activator staphylokinase (SAK) is a promising thrombolytic agent for treatment of myocardial infarction. It can specifically stimulate the thrombolysis of both erythrocyte-rich and platelet-rich clots. However, SAK lacks fibrin-binding and thrombin inhibitor activities, two functions which would supplement and potentially improve its thrombolytic potency. Creating a recombinant fusion protein is one approach for combining protein domains with complementary functions. To evaluate SAK for use in a translational fusion protein, both N- and C-terminal fusions to SAK were constructed by using hirudin as a fusion partner. Recombinant fusion proteins were secreted from Bacillus subtilis and purified from culture supernatants. The rate of plasminogen activation by SAK was not altered by the presence of an additional N- or C-terminal protein sequence. However, cleavage at N-terminal lysines within SAK rendered the N-terminal fusion unstable in the presence of plasmin. The results of site-directed mutagenesis of lysine 10 and lysine 11 in SAK suggested that a plasmin-resistant variant cannot be created without interfering with the plasmin processing necessary for activation of SAK. Although putative plasmin cleavage sites are located at the C-terminal end of SAK at lysine 135 and lysine 136, these sites were resistant to plasmin cleavage in vitro. Therefore, C-terminal fusions represent stable configurations for developing improved thrombolytic agents based on SAK as the plasminogen activator component. PMID:9925575

  19. Efficient recovery of recombinant proteins from cereal endosperm is affected by interaction with endogenous storage proteins.

    PubMed

    Peters, Jenny; Sabalza, Maite; Ramessar, Koreen; Christou, Paul; Capell, Teresa; Stöger, Eva; Arcalís, Elsa

    2013-10-01

    Cereal seeds are versatile platforms for the production of recombinant proteins because they provide a stable environment for protein accumulation. Endogenous seed storage proteins, however, include several prolamin-type polypeptides that aggregate and crosslink via intermolecular disulfide bridges, which could potentially interact with multimeric recombinant proteins such as antibodies, which assemble in the same manner. We investigated this possibility by sequentially extracting a human antibody expressed in maize endosperm, followed by precipitation in vitro with zein. We provide evidence that a significant proportion of the antibody pool interacts with zein and therefore cannot be extracted using non-reducing buffers. Immunolocalization experiments demonstrated that antibodies targeted for secretion were instead retained within zein bodies because of such covalent interactions. Our findings suggest that the production of soluble recombinant antibodies in maize could be enhanced by eliminating or minimizing interactions with endogenous storage proteins.

  20. Expression and export: recombinant protein production systems for Aspergillus.

    PubMed

    Fleissner, André; Dersch, Petra

    2010-07-01

    Several Aspergillus species, in particular Aspergillus niger and Aspergillus oryzae, are widely used as protein production hosts in various biotechnological applications. In order to improve the expression and secretion of recombinant proteins in these filamentous fungi, several novel genetic engineering strategies have been developed in recent years. This review describes state-of-the-art genetic manipulation technologies used for strain improvement, as well as recent advances in designing the most appropriate engineering strategy for a particular protein production process. Furthermore, current developments in identifying bottlenecks in the protein production and secretion pathways are described and novel approaches to overcome these limitations are introduced. An appropriate combination of expression vectors and optimized host strains will provide cell factories customized for each production process and expand the great potential of Aspergilli as biotechnology workhorses to more complex multi-step industrial applications.

  1. Recombinant proteins in newly developed foods: identification of allergenic activity.

    PubMed

    Lehrer, S B; Reese, G

    1997-01-01

    A number of agricultural crops are being modified for various purposes using recombinant DNA technology. Since transferred genes may code for proteins that are ordinarily not present, there is concern about the potential allergenicity of these new varieties. The safety evaluation of transgenic foods is relatively easy when the allergenicity of the gene source is known. Recombinant allergens in genetically engineered or altered foods can be identified using traditional immunological assays such as RAST or ELISA inhibition or immunoblotting procedures. Our recent studies of two corn proteins (10 kD and HSZ) used to alter grain amino acid composition and of transgenic soybeans with an altered fatty acid profile are examples of this approach. Both 10 kD and HSZ did not bind IgE antibodies from sera of corn-reactive subjects by immunoblotting. Studies of wild-type and transgenic soybeans with high oleic acidic content by RAST inhibition and immunoblotting with pooled sera of soy-allergic individuals demonstrated no difference in the allergen content of both extracts. In contrast to these studies, a recent investigation by Nordlee et al. (1996) of transgenic soybeans which expressed a methionine/cysteine-rich protein from Brazil nuts identified this protein as a major Brazil nut allergen. These studies indicate that, when the gene source is from a known allergen or if the recipient contains allergens, it is possible to determine whether the allergen content of the transgenic line is altered relative to the nontransgenic varieties.

  2. Recent advances in production of recombinant spider silk proteins.

    PubMed

    Chung, Hannah; Kim, Tae Yong; Lee, Sang Yup

    2012-12-01

    Spider silk has been drawing much attention as a great biomaterial having many applications in biotechnology and biomedicine owing to its several desired material characteristics such as outstanding strength, toughness, and elasticity as well as biodegradability and biocompatibility. With various applications foreseeable in industry, there has been much effort to produce recombinant spider silk protein in large amounts. However, owing to the difficulties in its production using spiders, alternative host systems and engineering methods have been investigated to develop suitable production systems that can efficiently produce spider silk protein. Here, we review recent advances in production of spider silk proteins in various heterologous host systems with focus given on the development of metabolic and cellular engineering strategies. PMID:22521455

  3. Generation of recombinant antibody fragments for membrane protein crystallization.

    PubMed

    Mir, Syed H; Escher, Claudia; Kao, Wei-Chun; Birth, Dominic; Wirth, Christophe; Hunte, Carola

    2015-01-01

    Membrane proteins are challenging targets for crystallization and structure determination by X-ray crystallography. Hurdles can be overcome by antibody-mediated crystallization. More than 25 unique structures of membrane protein:antibody complexes have already been determined. In the majority of cases, hybridoma-derived antibody fragments either in Fab or Fv fragment format were employed for these complexes. We will briefly introduce the background and current status of the strategy and describe in detail the current protocols of well-established methods for the immunization, the selection, and the characterization of antibodies, as well as the cloning, the production, and the purification of recombinant antibodies useful for structural analysis of membrane proteins.

  4. Recombinant human bone morphogenetic protein 2 in lateral ridge augmentation.

    PubMed

    Mehanna, Robert; Koo, Samuel; Kim, David M

    2013-01-01

    This case report describes the augmentation of severe lateral ridge defects in the maxilla and mandible using recombinant human bone morphogenetic protein 2 (rhBMP-2) on an absorbable collagen sponge (ACS). The surgical technique used tenting screws and a membrane to maintain space for the ACS. After 7 months of healing, the ridge width increased from 1 to 2 mm to 6 to 9 mm, thus allowing successful placement of dental implants. De novo bone formation through use of the surgical technique for space maintenance of rhBMP-2/ACS was demonstrated without the need for additional particulate bone grafting. PMID:23342352

  5. Expression of recombinant green fluorescent protein in Bacillus methanolicus.

    PubMed

    Nilasari, Dewi; Dover, Nir; Rech, Sabine; Komives, Claire

    2012-01-01

    Microbial biocatalysts are used in a wide range of industries to produce large scale quantities of proteins, amino acids, and commodity chemicals. While the majority of these processes use glucose or other low-cost sugars as the substrate, Bacillus methanolicus is one example of a biocatalyst that has shown sustained growth on methanol as a carbon source at elevated temperature (50-53°C optimum) resulting in reduced feed and utility costs. Specifically, the complete chemical process enabled by this approach takes methane from natural gas, and following a low-cost conversion to methanol, can be used for the production of high value products. In this study, production of recombinant green fluorescent protein (GFPuv) by B. methanolicus is explored. A plasmid was constructed that incorporates the methanol dehydrogenase (mdh) promoter of B. methanolicus MGA3 together with the GFPuv gene. The plasmid, pNW33N, was shown to be effective for expression in other Bacillus strains, although not previously in B. methanolicus. A published electroporation protocol for transformation of B. methanolicus was modified to result in expression of GFP using plasmid pNW33N-mdh-GFPuv (pNmG). Transformation was confirmed by both agarose gel electrophoresis and by observation of green fluorescence under UV light exposure. The mass yield of cells and protein were measured in shake flask experiments. The optimum concentration of methanol for protein production was found to be at 200 mM. Higher concentrations than 200 mM resulted in slightly higher biomass production but lower amounts of recombinant protein. PMID:22275315

  6. Thermal adaptability of Kluyveromyces marxianus in recombinant protein production

    PubMed Central

    2013-01-01

    Background Kluyveromyces marxianus combines the ease of genetic manipulation and fermentation with the ability to efficiently secrete high molecular weight proteins, performing eukaryotic post-translational modifications. It is able to grow efficiently in a wide range of temperatures. The secretion performances were analyzed in the host K. marxianus L3 in the range between 5°C and 40°C by means of 3 different reporter proteins, since temperature appears a key parameter for production and secretion of recombinant proteins. Results The recombinant strains were able to grow up to 40°C and, along the tested temperature interval (5-40°C), the specific growth rates (μ) were generally lower as compared to those of the untransformed strain. Biomass yields were slightly affected by temperature, with the highest values reached at 15°C and 30°C. The secretion of the endogenous β-fructofuranosidase, used as an internal control, was efficient in the range of the tested temperature, as evaluated by assaying the enzyme activity in the culture supernatants. The endogenous β-fructofuranosidase production was temperature dependent, with the highest yield at 30°C. The heterologous proteins HSA, GAA and Sod1p were all successfully produced and secreted between 5°C and 40°C, albeit each one presented a different optimal production temperature (15, 40, 5-30°C for HSA, GAA and Sod1p, respectively). Conclusions K. marxianus L3 has been identified as a promising and flexible cell factory. In a sole host, the optimization of growth temperatures for the efficient secretion of each individual protein can be carried out over a wide range of temperatures. PMID:23587421

  7. Accelerated protein engineering for chemical biotechnology via homologous recombination.

    PubMed

    Nordwald, Erik M; Garst, Andrew; Gill, Ryan T; Kaar, Joel L

    2013-12-01

    Protein engineering has traditionally relied on random mutagenesis strategies to generate diverse libraries, which require high-throughput screening or selection methods to identify rare variants. Alternatively, approaches to semi-rational library construction can be used to minimize the screening load and enhance the efficiency by which improved mutants may be identified. Such methods are typically limited to characterization of relatively few variants due to the difficulties in generating large rational libraries. New tools from synthetic biology, namely multiplexed DNA synthesis and homologous recombination, provide a promising avenue to rapidly construct large, rational libraries. These technologies also enable incorporation of synthetically encoded features that permit efficient characterization of the fitness of each mutant. Extension of these tools to protein library design could complement rational protein design cycles in an effort to more systematically search complex fitness landscapes. The highly parallelized nature with which such libraries can be generated also has the potential to expand directed protein evolution from single protein targets to protein networks whose concerted activities are required for the biological function of interest. PMID:23540421

  8. Accelerated protein engineering for chemical biotechnology via homologous recombination.

    PubMed

    Nordwald, Erik M; Garst, Andrew; Gill, Ryan T; Kaar, Joel L

    2013-12-01

    Protein engineering has traditionally relied on random mutagenesis strategies to generate diverse libraries, which require high-throughput screening or selection methods to identify rare variants. Alternatively, approaches to semi-rational library construction can be used to minimize the screening load and enhance the efficiency by which improved mutants may be identified. Such methods are typically limited to characterization of relatively few variants due to the difficulties in generating large rational libraries. New tools from synthetic biology, namely multiplexed DNA synthesis and homologous recombination, provide a promising avenue to rapidly construct large, rational libraries. These technologies also enable incorporation of synthetically encoded features that permit efficient characterization of the fitness of each mutant. Extension of these tools to protein library design could complement rational protein design cycles in an effort to more systematically search complex fitness landscapes. The highly parallelized nature with which such libraries can be generated also has the potential to expand directed protein evolution from single protein targets to protein networks whose concerted activities are required for the biological function of interest.

  9. Utilizing protein-lean coproducts from corn containing recombinant pharmaceutical proteins for ethanol production.

    PubMed

    Paraman, Ilankovan; Moeller, Lorena; Scott, M Paul; Wang, Kan; Glatz, Charles E; Johnson, Lawrence A

    2010-10-13

    Protein-lean fractions of corn (maize) containing recombinant (r) pharmaceutical proteins were evaluated as a potential feedstock to produce fuel ethanol. The levels of residual r-proteins in the coproduct, distillers dry grains with solubles (DDGS), were determined. Transgenic corn lines containing recombinant green fluorescence protein (r-GFP) and a recombinant subunit vaccine of Escherichia coli enterotoxin (r-LTB), primarily expressed in endosperm, and another two corn lines containing recombinant human collagen (r-CIα1) and r-GFP, primarily expressed in germ, were used as model systems. The kernels were either ground and used for fermentation or dry fractionated to recover germ-rich fractions prior to grinding for fermentation. The finished beers of whole ground kernels and r-protein-spent endosperm solids contained 127-139 and 138-155 g/L ethanol concentrations, respectively. The ethanol levels did not differ among transgenic and normal corn feedstocks, indicating the residual r-proteins did not negatively affect ethanol production. r-Protein extraction and germ removal also did not negatively affect fermentation of the remaining mass. Most r-proteins were inactivated during the mashing process used to prepare corn for fermentation. No functionally active r-GFP or r-LTB proteins were found after fermentation of the r-protein-spent solids; however, a small quantity of residual r-CIα1 was detected in DDGS, indicating that the safety of DDGS produced from transgenic grain for r-protein production needs to be evaluated for each event. Protease treatment during fermentation completely hydrolyzed the residual r-CIα1, and no residual r-proteins were detectable in DDGS.

  10. [Eukaryotic Expression and Immunogenic Research of Recombination Ebola Virus Membrane Protein Gp-Fc].

    PubMed

    Zhang, Xiaoguang; Yang, Ren; Wang, Jiao; Wang, Xuan; Hou, Mieling; An, Lina; Zhu, Ying; Cao, Yuxi; Zeng, Yi

    2016-01-01

    We used 293 cells to express the recombinant membrane protein of the Ebola virus. Then, the immunogenicity of the recombinant protein was studied by immunized BALB/c mice. According to the codon use frequency of humans, the gene encoding the extracellular domain of the Ebola virus membrane protein was optimized, synthesized, and inserted into the eukaryotic expression plasmid pXG-Fc to construct the human IgG Fc and Ebola GP fusion protein expression plasmid pXG-modGP-Fc. To achieve expression, the fusion protein expression vector was transfected into high-density 293 cells using transient transfection technology. The recombinant protein was purified by protein A affinity chromatography. BALB/c mice were immunized with the purified fusion protein, and serum antibody titers evaluated by an indirect enzyme-linked immunosorbent assay (ELISA). Purification and analyses of the protein revealed that the eukaryotic expression vector could express the recombinant protein GP-Fc effectively, and that the recombinant protein in the supernatant of the cell culture was present as a dimer. After immunization with the purified recombinant protein, a high titer of antigen-specific IgG could be detected in the serum of immunized mice by indirect ELISA, showing that the recombinant protein had good immunogenicity. These data suggest that we obtained a recombinant protein with good immunogenicity. Our study is the basis for development of a vaccine against the Ebola virus and for screening of monoclonal antibodies.

  11. Production of Recombinant Proteins in the Chloroplast of the Green Alga Chlamydomonas reinhardtii.

    PubMed

    Guzmán-Zapata, Daniel; Macedo-Osorio, Karla Soledad; Almaraz-Delgado, Alma Lorena; Durán-Figueroa, Noé; Badillo-Corona, Jesus Agustín

    2016-01-01

    Chloroplast transformation in the green algae Chlamydomonas reinhardtii can be used for the production of valuable recombinant proteins. Here, we describe chloroplast transformation of C. reinhardtii followed by protein detection. Genes of interest integrate stably by homologous recombination into the chloroplast genome following introduction by particle bombardment. Genes are inherited and expressed in lines recovered after selection in the presence of an antibiotic. Recombinant proteins can be detected by conventional techniques like immunoblotting and purified from liquid cultures.

  12. Production of Recombinant Proteins in the Chloroplast of the Green Alga Chlamydomonas reinhardtii.

    PubMed

    Guzmán-Zapata, Daniel; Macedo-Osorio, Karla Soledad; Almaraz-Delgado, Alma Lorena; Durán-Figueroa, Noé; Badillo-Corona, Jesus Agustín

    2016-01-01

    Chloroplast transformation in the green algae Chlamydomonas reinhardtii can be used for the production of valuable recombinant proteins. Here, we describe chloroplast transformation of C. reinhardtii followed by protein detection. Genes of interest integrate stably by homologous recombination into the chloroplast genome following introduction by particle bombardment. Genes are inherited and expressed in lines recovered after selection in the presence of an antibiotic. Recombinant proteins can be detected by conventional techniques like immunoblotting and purified from liquid cultures. PMID:26614282

  13. Production of a recombinant industrial protein using barley cell cultures.

    PubMed

    Ritala, A; Wahlström, E H; Holkeri, H; Hafren, A; Mäkeläinen, K; Baez, J; Mäkinen, K; Nuutila, A M

    2008-06-01

    The use of recombinant DNA-based protein production using genetically modified plants could provide a reproducible, consistent quality, safe, animal-component free, origin-traceable, and cost-effective source for industrial proteins required in large amounts (1000s of metric tons) and at low cost (below US$100/Kg). The aim of this work was to demonstrate the feasibility of using barley suspension cell culture to support timely testing of the genetic constructs and early product characterization to detect for example post-translational modifications within the industrial protein caused by the selected recombinant system. For this study the human Collagen I alpha 1 (CIa1) chain gene encoding the complete helical region of CIa1 optimized for monocot expression was fused to its N- and C-terminal telopeptide and to a bacteriophage T4 fibritin foldon peptide encoding sequences. The CIa1 accumulation was targeted to the endoplasmic reticulum (ER) by fusing the CIa1 gene to an ER-directing signal peptide sequence and an ER retention signal HDEL. The construct containing the CIa1 gene was then introduced into immature barley half embryos or barley cells by particle bombardment. Transgenic barley cells resulting from these transformations were grown as suspension cultures in flasks and in a Wave bioreactor producing CIa1 similar to CIa1 purified from the yeast Pichia pastoris based on Western blotting, pepsin resistance, and mass spectroscopy analysis. The barley cell culture derived-CIa1 intracellular accumulation levels ranged from 2 to 9 microg/l illustrating the need for further process improvement in order to use this technology to supply material for product development activities.

  14. Determination of enamel protein synthesized by recombined mouse molar tooth germs in organ culture.

    PubMed

    Baba, T; Terashima, T; Oida, S; Sasaki, S

    1996-02-01

    Epithelial-mesenchymal interaction is a prerequisite for tooth morphogenesis. To study this interaction, inner enamel epithelium and dental papilla mesenchyme of molar tooth germs from a 16.5-day mouse embryo were dissociated enzymatically and cultured alone or after recombination. Characteristic matrix protein synthesized and secreted by recombined tooth germ was determined quantitatively by enzyme-linked immunosorbent assay. The protein was detected in the culture of recombined tooth germ but not of dissociated enamel epithelium alone. The amount of enamel protein increased until 8 days in culture. Morphological differentiation of the recombined epithelial rudiment into ameloblasts and enamel protein production were confirmed.

  15. Molecular beacon aptamers for direct and universal quantitation of recombinant proteins from cell lysates.

    PubMed

    Tan, Xiaohong; Chen, Weijun; Lu, Shun; Zhu, Zhi; Chen, Tao; Zhu, Guizhi; You, Mingxu; Tan, Weihong

    2012-10-01

    Western blot, enzyme linked immunosorbent assay (ELISA), and fluorescent fusion proteins are currently the most common methods for detecting recombinant proteins. However, the former two are cumbersome and time-consuming, and the latter method may interfere with the trafficking and function of the fused recombinant proteins. We report here a rapid, inexpensive, and simple approach to detect and quantify recombinant proteins using an anti-His-tag molecular beacon aptamer (HMBA). We demonstrated the technique by detection and quantitation of expressed recombinant proteins directly from E. coli cell lysate. The amount of expressed P78-His was determined to be 1.49 μg from the 20 μg cell lysate proteins. To the best of our knowledge, this is the first example directly measuring the concentration and expression yield of recombinant proteins from cell lysate, and the entire procedure required only 5 min.

  16. Immunogenicity of recombinant feline infectious peritonitis virus spike protein in mice and kittens.

    PubMed

    Vennema, H; de Groot, R J; Harbour, D A; Dalderup, M; Gruffydd-Jones, T; Horzinek, M C; Spaan, W J

    1990-01-01

    The gene encoding the fusogenic spike protein of the coronavirus causing feline infectious peritonitis (FIPV) was recombined into the genome of vaccinia virus, strain WR. The recombinant induced spike protein specific, in vitro neutralizing antibodies in mice. When kittens were immunized with the recombinant, low titers of neutralizing antibodies were obtained. After challenge with FIPV, these animals succumbed earlier than the vWR-immunized control group ("early death syndrome").

  17. Cereal seed storage protein synthesis: fundamental processes for recombinant protein production in cereal grains.

    PubMed

    Kawakatsu, Taiji; Takaiwa, Fumio

    2010-12-01

    Cereal seeds provide an ideal production platform for high-value products such as pharmaceuticals and industrial materials because seeds have ample and stable space for the deposition of recombinant products without loss of activity at room. Seed storage proteins (SSPs) are predominantly synthesized and stably accumulated in maturing endosperm tissue. Therefore, understanding the molecular mechanisms regulating SSP expression and accumulation is expected to provide valuable information for producing higher amounts of recombinant products. SSP levels are regulated by several steps at the transcriptional (promoters, transcription factors), translational and post-translational levels (modification, processing trafficking, and deposition). Our objective is to develop a seed production platform capable of producing very high yields of recombinant product. Towards this goal, we review here the individual regulatory steps controlling SSP synthesis and accumulation.

  18. Annexin proteins PP4 and PP4-X. Comparative characterization of biological activities of placental and recombinant proteins.

    PubMed Central

    Römisch, J; Grote, M; Weithmann, K U; Heimburger, N; Amann, E

    1990-01-01

    The human placental proteins PP4 and PP4-X, belonging to the annexin protein family, were expressed in Escherichia coli at high yield. The proteins were purified to homogeneity. The physicochemical parameters of the recombinant proteins were determined and compared with those of their natural placental counterparts. Except for a minor change in the pI, the proteins appeared to be indistinguishable by several criteria. Both recombinant PP4 and recombinant PP4-X were biologically active in a thromboplastin inhibition test and in a phospholipase A2 inhibition test. Images Fig. 2. Fig. 3. Fig. 4. PMID:2148260

  19. Strain engineering to prevent norleucine incorporation during recombinant protein production in Escherichia coli.

    PubMed

    Veeravalli, Karthik; Laird, Michael W; Fedesco, Mark; Zhang, Yu; Yu, X Christopher

    2015-01-01

    Incorporation of norleucine in place of methionine residues during recombinant protein production in Escherichia coli is well known. Continuous feeding of methionine is commonly used in E. coli recombinant protein production processes to prevent norleucine incorporation. Although this strategy is effective in preventing norleucine incorporation, there are several disadvantages associated with continuous feeding. Continuous feeding increases the operational complexity and the overall cost of the fermentation process. In addition, the continuous feed leads to undesirable dilution of the fermentation medium possibly resulting in lower cell densities and recombinant protein yields. In this work, the genomes of three E. coli hosts were engineered by introducing chromosomal mutations that result in methionine overproduction in the cell. The recombinant protein purified from the fermentations using the methionine overproducing hosts had no norleucine incorporation. Furthermore, these studies demonstrated that the fermentations using one of the methionine overproducing hosts exhibited comparable fermentation performance as the control host in three different recombinant protein production processes. PMID:25315437

  20. Self-assembly studies of native and recombinant fibrous proteins

    NASA Astrophysics Data System (ADS)

    Wilson, Donna Lucille

    unmodified silk protein. A sequence block from the native primary structure of collagen IV, as well as sequences of selected collagen-modifying enzymes, were manipulated through recombinant DNA technology. Collagen IV is found primarily in the basement membrane of cells and typically characterized by a loose "chicken-mesh" network of individual molecules assembled via their end regions. (Abstract shortened by UMI.)

  1. A role for homologous recombination proteins in cell cycle regulation

    PubMed Central

    Kostyrko, Kaja; Bosshard, Sandra; Urban, Zuzanna; Mermod, Nicolas

    2015-01-01

    Eukaryotic cells respond to DNA breaks, especially double-stranded breaks (DSBs), by activating the DNA damage response (DDR), which encompasses DNA repair and cell cycle checkpoint signaling. The DNA damage signal is transmitted to the checkpoint machinery by a network of specialized DNA damage-recognizing and signal-transducing molecules. However, recent evidence suggests that DNA repair proteins themselves may also directly contribute to the checkpoint control. Here, we investigated the role of homologous recombination (HR) proteins in normal cell cycle regulation in the absence of exogenous DNA damage. For this purpose, we used Chinese Hamster Ovary (CHO) cells expressing the Fluorescent ubiquitination-based cell cycle indicators (Fucci). Systematic siRNA-mediated knockdown of HR genes in these cells demonstrated that the lack of several of these factors alters cell cycle distribution, albeit differentially. The knock-down of MDC1, Rad51 and Brca1 caused the cells to arrest in the G2 phase, suggesting that they may be required for the G2/M transition. In contrast, inhibition of the other HR factors, including several Rad51 paralogs and Rad50, led to the arrest in the G1/G0 phase. Moreover, reduced expression of Rad51B, Rad51C, CtIP and Rad50 induced entry into a quiescent G0-like phase. In conclusion, the lack of many HR factors may lead to cell cycle checkpoint activation, even in the absence of exogenous DNA damage, indicating that these proteins may play an essential role both in DNA repair and checkpoint signaling. PMID:26125600

  2. Comparative Evaluation of Recombinant Protein Production in Different Biofactories: The Green Perspective

    PubMed Central

    Capaldi, Stefano

    2014-01-01

    In recent years, the production of recombinant pharmaceutical proteins in heterologous systems has increased significantly. Most applications involve complex proteins and glycoproteins that are difficult to produce, thus promoting the development and improvement of a wide range of production platforms. No individual system is optimal for the production of all recombinant proteins, so the diversity of platforms based on plants offers a significant advantage. Here, we discuss the production of four recombinant pharmaceutical proteins using different platforms, highlighting from these examples the unique advantages of plant-based systems over traditional fermenter-based expression platforms. PMID:24745008

  3. Thermostable tag (TST) protein expression system: engineering thermotolerant recombinant proteins and vaccines.

    PubMed

    Luke, Jeremy M; Carnes, Aaron E; Sun, Ping; Hodgson, Clague P; Waugh, David S; Williams, James A

    2011-02-10

    Methods to increase temperature stability of vaccines and adjuvants are needed to reduce dependence on cold chain storage. We report herein creation and application of pVEX expression vectors to improve vaccine and adjuvant manufacture and thermostability. Defined media fermentation yields of 6g/L thermostable toll-like receptor 5 agonist flagellin were obtained using an IPTG inducible pVEX-flagellin expression vector. Alternative pVEX vectors encoding Pyrococcus furiosus maltodextrin-binding protein (pfMBP) as a fusion partner improved Influenza hemagglutinin antigen vaccine solubility and thermostability. A pfMBP hemagglutinin HA2 domain fusion protein was a potent immunogen. Manufacturing processes that combined up to 5 g/L defined media fermentation yields with rapid, selective, thermostable pfMBP fusion protein purification were developed. The pVEX pfMBP-based thermostable tag (TST) platform is a generic protein engineering approach to enable high yield manufacture of thermostable recombinant protein vaccine components.

  4. 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

  5. Sustained release emphasizing recombinant human bone morphogenetic protein-2.

    PubMed

    Hollinger; Uludag; Winn

    1998-05-01

    Bone homeostasis is a dynamic process involving a myriad of cells and substrates modulated by regulatory signals such as hormones, growth and differentiating factors. When this environment is damaged, the regenerative sequalae follows a programmed pattern, and the capacity for successful recovery is often dependent on the extent of the injury. Many bony deficits that are excessively traumatic will not result in complete recovery and require therapeutic intervention(s) such as autografting or grafting from banked bone. However, for numerous reasons, an unacceptably high rate of failure is associated with these conventional therapies. Thus, alternative approaches are under investigation. A class of osteogenic regulatory molecules, the bone morphogenetic proteins (BMPs), have been isolated, cloned and characterized as potent supplements to augment bone regeneration. Optimizing a therapeutic application for BMPs may be dependent upon localized sustained release which in kind relies on a safe and well characterized carrier system. This review will discuss the current status of BMPs in bone regeneration and specifically will present the potential for a clinical therapeutic role of recombinant human BMP-2 sustained release carrier systems. PMID:10837631

  6. Purification by reflux electrophoresis of whey proteins and of a recombinant protein expressed in Dictyostelium discoideum.

    PubMed

    Corthals, G L; Collins, B M; Mabbutt, B C; Williams, K L; Gooley, A A

    1997-06-27

    Protein purification that combines the use of molecular mass exclusion membranes with electrophoresis is particularly powerful as it uses properties inherent to both techniques. The use of membranes allows efficient processing and is easily scaled up, while electrophoresis permits high resolution separation under mild conditions. The Gradiflow apparatus combines these two technologies as it uses polyacrylamide membranes to influence electrokinetic separations. The reflux electrophoresis process consists of a series of cycles incorporating a forward phase and a reverse phase. The forward phase involves collection of a target protein that passes through a separation membrane before trailing proteins in the same solution. The forward phase is repeated following clearance of the membrane in the reverse phase by reversing the current. We have devised a strategy to establish optimal reflux separation parameters, where membranes are chosen for a particular operating range and protein transfer is monitored at different pH values. In addition, forward and reverse phase times are determined during this process. Two examples of the reflux method are described. In the first case, we described the purification strategy for proteins from a complex mixture which contains proteins of higher electrophoretic mobility than the target protein. This is a two-step procedure, where first proteins of higher mobility than the target protein are removed from the solution by a series of reflux cycles, so that the target protein remains as the leading fraction. In the second step the target protein is collected, as it has become the leading fraction of the remaining proteins. In the second example we report the development of a reflux strategy which allowed a rapid one-step preparative purification of a recombinant protein, expressed in Dictyostelium discoideum. These strategies demonstrate that the Gradiflow is amenable to a wide range of applications, as the protein of interest is not

  7. Western Blot Detection of Human Anti-Chikungunya Virus Antibody with Recombinant Envelope 2 Protein.

    PubMed

    Yang, Zhaoshou; Lee, Jihoo; Ahn, Hye-Jin; Chong, Chom-Kyu; Dias, Ronaldo F; Nam, Ho-Woo

    2016-04-01

    Chikungunya virus (CHIKV), a tropical pathogen, has re-emerged and has massive outbreaks abruptly all over the world. Containing many dominant epitopes, the envelope E2 protein of CHIKV has been explored for the vaccination or diagnosis. In the present study, the antigenicity of a recombinant expressed intrinsically disorder domain (IUD) of E2 was tested for the detection of the antibody against CHIKV through western blot method. The gene of the IUD of E2 was inserted into 2 different vectors and expressed as recombinant GST-E2 and recombinant MBP-E2 fusion protein, respectively. Two kinds of fusion proteins were tested with 30 CHIKV patient sera and 30 normal sera, respectively. Both proteins were detected by 25 patients sera (83.3%) and 1 normal serum (3.3%). This test showed a relatively high sensitivity and very high specificity of the recombinant E2 proteins to be used as diagnostic antigens against CHIKV infection. PMID:27180586

  8. Western Blot Detection of Human Anti-Chikungunya Virus Antibody with Recombinant Envelope 2 Protein

    PubMed Central

    Yang, Zhaoshou; Lee, Jihoo; Ahn, Hye-Jin; Chong, Chom-Kyu; Dias, Ronaldo F.; Nam, Ho-Woo

    2016-01-01

    Chikungunya virus (CHIKV), a tropical pathogen, has re-emerged and has massive outbreaks abruptly all over the world. Containing many dominant epitopes, the envelope E2 protein of CHIKV has been explored for the vaccination or diagnosis. In the present study, the antigenicity of a recombinant expressed intrinsically disorder domain (IUD) of E2 was tested for the detection of the antibody against CHIKV through western blot method. The gene of the IUD of E2 was inserted into 2 different vectors and expressed as recombinant GST-E2 and recombinant MBP-E2 fusion protein, respectively. Two kinds of fusion proteins were tested with 30 CHIKV patient sera and 30 normal sera, respectively. Both proteins were detected by 25 patients sera (83.3%) and 1 normal serum (3.3%). This test showed a relatively high sensitivity and very high specificity of the recombinant E2 proteins to be used as diagnostic antigens against CHIKV infection. PMID:27180586

  9. 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.

  10. Production of antigens in Chlamydomonas reinhardtii: green microalgae as a novel source of recombinant proteins.

    PubMed

    Fuhrmann, Markus

    2004-01-01

    Recombinant small-scale proteins are produced in a number of systems, from bacteria like Escherichia coli, through lower eukaryotes like baker's yeast, up to mammalian cell cultures. However, the need for safe and cheap sources of large amounts of recombinant proteins for different purposes, including material sciences, diagnostics, and, of course, medical therapy, has forced the development of alternative production systems. Green microalgae are cheap and easily grown and offer a high protein content, which would seem to make them ideal hosts for the large-scale sustainable production of recombinant proteins in the future. In selected species, recombinant DNA can be introduced into the genomes of the nucleus, the chloroplast, and even the mitochondria, and thus the system offers both prokaryotic (chloroplast, mitochondria) and eukaryotic translation systems for a tailored expression of virtually any protein.

  11. Expression, delivery and function of insecticidal proteins expressed by recombinant baculoviruses

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Since the development of methods for inserting and expressing genes in baculoviruses, a line of research has focused on developing recombinant baculoviruses that express insecticidal peptides and proteins. These recombinant viruses have been engineered with the goal of improving their pesticidal po...

  12. 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. PMID:25975624

  13. Fasting-induced G0/G1 switch gene 2 and FGF21 expression in the liver are under regulation of adipose tissue derived fatty acids

    PubMed Central

    Jaeger, Doris; Schoiswohl, Gabriele; Hofer, Peter; Schreiber, Renate; Schweiger, Martina; Eichmann, Thomas O.; Pollak, Nina M.; Poecher, Nadja; Grabner, Gernot F.; Zierler, Kathrin A.; Eder, Sandra; Kolb, Dagmar; Radner, Franz P.W.; Preiss-Landl, Karina; Lass, Achim; Zechner, Rudolf; Kershaw, Erin E.; Haemmerle, Guenter

    2015-01-01

    Background & Aims Adipose tissue (AT)-derived fatty acids (FAs) are utilized for hepatic triacylglycerol (TG) generation upon fasting. However, their potential impact as signaling molecules is not established. Herein we examined the role of exogenous AT-derived FAs in the regulation of hepatic gene expression by investigating mice with a defect in AT-derived FA supply to the liver. Methods Plasma FA levels, tissue TG hydrolytic activities and lipid content were determined in mice lacking the lipase co-activator comparative gene identification-58 (CGI-58) selectively in AT (CGI-58-ATko) applying standard protocols. Hepatic expression of lipases, FA oxidative genes, transcription factors, ER stress markers, hormones and cytokines were determined by qRT-PCR, Western blotting and ELISA. Results Impaired AT-derived FA supply upon fasting of CGI-58-ATko mice causes a marked defect in liver PPARα-signaling and nuclear CREBH translocation. This severely reduced the expression of respective target genes such as the ATGL inhibitor G0/G1 switch gene-2 (G0S2) and the endocrine metabolic regulator FGF21. These changes could be reversed by lipid administration and raising plasma FA levels. Impaired AT-lipolysis failed to induce hepatic G0S2 expression in fasted CGI-58-ATko mice leading to enhanced ATGL-mediated TG-breakdown strongly reducing hepatic TG deposition. On high fat diet, impaired AT-lipolysis counteracts hepatic TG accumulation and liver stress linked to improved systemic insulin sensitivity. Conclusions AT-derived FAs are a critical regulator of hepatic fasting gene expression required for the induction of G0S2-expression in the liver to control hepatic TG-breakdown. Interfering with AT-lipolysis or hepatic G0S2 expression represents an effective strategy for the treatment of hepatic steatosis. PMID:25733154

  14. Expression and purification of recombinant proteins in Escherichia coli tagged with the metal-binding protein CusF.

    PubMed

    Cantu-Bustos, J Enrique; Vargas-Cortez, Teresa; Morones-Ramirez, Jose Ruben; Balderas-Renteria, Isaias; Galbraith, David W; McEvoy, Megan M; Zarate, Xristo

    2016-05-01

    Production of recombinant proteins in Escherichia coli has been improved considerably through the use of fusion proteins, because they increase protein solubility and facilitate purification via affinity chromatography. In this article, we propose the use of CusF as a new fusion partner for expression and purification of recombinant proteins in E. coli. Using a cell-free protein expression system, based on the E. coli S30 extract, Green Fluorescent Protein (GFP) was expressed with a series of different N-terminal tags, immobilized on self-assembled protein microarrays, and its fluorescence quantified. GFP tagged with CusF showed the highest fluorescence intensity, and this was greater than the intensities from corresponding GFP constructs that contained MBP or GST tags. Analysis of protein production in vivo showed that CusF produces large amounts of soluble protein with low levels of inclusion bodies. Furthermore, fusion proteins can be exported to the cellular periplasm, if CusF contains the signal sequence. Taking advantage of its ability to bind copper ions, recombinant proteins can be purified with readily available IMAC resins charged with this metal ion, producing pure proteins after purification and tag removal. We therefore recommend the use of CusF as a viable alternative to MBP or GST as a fusion protein/affinity tag for the production of soluble recombinant proteins in E. coli. PMID:26805756

  15. Expression and purification of recombinant proteins in Escherichia coli tagged with the metal-binding protein CusF.

    PubMed

    Cantu-Bustos, J Enrique; Vargas-Cortez, Teresa; Morones-Ramirez, Jose Ruben; Balderas-Renteria, Isaias; Galbraith, David W; McEvoy, Megan M; Zarate, Xristo

    2016-05-01

    Production of recombinant proteins in Escherichia coli has been improved considerably through the use of fusion proteins, because they increase protein solubility and facilitate purification via affinity chromatography. In this article, we propose the use of CusF as a new fusion partner for expression and purification of recombinant proteins in E. coli. Using a cell-free protein expression system, based on the E. coli S30 extract, Green Fluorescent Protein (GFP) was expressed with a series of different N-terminal tags, immobilized on self-assembled protein microarrays, and its fluorescence quantified. GFP tagged with CusF showed the highest fluorescence intensity, and this was greater than the intensities from corresponding GFP constructs that contained MBP or GST tags. Analysis of protein production in vivo showed that CusF produces large amounts of soluble protein with low levels of inclusion bodies. Furthermore, fusion proteins can be exported to the cellular periplasm, if CusF contains the signal sequence. Taking advantage of its ability to bind copper ions, recombinant proteins can be purified with readily available IMAC resins charged with this metal ion, producing pure proteins after purification and tag removal. We therefore recommend the use of CusF as a viable alternative to MBP or GST as a fusion protein/affinity tag for the production of soluble recombinant proteins in E. coli.

  16. Physical studies of conformational plasticity in a recombinant prion protein.

    PubMed

    Zhang, H; Stockel, J; Mehlhorn, I; Groth, D; Baldwin, M A; Prusiner, S B; James, T L; Cohen, F E

    1997-03-25

    PrP(Sc) is known to be the major, if not the only, component of the infectious prion. Limited proteolysis of PrP(Sc) produces an N-terminally truncated polypeptide of about 142 residues, designated PrP 27-30. Recently, a recombinant protein (rPrP) of 142 residues corresponding to the Syrian hamster PrP 27-30 was expressed in Escherichia coli and purified (Mehlhorn et al., 1996). rPrP has been refolded into both alpha-helical and beta-sheet structures as well as various intermediates in aqueous buffers. The beta-sheet state and two pH-dependent alpha-helical states were characterized by CD and NMR. The alpha-helical conformation occurred only after the formation of an intramolecular disulfide bond, whereas the beta-sheet form was accessible either with or without the disulfide. Of the different alpha-helical forms studied, only those refolded in the pH range 5-8 were substantially soluble at physiological pH, exhibiting similar conformations and monomeric analytical sedimentation profiles throughout the above pH range. Furthermore, refolded alpha-rPrP showed NMR chemical shift dispersion typical of proteins with native conformations, although 2D NMR indicated large segments of conformational flexibility. It displayed a cooperative thermal denaturation transition; at elevated temperatures, it converted rapidly and irreversibly to the thermodynamically more stable beta-sheet form. Unfolding of alpha-rPrP by GdnHCl revealed a two-phase transition with a relatively stable folding intermediate at 2 M GdnHCl. The deltaG values were estimated to be 1.9 +/- 0.4 kcal/mol for the first phase and 6.5 +/- 1.2 kcal/mol for the second, consistent with a folding core surrounded by significant segments of flexible conformation. By NMR, alpha-rPrP(acid) isolated at pH 2 without refolding exhibited heterogeneous line widths, consistent with an acid-denatured molten globular state. We conclude that to the extent that rPrP constitutes a relevant folding domain of PrP(C), the various

  17. 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.

  18. 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.

  19. Recominant Pinoresino-Lariciresinol Reductase, Recombinant Dirigent Protein And Methods Of Use

    DOEpatents

    Lewis, Norman G.; Davin, Laurence B.; Dinkova-Kostova, Albena T.; Fujita, Masayuki , Gang; David R. , Sarkanen; Simo , Ford; Joshua D.

    2003-10-21

    Dirigent proteins and pinoresinol/lariciresinol reductases have been isolated, together with cDNAs encoding dirigent proteins and pinoresinol/lariciresinol reductases. Accordingly, isolated DNA sequences are provided from source species Forsythia intermedia, Thuja plicata, Tsuga heterophylla, Eucommia ulmoides, Linum usitatissimum, and Schisandra chinensis, which code for the expression of dirigent proteins and pinoresinol/lariciresinol reductases. In other aspects, replicable recombinant cloning vehicles are provided which code for dirigent proteins or pinoresinol/lariciresinol reductases or for a base sequence sufficiently complementary to at least a portion of dirigent protein or pinoresinol/lariciresinol reductase DNA or RNA to enable hybridization therewith. In yet other aspects, modified host cells are provided that have been transformed, transfected, infected and/or injected with a recombinant cloning vehicle and/or DNA sequence encoding dirigent protein or pinoresinol/lariciresinol reductase. Thus, systems and methods are provided for the recombinant expression of dirigent proteins and/or pinoresinol/lariciresinol reductases.

  20. Refolding techniques for recovering biologically active recombinant proteins from inclusion bodies.

    PubMed

    Yamaguchi, Hiroshi; Miyazaki, Masaya

    2014-02-20

    Biologically active proteins are useful for studying the biological functions of genes and for the development of therapeutic drugs and biomaterials in a biotechnology industry. Overexpression of recombinant proteins in bacteria, such as Escherichia coli, often results in the formation of inclusion bodies, which are protein aggregates with non-native conformations. As inclusion bodies contain relatively pure and intact proteins, protein refolding is an important process to obtain active recombinant proteins from inclusion bodies. However, conventional refolding methods, such as dialysis and dilution, are time consuming and, often, recovered yields of active proteins are low, and a trial-and-error process is required to achieve success. Recently, several approaches have been reported to refold these aggregated proteins into an active form. The strategies largely aim at reducing protein aggregation during the refolding procedure. This review focuses on protein refolding techniques using chemical additives and laminar flow in microfluidic chips for the efficient recovery of active proteins from inclusion bodies.

  1. Recombination hotspot activity of hypervariable minisatellite DNA requires minisatellite DNA binding proteins.

    PubMed

    Wahls, W P; Moore, P D

    1998-01-01

    Hypervariable minisatellite DNA repeats are found at tens of thousands of loci in the mammalian genome. These sequences stimulate homologous recombination in mammalian cells [Cell 60:95-103]. To test the hypothesis that protein-DNA interaction is required for hotspot function in vivo, we determined whether a second protein binding nearby could abolish hotspot activity. Intermolecular recombination between pairs of plasmid substrates was measured in the presence or absence of the cis-acting recombination hotspot and in the presence or absence of the second trans-acting DNA binding protein. Minisatellite DNA had hotspot activity in two cell lines, but lacked hotspot activity in two closely related cell lines expressing a site-specific helicase that bound to DNA adjacent to the hotspot. Suppression of hotspot function occurred for both replicating and non-replicating recombination substrates. These results indicate that hotspot activity in vivo requires site occupancy by minisatellite DNA binding proteins. PMID:9776980

  2. Five RecA-like proteins of Schizosaccharomyces pombe are involved in meiotic recombination.

    PubMed Central

    Grishchuk, A L; Kohli, J

    2003-01-01

    The genome of Schizosaccharomyces pombe contains five genes that code for proteins with sequence similarity to the Escherichia coli recombination protein RecA: rad51+, rhp55+, rhp57+, rlp1+, and dmc1+. We analyzed the effect of deletion of each of these genes on meiotic recombination and viability of spores. Meiotic recombination levels were different from wild type in all recA-related mutants in several genetic intervals, suggesting that all five RecA homologs of S. pombe are required for normal levels of meiotic recombination. Spore viability was reduced in rad51, rhp55, and rhp57 mutants, but not in rlp1 and dmc1. It is argued that reduction of crossover is not the only cause for the observed reduction of spore viability. Analysis of double and triple mutants revealed that Rad51 and Dmc1 play major and partially overlapping roles in meiotic recombination, while Rhp55, Rhp57, and Rlp1 play accessory roles. Remarkably, deletion of Rlp1 decreases the frequency of intergenic recombination (crossovers), but increases intragenic recombination (gene conversion). On the basis of our results, we present a model for the involvement of five RecA-like proteins of S. pombe in meiotic recombination and discuss their respective roles. PMID:14668362

  3. [Preparation and characterization of the recombinant protein containing immunomimetic peptide of benzo[a]pyrene].

    PubMed

    Apal'ko, S V; Lunin, V G; Filipenko, M L; Matveeva, V A; Liashchuk, A M; Lavrova, N V; Sherina, E A; Aver'ianov, A V; Kostianko, M V; Glushkov, A N

    2011-01-01

    Two recombinant plasmids were constructed. The first plasmid contained the hybrid gene composed of immunomimetic peptide of benzo[a]pyrene, of the protein pIII of bacteriophage M13 and of cellulose binding domain encoding sequences. The second plasmid contained the hybrid gene composed of the signal peptide of the protein pIII of bacteriophage M13, of immunomimetic peptide of benzo[a]pyrene, of the protein pill of bacteriophage M13 and of cellulose binding domain sequences. The obtained recombinant plasmids were used in expression of chimeric protein containing immunomimetic peptide ofbenzo[a]pyrene based on strain E. coli M15. The lack of the recombinant protein expression using first plasmid was demonstrated. In the same time, it was shown that accumulation of recombinant protein contained immunomimetic peptide with signal peptide of the protein pIIIl of bacteriophage was present. This chimeric protein was produced in "mature" (without signal peptide) and "unprocessing" (with signal peptide) forms. Using the Western-blot analysis, it was shown that the "mature" form only specifically bound to the B2 monoclonal antibody against benzo[a]pyrene. Thus, we expressed, purified, and characterized the recombinant protein containing immunomimetic peptide of benzo[a]pyrene.

  4. Fibroblast Growth Factor 21 Protects against Atherosclerosis via Fine-Tuning the Multiorgan Crosstalk

    PubMed Central

    Jin, Leigang; Lin, Zhuofeng

    2016-01-01

    Fibroblast growth factor 21 (FGF21) is a metabolic hormone with pleiotropic effects on energy metabolism and insulin sensitivity. Besides its antiobese and antidiabetic activity, FGF21 also possesses the protective effects against atherosclerosis. Circulating levels of FGF21 are elevated in patients with atherosclerosis, macrovascular and microvascular complications of diabetes, possibly due to a compensatory upregulation. In apolipoprotein E-deficient mice, formation of atherosclerotic plaques is exacerbated by genetic depletion of FGF21, but is attenuated upon replenishment with recombinant FGF21. However, the blood vessel is not the direct target of FGF21, and the antiatherosclerotic activity of FGF21 is attributed to its actions in adipose tissues and liver. In adipocytes, FGF21 promotes secretion of adiponectin, which in turn acts directly on blood vessels to reduce endothelial dysfunction, inhibit proliferation of smooth muscle cells and block conversion of macrophages to foam cells. Furthermore, FGF21 suppresses cholesterol biosynthesis and attenuates hypercholesterolemia by inhibiting the transcription factor sterol regulatory element-binding protein-2 in hepatocytes. The effects of FGF21 on elevation of adiponectin and reduction of hypercholesterolemia are also observed in a phase-1b clinical trial in patients with obesity and diabetes. Therefore, FGF21 exerts its protection against atherosclerosis by fine-tuning the interorgan crosstalk between liver, brain, adipose tissue, and blood vessels. PMID:26912152

  5. Systems and methods for the secretion of recombinant proteins in gram negative bacteria

    DOEpatents

    Withers, III, Sydnor T.; Dominguez, Miguel A; DeLisa, Matthew P.; Haitjema, Charles H.

    2016-08-09

    Disclosed herein are systems and methods for producing recombinant proteins utilizing mutant E. coli strains containing expression vectors carrying nucleic acids encoding the proteins, and secretory signal sequences to direct the secretion of the proteins to the culture medium. Host cells transformed with the expression vectors are also provided.

  6. Unexpected Deposition Patterns of Recombinant Proteins in Post-Endoplasmic Reticulum Compartments of Wheat Endosperm1

    PubMed Central

    Arcalis, Elsa; Marcel, Sylvain; Altmann, Friedrich; Kolarich, Daniel; Drakakaki, Georgia; Fischer, Rainer; Christou, Paul; Stoger, Eva

    2004-01-01

    Protein transport within cereal endosperm cells is complicated by the abundance of endoplasmic reticulum (ER)-derived and vacuolar protein bodies. For wheat storage proteins, two major transport routes run from the ER to the vacuole, one bypassing and one passing through the Golgi. Proteins traveling along each route converge at the vacuole and form aggregates. To determine the impact of this trafficking system on the fate of recombinant proteins expressed in wheat endosperm, we used confocal and electron microscopy to investigate the fate of three recombinant proteins containing different targeting information. KDEL-tagged recombinant human serum albumin, which is retrieved to the ER lumen in leaf cells, was deposited in prolamin aggregates within the vacuole of endosperm cells, most likely following the bulk of endogenous glutenins. Recombinant fungal phytase, a glycoprotein designed for secretion, was delivered to the same compartment, with no trace of the molecule in the apoplast. Glycan analysis revealed that this protein had passed through the Golgi. The localization of human serum albumin and phytase was compared to that of recombinant legumin, which contains structural targeting information directing it to the vacuole. Uniquely, legumin accumulated in the globulin inclusion bodies at the periphery of the prolamin bodies, suggesting a different mode of transport and/or aggregation. Our results demonstrate that recombinant proteins are deposited in an unexpected pattern within wheat endosperm cells, probably because of the unique storage properties of this tissue. Our data also confirm that recombinant proteins are invaluable tools for the analysis of protein trafficking in cereals. PMID:15489278

  7. 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. PMID:26021569

  8. Improving recombinant protein production in the Chlamydomonas reinhardtii chloroplast using vivid Verde Fluorescent Protein as a reporter.

    PubMed

    Braun-Galleani, Stephanie; Baganz, Frank; Purton, Saul

    2015-08-01

    Microalgae have potential as platforms for the synthesis of high-value recombinant proteins due to their many beneficial attributes including ease of cultivation, lack of pathogenic agents, and low-cost downstream processing. However, current recombinant protein levels are low compared to other microbial platforms and stable insertion of transgenes is available in only a few microalgal species. We have explored different strategies aimed at increasing growth rate and recombinant protein production in the Chlamydomonas reinhardtii chloroplast. A novel fluorescent protein (vivid Verde Fluorescent Protein, VFP) was expressed under the control of the native atpA promoter/5'UTR element. VFP levels were detected by western blotting, with increased protein levels observed when co-expressed with a gene encoding the Escherichia coli Spy chaperone. We used these transformant lines to study the effect of temperature, light and media on recombinant protein production and cell growth. VFP levels and fluorescence, assessed by flow cytometry, allowed a determination of improved cultivation conditions as 30°C under mixotrophic mode. These conditions were tested for the accumulation of an antimicrobial endolysin (Cpl-1) of potential commercial interest, observing that the outcome obtained for VFP could not be easily replicated for Cpl-1. This study suggests that recombinant protein expression is product-specific and needs to be optimized individually. PMID:26098300

  9. Improving recombinant protein production in the Chlamydomonas reinhardtii chloroplast using vivid Verde Fluorescent Protein as a reporter

    PubMed Central

    Baganz, Frank; Purton, Saul

    2015-01-01

    Abstract Microalgae have potential as platforms for the synthesis of high‐value recombinant proteins due to their many beneficial attributes including ease of cultivation, lack of pathogenic agents, and low‐cost downstream processing. However, current recombinant protein levels are low compared to other microbial platforms and stable insertion of transgenes is available in only a few microalgal species. We have explored different strategies aimed at increasing growth rate and recombinant protein production in the Chlamydomonas reinhardtii chloroplast. A novel fluorescent protein (vivid Verde Fluorescent Protein, VFP) was expressed under the control of the native atpA promoter/5'UTR element. VFP levels were detected by western blotting, with increased protein levels observed when co‐expressed with a gene encoding the Escherichia coli Spy chaperone. We used these transformant lines to study the effect of temperature, light and media on recombinant protein production and cell growth. VFP levels and fluorescence, assessed by flow cytometry, allowed a determination of improved cultivation conditions as 30°C under mixotrophic mode. These conditions were tested for the accumulation of an antimicrobial endolysin (Cpl‐1) of potential commercial interest, observing that the outcome obtained for VFP could not be easily replicated for Cpl‐1. This study suggests that recombinant protein expression is product‐specific and needs to be optimized individually. PMID:26098300

  10. Quantitative measurement of bitagged recombinant proteins using an immunometric assay: application to an anti-substance P recombinant antibody.

    PubMed

    Boquet, D; Créminon, C; Clément, G; Frobert, Y; Nevers, M C; Essono, S; Grassi, J

    2000-09-10

    We have developed two different immunometric assays to directly quantify both the total and the active fractions of a recombinant antibody (single chain fragment variable, or ScFv) as obtained in a crude extract from an Escherichia coli expression system. For total determination, the assay is based on the simultaneous recognition of two different peptide Tag sequences (Ha-Tag and Myc-Tag) at each of the N- and C-terminal extremities of the recombinant protein. A monoclonal antibody (mAb 12CA5, directed against Ha-Tag), coated on microtiter plates, is used for capture, and the mAb 9E10 (directed against Myc-Tag), labeled with acetylcholinesterase (AChE, EC 3.1.1.7), acts as tracer. In parallel, for the determination of the active fraction, the capture is performed using microtiter plates coated with the antigen, while solid-phase-immobilized ScFv is measured using the same 9E10 tracer mAb. A synthetic peptide in which the two Tag sequences were joined was used as a standard, thus avoiding the laborious purification of a recombinant protein as reference. The method was applied to the direct measurement, in periplasmic extracts, of the total and active fractions of an ScFv produced at different induction temperatures.

  11. Expression and purification of recombinant proteins in Escherichia coli tagged with a small metal-binding protein from Nitrosomonas europaea.

    PubMed

    Vargas-Cortez, Teresa; Morones-Ramirez, Jose Ruben; Balderas-Renteria, Isaias; Zarate, Xristo

    2016-02-01

    Escherichia coli is still the preferred organism for large-scale production of recombinant proteins. The use of fusion proteins has helped considerably in enhancing the solubility of heterologous proteins and their purification with affinity chromatography. Here, the use of a small metal-binding protein (SmbP) from Nitrosomonas europaea is described as a new fusion protein for protein expression and purification in E. coli. Fluorescent proteins tagged at the N-terminal with SmbP showed high levels of solubility, compared with those of maltose-binding protein and glutathione S-transferase, and low formation of inclusion bodies. Using commercially available IMAC resins charged with Ni(II), highly pure recombinant proteins were obtained after just one chromatography step. Proteins may be purified from the periplasm of E. coli if SmbP contains the signal sequence at the N-terminal. After removal of the SmbP tag from the protein of interest, high-yields are obtained since SmbP is a protein of just 9.9 kDa. The results here obtained suggest that SmbP is a good alternative as a fusion protein/affinity tag for the production of soluble recombinant proteins in E. coli.

  12. Purification of recombinant protein by cold-coacervation of fusion constructs incorporating resilin-inspired polypeptides.

    PubMed

    Lyons, Russell E; Elvin, Christopher M; Taylor, Karin; Lekieffre, Nicolas; Ramshaw, John A M

    2012-12-01

    Polypeptides containing between 4 and 32 repeats of a resilin-inspired sequence AQTPSSYGAP, derived from the mosquito Anopheles gambiae, have been used as tags on recombinant fusion proteins. These repeating polypeptides were inspired by the repeating structures that are found in resilins and sequence-related proteins from various insects. Unexpectedly, an aqueous solution of a recombinant resilin protein displays an upper critical solution temperature (cold-coacervation) when held on ice, leading to a separation into a protein rich phase, typically exceeding 200 mg/mL, and a protein-poor phase. We show that purification of recombinant proteins by cold-coacervation can be performed when engineered as a fusion partner to a resilin-inspired repeat sequence. In this study, we demonstrate the process by the recombinant expression and purification of enhanced Green fluorescent protein (EGFP) in E. coli. This facile purification system can produce high purity, concentrated protein solutions without the need for affinity chromatography or other time-consuming or expensive purification steps, and that it can be used with other bulk purification steps such as low concentration ammonium sulfate precipitation. Protein purification by cold-coacervation also minimizes the exposure of the target protein to enhanced proteolysis at higher temperature.

  13. Assessment of contraceptive vaccines based on recombinant mouse sperm protein PH20.

    PubMed

    Hardy, Christopher M; Clydesdale, Gavin; Mobbs, Karen J; Pekin, Jenny; Lloyd, Megan L; Sweet, Clive; Shellam, Geoffrey R; Lawson, Malcolm A

    2004-03-01

    Mouse PH20 (mPH20), the mouse homologue to guinea pig hyaluronidase protein PH20 (gpPH20), was used to produce contraceptive vaccines that target both sexes of mice. Previously, immunization with a female gamete antigen (the zona pellucida subunit 3 protein) delivered in a recombinant murine cytomegalovirus (MCMV), or as a purified recombinant protein, has been shown to induce infertility in female mice. There is evidence, however, that sperm protein antigens could provide broader contraceptive coverage by affecting both males and females, and the most promising has been gpPH20 when tested in a guinea pig model. Mice were therefore either inoculated with a recombinant MCMV expressing mPH20 or immunized directly with purified recombinant mPH20 protein fused to maltose-binding protein. Mice treated with either vaccine formulation developed serum antibodies that cross-reacted to a protein band of 55 kDa corresponding to mPH20 in Western blots of mouse sperm. However, there was no significant reduction in the fertility of males or females compared with control animals with either formulation. We conclude from our data that recombinant mPH20 is not a useful antigen for inclusion in immunocontraceptive vaccines that target mice.

  14. A novel method for the purification of low soluble recombinant C-type lectin proteins.

    PubMed

    Yin, Chunhui; Jia, Ying; Garcia, Carlos A

    2012-08-31

    Snake venoms contain a complex mixture of many biological molecules including proteins. The purification of recombinant proteins is a key step in studying their function and structure with affinity chromatography as the common method used in their purification. In bacterial expression systems, hydrophobic recombinant proteins are usually precipitated into inclusion bodies, and contaminants are typically associated with tagged proteins after purification. The purpose of this study was to develop a procedure to purify hydrophobic recombinant proteins without an affinity tag. Snake venom mature C-type lectin-like proteins (CLPs) with a tag were cloned, expressed, and purified by repeated sonication and wash steps. The effects of the signal peptide on the expression and solubility of the recombinant protein were investigated. The CLPs in washed inclusion bodies were solubilized and refolded by dialysis. The CLPs without a tag were successfully purified with a yield 38 times higher than the traditional method, and inhibited blood platelet aggregation with an IC(50) of 100.57 μM in whole blood. This novel procedure is a rapid, and inexpensive method to purify functional recombinant hydrophobic CLPs from snake venoms useful in the development of drug therapies. PMID:22867876

  15. Molecular design of performance proteins with repetitive sequences: recombinant flagelliform spider silk as basis for biomaterials.

    PubMed

    Vendrely, Charlotte; Ackerschott, Christian; Römer, Lin; Scheibel, Thomas

    2008-01-01

    Most performance proteins responsible for the mechanical stability of cells and organisms reveal highly repetitive sequences. Mimicking such performance proteins is of high interest for the design of nanostructured biomaterials. In this article, flagelliform silk is exemplary introduced to describe a general principle for designing genes of repetitive performance proteins for recombinant expression in Escherichia coli . In the first step, repeating amino acid sequence motifs are reversely transcripted into DNA cassettes, which can in a second step be seamlessly ligated, yielding a designed gene. Recombinant expression thereof leads to proteins mimicking the natural ones. The recombinant proteins can be assembled into nanostructured materials in a controlled manner, allowing their use in several applications. PMID:19031057

  16. Photosynthetic biomanufacturing in green algae; production of recombinant proteins for industrial, nutritional, and medical uses.

    PubMed

    Rasala, Beth A; Mayfield, Stephen P

    2015-03-01

    Recombinant proteins are widely used for industrial, nutritional, and medical applications. Green microalgae have attracted considerable attention recently as a biomanufacturing platform for the production of recombinant proteins for a number of reasons. These photosynthetic eukaryotic microorganisms are safe, scalable, easy to genetically modify through transformation, mutagenesis, or breeding, and inexpensive to grow. Many microalgae species are genetically transformable, but the green alga Chlamydomonas reinhardtii is the most widely used host for recombinant protein expression. An extensive suite of molecular genetic tools has been developed for C. reinhardtii over the last 25 years, including a fully sequenced genome, well-established methods for transformation, mutagenesis and breeding, and transformation vectors for high levels of recombinant protein accumulation and secretion. Here, we review recent successes in the development of C. reinhardtii as a biomanufacturing host for recombinant proteins, including antibodies and immunotoxins, hormones, industrial enzymes, an orally-active colostral protein for gastrointestinal health, and subunit vaccines. In addition, we review the biomanufacturing potential of other green algae from the genera Dunaliella and Chlorella.

  17. Capillary gel electrophoresis for the quantification and purity determination of recombinant proteins in inclusion bodies.

    PubMed

    Espinosa-de la Garza, Carlos E; Perdomo-Abúndez, Francisco C; Campos-García, Víctor R; Pérez, Néstor O; Flores-Ortiz, Luis F; Medina-Rivero, Emilio

    2013-09-01

    In this work, a high-resolution CGE method for quantification and purity determination of recombinant proteins was developed, involving a single-component inclusion bodies (IBs) solubilization solution. Different recombinant proteins expressed as IBs were used to show method capabilities, using recombinant interferon-β 1b as the model protein for method validation. Method linearity was verified in the range from 0.05 to 0.40 mg/mL and a determination coefficient (r(2) ) of 0.99 was obtained. The LOQs and LODs were 0.018 and 0.006 mg/mL, respectively. RSD for protein content repeatability test was 2.29%. In addition, RSD for protein purity repeatability test was 4.24%. Method accuracy was higher than 90%. Specificity was confirmed, as the method was able to separate recombinant interferon-β 1b monomer from other aggregates and impurities. Sample content and purity was demonstrated to be stable for up to 48 h. Overall, this method is suitable for the analysis of recombinant proteins in IBs according to the attributes established on the International Conference for Harmonization guidelines.

  18. Production of recombinant protein in Escherichia coli cultured in extract from waste product alga, Ulva lactuca.

    PubMed

    Rechtin, Tammy M; Hurst, Matthew; Potts, Tom; Hestekin, Jamie; Beitle, Robert; McLaughlin, John; May, Peter

    2014-01-01

    This study examined the potential for waste product alga, Ulva lactuca, to serve as a media component for recombinant protein production in Escherichia coli. To facilitate this investigation, U. lactuca harvested from Jamaica Bay was dried, and nutrients acid extracted for use as a growth media. The E. coli cell line BL21(DE3) was used to assess the effects on growth and production of recombinant green fluorescent protein (GFP). This study showed that media composed of acid extracts without further nutrient addition maintained E. coli growth and recombinant protein production. Extracts made from dried algae lots less than six-months-old were able to produce two-fold more GFP protein than traditional Lysogeny Broth media. PMID:24799463

  19. Transphosphorylation of E. coli proteins during production of recombinant protein kinases provides a robust system to characterize kinase specificity

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Protein kinase specificity is of fundamental importance to pathway regulation and signal transduction. Here, we report a convenient system to monitor the activity and specificity of recombinant protein kinases expressed in E.coli. We apply this to the study of the cytoplasmic domain of the plant rec...

  20. 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

    2014-09-01

    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.

  1. Overexpression and Enzymatic Assessment of Antigenic Fragments of Hyaluronidase Recombinant Protein From Streptococcus pyogenes

    PubMed Central

    Sadoogh Abbasian, Shabnam; Ghaznavi Rad, Ehsanollah; Akbari, Neda; Zolfaghari, Mohammad Reza; pakzad, Iraj; Abtahi, Hamid

    2014-01-01

    Background: Hyaluronidase catalyzes the hydrolysis of hyaluronan polymers to N-acetyl-D-glucosamine and D-glucuronic acid. This enzyme is a dimer of identical subunits. Hyaluronidase has different pharmaceutical and medical applications. Previously, we produced a recombinant hyaluronidase antigenic fragment of Streptococcus pyogenes. Objectives: This study aimed to improve the protein production and purity of hyaluronidase recombinant protein from S. pyogenes. In addition, the enzymatic activity of this protein was investigated. Materials and Methods: The expression of hyaluronidase antigenic fragments was optimized using IPTG concentration, time of induction, temperature, culture, and absorbance of 0.6-0.8-1 at 600 nm. Afterwards, the expressed proteins were purified and the enzymatic activity was assessed by turbid metric method. Results: Data indicated that maximum protein is produced in OD = 0.8, 0.5 mM Isopropyl β-D-1-thiogalactopyranoside (IPTG), 37ºC, NB 1.5x, without glucose, incubated for overnight. The enzymatic activity of the recombinant protein was similar to the commercial form of hyaluronidase. Conclusions: The results showed that an antigenic fragment of the recombinant hyaluronidase protein from S. pyogenes has a considerable enzymatic activity. It can be suggested to use it for medical purposes. In addition, applications of bioinformatics software would facilitate the production of a smaller protein with same antigenic properties and enzymatic activity. PMID:25789122

  2. Impact of Profiling Technologies in the Understanding of Recombinant Protein Production

    NASA Astrophysics Data System (ADS)

    Vijayendran, Chandran; Flaschel, Erwin

    Since expression profiling methods have been available in a high throughput fashion, the implication of these technologies in the field of biotechnology has increased dramatically. Microarray technology is one such unique and efficient methodology for simultaneous exploration of expression levels of numerous genes. Likewise, two-dimensional gel electrophoresis or multidimensional liquid chromatography coupled with mass spectrometry are extensively utilised for studying expression levels of numerous proteins. In the field of biotechnology these highly parallel analytical methods have paved the way to study and understand various biological phenomena depending on expression patterns. The next phenomenological level is represented by the metabolome and the (metabolic) fluxome. However, this chapter reviews gene and protein profiling and their impact on understanding recombinant protein production. We focus on the computational methods utilised for the analyses of data obtained from these profiling technologies as well as prominent results focusing on recombinant protein expression with Escherichia coli. Owing to the knowledge accumulated with respect to cellular signals triggered during recombinant protein production, this field is on the way to design strategies for developing improved processes. Both gene and protein profiling have exhibited a handful of functional categories to concentrate on in order to identify target genes and proteins, respectively, involved in the signalling network with major impact on recombinant protein production.

  3. The effects of protein solubility on the RNA Integrity Number (RIN) for recombinant Escherichia coli

    PubMed Central

    Baig, Faraz; Harcum, Sarah W.

    2013-01-01

    High quality, intact messenger RNA (mRNA) is required for DNA microarray and reverse transcriptase polymerase chain reaction analysis and is generally obtained from total RNA isolations. The most widely recognized measure of RNA integrity is the RNA Integrity Number (RIN) obtained from the Agilent Bioanalyzer, as it provides sizing, quantification, and quality control measures. This work describes comparisons of the RIN values obtained for recombinant E. coli. Uninduced recombinant E. coli cultures were examined, as well as induced cultures that produced either a soluble or insoluble recombinant protein. The uninduced cultures and the induced cultures producing soluble protein had higher RIN values than the induced cultures producing insoluble protein. These lower RIN values for E. coli producing the insoluble protein indicate that cellular degradation of the ribosomal RNA species is the likely cause of the lower RIN values. As the use of DNA microarrays and other gene expression tools increase in usage in the industrial recombinant protein production community, these results suggest the need for further studies to determine acceptable RIN ranges for gene expression analysis and effects of various culture conditions on RIN values for recombinant E. coli. PMID:24151430

  4. A Rapid Method for Determining the Concentration of Recombinant Protein Secreted from Pichia pastoris

    NASA Astrophysics Data System (ADS)

    Sun, L. W.; Zhao, Y.; Niu, L. P.; Jiang, R.; Song, Y.; Feng, H.; feng, K.; Qi, C.

    2011-02-01

    Pichia secretive expression system is one of powerful eukaryotic expression systems in genetic engineering, which is especially suitable for industrial utilization. Because of the low concentration of the target protein in initial experiment, the methods and conditions for expression of the target protein should be optimized according to the protein yield repetitively. It is necessary to set up a rapid, simple and convenient analysis method for protein expression levels instead of the generally used method such as ultrafiltration, purification, dialysis, lyophilization and so on. In this paper, acetone precipitation method was chosen to concentrate the recombinant protein firstly after comparing with four different protein precipitation methods systematically, and then the protein was analyzed by SDS-Polyacrylamide Gel Electrophoresis. The recombinant protein was determined with the feature of protein band by the Automated Image Capture and 1-D Analysis Software directly. With this method, the optimized expression conditions of basic fibroblast growth factor secreted from pichia were obtained, which is as the same as using traditional methods. Hence, a convenient tool to determine the optimized conditions for the expression of recombinant proteins in Pichia was established.

  5. A Library of Plasmodium vivax Recombinant Merozoite Proteins Reveals New Vaccine Candidates and Protein-Protein Interactions

    PubMed Central

    Hostetler, Jessica B.; Sharma, Sumana; Bartholdson, S. Josefin; Wright, Gavin J.; Fairhurst, Rick M.; Rayner, Julian C.

    2015-01-01

    suggesting that the proteins are natively folded and functional. This screen also identified two novel protein-protein interactions, between P12 and PVX_110945, and between MSP3.10 and MSP7.1, the latter of which was confirmed by surface plasmon resonance. Conclusions/Significance We produced a new library of recombinant full-length P. vivax ectodomains, established that the majority of them contain tertiary structure, and used them to identify predicted and novel protein-protein interactions. As well as identifying new interactions for further biological studies, this library will be useful in identifying P. vivax proteins with vaccine potential, and studying P. vivax malaria pathogenesis and immunity. Trial Registration ClinicalTrials.gov NCT00663546 PMID:26701602

  6. Bicistronic expression plasmid for the rapid production of recombinant fused proteins in Escherichia coli.

    PubMed

    Yero, Daniel; Pajón, Rolando; Niebla, Olivia; Sardiñas, Gretel; Vivar, Isbel; Perera, Yasser; García, Darien; Delgado, Maité; Cobas, Karem

    2006-04-01

    In the post-genomic era, every aspect of the production of proteins must be accelerated. In this way, several vectors are currently exploited for rapid production of recombinant proteins in Escherichia coli. N-terminal fusions to the first 47 amino acids of the LpdA (dihydrolipoamide dehydrogenase A) protein of Neisseria meningitidis have been shown to increase the expression of recombinant proteins. Consequently, we have constructed a modified N-terminal LpdA fusion vector, introducing the blue/white colony selection by exploiting a bicistronic gene organization. In the new vector, the sequence encoding the first 47 amino acids of meningococcal LpdA and the alpha-peptide sequence of beta-galactosidase were connected via a ribosome-binding site, and two MCSs (multiple cloning sites) were located surrounding the latter, allowing efficient cloning by colour selection of recombinants. The vector was also improved with the addition of a C-terminal polyhistidine tag, and an EKS (enterokinase recognition sequence) immediately after the LpdA fusion sequence. The new plasmid was employed in the expression and purification of six different bacterial polypeptides. One of these recombinant proteins, P6 protein from Haemophilus influenzae, was used as a model and its N-terminal fusion sequence was totally removed from the recombinant version after incubation with the enterokinase protease, while the polyhistidine tail successfully allowed the purification of the unfused protein from the protease reaction. Two completely new neisserial vaccine candidates, NMB0088 and NMB1126 proteins, were cloned, expressed and purified using this system. To our knowledge, this constitutes the first report of the cloning and expression of these proteins in E. coli.

  7. Expression of recombinant small hydrophobic protein for serospecific detection of avian pneumovirus subgroup C.

    PubMed

    Luo, Lizhong; Sabara, Marta I; Li, Yan

    2005-01-01

    The small hydrophobic (SH) gene of the avian pneumovirus (APV) Colorado isolate (CO), which belongs to subgroup C (APV/C), was expressed with a baculovirus vector. The recombinant SH protein was evaluated as a potential subgroup-specific diagnostic reagent in order to differentiate infections resulting from APV/C from those induced by APV/A, APV/B, and human metapneumovirus (hMPV). When the recombinant baculovirus was used to infect insect cells, a 31- to 38-kDa glycosylated form of the SH protein was produced and subsequently tested for reactivity with antibodies specific for APV/A, APV/B, APV/C, and hMPV. Western blot analysis showed that the expressed recombinant SH protein could only be recognized by APV/C-specific antibodies. This result was consistent with sequence analysis of the APV/C SH protein, which had very low (24%) amino acid identity with the corresponding protein of hMPV and no discernible identity with the SH protein of APV/A or APV/B. A recombinant SH protein-based enzyme-linked immunosorbent assay (ELISA) was developed, and it further confirmed the lack of reactivity of this protein with antisera raised to APV/A, APV/B, and hMPV and supported its designation as a subgroup-specific antigen. This finding indicated that the recombinant SH protein was a suitable antigen for ELISA-based detection of subgroup-specific antibodies in turkeys and could be used for serologically based differential diagnosis of APV and hMPV infections.

  8. Expression of Recombinant Small Hydrophobic Protein for Serospecific Detection of Avian Pneumovirus Subgroup C

    PubMed Central

    Luo, Lizhong; Sabara, Marta I.; Li, Yan

    2005-01-01

    The small hydrophobic (SH) gene of the avian pneumovirus (APV) Colorado isolate (CO), which belongs to subgroup C (APV/C), was expressed with a baculovirus vector. The recombinant SH protein was evaluated as a potential subgroup-specific diagnostic reagent in order to differentiate infections resulting from APV/C from those induced by APV/A, APV/B, and human metapneumovirus (hMPV). When the recombinant baculovirus was used to infect insect cells, a 31- to 38-kDa glycosylated form of the SH protein was produced and subsequently tested for reactivity with antibodies specific for APV/A, APV/B, APV/C, and hMPV. Western blot analysis showed that the expressed recombinant SH protein could only be recognized by APV/C-specific antibodies. This result was consistent with sequence analysis of the APV/C SH protein, which had very low (24%) amino acid identity with the corresponding protein of hMPV and no discernible identity with the SH protein of APV/A or APV/B. A recombinant SH protein-based enzyme-linked immunosorbent assay (ELISA) was developed, and it further confirmed the lack of reactivity of this protein with antisera raised to APV/A, APV/B, and hMPV and supported its designation as a subgroup-specific antigen. This finding indicated that the recombinant SH protein was a suitable antigen for ELISA-based detection of subgroup-specific antibodies in turkeys and could be used for serologically based differential diagnosis of APV and hMPV infections. PMID:15643005

  9. RecO protein initiates DNA recombination and strand annealing through two alternative DNA binding mechanisms.

    PubMed

    Ryzhikov, Mikhail; Gupta, Richa; Glickman, Michael; Korolev, Sergey

    2014-10-17

    Recombination mediator proteins (RMPs) are important for genome stability in all organisms. Several RMPs support two alternative reactions: initiation of homologous recombination and DNA annealing. We examined mechanisms of RMPs in both reactions with Mycobacterium smegmatis RecO (MsRecO) and demonstrated that MsRecO interacts with ssDNA by two distinct mechanisms. Zinc stimulates MsRecO binding to ssDNA during annealing, whereas the recombination function is zinc-independent and is regulated by interaction with MsRecR. Thus, different structural motifs or conformations of MsRecO are responsible for interaction with ssDNA during annealing and recombination. Neither annealing nor recombinase loading depends on MsRecO interaction with the conserved C-terminal tail of single-stranded (ss) DNA-binding protein (SSB), which is known to bind Escherichia coli RecO. However, similarly to E. coli proteins, MsRecO and MsRecOR do not dismiss SSB from ssDNA, suggesting that RMPs form a complex with SSB-ssDNA even in the absence of binding to the major protein interaction motif. We propose that alternative conformations of such complexes define the mechanism by which RMPs initiate the repair of stalled replication and support two different functions during recombinational repair of DNA breaks. PMID:25170075

  10. Application of Recombinant Proteins for Serodiagnosis of Visceral Leishmaniasis in Humans and Dogs

    PubMed Central

    Farahmand, Mahin; Nahrevanian, Hossein

    2016-01-01

    Visceral leishmaniasis (VL) is a zoonotic disease caused by leishmania species. Dogs are considered to be the main reservoir of VL. A number of methods and antigen-based assays are used for the diagnosis of leishmaniasis. However, currently available methods are mainly based on direct examination of tissues for the presence of parasites, which is highly invasive. A variety of serological tests are commonly applied for VL diagnosis, including indirect fluorescence antibody test, enzyme-linked immunosorbent assay (ELISA), dot-ELISA, direct agglutination test, Western-blotting, and immunochromatographic test. However, when soluble antigens are used, serological tests are less specific due to cross-reactivity with other parasitic diseases. Several studies have attempted to replace soluble antigens with recombinant proteins to improve the sensitivity and the specificity of the immunodiagnostic tests. Major technological advances in recombinant antigens as reagents for the serological diagnosis of VL have led to high sensitivity and specificity of these serological tests. A great number of recombinant proteins have been shown to be effective for the diagnosis of leishmania infection in dogs, the major reservoir of L. infantum. Although few recombinant proteins with high efficacy provide reasonable results for the diagnosis of human and canine VL, more optimization is still needed for the appropriate antigens to provide high-throughput performance. This review aims to explore the application of different recombinant proteins for the serodiagnosis of VL in humans and dogs. PMID:26883952

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

    PubMed

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

    2014-01-01

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

  12. Evaluation of the recombinant protein TpF1 of Treponema pallidum for serodiagnosis of syphilis.

    PubMed

    Jiang, Chuanhao; Zhao, Feijun; Xiao, Jinhong; Zeng, Tiebing; Yu, Jian; Ma, Xiaohua; Wu, Haiying; Wu, Yimou

    2013-10-01

    Syphilis is a chronic infection caused by Treponema pallidum subsp. pallidum, and diagnosis with sensitive and specific methods is a challenging process that is important for its prevention and treatment. In the present study, we established a recombinant protein TpF1-based indirect immunoglobulin G (IgG) enzyme-linked immunosorbent assay (ELISA) and a Western blot assay for human and rabbit sera. The 20-kDa recombinant protein TpF1 was detected by Western blotting performed with sera from rabbits immunized with recombinant TpF1 and infected with the T. pallidum Nichols strain and T. pallidum clinical isolates but was not detected by Western blotting with sera from uninfected rabbits. The sensitivity of the recombinant protein was determined by screening sera from individuals with primary, secondary, latent, and congenital syphilis (n = 82). The specificity of the recombinant protein was determined by screening sera from uninfected controls (n = 30) and individuals with potentially cross-reactive infections, including Lyme disease (n = 30) and leptospirosis (n = 5). The sensitivities of TpF1-based ELISAs were 93.3%, 100%, 100%, and 100% for primary, secondary, latent, and congenital syphilis, respectively, and the specificities were all 100% for sera from uninfected controls and individuals with potentially cross-reactive infections. In Western blot assays, the sensitivities and specificities of TpF1 for human sera were all 100%. The reactivities of TpF1 with syphilitic sera were proportional to the titers of the T. pallidum particle agglutination (TPPA) assay. These data indicate that the recombinant protein TpF1 is a highly immunogenic protein in human and rabbit infections and a promising marker for the screening of syphilis. PMID:23945159

  13. Detection of antibodies to caprine arthritis-encephalitis virus using recombinant gag proteins.

    PubMed

    Rimstad, E; East, N; DeRock, E; Higgins, J; Pedersen, N C

    1994-01-01

    The coding sequences of the core proteins p17 and p28 of caprine arthritis-encephalitis virus (CAEV) were amplified using the polymerase chain reaction and cloned into the plasmid expression vector p-GEX-2T. Both p17 and p28 were expressed as fusion proteins with glutathione S-transferase. The recombinant proteins were affinity purified from induced bacterial lysates using glutathione-agarose beads. The purified proteins were used in an enzyme-linked immunosorbent assay (ELISA) to detect antibodies against CAEV in goat sera and milk samples. Three different ELISA tests were developed based on p17, p28 or the combination of these two recombinant proteins (p17 + p28). A comparison was made to an ELISA based on purified whole virus particles and to agar immunodiffusion test (AGID). Sera with conflicting results in the different ELISA tests were examined by Western blotting. There was a high correlation between the ELISA tests based on p17 + p28 recombinant proteins and whole virus ELISA, with an estimated kappa value of 0.92. Only 72-75% of the sera that tested positive in these two ELISA tests were positive in AGID. Antibodies to CAEV were detected in significantly more animals when serum samples were tested compared to milk samples. Based on the time and materials required to prepare the reagents, the recombinant based ELISA test was less expensive than the whole virus ELISA.

  14. Micro-algae come of age as a platform for recombinant protein production

    PubMed Central

    Specht, Elizabeth; Miyake-Stoner, Shigeki

    2010-01-01

    A complete set of genetic tools is still being developed for the micro-alga Chlamydomonas reinhardtii. Yet even with this incomplete set, this photosynthetic single-celled plant has demonstrated significant promise as a platform for recombinant protein expression. In recent years, techniques have been developed that allow for robust expression of genes from both the nuclear and plastid genome. With these advances, many research groups have examined the pliability of this and other micro-algae as biological machines capable of producing recombinant peptides and proteins. This review describes recent successes in recombinant protein production in Chlamydomonas, including production of complex mammalian therapeutic proteins and monoclonal antibodies at levels sufficient for production at economic parity with existing production platforms. These advances have also shed light on the details of algal protein production at the molecular level, and provide insight into the next steps for optimizing micro-algae as a useful platform for the production of therapeutic and industrially relevant recombinant proteins. PMID:20556634

  15. Recombinant Sheep Pox Virus Proteins Elicit Neutralizing Antibodies

    PubMed Central

    Chervyakova, Olga V.; Zaitsev, Valentin L.; Iskakov, Bulat K.; Tailakova, Elmira T.; Strochkov, Vitaliy M.; Sultankulova, Kulyaisan T.; Sandybayev, Nurlan T.; Stanbekova, Gulshan E.; Beisenov, Daniyar K.; Abduraimov, Yergali O.; Mambetaliyev, Muratbay; Sansyzbay, Abylay R.; Kovalskaya, Natalia Y.; Nemchinov, Lev. G.; Hammond, Rosemarie W.

    2016-01-01

    The aim of this work was to evaluate the immunogenicity and neutralizing activity of sheep pox virus (SPPV; genus Capripoxvirus, family Poxviridae) structural proteins as candidate subunit vaccines to control sheep pox disease. SPPV structural proteins were identified by sequence homology with proteins of vaccinia virus (VACV) strain Copenhagen. Four SPPV proteins (SPPV-ORF 060, SPPV-ORF 095, SPPV-ORF 117, and SPPV-ORF 122), orthologs of immunodominant L1, A4, A27, and A33 VACV proteins, respectively, were produced in Escherichia coli. Western blot analysis revealed the antigenic and immunogenic properties of SPPV-060, SPPV-095, SPPV-117 and SPPV-122 proteins when injected with adjuvant into experimental rabbits. Virus-neutralizing activity against SPPV in lamb kidney cell culture was detected for polyclonal antisera raised to SPPV-060, SPPV-117, and SPPV-122 proteins. To our knowledge, this is the first report demonstrating the virus-neutralizing activities of antisera raised to SPPV-060, SPPV-117, and SPPV-122 proteins. PMID:27338444

  16. 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. PMID:26614293

  17. A protocol for the production of recombinant spider silk-like proteins for artificial fiber spinning

    PubMed Central

    Teulé, Florence; Cooper, Alyssa R; Furin, William A; Bittencourt, Daniela; Rech, Elibio L; Brooks, Amanda; Lewis, Randolph V

    2009-01-01

    The extreme strength and elasticity of spider silks originate from the modular nature of their repetitive proteins. To exploit such materials and mimic spider silks, comprehensive strategies to produce and spin recombinant fibrous proteins are necessary. This protocol describes silk gene design and cloning, protein expression in bacteria, recombinant protein purification and fiber formation. With an improved gene construction and cloning scheme, this technique is adaptable for the production of any repetitive fibrous proteins, and ensures the exact reproduction of native repeat sequences, analogs or chimeric versions. The proteins are solubilized in 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) at 25–30% (wt/vol) for extrusion into fibers. This protocol, routinely used to spin single micrometer-size fibers from several recombinant silk-like proteins from different spider species, is a powerful tool to generate protein libraries with corresponding fibers for structure–function relationship investigations in protein-based biomaterials. This protocol may be completed in 40 d. PMID:19229199

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

    SciTech Connect

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

    2012-02-03

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

  19. Expression and affinity purification of recombinant proteins from plants

    NASA Technical Reports Server (NTRS)

    Desai, Urvee A.; Sur, Gargi; Daunert, Sylvia; Babbitt, Ruth; Li, Qingshun

    2002-01-01

    With recent advances in plant biotechnology, transgenic plants have been targeted as an inexpensive means for the mass production of proteins for biopharmaceutical and industrial uses. However, the current plant purification techniques lack a generally applicable, economic, large-scale strategy. In this study, we demonstrate the purification of a model protein, beta-glucuronidase (GUS), by employing the protein calmodulin (CaM) as an affinity tag. In the proposed system, CaM is fused to GUS. In the presence of calcium, the calmodulin fusion protein binds specifically to a phenothiazine-modified surface of an affinity column. When calcium is removed with a complexing agent, e.g., EDTA, calmodulin undergoes a conformational change allowing the dissociation of the calmodulin-phenothiazine complex and, therefore, permitting the elution of the GUS-CaM fusion protein. The advantages of this approach are the fast, efficient, and economical isolation of the target protein under mild elution conditions, thus preserving the activity of the target protein. Two types of transformation methods were used in this study, namely, the Agrobacterium-mediated system and the viral-vector-mediated transformation system. Copyright 2002 Elsevier Science (USA).

  20. Recombinant methods in protein and whole-cell biosensing

    NASA Astrophysics Data System (ADS)

    Shetty, R. S.; Salins, Lyndon L.; Ramanathan, S.; Daunert, Sylvia

    1999-12-01

    In this paper, we investigate the use of fluorescently- labeled binding proteins and genetically engineered bacterial cells for sensing of phosphate, glucose, and L- arabinose. To optimize the performance of the labeled binding proteins for biosensing purposes, a few key considerations were taken into account. A site-selective labeling protocol of the fluorescent reporter to the protein was used to ensure that the probe reported from a specific domain of the protein. The labeling sites chosen were hypothesized to undergo a physicochemical change when the biorecognition element binds the analyte. Cysteine mutations were introduced into the binding proteins by site-directed mutagenesis using the polymerase chain reaction. The residues selected were all in close proximity to the binding cleft, a region that is affected the most by the conformational change that accompanies ligand binding. The cysteine residues were then labeled with environment- sensitive fluorophores and changes in the fluorescence properties of the conjugates were monitored and related to the amount of ligand present. The application of microorganisms in sensing systems represent new advances in the development of novel analytical techniques for the detection of a target analyte. In these systems, a genetically engineered organism generates an analytically useful signal when it encounters a specific target substance due to selective recognition and binding properties towards that particular compound. This concept has been demonstrated using an optical bacteria-based sensing system capable of detecting the monosaccharide L-arabinose that employed the green fluorescent protein as a reporter protein.

  1. Algorithms for selecting breakpoint locations to optimize diversity in protein engineering by site-directed protein recombination.

    PubMed

    Zheng, Wei; Ye, Xiaoduan; Friedman, Alan M; Bailey-Kellogg, Chris

    2007-01-01

    Protein engineering by site-directed recombination seeks to develop proteins with new or improved function, by accumulating multiple mutations from a set of homologous parent proteins. A library of hybrid proteins is created by recombining the parent proteins at specified breakpoint locations; subsequent screening/selection identifies hybrids with desirable functional characteristics. In order to improve the frequency of generating novel hybrids, this paper develops the first approach to explicitly plan for diversity in site-directed recombination, including metrics for characterizing the diversity of a planned hybrid library and efficient algorithms for optimizing experiments accordingly. The goal is to choose breakpoint locations to sample sequence space as uniformly as possible (which we argue maximizes diversity), under the constraints imposed by the recombination process and the given set of parents. A dynamic programming approach selects optimal breakpoint locations in polynomial time. Application of our method to optimizing breakpoints for an example biosynthetic enzyme, purE, demonstrates the significance of diversity optimization and the effectiveness of our algorithms.

  2. Complement receptor activity of recombinant porcine CR1-like protein expressed in a eukaryotic system.

    PubMed

    Yin, Wei; Wei, Xiaoming; Jiang, Junbing; Fan, Kuohai; Zhao, Junxing; Sun, Na; Wang, Zhiwei; Sun, Yaogui; Ma, Haili; Zhao, Xin; Li, Hongquan

    2016-08-01

    Primate complement receptor type 1 (CR1) protein, a single-chain transmembrane glycoprotein, plays an important role in immune adherence and clearing complement-opsonized immune complexes. Here, the mRNA of the porcine primate-like complement receptor (CR1-like) gene was analyzed, and two domain sequences with potential functions were cloned into the pwPICZalpha vector for expression in Pichia pastoris. The recombinant proteins were purified with both Protein Pure Ni-NTA resin and strong anion exchange resin. The activities of the purified recombinant proteins were evaluated by SDS-PAGE, western blotting, and complement receptor assays. The results indicated that two domains of the CR1-like protein, CCP36 and CCP811 with molecular weights of 29.8 kDa and 30 kDa, respectively, were successfully expressed in P. pastoris. These two recombinant proteins possess some of the functions of the primate CR1 protein. Using these two proteins coupled with an antibody blocking technique, we also showed that CR1-like is expressed on natural porcine erythrocytes. PMID:26903010

  3. Replication protein A is required for meiotic recombination in Saccharomyces cerevisiae.

    PubMed Central

    Soustelle, Christine; Vedel, Michèle; Kolodner, Richard; Nicolas, Alain

    2002-01-01

    In Saccharomyces cerevisiae, meiotic recombination is initiated by transient DNA double-stranded breaks (DSBs). These DSBs undergo a 5' --> 3' resection to produce 3' single-stranded DNA ends that serve to channel DSBs into the RAD52 recombinational repair pathway. In vitro studies strongly suggest that several proteins of this pathway--Rad51, Rad52, Rad54, Rad55, Rad57, and replication protein A (RPA)--play a role in the strand exchange reaction. Here, we report a study of the meiotic phenotypes conferred by two missense mutations affecting the largest subunit of RPA, which are localized in the protein interaction domain (rfa1-t11) and in the DNA-binding domain (rfa1-t48). We find that both mutant diploids exhibit reduced sporulation efficiency, very poor spore viability, and a 10- to 100-fold decrease in meiotic recombination. Physical analyses indicate that both mutants form normal levels of meiosis-specific DSBs and that the broken ends are processed into 3'-OH single-stranded tails, indicating that the RPA complex present in these rfa1 mutants is functional in the initial steps of meiotic recombination. However, the 5' ends of the broken fragments undergo extensive resection, similar to what is observed in rad51, rad52, rad55, and rad57 mutants, indicating that these RPA mutants are defective in the repair of the Spo11-dependent DSBs that initiate homologous recombination during meiosis. PMID:12072452

  4. Several recombinant capsid proteins of equine rhinitis a virus show potential as diagnostic antigens.

    PubMed

    Li, Fan; Stevenson, Rachel A; Crabb, Brendan S; Studdert, Michael J; Hartley, Carol A

    2005-06-01

    Equine rhinitis A virus (ERAV) is a significant pathogen of horses and is also closely related to Foot-and-mouth disease virus (FMDV). Despite these facts, knowledge of the prevalence and importance of ERAV infections remains limited, largely due to the absence of a simple, robust diagnostic assay. In this study, we compared the antigenicities of recombinant full-length and fragmented ERAV capsid proteins expressed in Escherichia coli by using sera from experimentally infected and naturally exposed horses. We found that, from the range of antigens tested, recombinant proteins encompassing the C-terminal region of VP1, full-length VP2, and the N-terminal region of VP2 reacted specifically with antibodies present in sera from each of the five experimentally infected horses examined. Antibodies to epitopes on VP2 (both native and recombinant forms) persisted longer postinfection (>105 days) than antibodies specific for epitopes on other fragments. Our data also suggest that B-cell epitopes within the C terminus of VP1 and N terminus of VP2 contribute to a large proportion of the total reactivity of recombinant VP1 and VP2, respectively. Importantly, the reactivity of these VP1 and VP2 recombinant proteins in enzyme-linked immunosorbent assays (ELISAs) correlated well with the results from a range of native antigen-based serological assays using sera from 12 field horses. This study provides promising candidates for development of a diagnostic ERAV ELISA.

  5. The recombinant expression and activity detection of MAF-1 fusion protein

    PubMed Central

    Fu, Ping; Wu, Jianwei; Gao, Song; Guo, Guo; Zhang, Yong; Liu, Jian

    2015-01-01

    This study establishes the recombinant expression system of MAF-1 (Musca domestica antifungal peptide-1) and demonstrates the antifungal activity of the expression product and shows the relationship between biological activity and structure. The gene segments on mature peptide part of MAF-1 were cloned, based on the primers designed according to the cDNA sequence of MAF-1. We constructed the recombinant prokaryotic expression plasmid using prokaryotic expression vector (pET-28a(+)) and converted it to the competent cell of BL21(DE3) to gain recombinant MAF-1 fusion protein with His tag sequence through purifying affinity chromatographic column of Ni-NTA. To conduct the Western Blotting test, recombinant MAF-1 fusion protein was used to produce the polyclonal antibody of rat. The antifungal activity of the expression product was detected using Candida albicans (ATCC10231) as the indicator. The MAF-1 recombinant fusion protein was purified to exhibit obvious antifungal activity, which lays the foundation for the further study of MAF-1 biological activity, the relationship between structure and function, as well as control of gene expression. PMID:26423137

  6. Evaluation of recombinant proteins of Burkholderia mallei for serodiagnosis of glanders.

    PubMed

    Pal, Vijai; Kumar, Subodh; Malik, Praveen; Rai, Ganga Prasad

    2012-08-01

    Glanders is a contagious disease caused by the Gram-negative bacillus Burkholderia mallei. The number of equine glanders outbreaks has increased steadily during the last decade. The disease must be reported to the Office International des Epizooties, Paris, France. Glanders serodiagnosis is hampered by the considerable number of false positives and negatives of the internationally prescribed tests. The major problem leading to the low sensitivity and specificity of the complement fixation test (CFT) and enzyme-linked immunosorbent assay (ELISA) has been linked to the test antigens currently used, i.e., crude preparations of whole cells. False-positive results obtained from other diagnostic tests utilizing crude antigens lead to financial losses to animal owners, and false-negative results can turn a risk into a possible threat. In this study, we report on the identification of diagnostic targets using bioinformatics tools for serodiagnosis of glanders. The identified gene sequences were cloned and expressed as recombinant proteins. The purified recombinant proteins of B. mallei were used in an indirect ELISA format for serodiagnosis of glanders. Two recombinant proteins, 0375H and 0375TH, exhibited 100% sensitivity and specificity for glanders diagnosis. The proteins also did not cross-react with sera from patients with the closely related disease melioidosis. The results of this investigation highlight the potential of recombinant 0375H and 0375TH proteins in specific and sensitive diagnosis of glanders.

  7. The Quaternary Structure of the Recombinant Bovine Odorant-Binding Protein Is Modulated by Chemical Denaturants

    PubMed Central

    Stepanenko, Olga V.; Stepanenko, Olesya V.; Staiano, Maria; Kuznetsova, Irina M.; Turoverov, Konstantin K.; D’Auria, Sabato

    2014-01-01

    A large group of odorant-binding proteins (OBPs) has attracted great scientific interest as promising building blocks in constructing optical biosensors for dangerous substances, such as toxic and explosive molecules. Native tissue-extracted bovine OBP (bOBP) has a unique dimer folding pattern that involves crossing the α-helical domain in each monomer over the other monomer’s β-barrel. In contrast, recombinant bOBP maintaining the high level of stability inherent to native tissue bOBP is produced in a stable native-like state with a decreased tendency for dimerization and is a mixture of monomers and dimers in a buffered solution. This work is focused on the study of the quaternary structure and the folding-unfolding processes of the recombinant bOBP in the absence and in the presence of guanidine hydrochloride (GdnHCl). Our results show that the recombinant bOBP native dimer is only formed at elevated GdnHCl concentrations (1.5 M). This process requires re-organizing the protein structure by progressing through the formation of an intermediate state. The bOBP dimerization process appears to be irreversible and it occurs before the protein unfolds. Though the observed structural changes for recombinant bOBP at pre-denaturing GdnHCl concentrations show a local character and the overall protein structure is maintained, such changes should be considered where the protein is used as a sensitive element in a biosensor system. PMID:24409322

  8. Toward an era of utilizing methionine overproducing hosts for recombinant protein production in Escherichia coli.

    PubMed

    Veeravalli, Karthik; Laird, Michael W

    2015-01-01

    Amino acid sequence variants, especially variants containing non-canonical amino acids such as norleucine and norvaline, are a concern during therapeutic protein production in microbial systems. Substitution of methionine residues with norleucine in recombinant proteins produced in Escherichia coli is well known. Continuous feeding of amino acids such as methionine is commonly used in E. coli fermentation processes to control incorporation of norleucine in the recombinant protein. There are several disadvantages associated with continuous feeding during a fermentation process. For example, a continuous feed increases the operational complexity and cost of a manufacturing process and results in dilution of culture medium which could result in lower cell densities and product yields. To overcome the limitations of existing approaches to prevent norleucine incorporation during E. coli fermentations, a new approach using an engineered host was developed that overproduces methionine in the cell to prevent norleucine incorporation without negatively impacting fermentation process performance and product yields. In this commentary, the results on using methionine overproducing hosts for recombinant protein production in E. coli and some "watch outs" when using these hosts for recombinant protein production are discussed.

  9. Toward an era of utilizing methionine overproducing hosts for recombinant protein production in Escherichia coli

    PubMed Central

    Veeravalli, Karthik; Laird, Michael W

    2015-01-01

    Amino acid sequence variants, especially variants containing non-canonical amino acids such as norleucine and norvaline, are a concern during therapeutic protein production in microbial systems. Substitution of methionine residues with norleucine in recombinant proteins produced in Escherichia coli is well known. Continuous feeding of amino acids such as methionine is commonly used in E. coli fermentation processes to control incorporation of norleucine in the recombinant protein. There are several disadvantages associated with continuous feeding during a fermentation process. For example, a continuous feed increases the operational complexity and cost of a manufacturing process and results in dilution of culture medium which could result in lower cell densities and product yields. To overcome the limitations of existing approaches to prevent norleucine incorporation during E. coli fermentations, a new approach using an engineered host was developed that overproduces methionine in the cell to prevent norleucine incorporation without negatively impacting fermentation process performance and product yields. In this commentary, the results on using methionine overproducing hosts for recombinant protein production in E. coli and some “watch outs” when using these hosts for recombinant protein production are discussed. PMID:25801611

  10. Recombinant Flagellin-Porcine Circovirus Type 2 Cap Fusion Protein Promotes Protective Immune Responses in Mice

    PubMed Central

    Zhang, Chunyan; Zhu, Shanshan; Wei, Li; Yan, Xu; Wang, Jing; Quan, Rong; She, Ruiping; Hu, Fengjiao; Liu, Jue

    2015-01-01

    The Cap protein of porcine circovirus type 2 (PCV2) that serves as a major host-protective immunogen was used to develop recombinant vaccines for control of PCV2-associated diseases. Growing research data have demonstrated the high effectiveness of flagellin as an adjuvant for humoral and cellular immune responses. Here, a recombinant protein was designed by fusing a modified version of bacterial flagellin to PCV2 Cap protein and expressed in a baculovirus system. When administered without adjuvant to BALB/c mice, the flagellin-Cap fusion protein elicited stronger PCV2-specific IgG antibody response, higher neutralizing antibody levels, milder histopathological changes and lower viremia, as well as higher secretion of cytokines such as TNF-α and IFN-γ that conferred better protection against virus challenge than those in the recombinant Cap alone-inoculated mice. These results suggest that the recombinant Cap protein when fused to flagellin could elicit better humoral and cellular immune responses against PCV2 infection in a mouse model, thereby acting as an attractive candidate vaccine for control of the PCV2-associated diseases. PMID:26070075

  11. Expression of functional Bunyamwera virus L protein by recombinant vaccinia viruses.

    PubMed

    Jin, H; Elliott, R M

    1991-08-01

    A cDNA containing the complete coding sequence of the Bunyamwera virus (family Bunyaviridae) L genome segment has been constructed and cloned into two recombinant vaccinia virus expression systems. In the first, the L gene is under control of vaccinia virus P7.5 promoter; in the second, the L gene is under control of the bacteriophage T7 phi 10 promoter, and expression of the L gene requires coinfection with a second recombinant vaccinia virus which synthesizes T7 RNA polymerase. Both systems express a protein which is the same size as the Bunyamwera virus L protein and is recognized by a monospecific L antiserum. The expressed L protein was shown to be functional in synthesizing Bunyamwera virus RNA in a nucleocapsid transfection assay: recombinant vaccinia virus-infected cells were transfected with purified Bunyamwera virus nucleocapsids, and subsequently, total cellular RNA was analyzed by Northern (RNA) blotting. No Bunyamwera virus RNA was detected in control transfections, but in cells which had previously been infected with recombinant vaccinia viruses expressing the L protein, both positive- and negative-sense Bunyamwera virus S segment RNA was detected. The suitability of this system to delineate functional domains within the Bunyamwera virus L protein is discussed.

  12. The Use of Affinity Tags to Overcome Obstacles in Recombinant Protein Expression and Purification.

    PubMed

    Amarasinghe, Chinthaka; Jin, Jian-Ping

    2015-01-01

    Research and industrial demands for recombinant proteins continue to increase over time for their broad applications in structural and functional studies and as therapeutic agents. These applications often require large quantities of recombinant protein at desirable purity, which highlights the importance of developing and improving production approaches that provide high level expression and readily achievable purity of recombinant protein. E. coli is the most widely used host for the expression of a diverse range of proteins at low cost. However, there are common pitfalls that can severely limit the expression of exogenous proteins, such as stability, low solubility and toxicity to the host cell. To overcome these obstacles, one strategy that has found to be promising is the use of affinity tags or carrier peptide to aid in the folding of the target protein, increase solubility, lower toxicity and increase the level of expression. In the meantime, the tags and fusion proteins can be designed to facilitate affinity purification. Since the fusion protein may not exhibit the native conformation of the target protein, various strategies have been developed to remove the tag during or after purification to avoid potential complications in structural and functional studies and to obtain native biological activities. Despite extensive research and rapid development along these lines, there are unsolved problems and imperfect applications. This focused review compares and contrasts various strategies that employ affinity tags to improve bacterial expression and to facilitate purification of recombinant proteins. The pros and cons of the approaches are discussed for more effective applications and new directions of future improvement. PMID:26216265

  13. A Library of Functional Recombinant Cell-surface and Secreted P. falciparum Merozoite Proteins*

    PubMed Central

    Crosnier, Cécile; Wanaguru, Madushi; McDade, Brian; Osier, Faith H.; Marsh, Kevin; Rayner, Julian C.; Wright, Gavin J.

    2013-01-01

    Malaria, an infectious disease caused by parasites of the Plasmodium genus, is one of the world's major public health concerns causing up to a million deaths annually, mostly because of P. falciparum infections. All of the clinical symptoms are associated with the blood stage of the disease, an obligate part of the parasite life cycle, when a form of the parasite called the merozoite recognizes and invades host erythrocytes. During erythrocyte invasion, merozoites are directly exposed to the host humoral immune system making the blood stage of the parasite a conceptually attractive therapeutic target. Progress in the functional and molecular characterization of P. falciparum merozoite proteins, however, has been hampered by the technical challenges associated with expressing these proteins in a biochemically active recombinant form. This challenge is particularly acute for extracellular proteins, which are the likely targets of host antibody responses, because they contain structurally critical post-translational modifications that are not added by some recombinant expression systems. Here, we report the development of a method that uses a mammalian expression system to compile a protein resource containing the entire ectodomains of 42 P. falciparum merozoite secreted and cell surface proteins, many of which have not previously been characterized. Importantly, we are able to recapitulate known biochemical activities by showing that recombinant MSP1-MSP7 and P12-P41 directly interact, and that both recombinant EBA175 and EBA140 can bind human erythrocytes in a sialic acid-dependent manner. Finally, we use sera from malaria-exposed immune adults to profile the relative immunoreactivity of the proteins and show that the majority of the antigens contain conformational (heat-labile) epitopes. We envisage that this resource of recombinant proteins will make a valuable contribution toward a molecular understanding of the blood stage of P. falciparum infections and

  14. Contraceptive efficacy of recombinant fusion protein comprising zona pellucida glycoprotein-3 fragment and gonadotropin releasing hormone.

    PubMed

    Arukha, Ananta Prasad; Minhas, Vidisha; Shrestha, Abhinav; Gupta, Satish Kumar

    2016-04-01

    Contraceptive vaccines have been used for the management of wildlife population. In the present study, we have examined the contraceptive potential of Escherichia coli-expressed recombinant fusion protein comprising of 'promiscuous' T cell epitope of tetanus toxoid [TT; amino acid (aa) residues 830-844] followed by dilysine linker (KK), dog ZP3 fragment (aa residues 307-346), triglycine spacer (GGG), T cell epitope of bovine RNase (bRNase; aa residues 94-104), GnRH, T cell epitope of circumsporozoite protein of Plasmodium falciparum (CSP; aa residues 362-383), and GnRH. SDS-PAGE analysis of the purified refolded protein revealed a dominant ∼12 kDa band, which in Western blot reacted with mouse polyclonal antibodies against dog ZP3 fragment and mouse monoclonal antibodies against GnRH. Immunization of female FvB/J mice following two booster schedule with the above recombinant protein supplemented with alum led to high antibody titres against the immunogen as well as ZP3 and GnRH as determined by ELISA. The immune sera reacted with zona pellucida of mouse oocyte and also inhibited in-vitro fertilization. The qRT-PCR studies showed decrease in the ovarian GnRH receptor in mice immunized with the recombinant fusion protein. Mating studies revealed high contraceptive efficacy of the recombinant protein as in two independent experiments, 90% of the immunized female mice failed to conceive. Following one booster immunization schedule, 50% of the immunized female mice failed to conceive. However, in adjuvanted controls, all the female mice became pregnant. To conclude, the recombinant protein described herein has a good potential to be developed as candidate contraceptive vaccine. PMID:26859695

  15. Recent advances in recombinant protein-based malaria vaccines.

    PubMed

    Draper, Simon J; Angov, Evelina; Horii, Toshihiro; Miller, Louis H; Srinivasan, Prakash; Theisen, Michael; Biswas, Sumi

    2015-12-22

    Plasmodium parasites are the causative agent of human malaria, and the development of a highly effective vaccine against infection, disease and transmission remains a key priority. It is widely established that multiple stages of the parasite's complex lifecycle within the human host and mosquito vector are susceptible to vaccine-induced antibodies. The mainstay approach to antibody induction by subunit vaccination has been the delivery of protein antigen formulated in adjuvant. Extensive efforts have been made in this endeavor with respect to malaria vaccine development, especially with regard to target antigen discovery, protein expression platforms, adjuvant testing, and development of soluble and virus-like particle (VLP) delivery platforms. The breadth of approaches to protein-based vaccines is continuing to expand as innovative new concepts in next-generation subunit design are explored, with the prospects for the development of a highly effective multi-component/multi-stage/multi-antigen formulation seeming ever more likely. This review will focus on recent progress in protein vaccine design, development and/or clinical testing for a number of leading malaria antigens from the sporozoite-, merozoite- and sexual-stages of the parasite's lifecycle-including PfCelTOS, PfMSP1, PfAMA1, PfRH5, PfSERA5, PfGLURP, PfMSP3, Pfs48/45 and Pfs25. Future prospects and challenges for the development, production, human delivery and assessment of protein-based malaria vaccines are discussed.

  16. Recent advances in recombinant protein-based malaria vaccines

    PubMed Central

    Draper, Simon J.; Angov, Evelina; Horii, Toshihiro; Miller, Louis H.; Srinivasan, Prakash; Theisen, Michael; Biswas, Sumi

    2015-01-01

    Plasmodium parasites are the causative agent of human malaria, and the development of a highly effective vaccine against infection, disease and transmission remains a key priority. It is widely established that multiple stages of the parasite's complex lifecycle within the human host and mosquito vector are susceptible to vaccine-induced antibodies. The mainstay approach to antibody induction by subunit vaccination has been the delivery of protein antigen formulated in adjuvant. Extensive efforts have been made in this endeavor with respect to malaria vaccine development, especially with regard to target antigen discovery, protein expression platforms, adjuvant testing, and development of soluble and virus-like particle (VLP) delivery platforms. The breadth of approaches to protein-based vaccines is continuing to expand as innovative new concepts in next-generation subunit design are explored, with the prospects for the development of a highly effective multi-component/multi-stage/multi-antigen formulation seeming ever more likely. This review will focus on recent progress in protein vaccine design, development and/or clinical testing for a number of leading malaria antigens from the sporozoite-, merozoite- and sexual-stages of the parasite's lifecycle–including PfCelTOS, PfMSP1, PfAMA1, PfRH5, PfSERA5, PfGLURP, PfMSP3, Pfs48/45 and Pfs25. Future prospects and challenges for the development, production, human delivery and assessment of protein-based malaria vaccines are discussed. PMID:26458807

  17. Synthesizing a Cellulase like Chimeric Protein by Recombinant Molecular Biology Techniques

    PubMed Central

    Banerjee, Hirendra Nath; Krauss, Christopher; Smith, Valerie; Mahaffey, Kelly; Boston, Ava

    2016-01-01

    In order to meet the Renewable Fuels Standard demands for 30 billion gallons of biofuels by the end of 2020, new technologies for generation of cellulosic ethanol must be exploited. Breaking down cellulose by cellulase enzyme is very important for this purpose but this is not thermostable and degrades at higher temperatures in bioreactors. Towards creation of a more ecologically friendly method of rendering bioethanol from cellulosic waste, we attempted to produce recombinant higher temperature resistant cellulases for use in bioreactors. The project involved molecular cloning of genes for cellulose-degrading enzymes based on bacterial source, expressing the recombinant proteins in E. coli and optimizing enzymatic activity. We were able to generate in vitro bacterial expression systems to produce recombinant His-tag purified protein which showed cellulase like activity. PMID:27468362

  18. Rapid screening for the robust expression of recombinant proteins in algal plastids.

    PubMed

    Barrera, Daniel; Gimpel, Javier; Mayfield, Stephen

    2014-01-01

    Chlamydomonas reinhardtii has many advantages as a photosynthetic model organism. One of these is facile, targeted chloroplast transformation by particle bombardment. Functional recombinant proteins can be expressed to significant levels in this system, potentially outperforming higher plants in speed of scaling, cost, and space requirements. Several strategies and regulatory regions can be used for achieving transgene expression. Here we present two of those strategies: one makes use of the psbD promoter for expressing moderate levels of the recombinant protein in a photosynthetic background. The other strategy is based on the strong psbA promoter for obtaining high yields of the recombinant product in a non-photosynthetic strain. We herein describe the vectors, transformation procedures, and screening methods associated with these two strategies. PMID:24599869

  19. Production and Application of Polyclonal Antibodies Against Recombinant Capsid Protein of Extra Small Virus of Macrobrachium rosenbergii.

    PubMed

    Neethi, V; Sivakumar, N; Kumar, Kundan; Rajendran, K V; Makesh, M

    2012-12-01

    Macrobrachium rosenbergii nodavirus along with a satellite virus, extra small virus (XSV) causes white tail disease (WTD) in the giant freshwater prawn M. rosenbergii. Infected M. rosenbergii postlarvae were collected from a hatchery in Kakinada, Andhra Pradesh. The gene coding the capsid protein of XSV was cloned in a bacterial expression vector pRSET A and the recombinant protein was expressed in Escherichia coli BL21(DE3)pLysS cells. The recombinant protein was purified by Nickel affinity chromatography. Polyclonal antibodies were produced in mice against the recombinant protein and the antibodies reacted specifically with the recombinant protein and XSV in WTD-infected tissues. This is the first report of detection of XSV using antibodies against recombinant capsid protein. PMID:24293828

  20. Production and Application of Polyclonal Antibodies Against Recombinant Capsid Protein of Extra Small Virus of Macrobrachium rosenbergii.

    PubMed

    Neethi, V; Sivakumar, N; Kumar, Kundan; Rajendran, K V; Makesh, M

    2012-12-01

    Macrobrachium rosenbergii nodavirus along with a satellite virus, extra small virus (XSV) causes white tail disease (WTD) in the giant freshwater prawn M. rosenbergii. Infected M. rosenbergii postlarvae were collected from a hatchery in Kakinada, Andhra Pradesh. The gene coding the capsid protein of XSV was cloned in a bacterial expression vector pRSET A and the recombinant protein was expressed in Escherichia coli BL21(DE3)pLysS cells. The recombinant protein was purified by Nickel affinity chromatography. Polyclonal antibodies were produced in mice against the recombinant protein and the antibodies reacted specifically with the recombinant protein and XSV in WTD-infected tissues. This is the first report of detection of XSV using antibodies against recombinant capsid protein.

  1. Cell culture process operations for recombinant protein production.

    PubMed

    Abu-Absi, Susan; Xu, Sen; Graham, Hugh; Dalal, Nimish; Boyer, Marcus; Dave, Kedar

    2014-01-01

    The market for protein therapeutics has grown significantly over the past two decades and the pace of development continues to increase. It is a challenge to the industry to maintain the desired quality attributes while accelerating delivery to patients, reducing the cost of goods, and providing production flexibility. Efficient manufacturing scale production of protein therapeutics is required to continue to meet the needs of the patients and stockholders. This chapter describes batch, fed-batch, and perfusion processes and their utilization in the production of monoclonal antibodies and other therapeutic proteins. In addition, we have provided detailed discussions of the ongoing challenges of lactate metabolism and the future prospects of process monitoring and control. PMID:24153406

  2. Cell culture process operations for recombinant protein production.

    PubMed

    Abu-Absi, Susan; Xu, Sen; Graham, Hugh; Dalal, Nimish; Boyer, Marcus; Dave, Kedar

    2014-01-01

    The market for protein therapeutics has grown significantly over the past two decades and the pace of development continues to increase. It is a challenge to the industry to maintain the desired quality attributes while accelerating delivery to patients, reducing the cost of goods, and providing production flexibility. Efficient manufacturing scale production of protein therapeutics is required to continue to meet the needs of the patients and stockholders. This chapter describes batch, fed-batch, and perfusion processes and their utilization in the production of monoclonal antibodies and other therapeutic proteins. In addition, we have provided detailed discussions of the ongoing challenges of lactate metabolism and the future prospects of process monitoring and control.

  3. Recombinant Expression, Purification, and Functional Characterisation of Connective Tissue Growth Factor and Nephroblastoma-Overexpressed Protein

    PubMed Central

    Bohr, Wilhelm; Kupper, Michael; Hoffmann, Kurt; Weiskirchen, Ralf

    2010-01-01

    The CCN family of proteins, especially its prominent member, the Connective tissue growth factor (CTGF/CCN2) has been identified as a possible biomarker for the diagnosis of fibrotic diseases. As a downstream mediator of TGF-β1 signalling, it is involved in tissue scarring, stimulates interstitial deposition of extracellular matrix proteins, and promotes proliferation of several cell types. Another member of this family, the Nephroblastoma-Overexpressed protein (NOV/CCN3), has growth-inhibiting properties. First reports further suggest that these two CCN family members act opposite to each other in regulating extracellular matrix protein expression and reciprocally influence their own expression when over-expressed. We have established stable HEK and Flp-In-293 clones as productive sources for recombinant human CCN2/CTGF. In addition, we generated an adenoviral vector for recombinant expression of rat NOV and established protocols to purify large quantities of these CCN proteins. The identity of purified human CCN2/CTGF and rat CCN3/NOV was proven by In-gel digest followed by ESI-TOF/MS mass spectrometry. The biological activity of purified proteins was demonstrated using a Smad3-sensitive reporter gene and BrdU proliferation assay in permanent cell line EA•hy 926 cells. We further demonstrate for the first time that both recombinant CCN proteins are N-glycosylated. PMID:21209863

  4. Endotoxin depletion of recombinant protein preparations through their preferential binding to histidine tags.

    PubMed

    Mack, Laura; Brill, Boris; Delis, Natalia; Groner, Bernd

    2014-12-01

    The presence of endotoxins in preparations of recombinantly produced therapeutic proteins poses serious problems for patients. Endotoxins can cause fever, respiratory distress syndromes, intravascular coagulation, or endotoxic shock. A number of methods have been devised to remove endotoxins from protein preparations using separation procedures based on molecular mass or charge properties. Most of the methods are limited in their endotoxin removal capacities and lack general applicability. We are describing a biotechnological approach for endotoxin removal. This strategy exploits the observation that endotoxins form micelles that expose negative charges on their surface, leading to preferential binding of endotoxins to cationic surfaces, allowing the separation from their resident protein. Endotoxins exhibit high affinity to stretches of histidines, which are widely used tools to facilitate the purification of recombinant proteins. They bind to nickel ions and are the basis for protein purification from cellular extracts by immobilized metal affinity chromatography. We show that the thrombin-mediated cleavage of two histidine tags from the purified recombinant protein and the adsorption of these histidine tags and their associated endotoxins to a nickel affinity column result in an appreciable depletion of the endotoxins in the purified protein fraction.

  5. The role of Deinococcus radiodurans RecFOR proteins in homologous recombination.

    PubMed

    Satoh, Katsuya; Kikuchi, Masahiro; Ishaque, Abu M; Ohba, Hirofumi; Yamada, Mitsugu; Tejima, Kouhei; Onodera, Takefumi; Narumi, Issay

    2012-04-01

    Deinococcus radiodurans exhibits extraordinary resistance to the lethal effect of DNA-damaging agents, a characteristic attributed to its highly proficient DNA repair capacity. Although the D. radiodurans genome is clearly devoid of recBC and addAB counterparts as RecA mediators, the genome possesses all genes associated with the RecFOR pathway. In an effort to gain insights into the role of D. radiodurans RecFOR proteins in homologous recombination, we generated recF, recO and recR disruptant strains and characterized the disruption effects. All the disruptant strains exhibited delayed growth relative to the wild-type, indicating that the RecF, RecO and RecR proteins play an important role in cell growth under normal growth conditions. A slight reduction in transformation efficiency was observed in the recF and recO disruptant strains compared to the wild-type strain. Interestingly, disruption of recR resulted in severe reduction of the transformation efficiency. On the other hand, the recF disruptant strain was the most sensitive phenotype to γ rays, UV irradiation and mitomycin C among the three disruptants. In the recF disruptant strain, the intracellular level of the LexA1 protein did not decrease following γ irradiation, suggesting that a large amount of the RecA protein remains inactive despite being induced. These results demonstrate that the RecF protein plays a crucial role in the homologous recombination repair process by facilitating RecA activation in D. radiodurans. Thus, the RecF and RecR proteins are involved in the RecA activation and the stability of incoming DNA, respectively, during RecA-mediated homologous recombination processes that initiated the ESDSA pathway in D. radiodurans. Possible mechanisms that involve the RecFOR complex in homologous intermolecular recombination and homologous recombination repair processes are also discussed. PMID:22321371

  6. Expression of Functional Recombinant Mussel Adhesive Protein Mgfp-5 in Escherichia coli

    PubMed Central

    Hwang, Dong Soo; Yoo, Hyo Jin; Jun, Jong Hyub; Moon, Won Kyu; Cha, Hyung Joon

    2004-01-01

    Mussel adhesive proteins have been suggested as a basis for environmentally friendly adhesives for use in aqueous conditions and in medicine. However, attempts to produce functional and economical recombinant mussel adhesive proteins (mainly foot protein type 1) in several systems have failed. Here, the cDNA coding for Mytilus galloprovincialis foot protein type 5 (Mgfp-5) was isolated for the first time. Using this cDNA, we produced a recombinant Mgfp-5 fused with a hexahistidine affinity ligand, which was expressed in a soluble form in Escherichia coli and was highly purified using affinity chromatography. The adhesive properties of purified recombinant Mgfp-5 were compared with the commercial extracted mussel adhesive Cell-Tak by investigating adhesion force using atomic force microscopy, material surface coating, and quartz crystal microbalance. Even though further macroscale assays are needed, these microscale assays showed that recombinant Mgfp-5 has significant adhesive ability and may be useful as a bioadhesive in medical or underwater environments. PMID:15184131

  7. Antigenic Profiles of Recombinant Proteins from Mycobacterium avium subsp paratuberculosis in Sheep with Johne's Disease

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Methods to improve the ELISA test to detect Mycobacterium avium subsp paratuberculosis have been explored over several years. Previously, selected recombinant proteins of M. avium subspecies paratuberculosis were found to be immunogenic in cattle with Johne’s disease. In the present study, antibo...

  8. Indirect enzyme-linked immunosorbent assay for detection of immunoglobulin G reactive with a recombinant protein expressed from the gene encoding the 116-kilodalton protein of Mycoplasma pneumoniae.

    PubMed

    Duffy, M F; Whithear, K G; Noormohammadi, A H; Markham, P F; Catton, M; Leydon, J; Browning, G F

    1999-04-01

    Serology remains the method of choice for laboratory diagnosis of Mycoplasma pneumoniae infection. Currently available serological tests employ complex cellular fractions of M. pneumoniae as antigen. To improve the specificity of M. pneumoniae diagnosis, a recombinant protein was assessed as a serodiagnostic reagent. A panel of recombinant proteins were expressed from a cloned M. pneumoniae gene that encodes a 116-kDa surface protein antigen. The recombinant proteins were assessed for reactivity with patient sera and the most antigenic was further assessed for its serodiagnostic potential by indirect enzyme-linked immunosorbent assay (ELISA). The ELISA based on the recombinant protein was equivalent in sensitivity to the commercial test (Serodia Myco II; Fujirebio Inc.) to which it was compared. Southern and Western blotting data suggested that the recombinant protein derived from the 116-kDa protein of M. pneumoniae could provide a species-specific diagnostic tool, although further assessment is required.

  9. Beyond the cytoplasm of Escherichia coli: localizing recombinant proteins where you want them.

    PubMed

    Boock, Jason T; Waraho-Zhmayev, Dujduan; Mizrachi, Dario; DeLisa, Matthew P

    2015-01-01

    Recombinant protein expression in Escherichia coli represents a cornerstone of the biotechnology enterprise. While cytoplasmic expression in this host has received the most attention, achieving substantial yields of correctly folded proteins in this compartment can sometimes be met with difficulties. These issues can often be overcome by targeting protein expression to extracytoplasmic compartments (e.g., membrane, periplasm) or to the culture medium. This chapter discusses various strategies for exporting proteins out of the cytoplasm as well as tools for monitoring and optimizing these different export mechanisms. PMID:25447860

  10. Recombinant Dragline Silk-Like Proteins-Expression and Purification.

    PubMed

    Gaines, William A; Marcotte, William R

    2011-03-01

    Spider dragline silk is a proteinaceous fiber with impressive physical characteristics making it attractive for use in advanced materials. The fiber is composed of two proteins (spidroins MaSp1 and MaSp2), each of which contains a large central repeat array flanked by non-repetitive N- and C-terminal domains. The repeat arrays appear to be largely responsible for the tensile properties of the fiber, suggesting that the N- and C-terminal domains may be involved in self-assembly. We recently isolated the MaSp1 and MaSp2 N-terminal domains from Nephila clavipes and have incorporated these into mini-silk genes for expression in transgenic systems. Current efforts involve the development of expression vectors that will allow purification using a removable affinity tag for scalable protein purification.

  11. Preparative Purification of Recombinant Proteins: Current Status and Future Trends

    PubMed Central

    Saraswat, Mayank; Ravidá, Alessandra; Holthofer, Harry

    2013-01-01

    Advances in fermentation technologies have resulted in the production of increased yields of proteins of economic, biopharmaceutical, and medicinal importance. Consequently, there is an absolute requirement for the development of rapid, cost-effective methodologies which facilitate the purification of such products in the absence of contaminants, such as superfluous proteins and endotoxins. Here, we provide a comprehensive overview of a selection of key purification methodologies currently being applied in both academic and industrial settings and discuss how innovative and effective protocols such as aqueous two-phase partitioning, membrane chromatography, and high-performance tangential flow filtration may be applied independently of or in conjunction with more traditional protocols for downstream processing applications. PMID:24455685

  12. Liposomes containing recombinant E protein vaccine against duck Tembusu virus in ducks.

    PubMed

    Ma, Tengfei; Liu, Yongxia; Cheng, Jia; Liu, Yanhan; Fan, Wentao; Cheng, Ziqiang; Niu, Xudong; Liu, Jianzhu

    2016-04-27

    To obtain an effective vaccine candidate against duck Tembusu viral (DTMUV) disease which causes egg-drop and great economical loss in the Chinese duck industry, liposome vaccines containing recombinant E protein were prepared and assessed in this study. The recombinant plasmid (PET28a-E) was constructed and transformed into BL21 (DE3) cells to produce E proteins. The recombinant E proteins were purified and entrapped by liposomes through reverse-phase evaporation. Eighty-four cherry valley ducks were randomly divided into seven groups and inoculated intramuscularly at one- or seven-day-old with liposomes-E protein or Freund's adjuvant-E protein vaccine. Blood samples were collected from the first week to the tenth week for serum antibody, plasma for viremia, as well as oropharyngeal and cloacal swabs for virus shedding analyses after being challenged with a 10(2.4) 50% tissue culture infective dose (TCID50) of duck Tembusu virus. Results showed that serum antibody level of the liposomes vaccine was higher than the Freund's adjuvant vaccine, and inoculating twice was superior to once; furthermore, the viremia and virus shedding tests also proved that the liposomes vaccine can provide complete protection against DTMUV challenge. These results demonstrated that the liposomes-E protein vaccine could be used as a potential candidate vaccine to prevent DTMUV infection in ducks. PMID:27016654

  13. Cryptocaryon irritans recombinant proteins as potential antigens for sero-surveillance of cryptocaryonosis.

    PubMed

    Lokanathan, Y; Mohd-Adnan, A; Kua, B-C; Nathan, S

    2016-09-01

    Cryptocaryonosis is a major problem for mariculture, and the absence of suitable sero-surveillance tools for the detection of cryptocaryonosis makes it difficult to screen Cryptocaryon irritans-infected fish, particularly asymptomatic fish. In this study, we proposed a serum-based assay using selected C. irritans proteins to screen infected and asymptomatic fish. Eight highly expressed genes were chosen from an earlier study on C. irritans expressed sequence tags and ciliate glutamine codons were converted to universal glutamine codons. The chemically synthesized C. irritans genes were then expressed in an Escherichia coli expression host under optimized conditions. Five C. irritans proteins were successfully expressed in E. coli and purified by affinity chromatography. These proteins were used as antigens in an enzyme-linked immunosorbent assay (ELISA) to screen sera from experimentally immunized fish and naturally infected fish. Sera from both categories of fish reacted equally well with the expressed C. irritans recombinant proteins as well as with sonicated theronts. This study demonstrated the utility of producing ciliate recombinant proteins in a heterologous expression host. An ELISA was successfully developed to diagnose infected and asymptomatic fish using the recombinant proteins as antigens. PMID:27086498

  14. 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.

  15. IMAC capture of recombinant protein from unclarified mammalian cell feed streams

    PubMed Central

    Kinna, Alexander; Tolner, Berend; Rota, Enrique Miranda; Titchener‐Hooker, Nigel; Nesbeth, Darren

    2015-01-01

    ABSTRACT Fusion‐tag affinity chromatography is a key technique in recombinant protein purification. Current methods for protein recovery from mammalian cells are hampered by the need for feed stream clarification. We have developed a method for direct capture using immobilized metal affinity chromatography (IMAC) of hexahistidine (His6) tagged proteins from unclarified mammalian cell feed streams. The process employs radial flow chromatography with 300–500 μm diameter agarose resin beads that allow free passage of cells but capture His‐tagged proteins from the feed stream; circumventing expensive and cumbersome centrifugation and/or filtration steps. The method is exemplified by Chinese Hamster Ovary (CHO) cell expression and subsequent recovery of recombinant His‐tagged carcinoembryonic antigen (CEA); a heavily glycosylated and clinically relevant protein. Despite operating at a high NaCl concentration necessary for IMAC binding, cells remained over 96% viable after passage through the column with host cell proteases and DNA detected at ∼8 U/mL and 2 ng/μL in column flow‐through, respectively. Recovery of His‐tagged CEA from unclarified feed yielded 71% product recovery. This work provides a basis for direct primary capture of fully glycosylated recombinant proteins from unclarified mammalian cell feed streams. Biotechnol. Bioeng. 2016;113: 130–140. © 2015 Wiley Periodicals, Inc. PMID:26174988

  16. IMAC capture of recombinant protein from unclarified mammalian cell feed streams.

    PubMed

    Kinna, Alexander; Tolner, Berend; Rota, Enrique Miranda; Titchener-Hooker, Nigel; Nesbeth, Darren; Chester, Kerry

    2016-01-01

    Fusion-tag affinity chromatography is a key technique in recombinant protein purification. Current methods for protein recovery from mammalian cells are hampered by the need for feed stream clarification. We have developed a method for direct capture using immobilized metal affinity chromatography (IMAC) of hexahistidine (His6) tagged proteins from unclarified mammalian cell feed streams. The process employs radial flow chromatography with 300-500 μm diameter agarose resin beads that allow free passage of cells but capture His-tagged proteins from the feed stream; circumventing expensive and cumbersome centrifugation and/or filtration steps. The method is exemplified by Chinese Hamster Ovary (CHO) cell expression and subsequent recovery of recombinant His-tagged carcinoembryonic antigen (CEA); a heavily glycosylated and clinically relevant protein. Despite operating at a high NaCl concentration necessary for IMAC binding, cells remained over 96% viable after passage through the column with host cell proteases and DNA detected at ∼ 8 U/mL and 2 ng/μL in column flow-through, respectively. Recovery of His-tagged CEA from unclarified feed yielded 71% product recovery. This work provides a basis for direct primary capture of fully glycosylated recombinant proteins from unclarified mammalian cell feed streams.

  17. Liposomes containing recombinant E protein vaccine against duck Tembusu virus in ducks.

    PubMed

    Ma, Tengfei; Liu, Yongxia; Cheng, Jia; Liu, Yanhan; Fan, Wentao; Cheng, Ziqiang; Niu, Xudong; Liu, Jianzhu

    2016-04-27

    To obtain an effective vaccine candidate against duck Tembusu viral (DTMUV) disease which causes egg-drop and great economical loss in the Chinese duck industry, liposome vaccines containing recombinant E protein were prepared and assessed in this study. The recombinant plasmid (PET28a-E) was constructed and transformed into BL21 (DE3) cells to produce E proteins. The recombinant E proteins were purified and entrapped by liposomes through reverse-phase evaporation. Eighty-four cherry valley ducks were randomly divided into seven groups and inoculated intramuscularly at one- or seven-day-old with liposomes-E protein or Freund's adjuvant-E protein vaccine. Blood samples were collected from the first week to the tenth week for serum antibody, plasma for viremia, as well as oropharyngeal and cloacal swabs for virus shedding analyses after being challenged with a 10(2.4) 50% tissue culture infective dose (TCID50) of duck Tembusu virus. Results showed that serum antibody level of the liposomes vaccine was higher than the Freund's adjuvant vaccine, and inoculating twice was superior to once; furthermore, the viremia and virus shedding tests also proved that the liposomes vaccine can provide complete protection against DTMUV challenge. These results demonstrated that the liposomes-E protein vaccine could be used as a potential candidate vaccine to prevent DTMUV infection in ducks.

  18. 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. PMID:25115849

  19. An Efficient Genome-Wide Fusion Partner Screening System for Secretion of Recombinant Proteins in Yeast

    PubMed Central

    Bae, Jung-Hoon; Hyun Sung, Bong; Kim, Hyun-Jin; Park, Soon-Ho; Lim, Kwang-Mook; Kim, Mi-Jin; Lee, Cho-Ryong; Sohn, Jung-Hoon

    2015-01-01

    To produce rarely secreted recombinant proteins in the yeast Saccharomyces cerevisiae, we developed a novel genome-wide optimal translational fusion partner (TFP) screening system that involves recruitment of an optimal secretion signal and fusion partner. A TFP library was constructed from a genomic and truncated cDNA library by using the invertase-based signal sequence trap technique. The efficiency of the system was demonstrated using two rarely secreted proteins, human interleukin (hIL)-2 and hIL-32. Optimal TFPs for secretion of hIL-2 and hIL-32 were easily selected, yielding secretion of these proteins up to hundreds of mg/L. Moreover, numerous uncovered yeast secretion signals and fusion partners were identified, leading to efficient secretion of various recombinant proteins. Selected TFPs were found to be useful for the hypersecretion of other recombinant proteins at yields of up to several g/L. This screening technique could provide new methods for the production of various types of difficult-to-express proteins. PMID:26195161

  20. Immunization against Rumen Methanogenesis by Vaccination with a New Recombinant Protein.

    PubMed

    Zhang, Litai; Huang, Xiaofeng; Xue, Bai; Peng, Quanhui; Wang, Zhisheng; Yan, Tianhai; Wang, Lizhi

    2015-01-01

    Vaccination through recombinant proteins against rumen methanogenesis provides a mitigation approach to reduce enteric methane (CH4) emissions in ruminants. The objective of present study was to evaluate the in vivo efficacy of a new vaccine candidate protein (EhaF) on methanogenesis and microbial population in the rumen of goats. We amplified the gene mru 1407 encoding protein EhaF using fresh rumen fluid samples of mature goats and successfully expressed recombinant protein (EhaF) in Escherichia coli Rosetta. This product was evaluated using 12 mature goats with half for control and other half injected with 400ug/goat the purified recombinant protein in day 1 and two subsequent booster immunizations in day 35 and 49. All measurements were undertaken from 63 to 68 days after the initial vaccination, with CH4 emissions determined using respiration calorimeter chambers. The results showed that the vaccination caused intensive immune responses in serum and saliva, although it had no significant effect on total enteric CH4 emissions and methanogen population in the rumen, when compared with the control goats. However, the vaccination altered the composition of rumen bacteria, especially the abundance of main phylum Firmicutes and genus Prevotella. The results indicate that protein EhaF might not be an effective vaccine to reduce enteric CH4 emissions but our vaccine have potential to influence the rumen ecosystem of goats. PMID:26445479

  1. Expression, Delivery and Function of Insecticidal Proteins Expressed by Recombinant Baculoviruses

    PubMed Central

    Kroemer, Jeremy A.; Bonning, Bryony C.; Harrison, Robert L.

    2015-01-01

    Since the development of methods for inserting and expressing genes in baculoviruses, a line of research has focused on developing recombinant baculoviruses that express insecticidal peptides and proteins. These recombinant viruses have been engineered with the goal of improving their pesticidal potential by shortening the time required for infection to kill or incapacitate insect pests and reducing the quantity of crop damage as a consequence. A wide variety of neurotoxic peptides, proteins that regulate insect physiology, degradative enzymes, and other potentially insecticidal proteins have been evaluated for their capacity to reduce the survival time of baculovirus-infected lepidopteran host larvae. Researchers have investigated the factors involved in the efficient expression and delivery of baculovirus-encoded insecticidal peptides and proteins, with much effort dedicated to identifying ideal promoters for driving transcription and signal peptides that mediate secretion of the expressed target protein. Other factors, particularly translational efficiency of transcripts derived from recombinant insecticidal genes and post-translational folding and processing of insecticidal proteins, remain relatively unexplored. The discovery of RNA interference as a gene-specific regulation mechanism offers a new approach for improvement of baculovirus biopesticidal efficacy through genetic modification. PMID:25609310

  2. Immunization against Rumen Methanogenesis by Vaccination with a New Recombinant Protein

    PubMed Central

    Zhang, Litai; Huang, Xiaofeng; Xue, Bai; Peng, Quanhui; Wang, Zhisheng; Yan, Tianhai; Wang, Lizhi

    2015-01-01

    Vaccination through recombinant proteins against rumen methanogenesis provides a mitigation approach to reduce enteric methane (CH4) emissions in ruminants. The objective of present study was to evaluate the in vivo efficacy of a new vaccine candidate protein (EhaF) on methanogenesis and microbial population in the rumen of goats. We amplified the gene mru 1407 encoding protein EhaF using fresh rumen fluid samples of mature goats and successfully expressed recombinant protein (EhaF) in Escherichia coli Rosetta. This product was evaluated using 12 mature goats with half for control and other half injected with 400ug/goat the purified recombinant protein in day 1 and two subsequent booster immunizations in day 35 and 49. All measurements were undertaken from 63 to 68 days after the initial vaccination, with CH4 emissions determined using respiration calorimeter chambers. The results showed that the vaccination caused intensive immune responses in serum and saliva, although it had no significant effect on total enteric CH4 emissions and methanogen population in the rumen, when compared with the control goats. However, the vaccination altered the composition of rumen bacteria, especially the abundance of main phylum Firmicutes and genus Prevotella. The results indicate that protein EhaF might not be an effective vaccine to reduce enteric CH4 emissions but our vaccine have potential to influence the rumen ecosystem of goats. PMID:26445479

  3. Immunization against Rumen Methanogenesis by Vaccination with a New Recombinant Protein.

    PubMed

    Zhang, Litai; Huang, Xiaofeng; Xue, Bai; Peng, Quanhui; Wang, Zhisheng; Yan, Tianhai; Wang, Lizhi

    2015-01-01

    Vaccination through recombinant proteins against rumen methanogenesis provides a mitigation approach to reduce enteric methane (CH4) emissions in ruminants. The objective of present study was to evaluate the in vivo efficacy of a new vaccine candidate protein (EhaF) on methanogenesis and microbial population in the rumen of goats. We amplified the gene mru 1407 encoding protein EhaF using fresh rumen fluid samples of mature goats and successfully expressed recombinant protein (EhaF) in Escherichia coli Rosetta. This product was evaluated using 12 mature goats with half for control and other half injected with 400ug/goat the purified recombinant protein in day 1 and two subsequent booster immunizations in day 35 and 49. All measurements were undertaken from 63 to 68 days after the initial vaccination, with CH4 emissions determined using respiration calorimeter chambers. The results showed that the vaccination caused intensive immune responses in serum and saliva, although it had no significant effect on total enteric CH4 emissions and methanogen population in the rumen, when compared with the control goats. However, the vaccination altered the composition of rumen bacteria, especially the abundance of main phylum Firmicutes and genus Prevotella. The results indicate that protein EhaF might not be an effective vaccine to reduce enteric CH4 emissions but our vaccine have potential to influence the rumen ecosystem of goats.

  4. Development of a SARS Coronavirus Vaccine from Recombinant Spike Protein Plus Delta Inulin Adjuvant.

    PubMed

    McPherson, Clifton; Chubet, Richard; Holtz, Kathy; Honda-Okubo, Yoshikazu; Barnard, Dale; Cox, Manon; Petrovsky, Nikolai

    2016-01-01

    Given periodic outbreaks of fatal human infections caused by coronaviruses, development of an optimal coronavirus vaccine platform capable of rapid production is an ongoing priority. This chapter describes the use of an insect cell expression system for rapid production of a recombinant vaccine against severe acute respiratory syndrome coronavirus (SARS). Detailed methods are presented for expression, purification, and release testing of SARS recombinant spike protein antigen, followed by adjuvant formulation and animal testing. The methods herein described for rapid development of a highly protective SARS vaccine are equally suited to rapid development of vaccines against other fatal human coronavirus infections, e.g., the MERS coronavirus. PMID:27076136

  5. RIG-I ligand enhances the immunogenicity of recombinant H7HA protein.

    PubMed

    Cao, Weiping; Liepkalns, Justine S; Kamal, Ram P; Reber, Adrian J; Kim, Jin Hyang; Hofstetter, Amelia R; Amoah, Samuel; Stevens, James; Ranjan, Priya; Gangappa, Shivaprakash; York, Ian A; Sambhara, Suryaprakash

    2016-01-01

    Avian H7N9 influenza virus infection with fatal outcomes continues to pose a pandemic threat and highly immunogenic vaccines are urgently needed. In this report we show that baculovirus-derived recombinant H7 hemagglutinin protein, when delivered with RIG-I ligand, induced enhanced antibody and T cell responses and conferred protection against lethal challenge with a homologous H7N9 virus. These findings indicate the potential utility of RIG-I ligands as vaccine adjuvants to increase the immunogenicity of recombinant H7 hemagglutinin.

  6. Development of a SARS Coronavirus Vaccine from Recombinant Spike Protein Plus Delta Inulin Adjuvant.

    PubMed

    McPherson, Clifton; Chubet, Richard; Holtz, Kathy; Honda-Okubo, Yoshikazu; Barnard, Dale; Cox, Manon; Petrovsky, Nikolai

    2016-01-01

    Given periodic outbreaks of fatal human infections caused by coronaviruses, development of an optimal coronavirus vaccine platform capable of rapid production is an ongoing priority. This chapter describes the use of an insect cell expression system for rapid production of a recombinant vaccine against severe acute respiratory syndrome coronavirus (SARS). Detailed methods are presented for expression, purification, and release testing of SARS recombinant spike protein antigen, followed by adjuvant formulation and animal testing. The methods herein described for rapid development of a highly protective SARS vaccine are equally suited to rapid development of vaccines against other fatal human coronavirus infections, e.g., the MERS coronavirus.

  7. Chloroplast-Based Expression of Recombinant Proteins by Gateway® Cloning Technology.

    PubMed

    Gottschamel, Johanna; Lössl, Andreas

    2016-01-01

    Plastid transformation for the expression of recombinant proteins and entire enzymatic pathways has become a promising tool for plant biotechnology in the past decade. Several improvements of the technology have turned plant plastids into robust and dependable expression platforms for multiple high value compounds. In this chapter, we describe our current methodology based on Gateway(®) recombinant cloning, which we have adapted for plastid transformation. We describe the steps required for cloning, biolistic transformation, identification, and regeneration of transplastomic plant lines and Western blot analysis. PMID:26614278

  8. Construction and characterization of a recombinant reticuloendotheliosis virus expressing enhanced green fluorescent protein.

    PubMed

    Deng, Xiaoyun; Hu, Feng; Qi, Xiaole; Gao, Li; Li, Kai; Gao, Honglei; Gao, Yulong; Wang, Yongqiang; Shen, Nan; Hua, Yuping; Wang, Xiaomei

    2015-09-01

    Reticuloendotheliosis virus (REV) causes an immunosuppressive and oncogenic disease in chickens and other birds. In this study, based on an infectious clone of REV, named HLJR0901, a recombinant virus containing the enhanced green fluorescence protein (EGFP) gene was constructed by inserting the EGFP expression cassette downstream of the 3' terminus of the viral env gene. An EGFP-tagged REV that stably expresses EGFP was rescued. This visible recombinant REV could contribute to the further understanding of the molecular mechanism involved in the replication and pathogenicity of REV.

  9. Tunable recombinant protein expression with E. coli in a mixed-feed environment.

    PubMed

    Sagmeister, Patrick; Schimek, Clemens; Meitz, Andrea; Herwig, Christoph; Spadiut, Oliver

    2014-04-01

    Controlling the recombinant protein production rate in Escherichia coli is of utmost importance to ensure product quality and quantity. Up to now, only the genetic construct, introduced into E. coli, and the specific growth rate of the culture were used to influence and stir the productivity. However, bioprocess technological means to control or even tune the productivity of E. coli are scarce. Here, we present a novel method for the process-technological control over the recombinant protein expression rate in E. coli. A mixed-feed fed-batch bioprocess based on the araBAD promoter expression system using both D-glucose and L-arabinose as assimilable C-sources was designed. Using the model product green fluorescent protein, we show that the specific product formation rate can be efficiently tuned even on the cellular level only via the uptake rate of L-arabinose. This novel approach introduces an additional degree of freedom for the design of recombinant bioprocesses with E. coli. We anticipate that the presented method will result in significant quality and robustness improvement as well as cost and process time reduction for recombinant bacterial bioprocesses in the future.

  10. RAC-tagging: Recombineering And Cas9-assisted targeting for protein tagging and conditional analyses

    PubMed Central

    Baker, Oliver; Gupta, Ashish; Obst, Mandy; Zhang, Youming; Anastassiadis, Konstantinos; Fu, Jun; Stewart, A. Francis

    2016-01-01

    A fluent method for gene targeting to establish protein tagged and ligand inducible conditional loss-of-function alleles is described. We couple new recombineering applications for one-step cloning of gRNA oligonucleotides and rapid generation of short-arm (~1 kb) targeting constructs with the power of Cas9-assisted targeting to establish protein tagged alleles in embryonic stem cells at high efficiency. RAC (Recombineering And Cas9)-tagging with Venus, BirM, APEX2 and the auxin degron is facilitated by a recombineering-ready plasmid series that permits the reuse of gene-specific reagents to insert different tags. Here we focus on protein tagging with the auxin degron because it is a ligand-regulated loss-of-function strategy that is rapid and reversible. Furthermore it includes the additional challenge of biallelic targeting. Despite high frequencies of monoallelic RAC-targeting, we found that simultaneous biallelic targeting benefits from long-arm (>4 kb) targeting constructs. Consequently an updated recombineering pipeline for fluent generation of long arm targeting constructs is also presented. PMID:27216209

  11. Lysostaphin: immunogenicity of locally administered recombinant protein used in mastitis therapy.

    PubMed

    Daley, M J; Oldham, E R

    1992-03-01

    A recombinant bactericidal protein, recombinant lysostaphin (r-lysostaphin), that may be useful as an intramammary therapeutic for Staphylococcus aureus mastitis in dairy cattle, was evaluated for immunogenicity to various hosts. Although immunogenicity could be demonstrated in a variety of other species when administered parenterally, oral administration failed to elicit a significant immunological response. Similarly, intramammary infusion of r-lysostaphin failed to elicit significant serum titers in the bovine until 18-21 infusions were administered (total administered dose of 2-3 g of protein). Antibody titers from dairy cattle which did develop an immune response were predominantly of the IgG1 subclass. Dairy cattle with significant anti-lysostaphin titers showed no deleterious symptoms (anaphylaxis, etc.) upon subsequent infusion, and these titers did not effect the in vitro bacteriostatic activity of r-lysostaphin. Intramammary infusion of r-lysostaphin does not elicit any observable effects on the host animal or on the potential efficacy of the recombinant molecule. Intramammary recombinant proteins may be suitable effective and safe infusion products that provide an alternative to classical antibiotic therapy.

  12. RAC-tagging: Recombineering And Cas9-assisted targeting for protein tagging and conditional analyses.

    PubMed

    Baker, Oliver; Gupta, Ashish; Obst, Mandy; Zhang, Youming; Anastassiadis, Konstantinos; Fu, Jun; Stewart, A Francis

    2016-01-01

    A fluent method for gene targeting to establish protein tagged and ligand inducible conditional loss-of-function alleles is described. We couple new recombineering applications for one-step cloning of gRNA oligonucleotides and rapid generation of short-arm (~1 kb) targeting constructs with the power of Cas9-assisted targeting to establish protein tagged alleles in embryonic stem cells at high efficiency. RAC (Recombineering And Cas9)-tagging with Venus, BirM, APEX2 and the auxin degron is facilitated by a recombineering-ready plasmid series that permits the reuse of gene-specific reagents to insert different tags. Here we focus on protein tagging with the auxin degron because it is a ligand-regulated loss-of-function strategy that is rapid and reversible. Furthermore it includes the additional challenge of biallelic targeting. Despite high frequencies of monoallelic RAC-targeting, we found that simultaneous biallelic targeting benefits from long-arm (>4 kb) targeting constructs. Consequently an updated recombineering pipeline for fluent generation of long arm targeting constructs is also presented. PMID:27216209

  13. Construction of green fluorescent protein-tagged recombinant iridovirus to assess viral replication.

    PubMed

    Huang, Youhua; Huang, Xiaohong; Cai, Jia; Ye, Fuzhou; Guan, Liya; Liu, Hong; Qin, Qiwei

    2011-09-01

    Green fluorescent protein-tagged recombinant virus has been successfully applied to observing the infective dynamics and evaluating viral replication. Here, we identified soft-shelled turtle iridovirus (STIV) ORF55 as an envelope protein (VP55), and developed a recombinant STIV expressing an enhanced green fluorescent protein (EGFP) fused to VP55 (EGFP-STIV). Recombinant EGFP-STIV shared similar single-step growth curves and ultrastructural morphology with wild type STIV (wt-STIV). The green fluorescence distribution during EGFP-STIV infection was consistent with the intracellular distribution of VP55 which was mostly co-localized with virus assembly sites. Furthermore, EGFP-STIV could be used to evaluate viral replication conveniently under drug treatment, and the result showed that STIV replication was significantly inhibited after the addition of antioxidant pyrrolidine dithiocarbamate (PDTC). Thus, the EGFP-tagged recombinant iridovirus will not only be useful for further investigations on the viral replicative dynamics, but also provide an alternative simple strategy to screen for antiviral substances.

  14. Essential bacterial helicases that counteract the toxicity of recombination proteins.

    PubMed

    Petit, Marie-Agnès; Ehrlich, Dusko

    2002-06-17

    PcrA, Rep and UvrD are three closely related bacterial helicases with a DExx signature. PcrA is encoded by Gram-positive bacteria and is essential for cell growth. Rep and UvrD are encoded by Gram-negative bacteria, and mutants lacking both helicases are also not viable. To understand the non-viability of the helicase mutants, we characterized spontaneous extragenic suppressors of a Bacillus subtilis pcrA null mutation. Here we report that one of these suppressors maps in recF and that previously isolated mutations in B.subtilis recF, recL, recO and recR, which belong to the same complementation group, all suppress the lethality of a pcrA mutation. Similarly, recF, recO or recR mutations suppress the lethality of the Escherichia coli rep uvrD double mutant. We conclude that RecFOR proteins are toxic in cells devoid of PcrA in Gram-positive bacteria, or Rep and UvrD in Gram-negative bacteria, and propose that the RecFOR proteins interfere with an essential cellular process, possibly replication, when DExx helicases PcrA, or Rep and UvrD are absent. PMID:12065426

  15. Multiplexed expression and screening for recombinant protein production in mammalian cells

    PubMed Central

    Chapple, Susan DJ; Crofts, Anna M; Shadbolt, S Paul; McCafferty, John; Dyson, Michael R

    2006-01-01

    Background A variety of approaches to understanding protein structure and function require production of recombinant protein. Mammalian based expression systems have advantages over bacterial systems for certain classes of protein but can be slower and more laborious. Thus the availability of a simple system for production and rapid screening of constructs or conditions for mammalian expression would be of great benefit. To this end we have coupled an efficient recombinant protein production system based on transient transfection in HEK-293 EBNA1 (HEK-293E) suspension cells with a dot blot method allowing pre-screening of proteins expressed in cells in a high throughput manner. Results A nested PCR approach was used to clone 21 extracellular domains of mouse receptors as CD4 fusions within a mammalian GATEWAY expression vector system. Following transient transfection, HEK-293E cells grown in 2 ml cultures in 24-deep well blocks showed similar growth kinetics, viability and recombinant protein expression profiles, to those grown in 50 ml shake flask cultures as judged by western blotting. Following optimisation, fluorescent dot blot analysis of transfection supernatants was shown to be a rapid method for analysing protein expression yielding similar results as western blot analysis. Addition of urea enhanced the binding of glycoproteins to a nitrocellulose membrane. A good correlation was observed between the results of a plate based small scale transient transfection dot blot pre-screen and successful purification of proteins expressed at the 50 ml scale. Conclusion The combination of small scale multi-well plate culture and dot blotting described here will allow the multiplex analysis of different mammalian expression experiments enabling a faster identification of high yield expression constructs or conditions prior to large scale protein production. The methods for parallel GATEWAY cloning and expression of multiple constructs in cell culture will also be useful

  16. Crystallization and preliminary crystallographic analysis of recombinant immunoglobulin G-binding protein from Streptococcus suis

    SciTech Connect

    Khan, Abdul Hamid; Chu, Fuliang; Feng, Youjun; Zhang, Qinagmin; Qi, Jianxun; Gao, George Fu

    2008-08-01

    Crystallization of recombinant IgG-binding protein expressed in Escherichia coli using the hanging-drop vapour-diffusion method is described. The crystals belonged to space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 38.98, b = 43.94, c = 78.17 Å. Streptococcus suis, an important zoonotic pathogen, expresses immunoglobulin G-binding protein, which is thought to be helpful to the organism in eluding the host defence system. Recombinant IgG-binding protein expressed in Escherichia coli has been crystallized using the hanging-drop vapour-diffusion method. The crystals belonged to space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 38.98, b = 43.94, c = 78.17 Å and one molecule in the asymmetric unit. Diffraction data were collected to 2.60 Å resolution.

  17. Recombinant plant-derived pharmaceutical proteins: current technical and economic bottlenecks.

    PubMed

    Sabalza, Maite; Christou, Paul; Capell, Teresa

    2014-12-01

    Molecular pharming is a cost-effective platform for the production of recombinant proteins in plants. Although the biopharmaceutical industry still relies on a small number of standardized fermentation-based technologies for the production of recombinant proteins there is now a greater awareness of the advantages of molecular pharming particularly in niche markets. Here we discuss some of the technical, economic and regulatory barriers that constrain the clinical development and commercialization of plant-derived pharmaceutical proteins. We also discuss strategies to increase productivity and product quality/homogeneity. The advantages of whole plants should be welcomed by the industry because this will help to reduce the cost of goods and therefore expand the biopharmaceutical market into untapped sectors.

  18. Recombinant plant-derived pharmaceutical proteins: current technical and economic bottlenecks.

    PubMed

    Sabalza, Maite; Christou, Paul; Capell, Teresa

    2014-12-01

    Molecular pharming is a cost-effective platform for the production of recombinant proteins in plants. Although the biopharmaceutical industry still relies on a small number of standardized fermentation-based technologies for the production of recombinant proteins there is now a greater awareness of the advantages of molecular pharming particularly in niche markets. Here we discuss some of the technical, economic and regulatory barriers that constrain the clinical development and commercialization of plant-derived pharmaceutical proteins. We also discuss strategies to increase productivity and product quality/homogeneity. The advantages of whole plants should be welcomed by the industry because this will help to reduce the cost of goods and therefore expand the biopharmaceutical market into untapped sectors. PMID:25048244

  19. Visualization of Iron-Binding Micelles in Acidic Recombinant Biomineralization Protein, MamC

    DOE PAGES

    Kashyap, Sanjay; Woehl, Taylor; Valverde-Tercedor, Carmen; Sánchez-Quesada, Miguel; Jiménez López, Concepción; Prozorov, Tanya

    2014-01-01

    Biological macromolecules are utilized in low-temperature synthetic methods to exert precise control over nanoparticle nucleation and placement. They enable low-temperature formation of a variety of functional nanostructured materials with properties often not achieved via conventional synthetic techniques. Here we report on the in situ visualization of a novel acidic bacterial recombinant protein, MamC, commonly present in the magnetosome membrane of several magnetotactic bacteria, including Magnetococcus marinus , strain MC-1. Our findings provide an insight into the self-assembly of MamC and point to formation of the extended protein surface, which is assumed to play an important role in the formationmore » of biotemplated inorganic nanoparticles. The self-organization of MamC is compared to the behavior of another acidic recombinant iron-binding protein, Mms6.« less

  20. Simultaneous targeting of Requiem & Alg-2 in Chinese hamster ovary cells for improved recombinant protein production.

    PubMed

    Lim, Yiping; Mantalaris, Athanasios; Yap, Miranda G S; Wong, Danny C F

    2010-11-01

    Apoptosis is known to be the main cause of cell death in the bioreactor environment, leading to the loss of recombinant protein productivity. In a previous study, transcriptional profiling was used to identify and target four early apoptosis-signaling genes: FADD, FAIM, Alg-2, and Requiem. The resulting cell lines had increased viable cell numbers and extended culture viability, which translated to increased protein productivity. Combinatorial targeting of two genes simultaneously has previously been shown to be more effective than targeting one gene alone. In this study, we sought to determine if targeting Requiem and Alg-2 was more effective than targeting Requiem alone. We found that targeting Requiem and Alg-2 did not result in extended culture viability, but resulted in an increase in maximum viable cell numbers and cumulative IVCD under fed-batch conditions. This in turn led to an approximately 1.5-fold increase in recombinant protein productivity.

  1. Visualization of Iron-Binding Micelles in Acidic Recombinant Biomineralization Protein, MamC

    SciTech Connect

    Kashyap, Sanjay; Woehl, Taylor; Valverde-Tercedor, Carmen; Sanchez-Quesada, Miguel; Lopez, Concepcion Jimenez; Prozorov, Tanya

    2014-03-07

    Biological macromolecules are utilized in low-temperature synthetic methods to exert precise control over nanoparticle nucleation and placement. They enable low-temperature formation of a variety of functional nanostructured materials with properties often not achieved via conventional synthetic techniques. Here we report on the in situ visualization of a novel acidic bacterial recombinant protein, MamC, commonly present in the magnetosome membrane of several magnetotactic bacteria, including Magnetococcus marinus, strain MC-1. Our findings provide an insight into the self-assembly of MamC and point to formation of the extended protein surface, which is assumed to play an important role in the formation of biotemplated inorganic nanoparticles. The self-organization of MamC is compared to the behavior of another acidic recombinant iron-binding protein, Mms6.

  2. Autonomous induction of recombinant proteins by minimally rewiring native quorum sensing regulon of E. coli.

    PubMed

    Tsao, Chen-Yu; Hooshangi, Sara; Wu, Hsuan-Chen; Valdes, James J; Bentley, William E

    2010-05-01

    Quorum sensing (QS) enables an individual bacterium's metabolic state to be communicated to and ultimately control the phenotype of an emerging population. Harnessing the hierarchical nature of this signal transduction process may enable the exploitation of individual cell characteristics to direct or "program" entire populations of cells. We re-engineered the native QS regulon so that individual cell signals (autoinducers) are used to guide high level expression of recombinant proteins in E. coli populations. Specifically, the autoinducer-2 (AI-2) QS signal initiates and guides the overexpression of green fluorescent protein (GFP), chloramphenicol acetyl transferase (CAT) and beta-galactosidase (LacZ). The new process requires no supervision or input (e.g., sampling for optical density measurement, inducer addition, or medium exchange) and represents a low-cost, high-yield platform for recombinant protein production. Moreover, rewiring a native signal transduction circuit exemplifies an emerging class of metabolic engineering approaches that target regulatory functions. PMID:20060924

  3. [Hepatitis E virus capsid protein production by high cell density culture of recombinant Escherichia coli].

    PubMed

    Liu, Ru-Shi; He, Zhi-Qiang; Li, Shao-Wei; Yang, Kun-Yu; Xian, Yang-Ling; Pang, Shu-Qiang; Zhang, Jun; Li, Yi-Min; Xia, Ning-Shao

    2004-05-01

    Production of Hepatitis E Virus capsid protein by high cell density culture in recombinant E. coli has been studied in 10L and 30L fermentors. The effects of different factors on growth and producing recombinant protein of E. coli have been studied by batch culture, such as different media, the ratio of phosphate and Magnesium sulfate. Comparison of fermentation performance for recombinant E. coli in different fed-methods culture has been investigated by fed-batch culture. The effects of inducing at different stages of growth and time of inducing on growth and producing recombinant protein, also obtained by fed-batch culture. At last, the solubility of inclusion body in different urea concentrations also has been obtained by fed-batch culture. The results show that the concentration of phosphate and Magnesium sulfate in the optimal media is 80mmol/L and 20mmol/L in batch culture respectively, that induction with 1.0mmol/L IPTG at mid log phase (about 45 OD at 600nm) is suitable for growth and recombinant protein expression, the cells were approaching stationary growth phase and the maximum cell OD at 600nm of 80 was achieved in 5h of fed-batch culture, and the expression level is 29.74%. The results also indicate that the solubility of inclusion body in 4mol/L urea solution induced at 37 degrees C reaches 14mg/mL, over 80% inclusion body was resolved. The culture process achieved in 10L fermentor could be successfully scaled up to 30L fenmentor with good reproducibility. PMID:15971623

  4. Liposomes containing recombinant gp85 protein vaccine against ALV-J in chickens.

    PubMed

    Zhang, Limei; Cai, Dongjie; Zhao, Xiaona; Cheng, Ziqiang; Guo, Huijun; Qi, Chunhua; Liu, Jianzhu; Xu, Ruixue; Zhao, Peng; Cui, Zhizhong

    2014-05-01

    To study the potential of liposome vaccines in the clinical prevention of ALV-J, the effect of recombinant gp85 protein of subgroup J avian leukosis virus (ALV-J) entrapped by liposomes in chickens against ALV-J infection was investigated in this paper. A recombinant plasmid (PET28a-gp85) containing the PET28a vector and gp85 gene was constructed and then expressed in Rosetta (DE3) cells with 0.5mM IPTG to produce recombinant gp85 proteins that could be entrapped by liposomes through reverse-phase evaporation. The chickens were inoculated intramuscularly either once or twice with the liposomes or with Freund's adjuvant emulsion containing recombinant gp85 protein. Sixty chickens were raised to one week old for the first inoculation and to three weeks old for the second inoculation. Chickens raised to five weeks old were challenged with a 10(2.4) 50% tissue culture infective dose (TCID50) of ALV-J. Blood samples were collected from each chicken at weekly intervals for serum antibody and viremia analyses. Changes in serum antibodies showed that positive serum antibodies (S/P value >0.6) could be induced in all groups regardless of the frequency of inoculation but improved significantly in the twice-inoculated groups. As well, high levels of antibodies emerged earlier in the Freund's adjuvant groups but persisted longer in the liposome groups. Detection of viremia indicated that the liposomes provide better protection against ALV-J than Freund's adjuvant emulsion and that this protection is directly influenced by serum antibody levels. Overall, this study reveals the potential of liposome vaccines containing recombinant gp85 protein in the clinical prevention of ALV-J.

  5. Recombinant TNF-binding protein from variola virus as a novel potential TNF antagonist.

    PubMed

    Gileva, I P; Nepomnyashchikh, T S; Ryazankin, I A; Shchelkunov, S N

    2009-12-01

    Gel-filtration chromatographic separation of the lysate of Sf21 insect cells infected with recombinant baculovirus BVi67 containing the gene for TNF-binding protein (CrmB) of variola virus (VARV) revealed that hTNF-cytotoxicity neutralization activity is associated with a fraction corresponding mainly to high molecular weight proteins (above 500 kDa) and less with fractions corresponding to proteins of 270 or 90 kDa. The recombinant VARV-CrmB protein has been purified by affinity chromatography. Difference in the experimentally determined and estimated (according to amino acid composition) VARV-CrmB molecular weight is due to glycosylation of the recombinant protein expressed in the insect cells. VARV-CrmB neutralizes in vitro the cytotoxic effect of hTNF and hLTalpha, and its TNF-neutralizing activity is two to three orders of magnitude higher compared to the analogous effects of type I and II soluble TNF receptors, comparable with the activity of mAb MAK195, and somewhat lower than the effect of the commercial drug Remicade.

  6. Reflectometric interference spectroscopy-based immunosensing using immobilized antibody via His-tagged recombinant protein A.

    PubMed

    Choi, Hyung Woo; Sakata, Yasuhiko; Ooya, Tooru; Takeuchi, Toshifumi

    2015-02-01

    The proposed approach demonstrated in this study provides an immunosensing system based on reflectometric interference spectroscopy (RIfS) in combination with an antibody immobilization method using histidine-tagged recombinant protein A. Carboxymethyldextran (CMD) was immobilized on a 3-aminopropyltriethoxysilane-treated a silicon nitride-coated silicon wafer, followed by chelating histidine-tagged recombinant protein A with copper (II) ions. The CMD-layer was found to be advantageous in terms of not only immobilization of histidine-tagged recombinant protein A-mediated an antibody against myoglobin (anti-Myo) but also prevention of non-specific binding of myoglobin. Myoglobin was repeatedly detected, and the apparent detection limit was 0.1 μg mL(-1). The proposed RIfS-based protein sensing system, in conjunction with the easy preparation of silicon-based inexpensive immunosensing chips, is expected to be applicable for label-free optical detection for other proteins in various fields.

  7. Monitoring the centrifugal recovery of recombinant protein inclusion bodies.

    PubMed

    Middelberg, A P; O'Neill, B K

    1991-04-01

    The industrial processing of proteins expressed as insoluble inclusion bodies employs a reasonably standard sequence of unit operations. One of these is centrifugation, which serves to concentrate the inclusion bodies after disruption of the host microorganism, and also separates the inclusion bodies from other cellular debris. Monitoring the performance of the centrifuge is essential if excessive product and hence financial loss is to be avoided and a reasonable separation obtained. The analytical disc centrifuge may be used to monitor the centrifugation. This instrument returns the sample size distribution with high resolution and without fouling. By obtaining size distributions of the centrifuge feed, supernatant and concentrate, the fractional collection efficiency of the centrifuge may be determined as a function of the Stokes diameter, and a mass balance constructed. PMID:1367325

  8. Nanopatterning of recombinant proteins and viruses using block copolymer templates

    NASA Astrophysics Data System (ADS)

    Cresce, Arthur Von Wald

    The study of interfaces is important in understanding biological interactions, including cellular signaling and virus infection. This thesis is an original effort to examine the interaction between a block copolymer and both a protein and a virus. Block copolymers intrinsically form nanometer-scale structures over large areas without expensive processing, making them ideal for the synthesis of the nanopatterned surfaces used in this study. The geometry of these nanostructures can be easily tuned for different applications by altering the block ratio and composition of the block copolymer. Block copolymers can be used for controlled uptake of metal ions, where one block selectively binds metal ions while the other does not. 5-norbornene-2,3-dicarboxylic acid is synthesized through ring-opening metathesis polymerization. It formed spherical domains with spheres approximately 30 nm in diameter, and these spheres were then subsequently loaded with nickel ion. This norbornene block copolymer was tested for its ability to bind histidine-tagged green fluorescent protein (hisGFP), and it was found that the nickel-loaded copolymer was able to retain hisGFP through chelation between the histidine tag and the metal-containing portions of the copolymer surface. Poly(styrene-b-4-vinylpyridine) (PS/P4VP) was also loaded with nickel, forming a cylindrical microstructure. The binding of Tobacco mosaic virus and Tobacco necrosis virus was tested through Tween 20 detergent washes. Electron microscopy allowed for observation of both block copolymer nanostructures and virus particles. Results showed that Tween washes could not remove bound Tobacco mosaic virus from the surface of PS/P4VP. It was also seen that the size and tunability of block copolymers and the lack of processing needed to attain different structures makes them attractive for many applications, including microfluidic devices, surfaces to influence cellular signaling and growth, and as a nanopatterning surface for

  9. Protection against myxomatosis and rabbit viral hemorrhagic disease with recombinant myxoma viruses expressing rabbit hemorrhagic disease virus capsid protein.

    PubMed Central

    Bertagnoli, S; Gelfi, J; Le Gall, G; Boilletot, E; Vautherot, J F; Rasschaert, D; Laurent, S; Petit, F; Boucraut-Baralon, C; Milon, A

    1996-01-01

    Two myxoma virus-rabbit hemorrhagic disease virus (RHDV) recombinant viruses were constructed with the SG33 strain of myxoma virus to protect rabbits against myxomatosis and rabbit viral hemorrhagic disease. These recombinant viruses expressed the RHDV capsid protein (VP60). The recombinant protein, which is 60 kDa in size, was antigenic, as revealed by its reaction in immunoprecipitation with antibodies raised against RHDV. Both recombinant viruses induced high levels of RHDV- and myxoma virus-specific antibodies in rabbits after immunization. Inoculations by the intradermal route protected animals against virulent RHDV and myxoma virus challenges. PMID:8764013

  10. Construction of a recombinant herpesvirus expressing the jellyfish green fluorescent protein.

    PubMed

    Boldogköi, Z; Erdélyi, F; Sik, A; Freund, T F; Fodor, I

    1999-01-01

    Here we report the insertion of a synthetic version of the cDNA encoding the jellyfish (Aequorea victoria) green fluorescent protein (gfph ) into the genome of pseudorabies (Aujeszky's disease) virus (PrV). A putative latency promoter (PLAT) located at the inverted repeat region of the PrV genome was chosen as the target site for the insertion. Recombinant viral DNA designated as vLAT-gfp was generated as a result of homologous recombination between the transfected viral DNA and a plasmid containing the GFP-expression cassette flanked by viral sequences homologous to the target region. Plaques containing recombinant virus were selected visually using a fluorescent microscope. We demonstrated a GFP-expression in infected neurons of rat brain which showed normal morphology at early stage of viral infection by monitoring fluorescent light emission. PMID:10398563

  11. Making recombinant proteins in filamentous fungi- are we expecting too much?

    PubMed

    Nevalainen, Helena; Peterson, Robyn

    2014-01-01

    Hosts used for the production of recombinant proteins are typically high-protein secreting mutant strains that have been selected for a specific purpose, such as efficient production of cellulose-degrading enzymes. Somewhat surprisingly, sequencing of the genomes of a series of mutant strains of the cellulolytic Trichoderma reesei, widely used as an expression host for recombinant gene products, has shed very little light on the nature of changes that boost high-level protein secretion. While it is generally agreed and shown that protein secretion in filamentous fungi occurs mainly through the hyphal tip, there is growing evidence that secretion of proteins also takes place in sub-apical regions. Attempts to increase correct folding and thereby the yields of heterologous proteins in fungal hosts by co-expression of cellular chaperones and foldases have resulted in variable success; underlying reasons have been explored mainly at the transcriptional level. The observed physiological changes in fungal strains experiencing increasing stress through protein overexpression under strong gene promoters also reflect the challenge the host organisms are experiencing. It is evident, that as with other eukaryotes, fungal endoplasmic reticulum is a highly dynamic structure. Considering the above, there is an emerging body of work exploring the use of weaker expression promoters to avoid undue stress. Filamentous fungi have been hailed as candidates for the production of pharmaceutically relevant proteins for therapeutic use. One of the biggest challenges in terms of fungally produced heterologous gene products is their mode of glycosylation; fungi lack the functionally important terminal sialylation of the glycans that occurs in mammalian cells. Finally, exploration of the metabolic pathways and fluxes together with the development of sophisticated fermentation protocols may result in new strategies to produce recombinant proteins in filamentous fungi.

  12. A liver stress-endocrine nexus promotes metabolic integrity during dietary protein dilution.

    PubMed

    Maida, Adriano; Zota, Annika; Sjøberg, Kim A; Schumacher, Jonas; Sijmonsma, Tjeerd P; Pfenninger, Anja; Christensen, Marie M; Gantert, Thomas; Fuhrmeister, Jessica; Rothermel, Ulrike; Schmoll, Dieter; Heikenwälder, Mathias; Iovanna, Juan L; Stemmer, Kerstin; Kiens, Bente; Herzig, Stephan; Rose, Adam J

    2016-09-01

    Dietary protein intake is linked to an increased incidence of type 2 diabetes (T2D). Although dietary protein dilution (DPD) can slow the progression of some aging-related disorders, whether this strategy affects the development and risk for obesity-associated metabolic disease such as T2D is unclear. Here, we determined that DPD in mice and humans increases serum markers of metabolic health. In lean mice, DPD promoted metabolic inefficiency by increasing carbohydrate and fat oxidation. In nutritional and polygenic murine models of obesity, DPD prevented and curtailed the development of impaired glucose homeostasis independently of obesity and food intake. DPD-mediated metabolic inefficiency and improvement of glucose homeostasis were independent of uncoupling protein 1 (UCP1), but required expression of liver-derived fibroblast growth factor 21 (FGF21) in both lean and obese mice. FGF21 expression and secretion as well as the associated metabolic remodeling induced by DPD also required induction of liver-integrated stress response-driven nuclear protein 1 (NUPR1). Insufficiency of select nonessential amino acids (NEAAs) was necessary and adequate for NUPR1 and subsequent FGF21 induction and secretion in hepatocytes in vitro and in vivo. Taken together, these data indicate that DPD promotes improved glucose homeostasis through an NEAA insufficiency-induced liver NUPR1/FGF21 axis. PMID:27548521

  13. Polar Fixation of Plasmids during Recombinant Protein Production in Bacillus megaterium Results in Population Heterogeneity.

    PubMed

    Münch, Karin M; Müller, Johannes; Wienecke, Sarah; Bergmann, Simone; Heyber, Steffi; Biedendieck, Rebekka; Münch, Richard; Jahn, Dieter

    2015-09-01

    During the past 2 decades, Bacillus megaterium has been systematically developed for the gram-per-liter scale production of recombinant proteins. The plasmid-based expression systems employed use a xylose-controlled promoter. Protein production analyses at the single-cell level using green fluorescent protein as a model product revealed cell culture heterogeneity characterized by a significant proportion of less productive bacteria. Due to the enormous size of B. megaterium, such bistable behavior seen in subpopulations was readily analyzed by time lapse microscopy and flow cytometry. Cell culture heterogeneity was not caused simply by plasmid loss: instead, an asymmetric distribution of plasmids during cell division was detected during the exponential-growth phase. Multicopy plasmids are generally randomly distributed between daughter cells. However, in vivo and in vitro experiments demonstrated that under conditions of strong protein production, plasmids are retained at one of the cell poles. Furthermore, it was found that cells with accumulated plasmids and high protein production ceased cell division. As a consequence, the overall protein production of the culture was achieved mainly by the subpopulation with a sufficient plasmid copy number. Based on our experimental data, we propose a model whereby the distribution of multicopy plasmids is controlled by polar fixation under protein production conditions. Thereby, cell lines with fluctuating plasmid abundance arise, which results in population heterogeneity. Our results provide initial insights into the mechanism of cellular heterogeneity during plasmid-based recombinant protein production in a Bacillus species. PMID:26116677

  14. Polar Fixation of Plasmids during Recombinant Protein Production in Bacillus megaterium Results in Population Heterogeneity

    PubMed Central

    Münch, Karin M.; Müller, Johannes; Wienecke, Sarah; Bergmann, Simone; Heyber, Steffi; Biedendieck, Rebekka; Jahn, Dieter

    2015-01-01

    During the past 2 decades, Bacillus megaterium has been systematically developed for the gram-per-liter scale production of recombinant proteins. The plasmid-based expression systems employed use a xylose-controlled promoter. Protein production analyses at the single-cell level using green fluorescent protein as a model product revealed cell culture heterogeneity characterized by a significant proportion of less productive bacteria. Due to the enormous size of B. megaterium, such bistable behavior seen in subpopulations was readily analyzed by time lapse microscopy and flow cytometry. Cell culture heterogeneity was not caused simply by plasmid loss: instead, an asymmetric distribution of plasmids during cell division was detected during the exponential-growth phase. Multicopy plasmids are generally randomly distributed between daughter cells. However, in vivo and in vitro experiments demonstrated that under conditions of strong protein production, plasmids are retained at one of the cell poles. Furthermore, it was found that cells with accumulated plasmids and high protein production ceased cell division. As a consequence, the overall protein production of the culture was achieved mainly by the subpopulation with a sufficient plasmid copy number. Based on our experimental data, we propose a model whereby the distribution of multicopy plasmids is controlled by polar fixation under protein production conditions. Thereby, cell lines with fluctuating plasmid abundance arise, which results in population heterogeneity. Our results provide initial insights into the mechanism of cellular heterogeneity during plasmid-based recombinant protein production in a Bacillus species. PMID:26116677

  15. Polar Fixation of Plasmids during Recombinant Protein Production in Bacillus megaterium Results in Population Heterogeneity.

    PubMed

    Münch, Karin M; Müller, Johannes; Wienecke, Sarah; Bergmann, Simone; Heyber, Steffi; Biedendieck, Rebekka; Münch, Richard; Jahn, Dieter

    2015-09-01

    During the past 2 decades, Bacillus megaterium has been systematically developed for the gram-per-liter scale production of recombinant proteins. The plasmid-based expression systems employed use a xylose-controlled promoter. Protein production analyses at the single-cell level using green fluorescent protein as a model product revealed cell culture heterogeneity characterized by a significant proportion of less productive bacteria. Due to the enormous size of B. megaterium, such bistable behavior seen in subpopulations was readily analyzed by time lapse microscopy and flow cytometry. Cell culture heterogeneity was not caused simply by plasmid loss: instead, an asymmetric distribution of plasmids during cell division was detected during the exponential-growth phase. Multicopy plasmids are generally randomly distributed between daughter cells. However, in vivo and in vitro experiments demonstrated that under conditions of strong protein production, plasmids are retained at one of the cell poles. Furthermore, it was found that cells with accumulated plasmids and high protein production ceased cell division. As a consequence, the overall protein production of the culture was achieved mainly by the subpopulation with a sufficient plasmid copy number. Based on our experimental data, we propose a model whereby the distribution of multicopy plasmids is controlled by polar fixation under protein production conditions. Thereby, cell lines with fluctuating plasmid abundance arise, which results in population heterogeneity. Our results provide initial insights into the mechanism of cellular heterogeneity during plasmid-based recombinant protein production in a Bacillus species.

  16. Ca2+ activates human homologous recombination protein Rad51 by modulating its ATPase activity

    PubMed Central

    Bugreev, Dmitry V.; Mazin, Alexander V.

    2004-01-01

    Human Rad51 (hRad51) protein plays a key role in homologous recombination and DNA repair. hRad51 protein forms a helical filament on single-stranded DNA (ssDNA), which performs the basic steps of homologous recombination: a search for homologous double-stranded DNA (dsDNA) and DNA strand exchange. hRad51 protein possesses DNA-dependent ATPase activity; however, the role of this activity has not been understood. Our current results show that Ca2+ greatly stimulates DNA strand exchange activity of hRad51 protein. We found that Ca2+ exerts its stimulatory effect by modulating the ATPase activity of hRad51 protein. Our data demonstrate that, in the presence of Mg2+, the hRad51-ATP-ssDNA filament is quickly converted to an inactive hRad51-ADP-ssDNA form, due to relatively rapid ATP hydrolysis and slow dissociation of ADP. Ca2+ maintains the active hRad51-ATP-ssDNA filament by reducing the ATP hydrolysis rate. These findings demonstrate a crucial role of the ATPase activity in regulation of DNA strand exchange activity of hRad51 protein. This mechanism of Rad51 protein regulation by modulating its ATPase activity is evolutionarily recent; we found no such mechanism for yeast Rad51 (yRad51) protein. PMID:15226506

  17. Substrate oscillations boost recombinant protein release from Escherichia coli.

    PubMed

    Jazini, Mohammadhadi; Herwig, Christoph

    2014-05-01

    Intracellular production of recombinant proteins in prokaryotes necessitates subsequent disruption of cells for protein recovery. Since the cell disruption and subsequent purification steps largely contribute to the total production cost, scalable tools for protein release into the extracellular space is of utmost importance. Although there are several ways for enhancing protein release, changing culture conditions is rather a simple and scalable approach compared to, for example, molecular cell design. This contribution aimed at quantitatively studying process technological means to boost protein release of a periplasmatic recombinant protein (alkaline phosphatase) from E. coli. Quantitative analysis of protein in independent bioreactor runs could demonstrate that a defined oscillatory feeding profile was found to improve protein release, about 60 %, compared to the conventional constant feeding rate. The process technology included an oscillatory post-induction feed profile with the frequency of 4 min. The feed rate was oscillated triangularly between a maximum (1.3-fold of the maximum feed rate achieved at the end of the fed-batch phase) and a minimum (45 % of the maximum). The significant improvement indicates the potential to maximize the production rate, while this oscillatory feed profile can be easily scaled to industrial processes. Moreover, quantitative analysis of the primary metabolism revealed that the carbon dioxide yield can be used to identify the preferred feeding profile. This approach is therefore in line with the initiative of process analytical technology for science-based process understanding in process development and process control strategies.

  18. A systematic investigation of production of synthetic prions from recombinant prion protein.

    PubMed

    Schmidt, Christian; Fizet, Jeremie; Properzi, Francesca; Batchelor, Mark; Sandberg, Malin K; Edgeworth, Julie A; Afran, Louise; Ho, Sammy; Badhan, Anjna; Klier, Steffi; Linehan, Jacqueline M; Brandner, Sebastian; Hosszu, Laszlo L P; Tattum, M Howard; Jat, Parmjit; Clarke, Anthony R; Klöhn, Peter C; Wadsworth, Jonathan D F; Jackson, Graham S; Collinge, John

    2015-12-01

    According to the protein-only hypothesis, infectious mammalian prions, which exist as distinct strains with discrete biological properties, consist of multichain assemblies of misfolded cellular prion protein (PrP). A critical test would be to produce prion strains synthetically from defined components. Crucially, high-titre 'synthetic' prions could then be used to determine the structural basis of infectivity and strain diversity at the atomic level. While there have been multiple reports of production of prions from bacterially expressed recombinant PrP using various methods, systematic production of high-titre material in a form suitable for structural analysis remains a key goal. Here, we report a novel high-throughput strategy for exploring a matrix of conditions, additives and potential cofactors that might generate high-titre prions from recombinant mouse PrP, with screening for infectivity using a sensitive automated cell-based bioassay. Overall, approximately 20,000 unique conditions were examined. While some resulted in apparently infected cell cultures, this was transient and not reproducible. We also adapted published methods that reported production of synthetic prions from recombinant hamster PrP, but again did not find evidence of significant infectious titre when using recombinant mouse PrP as substrate. Collectively, our findings are consistent with the formation of prion infectivity from recombinant mouse PrP being a rare stochastic event and we conclude that systematic generation of prions from recombinant PrP may only become possible once the detailed structure of authentic ex vivo prions is solved.

  19. Biologically produced bifunctional recombinant protein nanoparticles for immunoassays.

    PubMed

    Jääskeläinen, Anu; Harinen, Reija-Riitta; Soukka, Tero; Lamminmäki, Urpo; Korpimäki, Teemu; Virta, Marko

    2008-02-01

    Nanoparticles are increasingly used as labels for analytical purposes. In general, nanoparticles need to be functionalized with binding molecules (mostly antibodies or fragments thereof) and label substances using a multistep process that requires several manufacturing and purification steps. Here, we present a biological method of producing functionalized nanoparticles for effective use as label agents in a bioaffinity assay. The particles are based on the globular protein shell of human ferritin. A single chain Fv fragment (scFv) of an antibody is used as the binding moiety and Eu3+ ions as the label substance. Conventional chemical conjugation of the particle and antibody fragment is replaced with genetic fusion between the ferritin subunit and scFv genes. The material, for example, the fusion construct is produced in a single bacterial culture as insoluble forms that are easily purified by centrifugations. The subunits are solubilized and self-assembled, and label ions are introduced by shifting the pH. The functionality of these particles is demonstrated with a bioaffinity assay. This method of producing nanoparticles with inherent antigen binding activity presents several possibilities for the simple production of specific, functional nanoparticles. Production is fast, economical, and environmentally sustainable, making the system advantageous, particularly in applications requiring large quantities of specific nanoparticles. PMID:18179181

  20. Protection against Asiatic Taenia solium induced by a recombinant 45W-4B protein.

    PubMed

    Luo, Xuenong; Zheng, Yadong; Hou, Junling; Zhang, Shaohua; Cai, Xuepeng

    2009-02-01

    Taenia solium is a great threat not only to human health but also to the pig-raising industry. Oncospheral stage-specific 45W proteins are good candidates for the development of anticysticercosis vaccines. In this study, a recombinant 45W-4B protein was highly produced and used for vaccination. Two animal trials resulted in a significant reduction in parasite burden induced by the definite protein against Asiatic T. solium, up to 97.0% and 98.4%, respectively. These provide informative results for the development of effective 45W-4B vaccines against cysticercosis caused by both Chinese and Mexican T. solium isolates and even by other isolates.

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

    PubMed Central

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

    2013-01-01

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

  2. Environment Control to Improve Recombinant Protein Yields in Plants Based on Agrobacterium-Mediated Transient Gene Expression.

    PubMed

    Fujiuchi, Naomichi; Matoba, Nobuyuki; Matsuda, Ryo

    2016-01-01

    Agrobacterium-mediated transient expression systems enable plants to produce a wide range of recombinant proteins on a rapid timescale. To achieve economically feasible upstream production and downstream processing, two yield parameters should be considered: (1) recombinant protein content per unit biomass and (2) recombinant protein productivity per unit area-time at the end of the upstream production. Because environmental factors in the upstream production have impacts on these parameters, environment control is important to maximize the recombinant protein yield. In this review, we summarize the effects of pre- and postinoculation environmental factors in the upstream production on the yield parameters and discuss the basic concept of environment control for plant-based transient expression systems. Preinoculation environmental factors associated with planting density, light quality, and nutrient supply affect plant characteristics, such as biomass and morphology, which in turn affect recombinant protein content and productivity. Accordingly, environment control for such plant characteristics has significant implications to achieve a high yield. On the other hand, postinoculation environmental factors, such as temperature, light intensity, and humidity, have been shown to affect recombinant protein content. Considering that recombinant protein production in Agrobacterium-mediated transient expression systems is a result of a series of complex biological events starting from T-DNA transfer from Agrobacterium tumefaciens to protein biosynthesis and accumulation in leaf tissue, we propose that dynamic environment control during the postinoculation process, i.e., changing environmental conditions at an appropriate timing for each event, may be a promising approach to obtain a high yield. Detailed descriptions of plant growth conditions and careful examination of environmental effects will significantly contribute to our knowledge to stably obtain high recombinant

  3. Environment Control to Improve Recombinant Protein Yields in Plants Based on Agrobacterium-Mediated Transient Gene Expression

    PubMed Central

    Fujiuchi, Naomichi; Matoba, Nobuyuki; Matsuda, Ryo

    2016-01-01

    Agrobacterium-mediated transient expression systems enable plants to produce a wide range of recombinant proteins on a rapid timescale. To achieve economically feasible upstream production and downstream processing, two yield parameters should be considered: (1) recombinant protein content per unit biomass and (2) recombinant protein productivity per unit area–time at the end of the upstream production. Because environmental factors in the upstream production have impacts on these parameters, environment control is important to maximize the recombinant protein yield. In this review, we summarize the effects of pre- and postinoculation environmental factors in the upstream production on the yield parameters and discuss the basic concept of environment control for plant-based transient expression systems. Preinoculation environmental factors associated with planting density, light quality, and nutrient supply affect plant characteristics, such as biomass and morphology, which in turn affect recombinant protein content and productivity. Accordingly, environment control for such plant characteristics has significant implications to achieve a high yield. On the other hand, postinoculation environmental factors, such as temperature, light intensity, and humidity, have been shown to affect recombinant protein content. Considering that recombinant protein production in Agrobacterium-mediated transient expression systems is a result of a series of complex biological events starting from T-DNA transfer from Agrobacterium tumefaciens to protein biosynthesis and accumulation in leaf tissue, we propose that dynamic environment control during the postinoculation process, i.e., changing environmental conditions at an appropriate timing for each event, may be a promising approach to obtain a high yield. Detailed descriptions of plant growth conditions and careful examination of environmental effects will significantly contribute to our knowledge to stably obtain high recombinant

  4. Recombinant Protein-Stabilized Monodisperse Microbubbles with Tunable Size Using a Valve-Based Microfluidic Device

    PubMed Central

    2015-01-01

    Microbubbles are used as contrast enhancing agents in ultrasound sonography and more recently have shown great potential as theranostic agents that enable both diagnostics and therapy. Conventional production methods lead to highly polydisperse microbubbles, which compromise the effectiveness of ultrasound imaging and therapy. Stabilizing microbubbles with surfactant molecules that can impart functionality and properties that are desirable for specific applications would enhance the utility of microbubbles. Here we generate monodisperse microbubbles with a large potential for functionalization by combining a microfluidic method and recombinant protein technology. Our microfluidic device uses an air-actuated membrane valve that enables production of monodisperse microbubbles with narrow size distribution. The size of microbubbles can be precisely tuned by dynamically changing the dimension of the channel using the valve. The microbubbles are stabilized by an amphiphilic protein, oleosin, which provides versatility in controlling the functionalization of microbubbles through recombinant biotechnology. We show that it is critical to control the composition of the stabilizing agents to enable formation of highly stable and monodisperse microbubbles that are echogenic under ultrasound insonation. Our protein-shelled microbubbles based on the combination of microfluidic generation and recombinant protein technology provide a promising platform for ultrasound-related applications. PMID:25265041

  5. Comparison of Immunoprotection of Leptospira Recombinant Proteins with conventional vaccine in experimental animals.

    PubMed

    Parthiban, M; Kumar, S Senthil; Balachandran, C; Kumanan, K; Aarthi, K S; Nireesha, G

    2015-12-01

    Leptospirosis is a bacterial disease caused by bacteria of the genus Leptospira affecting humans and animals. Untreated leptospirosis may result in severe kidney damage, meningitis, liver failure, respiratory distress, and even death. Virulent leptospirosis can rapidly enter kidney fibroblasts and induce a programmed cell death. Thus, it is a challenge for immunologists to develop an effective and safe leptospirosis vaccine. Here, we compared the commercial canine leptospira vaccine and recombinant proteins (OmpL1 and LipL41) with and without adjuvant in terms of immune response and challenge studies in hamsters and immune response studies alone in experimental dogs. The outer membrane proteins viz., lipL41 and OmpL1 of leptospira interrogans serovars icterohaemorrhagiae were amplified. The primers were designed in such a way that amplified products of OmpL1 and lipL41 were ligated and cloned simultaneously into a single vector. The cloned products were expressed in E. coli BL21 cells. The immunoprotection studies were conducted for both recombinant proteins and commercial vaccine. The challenge experiment studies revealed that combination of both rLip41 and rOmpL1 and commercial vaccine gave 83% and 87% protection, respectively. Histopathological investigation revealed mild sub lethal changes were noticed in liver and kidney in commercially vaccinated group alone. The immune responses against recombinant leptospiral proteins were also demonstrated in dogs. PMID:26742322

  6. Overexpression of microRNAs enhances recombinant protein production in Chinese hamster ovary cells.

    PubMed

    Loh, Wan Ping; Loo, Bernard; Zhou, Lihan; Zhang, Peiqing; Lee, Dong-Yup; Yang, Yuansheng; Lam, Kong Peng

    2014-09-01

    MicroRNAs (miRNAs) are short, non-coding RNAs that can negatively regulate expression of multiple genes at post-transcriptional levels. Using miRNAs to target multiple genes and pathways is a promising cell-engineering strategy to increase recombinant protein production in mammalian cells. Here, we identified miRs-17, -19b, -20a, and -92a to be differentially expressed between high- and low- monoclonal antibody-producing Chinese hamster ovary (CHO) cell clones using next-generation sequencing and quantitative real-time PCR. These miRNAs were stably overexpressed individually and in combination in a high-producing clone to assess their effects on CHO cell growth, recombinant protein productivity and product quality. Stably transfected pools demonstrated 24-34% increases in specific productivity (qP) and 21-31% increases in titer relative to the parental clone, without significant alterations in proliferation rates. The highest protein-producing clones isolated from these pools exhibited 130-140% increases in qP and titer compared to the parental clone, without major changes in product aggregation and N-glycosylation profile. From our clonal data, correlations between enhanced qP/titer and increased levels of miRs-17, -19b, and -92a were observed. Our results demonstrate the potential of miRs-17, -19b, and -92a as cell-engineering targets to increase recombinant protein production in mammalian cells. PMID:24819042

  7. Removal of N-terminal methionine from recombinant proteins by engineered E. coli methionine aminopeptidase

    PubMed Central

    Liao, You-Di; Jeng, Jen-Chong; Wang, Chiu-Feng; Wang, Sui-Chi; Chang, Shu-Ting

    2004-01-01

    The removal of N-terminal translation initiator Met by methionine aminopeptidase (MetAP) is often crucial for the function and stability of proteins. On the basis of crystal structure and sequence alignment of MetAPs, we have engineered Escherichia coli MetAP by the mutation of three residues, Y168G, M206T, Q233G, in the substrate-binding pocket. Our engineered MetAPs are able to remove the Met from bulky or acidic penultimate residues, such as Met, His, Asp, Asn, Glu, Gln, Leu, Ile, Tyr, and Trp, as well as from small residues. The penultimate residue, the second residue after Met, was further removed if the antepenultimate residue, the third residue after Met, was small. By the coexpression of engineered MetAP in E. coli through the same or a separate vector, we have successfully produced recombinant proteins possessing an innate N terminus, such as onconase, an antitumor ribonuclease from the frog Rana pipiens. The N-terminal pyroglutamate of recombinant onconase is critical for its structural integrity, catalytic activity, and cyto-toxicity. On the basis of N-terminal sequence information in the protein database, 85%–90% of recombinant proteins should be produced in authentic form by our engineered MetAPs. PMID:15215523

  8. Theoretical and experimental investigation of chaperone effects on soluble recombinant proteins in Escherichia coli: effect of free DnaK level on temperature-induced recombinant streptokinase production.

    PubMed

    Balagurunathan, Balaji; Jayaraman, Guhan

    2008-06-01

    Modeling and analysis of genetic networks have become increasingly important in the investigation of cellular processes. The genetic networks involved in cellular stress response can have a critical effect on the productivity of recombinant proteins. In this work, it was found that the temperature-inducible expression system for the production of soluble recombinant streptokinase in Escherichia coli resulted in a lower productivity compared to the chemically-induced system. To investigate the effect of the induced cellular response due to temperature up-shift a model-based approach is adopted. The role played by the major molecular chaperone teams DnaK-DnaJ-GrpE and GroEL-GroES on the productivity of recombinant streptokinase was experimentally determined. Based on these investigations, a detailed mechanistic mathematical model was developed for the cellular response during the temperature-induced recombinant streptokinase production. The model simulations were found to have a good qualitative agreement with the experimental results. The mechanistic mathematical model was validated with the experiments conducted on a sigma(32) mutant strain. Detailed analysis of the parameter sensitivities of the model indicated that the level of free DnaK chaperone in the cell has the major effect on the productivity of recombinant streptokinase during temperature induction. Analysis of the model simulations also shows that down regulation or selective redirection of the heat shock proteins could be a better way of manipulating the cellular stress response than overexpression or deletion. In other words, manipulating the system properties resulting from the interaction of the components is better than manipulating the individual components. Although our results are specific to a recombinant protein (streptokinase) and the expression system (E. coli), we believe that such a systems-biological approach has several advantages over conventional experimental approaches and could be in

  9. 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.

  10. Immunotherapeutic potential of recombinant ESAT-6 protein in mouse model of experimental tuberculosis.

    PubMed

    Mir, Shabir Ahmad; Verma, Indu; Sharma, Sadhna

    2014-01-01

    Recent understanding of the pathogenesis of tuberculosis allows the possible application of immunotherapy for the treatment of tuberculosis. Therapies that would upregulate the host antimycobacterial immune response and/or attenuate T-cell suppressive and macrophage-deactivating cytokines may prove to be useful in the treatment of tuberculosis. ESAT6, 6-kDa early secreted antigenic target, is a potent protective antigen and is considered as major target for long-lived memory cells. In the present study the immunotherapeutic potential of ESAT-6 has been evaluated in mouse model of experimental tuberculosis. In the present study the ESAT-6 protein was cloned in Escherichia coli using pET23a(+) plasmid and purified by Ni(2+)-NTA chromatography. Further, the immunotherapeutic potential of the recombinant ESAT-6 (in terms of CFU enumeration in the target organs and histopathological analysis of lungs) was evaluated against experimental tuberculosis. The recombinant ESAT-6 with C-terminal histidine-tag and free N-terminus mimics the natural form of ESAT-6 has been successfully cloned and purified. The recombinant ESAT-6 protein adjuvanted with dimethyl dioctadecylammonium bromide (DDA) moderately reduced the bacterial load in the target organs of infected mice. Further, the formulation (ESAT-6-DDA) was able to act synergistically when given in combination with antituberculosis drugs. This recombinant ESAT-6 showed good immunotherapeutic potential against experimental tuberculosis and can be used as an adjunct to the conventional antituberculosis chemotherapy. PMID:24345702

  11. Murine T-cell response to native and recombinant protein antigens of Rickettsia tsutsugamushi.

    PubMed Central

    Hickman, C J; Stover, C K; Joseph, S W; Oaks, E V

    1993-01-01

    A polyclonal T-cell line with TH1 characteristics was used to assess the murine cellular immune response to native and recombinant Rickettsia tsutsugamushi antigens. Proliferation of this T-cell line was observed in response to numerous native antigen fractions, which indicates that the murine T-helper-cell response is directed at multiple scrub typhus antigens with no apparent antigenic immunodominance. Subsequent analysis of recombinant R. tsutsugamushi antigens made it possible to identify a 47-kDa scrub typhus antigen (Sta47) that was stimulatory for the polyclonal T-cell line. Recombinant clones encoding 56-, 58-, and 110-kDa antigens (Sta56, Sta58, and Sta110, respectively) were unable to induce proliferation of this T-cell line. DNA sequence analysis of the cloned rickettsial insert encoding the Sta47 protein revealed the presence of four open reading frames potentially encoding proteins of 47, 30, 18, and 13 kDa. Analysis of sodium dodecyl sulfate-polyacrylamide gel electrophoresis-separated and eluted fractions of lysates from the recombinant HB101(pRTS47B4.3) demonstrated that the fractions containing the 47-kDa protein as well as those containing proteins less than 18 kDa were stimulatory. Selected synthetic amphipathic peptides derived from the Sta47 antigen sequence identified a 20-amino-acid peptide that gave a 10-fold increase in T-cell proliferation over a control malarial peptide of similar length. Recognition of the 47-kDa antigen by a T-cell line with TH1 characteristics implicates this protein as one of potential importance in protection studies and future vaccine development. Images PMID:8478055

  12. Engineering formation of multiple recombinant Eut protein nanocompartments in E. coli

    PubMed Central

    Held, Mark; Kolb, Alexander; Perdue, Sarah; Hsu, Szu-Yi; Bloch, Sarah E.; Quin, Maureen B.; Schmidt-Dannert, Claudia

    2016-01-01

    Compartmentalization of designed metabolic pathways within protein based nanocompartments has the potential to increase reaction efficiency in multi-step biosynthetic reactions. We previously demonstrated proof-of-concept of this aim by targeting a functional enzyme to single cellular protein nanocompartments, which were formed upon recombinant expression of the Salmonella enterica LT2 ethanolamine utilization bacterial microcompartment shell proteins EutS or EutSMNLK in Escherichia coli. To optimize this system, increasing overall encapsulated enzyme reaction efficiency, factor(s) required for the production of more than one nanocompartment per cell must be identified. In this work we report that the cupin domain protein EutQ is required for assembly of more than one nanocompartment per cell. Overexpression of EutQ results in multiple nanocompartment assembly in our recombinant system. EutQ specifically interacts with the shell protein EutM in vitro via electrostatic interactions with the putative cytosolic face of EutM. These findings lead to the theory that EutQ could facilitate multiple nanocompartment biogenesis by serving as an assembly hub for shell proteins. This work offers insights into the biogenesis of Eut bacterial microcompartments, and also provides an improved platform for the production of protein based nanocompartments for targeted encapsulation of enzyme pathways. PMID:27063436

  13. Specific Genetic Immunotherapy Induced by Recombinant Vaccine Alpha-Fetoprotein-Heat Shock Protein 70 Complex

    NASA Astrophysics Data System (ADS)

    Wang, Xiaoping; Lin, Huanping; Wang, Qiaoxia

    Purposes: To construct a recombinant vaccine alpha-fetoprotein (AFP)-heat shock protein (HSP70) complex, and study its ability to induce specific CTL response and its protective effect against AFP-producing tumor. Material/Methods: A recombinant vaccine was constructed by conjugating mouse alpha-fetoprotein to heat shock protein 70. By way of intracutaneous injection, mice were primed and boosted with recombinant vaccine mAFP/HSP70, whereas single mAFP or HSP70 injection as controls. The ELISPOT and ELISA were used to measure the frequency of cells producing the cytokine IFN-γ in splenocytes and the level of anti-AFP antibody of serum from immunized mice respectively. In vivo tumor challenge were carried out to assess the immune effect of the recombinant vaccine. Results: By recombinant mAFP/HSP70 vaccine immunization, the results of ELISPOT and ELISA showed that the number of splenic cells producing IFN-γ and the level of anti-AFP antibody of serum were significantly higher in mAFP/HSP70 group than those in mAFP and HSP70 groups (108.50±11.70 IFN-γ spots/106 cells vs 41.60±10.40 IFN-γ spots/106 cells, 7.32±3.14 IFN-γ spots/106 cells, P<0.01; 156.32±10.42 μg/mL vs 66.52±7.35 μg/mL, 5.73±2.89 μg/mL, P<0.01). The tumor volume in mAFP/HSP70 group was significantly smaller than that in mAFP and HSP70 groups (42.44±7.14 mm3 vs 392.23±12.46 mm3, 838.63±13.84 mm3, P<0.01). Conclusions: The study further confirmed the function of heat shock protein 70's immune adjuvant. Sequential immunization with recombinant mAFP/HSP70 vaccine could generate effective antitumor immunity on AFP-producing tumor. The recombined mAFP/HSP70 vaccine may be suitable for serving as an immunotherapy for hepatocellular carcinoma.

  14. Altering the ribosomal subunit ratio in yeast maximizes recombinant protein yield

    PubMed Central

    Bonander, Nicklas; Darby, Richard AJ; Grgic, Ljuban; Bora, Nagamani; Wen, Jikai; Brogna, Saverio; Poyner, David R; O'Neill, Michael AA; Bill, Roslyn M

    2009-01-01

    Background The production of high yields of recombinant proteins is an enduring bottleneck in the post-genomic sciences that has yet to be addressed in a truly rational manner. Typically eukaryotic protein production experiments have relied on varying expression construct cassettes such as promoters and tags, or culture process parameters such as pH, temperature and aeration to enhance yields. These approaches require repeated rounds of trial-and-error optimization and cannot provide a mechanistic insight into the biology of recombinant protein production. We published an early transcriptome analysis that identified genes implicated in successful membrane protein production experiments in yeast. While there has been a subsequent explosion in such analyses in a range of production organisms, no one has yet exploited the genes identified. The aim of this study was to use the results of our previous comparative transcriptome analysis to engineer improved yeast strains and thereby gain an understanding of the mechanisms involved in high-yielding protein production hosts. Results We show that tuning BMS1 transcript levels in a doxycycline-dependent manner resulted in optimized yields of functional membrane and soluble protein targets. Online flow microcalorimetry demonstrated that there had been a substantial metabolic change to cells cultured under high-yielding conditions, and in particular that high yielding cells were more metabolically efficient. Polysome profiling showed that the key molecular event contributing to this metabolically efficient, high-yielding phenotype is a perturbation of the ratio of 60S to 40S ribosomal subunits from approximately 1:1 to 2:1, and correspondingly of 25S:18S ratios from 2:1 to 3:1. This result is consistent with the role of the gene product of BMS1 in ribosome biogenesis. Conclusion This work demonstrates the power of a rational approach to recombinant protein production by using the results of transcriptome analysis to engineer

  15. Type I shorthorn sculpin antifreeze protein: recombinant synthesis, solution conformation, and ice growth inhibition studies.

    PubMed

    Fairley, Kayesh; Westman, Belinda J; Pham, Linda H; Haymet, A D J; Harding, Margaret M; Mackay, Joel P

    2002-07-01

    A number of structurally diverse classes of "antifreeze" proteins that allow fish to survive in sub-zero ice-laden waters have been isolated from the blood plasma of cold water teleosts. However, despite receiving a great deal of attention, the one or more mechanisms through which these proteins act are not fully understood. In this report we have synthesized a type I antifreeze polypeptide (AFP) from the shorthorn sculpin Myoxocephalus scorpius using recombinant methods. Construction of a synthetic gene with optimized codon usage and expression as a glutathione S-transferase fusion protein followed by purification yielded milligram amounts of polypeptide with two extra residues appended to the N terminus. Circular dichroism and NMR experiments, including residual dipolar coupling measurements on a 15N-labeled recombinant polypeptide, show that the polypeptides are alpha-helical with the first four residues being more flexible than the remainder of the sequence. Both the recombinant and synthetic polypeptides modify ice growth, forming facetted crystals just below the freezing point, but display negligible thermal hysteresis. Acetylation of Lys-10, Lys-20, and Lys-21 as well as the N terminus of the recombinant polypeptide gave a derivative that displays both thermal hysteresis (0.4 degrees C at 15 mg/ml) and ice crystal faceting. These results confirm that the N terminus of wild-type polypeptide is functionally important and support our previously proposed mechanism for all type I proteins, in which the hydrophobic face is oriented toward the ice at the ice/water interface.

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2016-06-09

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

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

    PubMed Central

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

    2015-01-01

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

  19. Inflammatory response of endothelial cells to hepatitis C virus recombinant envelope glycoprotein 2 protein exposure

    PubMed Central

    Urbaczek, Ana Carolina; Ribeiro, Lívia Carolina de Abreu; Ximenes, Valdecir Farias; Afonso, Ana; Nogueira, Camila Tita; Generoso, Wesley Cardoso; Alberice, Juliana Vieira; Rudnicki, Martina; Ferrer, Renila; da Fonseca, Luiz Marcos; da Costa, Paulo Inácio

    2014-01-01

    The hepatitis C virus (HCV) encodes approximately 10 different structural and non-structural proteins, including the envelope glycoprotein 2 (E2). HCV proteins, especially the envelope proteins, bind to cell receptors and can damage tissues. Endothelial inflammation is the most important determinant of fibrosis progression and, consequently, cirrhosis. The aim of this study was to evaluate and compare the inflammatory response of endothelial cells to two recombinant forms of the HCV E2 protein produced in different expression systems (Escherichia coli and Pichia pastoris). We observed the induction of cell death and the production of nitric oxide, hydrogen peroxide, interleukin-8 and vascular endothelial growth factor A in human umbilical vein endothelial cells (HUVECs) stimulated by the two recombinant E2 proteins. The E2-induced apoptosis of HUVECs was confirmed using the molecular marker PARP. The apoptosis rescue observed when the antioxidant N-acetylcysteine was used suggests that reactive oxygen species are involved in E2-induced apoptosis. We propose that these proteins are involved in the chronic inflammation caused by HCV. PMID:25317702

  20. Mutant strains of Pichia pastoris with enhanced secretion of recombinant proteins.

    PubMed

    Larsen, Sasha; Weaver, Jun; de Sa Campos, Katherine; Bulahan, Rhobe; Nguyen, Jackson; Grove, Heather; Huang, Amy; Low, Lauren; Tran, Namphuong; Gomez, Seth; Yau, Jennifer; Ilustrisimo, Thomas; Kawilarang, Jessica; Lau, Jonathan; Tranphung, Maivi; Chen, Irene; Tran, Christina; Fox, Marcia; Lin-Cereghino, Joan; Lin-Cereghino, Geoff P

    2013-11-01

    Although Pichia pastoris is a popular protein expression system, it exhibits limitations in its ability to secrete heterologous proteins. Therefore, a REMI (restriction enzyme mediated insertion) strategy was utilized to select mutant beta-g alactosidase s upersecretion (bgs) strains that secreted increased levels of a β-galactosidase reporter. Many of the twelve BGS genes may have functions in intracellular signaling or vesicle transport. Several of these strains also appeared to contain a more permeable cell wall. Preliminary characterization of four bgs mutants showed that they differed in the ability to enhance the export of other reporter proteins. bgs13, which has a disruption in a gene homologous to Saccharomyces cerevisiae protein kinase C (PKC1), gave enhanced secretion of most recombinant proteins that were tested, raising the possibility that it has the universal super-secreter phenotype needed in an industrial production strain of P. pastoris. PMID:23881328

  1. High-throughput recombinant protein expression in Escherichia coli: current status and future perspectives.

    PubMed

    Jia, Baolei; Jeon, Che Ok

    2016-08-01

    The ease of genetic manipulation, low cost, rapid growth and number of previous studies have made Escherichia coli one of the most widely used microorganism species for producing recombinant proteins. In this post-genomic era, challenges remain to rapidly express and purify large numbers of proteins for academic and commercial purposes in a high-throughput manner. In this review, we describe several state-of-the-art approaches that are suitable for the cloning, expression and purification, conducted in parallel, of numerous molecules, and we discuss recent progress related to soluble protein expression, mRNA folding, fusion tags, post-translational modification and production of membrane proteins. Moreover, we address the ongoing efforts to overcome various challenges faced in protein expression in E. coli, which could lead to an improvement of the current system from trial and error to a predictable and rational design. PMID:27581654

  2. A Recombinant Collagen-mRNA Platform for Controllable Protein Synthesis.

    PubMed

    Sun, Liping; Xiong, Yunjing; Bashan, Anat; Zimmerman, Ella; Shulman Daube, Shirley; Peleg, Yoav; Albeck, Shira; Unger, Tamar; Yonath, Hagith; Krupkin, Miri; Matzov, Donna; Yonath, Ada

    2015-07-01

    We have developed a collagen-mRNA platform for controllable protein production that is intended to be less prone to the problems associated with commonly used mRNA therapy as well as with collagen skin-healing procedures. A collagen mimic was constructed according to a recombinant method and was used as scaffold for translating mRNA chains into proteins. Cysteines were genetically inserted into the collagen chain at positions allowing efficient ribosome translation activity while minimizing mRNA misfolding and degradation. Enhanced green fluorescence protein (eGFP) mRNA bound to collagen was successfully translated by cell-free Escherichia coli ribosomes. This system enabled an accurate control of specific protein synthesis by monitoring expression time and level. Luciferase-mRNA was also translated on collagen scaffold by eukaryotic cell extracts. Thus we have demonstrated the feasibility of controllable protein synthesis on collagen scaffolds by ribosomal machinery.

  3. Robotic high-throughput purification of affinity-tagged recombinant proteins.

    PubMed

    Wiesler, Simone C; Weinzierl, Robert O J

    2015-01-01

    Affinity purification of recombinant proteins has become the method of choice to obtain good quantities and qualities of proteins for a variety of downstream biochemical applications. While manual or FPLC-assisted purification techniques are generally time-consuming and labor-intensive, the advent of high-throughput technologies and liquid handling robotics has simplified and accelerated this process significantly. Additionally, without the human factor as a potential source of error, automated purification protocols allow for the generation of large numbers of proteins simultaneously and under directly comparable conditions. The delivered material is ideal for activity comparisons of different variants of the same protein. Here, we present our strategy for the simultaneous purification of up to 24 affinity-tagged proteins for activity measurements in biochemical assays. The protocol described is suitable for the scale typically required in individual research laboratories. PMID:25749949

  4. High-throughput recombinant protein expression in Escherichia coli: current status and future perspectives

    PubMed Central

    2016-01-01

    The ease of genetic manipulation, low cost, rapid growth and number of previous studies have made Escherichia coli one of the most widely used microorganism species for producing recombinant proteins. In this post-genomic era, challenges remain to rapidly express and purify large numbers of proteins for academic and commercial purposes in a high-throughput manner. In this review, we describe several state-of-the-art approaches that are suitable for the cloning, expression and purification, conducted in parallel, of numerous molecules, and we discuss recent progress related to soluble protein expression, mRNA folding, fusion tags, post-translational modification and production of membrane proteins. Moreover, we address the ongoing efforts to overcome various challenges faced in protein expression in E. coli, which could lead to an improvement of the current system from trial and error to a predictable and rational design. PMID:27581654

  5. Spider silks: recombinant synthesis, assembly, spinning, and engineering of synthetic proteins

    PubMed Central

    Scheibel, Thomas

    2004-01-01

    Since thousands of years humans have utilized insect silks for their own benefit and comfort. The most famous example is the use of reeled silkworm silk from Bombyx mori to produce textiles. In contrast, despite the more promising properties of their silk, spiders have not been domesticated for large-scale or even industrial applications, since farming the spiders is not commercially viable due to their highly territorial and cannibalistic nature. Before spider silks can be copied or mimicked, not only the sequence of the underlying proteins but also their functions have to be resolved. Several attempts to recombinantly produce spider silks or spider silk mimics in various expression hosts have been reported previously. A new protein engineering approach, which combines synthetic repetitive silk sequences with authentic silk domains, reveals proteins that closely resemble silk proteins and that can be produced at high yields, which provides a basis for cost-efficient large scale production of spider silk-like proteins. PMID:15546497

  6. Glucose-induced production of recombinant proteins in Hansenula polymorpha mutants deficient in catabolite repression.

    PubMed

    Krasovska, Olena S; Stasyk, Olena G; Nahorny, Viktor O; Stasyk, Oleh V; Granovski, Nikolai; Kordium, Vitaliy A; Vozianov, Oleksandr F; Sibirny, Andriy A

    2007-07-01

    The most commonly used expression platform for production of recombinant proteins in the methylotrophic yeast Hansenula polymorpha relies on the strong and strictly regulated promoter from the gene encoding peroxisomal enzyme alcohol (or methanol) oxidase (P(MOX)). Expression from P(MOX) is induced by methanol and is partially derepressed in glycerol or xylose medium, whereas in the presence of hexoses, disaccharides or ethanol, it is repressed. The need for methanol for maximal induction of gene expression in large-scale fermentation is a significant drawback, as this compound is toxic, flammable, supports a slow growth rate and requires extensive aeration. We isolated H. polymorpha mutants deficient in glucose repression of P(MOX) due to an impaired HpGCR1 gene, and other yet unidentified secondary mutations. The mutants exhibited pronounced defects in P(MOX) regulation only by hexoses and xylose, but not by disaccharides or ethanol. With one of these mutant strains as hosts, we developed a modified two-carbon source mode expression platform that utilizes convenient sugar substrates for growth (sucrose) and induction of recombinant protein expression (glucose or xylose). We demonstrate efficient regulatable by sugar carbon sources expression of three recombinant proteins: a secreted glucose oxidase from the fungus Aspergillus niger, a secreted mini pro-insulin, and an intracellular hepatitis B virus surface antigen in these mutant hosts. The modified expression platform preserves the favorable regulatable nature of P(MOX) without methanol, making a convenient alternative to the traditional system. PMID:17163508

  7. Dunaliella salina as a novel host for the production of recombinant proteins.

    PubMed

    Feng, Shuying; Li, Xuebing; Xu, Zhengshun; Qi, Jingjiao

    2014-05-01

    Although several expression systems are currently available for the production of recombinant proteins, they still have some inherent disadvantages, thereby resulting in the desire to explore a novel expression system for producing recombinant proteins. Dunaliella salina (D. salina) has been exploited as a novel expression system for the field of genetic engineering because of its distinct advantages, including low production cost, fast growth, easy culture, ease of transgenic manipulation, and modified abilities of transcription and translation. Thus far, studies on D. salina host have made great development and significant progress. This paper presents a comprehensive summary of the achievements of D. salina host from the following aspects: the advantages of D. salina cells, transformation methods, cloning of D. salina genes, and expression of exogenous genes into D. salina. Furthermore, the authors identified the current main obstacles and future application prospects for the recombinant proteins produced by D. salina, which could be used as a basis for the future maturation of D. salina expression system. PMID:24643734

  8. Tunable recombinant protein expression in E. coli: enabler for continuous processing?

    PubMed

    Marschall, Lukas; Sagmeister, Patrick; Herwig, Christoph

    2016-07-01

    Tuning of transcription is a powerful process technological tool for efficient recombinant protein production in Escherichia coli. Many challenges such as product toxicity, formation of inclusion bodies, cell death, and metabolic burden are associated with non-suitable (too high or too low) levels of recombinant protein expression. Tunable expression systems allow adjusting the recombinant protein expression using process technological means. This enables to exploit the cell's metabolic capacities to a maximum. Within this article, we review genetic and process technological aspects of tunable expression systems in E. coli, providing a roadmap for the industrial exploitation of the reviewed technologies. We attempt to differentiate the term "expression tuning" from its inflationary use by providing a concise definition and highlight interesting fields of application for this versatile new technology. Dependent on the type of inducer (metabolizable or non-metabolizable), different process strategies are required in order to achieve tuning. To fully profit from the benefits of tunable systems, an independent control of growth rate and expression rate is indispensable. Being able to tackle problems such as long-term culture stability and constant product quality expression tuning is a promising enabler for continuous processing in biopharmaceutical production. PMID:27170324

  9. Dunaliella salina as a novel host for the production of recombinant proteins.

    PubMed

    Feng, Shuying; Li, Xuebing; Xu, Zhengshun; Qi, Jingjiao

    2014-05-01

    Although several expression systems are currently available for the production of recombinant proteins, they still have some inherent disadvantages, thereby resulting in the desire to explore a novel expression system for producing recombinant proteins. Dunaliella salina (D. salina) has been exploited as a novel expression system for the field of genetic engineering because of its distinct advantages, including low production cost, fast growth, easy culture, ease of transgenic manipulation, and modified abilities of transcription and translation. Thus far, studies on D. salina host have made great development and significant progress. This paper presents a comprehensive summary of the achievements of D. salina host from the following aspects: the advantages of D. salina cells, transformation methods, cloning of D. salina genes, and expression of exogenous genes into D. salina. Furthermore, the authors identified the current main obstacles and future application prospects for the recombinant proteins produced by D. salina, which could be used as a basis for the future maturation of D. salina expression system.

  10. Antigenic assessment of a recombinant human CD90 protein expressed in prokaryotic expression system.

    PubMed

    Yousefi-Rad, Narges; Shokrgozar, Mohammad Ali; Behdani, Mahdi; Moradi-Kalbolandi, Shima; Motamedi-Rad, Mahdieh; Habibi-Anbouhi, Mahdi

    2015-12-01

    Cluster of Differentiation 90 (CD90, Thy-1) has been proposed as one of the most important biomarkers in several cancer cells including cancer stem cells (CSCs). CD90 is considered as a potential normal stem cell and CSCs biomarker and also has been identified in lung cancer stem cells, hepatocellular carcinoma cells and high-grade gliomas. Using eukaryotic host systems involves complex procedures and frequently results in low protein yields. The expression of recombinant proteins in Escherichia coli is comparatively easier than eukaryotic host cells. The potential of large scale production of recombinant protein has made this system an economic production platform. In this study we expressed the extra-membrane domain of human CD90 (exCD90) antigen (Gln15-Cys130) in E. coli expression host cells. The epitope integrity of purified recombinant antigen was confirmed by antibody-antigen interaction using 5E10 anti-CD90 monoclonal antibody and binding study through ELISA and florescent staining of CD90(+) cells in a flow cytometry experiment. PMID:26297626

  11. Antigenic assessment of a recombinant human CD90 protein expressed in prokaryotic expression system.

    PubMed

    Yousefi-Rad, Narges; Shokrgozar, Mohammad Ali; Behdani, Mahdi; Moradi-Kalbolandi, Shima; Motamedi-Rad, Mahdieh; Habibi-Anbouhi, Mahdi

    2015-12-01

    Cluster of Differentiation 90 (CD90, Thy-1) has been proposed as one of the most important biomarkers in several cancer cells including cancer stem cells (CSCs). CD90 is considered as a potential normal stem cell and CSCs biomarker and also has been identified in lung cancer stem cells, hepatocellular carcinoma cells and high-grade gliomas. Using eukaryotic host systems involves complex procedures and frequently results in low protein yields. The expression of recombinant proteins in Escherichia coli is comparatively easier than eukaryotic host cells. The potential of large scale production of recombinant protein has made this system an economic production platform. In this study we expressed the extra-membrane domain of human CD90 (exCD90) antigen (Gln15-Cys130) in E. coli expression host cells. The epitope integrity of purified recombinant antigen was confirmed by antibody-antigen interaction using 5E10 anti-CD90 monoclonal antibody and binding study through ELISA and florescent staining of CD90(+) cells in a flow cytometry experiment.

  12. Canine Fibroblast Growth Factor 21 Ameliorates Hyperglycemia Associated with Inhibiting Hepatic Gluconeogenesis and Improving Pancreatic Beta-Cell Survival in Diabetic Mice and Dogs.

    PubMed

    Xu, Pengfei; Zhang, Yingjie; Jiang, Xinghao; Li, Junyan; Song, Liying; Khoso, Mir Hasson; Liu, Yunye; Wu, Qiang; Ren, Guiping; Li, Deshan

    2016-01-01

    Diabetes mellitus is a common endocrinopathy in dog. Fibroblast growth factor 21 (FGF-21) is a secreted protein, which is involved in glucose homeostasis. We speculate that the recombinant canine FGF-21 (cFGF-21) has the potential to become a powerful therapeutics to treat canine diabetes. The cFGF-21 gene was cloned and expressed in E. coli Rosetta (DE3). After purification, a cFGF-21 protein with the purity exceeding 95% was obtained. Mouse 3T3-L1 adipocytes and type 1 diabetic mice/dogs induced by STZ were used to examine the biological activity of cFGF-21 in vitro and in vivo, respectively. Results showed that cFGF-21 stimulated glucose uptake in adipocytes significantly in a dose-dependent manner, and reduced plasma glucose significantly in diabetic mice/dogs. After treatment with cFGF-21, the serum insulin level, glycosylated hemoglobin (HbA1c) level and the expressions of the hepatic gluconeogenesis genes (glucose-6-phosphatase, G6Pase and phosphoenolpyruvate carboxykinase, PCK) of the diabetic mice/dogs were attenuated significantly. In the mouse experiment, we also found that the phosphorylation of signal transducer and activator of transcription 3 (STAT3) and the expression of suppressor of cytokine signaling 3 (SOCS3) were up-regulated significantly in the livers after treatment. Histopathological and immunohistochemical results showed that treatment with cFGF-21 promoted recovery of pancreatic islets from STZ-induced apoptosis. Besides, we also found that treatment with cFGF-21 protected liver against STZ or hyperglycemia induced damage and the mechanism of this action associated with inhibiting oxidative stress. In conclusion, cFGF-21 represents a promising candidate for canine diabetes therapeutics. The mechanism of cFGF-21 ameliorates hyperglycemia associated with inhibiting hepatic gluconeogenesis by regulation of STAT3 signal pathway and improving pancreatic beta-cell survival. PMID:27203422

  13. Canine Fibroblast Growth Factor 21 Ameliorates Hyperglycemia Associated with Inhibiting Hepatic Gluconeogenesis and Improving Pancreatic Beta-Cell Survival in Diabetic Mice and Dogs

    PubMed Central

    Xu, Pengfei; Zhang, Yingjie; Jiang, Xinghao; Li, Junyan; Song, Liying; Khoso, Mir Hasson; Liu, Yunye; Wu, Qiang; Ren, Guiping; Li, Deshan

    2016-01-01

    Diabetes mellitus is a common endocrinopathy in dog. Fibroblast growth factor 21 (FGF-21) is a secreted protein, which is involved in glucose homeostasis. We speculate that the recombinant canine FGF-21 (cFGF-21) has the potential to become a powerful therapeutics to treat canine diabetes. The cFGF-21 gene was cloned and expressed in E. coli Rosetta (DE3). After purification, a cFGF-21 protein with the purity exceeding 95% was obtained. Mouse 3T3-L1 adipocytes and type 1 diabetic mice/dogs induced by STZ were used to examine the biological activity of cFGF-21 in vitro and in vivo, respectively. Results showed that cFGF-21 stimulated glucose uptake in adipocytes significantly in a dose-dependent manner, and reduced plasma glucose significantly in diabetic mice/dogs. After treatment with cFGF-21, the serum insulin level, glycosylated hemoglobin (HbA1c) level and the expressions of the hepatic gluconeogenesis genes (glucose-6-phosphatase, G6Pase and phosphoenolpyruvate carboxykinase, PCK) of the diabetic mice/dogs were attenuated significantly. In the mouse experiment, we also found that the phosphorylation of signal transducer and activator of transcription 3 (STAT3) and the expression of suppressor of cytokine signaling 3 (SOCS3) were up-regulated significantly in the livers after treatment. Histopathological and immunohistochemical results showed that treatment with cFGF-21 promoted recovery of pancreatic islets from STZ-induced apoptosis. Besides, we also found that treatment with cFGF-21 protected liver against STZ or hyperglycemia induced damage and the mechanism of this action associated with inhibiting oxidative stress. In conclusion, cFGF-21 represents a promising candidate for canine diabetes therapeutics. The mechanism of cFGF-21 ameliorates hyperglycemia associated with inhibiting hepatic gluconeogenesis by regulation of STAT3 signal pathway and improving pancreatic beta-cell survival. PMID:27203422

  14. Enhanced expression of the Epstein-Barr virus latent membrane protein by a recombinant vaccinia virus.

    PubMed

    Stewart, J P; Hampson, I N; Heinrich, H W; Mackett, M; Arrand, J R

    1989-05-01

    The complete coding sequence of the Epstein-Barr virus strain B95-8 latent membrane protein (LMP) was cloned using a Raji cell cDNA library and genomic B95-8 DNA. The clone was characterized by sequencing and then used to make a recombinant vaccinia virus. This virus (VLMP) was shown to express a relatively high level of LMP in an authentic fashion. Antisera raised in rabbits against VLMP were shown to react with B95-8 LMP as well as cross-reacting with a 50K cellular protein.

  15. Recombinant human bone morphogenetic protein-9 potently induces osteogenic differentiation of human periodontal ligament fibroblasts.

    PubMed

    Fuchigami, Sawako; Nakamura, Toshiaki; Furue, Kirara; Sena, Kotaro; Shinohara, Yukiya; Noguchi, Kazuyuki

    2016-04-01

    To accomplish effective periodontal regeneration for periodontal defects, several regenerative methods using growth and differentiation factors, including bone morphogenetic proteins (BMPs), have been developed. Bone morphogenetic protein-9 exhibits the most potent osteogenic activity of this growth factor family. However, it is unclear whether exogenous BMP-9 can induce osteogenic differentiation in human periodontal ligament (PDL) fibroblasts. Here, we examined the effects of recombinant human (rh) BMP-9 on osteoblastic differentiation in human PDL fibroblasts in vitro, compared with rhBMP-2. Recombinant human BMP-9 potently induced alkaline phosphatase (ALP) activity, mineralization, and increased expression of runt-related transcription factor-2/core binding factor alpha 1 (RUNX2/CBFA1), osterix, inhibitor of DNA binding/differentiation-1 (ID1), osteopontin, and bone sialoprotein genes, compared with rhBMP-2. The levels of rhBMP-9-induced osterix and ALP mRNA were significantly reduced in activin receptor-like kinase-1 and -2 small interfering RNA (siRNA)-transfected human PDL fibroblasts. Recombinant human BMP-9-induced ALP activity was not inhibited by noggin, in contrast to rhBMP-2 induced ALP activity, which was. Phosphorylation of SMAD1/5/8 in human PDL fibroblasts was induced by addition of rhBMP-9. Recombinant human BMP-9-induced ALP activity was suppressed by SB203580, SP600125, and U0126, which are inhibitors of p38, c-Jun N-terminal kinase (JNK), and extracellular signal-regulated kinase 1/2 (ERK1/2), respectively. Our data suggest that rhBMP-9 is a potent inducer of the differentiation of human PDL fibroblasts into osteoblast-like cells and that this may be mediated by the SMAD and mitogen-activated protein kinase (p38, ERK1/2, and JNK) pathways. PMID:26879145

  16. Photochemical degradation of citrate buffers leads to covalent acetonation of recombinant protein therapeutics

    PubMed Central

    Valliere-Douglass, John F; Connell-Crowley, Lisa; Jensen, Randy; Schnier, Paul D; Trilisky, Egor; Leith, Matt; Follstad, Brian D; Kerr, Jennifer; Lewis, Nathan; Vunnum, Suresh; Treuheit, Michael J; Balland, Alain; Wallace, Alison

    2010-01-01

    Novel acetone and aldimine covalent adducts were identified on the N-termini and lysine side chains of recombinant monoclonal antibodies. Photochemical degradation of citrate buffers, in the presence of trace levels of iron, is demonstrated as the source of these modifications. The link between degradation of citrate and the observed protein modifications was conclusively established by tracking the citrate decomposition products and protein adducts resulting from photochemical degradation of isotope labeled 13C citrate by mass spectrometry. The structure of the acetone modification was determined by nuclear magnetic resonance (NMR) spectroscopy on modified–free glycine and found to correspond to acetone linked to the N-terminus of the amino acid through a methyl carbon. Results from mass spectrometric fragmentation of glycine modified with an acetone adduct derived from 13C labeled citrate indicated that the three central carbons of citrate are incorporated onto protein amines in the presence of iron and light. While citrate is known to stoichiometrically decompose to acetone and CO2 through various intermediates in photochemical systems, it has never been shown to be a causative agent in protein carbonylation. Our results point to a previously unknown source for the generation of reactive carbonyl species. This work also highlights the potential deleterious impact of trace metals on recombinant protein therapeutics formulated in citrate buffers. PMID:20836085

  17. Model based engineering of Pichia pastoris central metabolism enhances recombinant protein production

    PubMed Central

    Nocon, Justyna; Steiger, Matthias G.; Pfeffer, Martin; Sohn, Seung Bum; Kim, Tae Yong; Maurer, Michael; Rußmayer, Hannes; Pflügl, Stefan; Ask, Magnus; Haberhauer-Troyer, Christina; Ortmayr, Karin; Hann, Stephan; Koellensperger, Gunda; Gasser, Brigitte; Lee, Sang Yup; Mattanovich, Diethard

    2014-01-01

    The production of recombinant proteins is frequently enhanced at the levels of transcription, codon usage, protein folding and secretion. Overproduction of heterologous proteins, however, also directly affects the primary metabolism of the producing cells. By incorporation of the production of a heterologous protein into a genome scale metabolic model of the yeast Pichia pastoris, the effects of overproduction were simulated and gene targets for deletion or overexpression for enhanced productivity were predicted. Overexpression targets were localized in the pentose phosphate pathway and the TCA cycle, while knockout targets were found in several branch points of glycolysis. Five out of 9 tested targets led to an enhanced production of cytosolic human superoxide dismutase (hSOD). Expression of bacterial β-glucuronidase could be enhanced as well by most of the same genetic modifications. Beneficial mutations were mainly related to reduction of the NADP/H pool and the deletion of fermentative pathways. Overexpression of the hSOD gene itself had a strong impact on intracellular fluxes, most of which changed in the same direction as predicted by the model. In vivo fluxes changed in the same direction as predicted to improve hSOD production. Genome scale metabolic modeling is shown to predict overexpression and deletion mutants which enhance recombinant protein production with high accuracy. PMID:24853352

  18. Immunocontraception of Eastern Grey kangaroos (Macropus giganteus) with recombinant brushtail possum (Trichosurus vulpecula) ZP3 protein.

    PubMed

    Kitchener, Anne L; Harman, Amanda; Kay, David J; McCartney, Carmen A; Mate, Karen E; Rodger, John C

    2009-01-01

    This study examined the potential of a recombinant marsupial zona pellucida 3 protein as a contraceptive vaccine for the Eastern Grey kangaroo, a marsupial that is locally overabundant in several regions of eastern Australia. First, a pilot study using porcine zona pellucidae (PZP) demonstrated that ZP proteins, primarily the ZP3 component of PZP, are highly immunogenic in the grey kangaroo and produce a long-lasting humoral response to a single immunisation, as found in other marsupials. Immunisation with 300 microg of a non-glycosylated recombinant brushtail possum ZP3 (recBP-ZP3) protein in complete Freund's adjuvant produced a similar, significant and sustained antibody response, and none of the immunised kangaroos (n=7) produced offspring during the following breeding season compared with four out of the six control animals. An epitope analysis of the B-cell response to recBP-ZP3 using a brushtail possum ZP3 identified numerous B-cell epitope regions clustered around the N- and C-terminal regions of the protein. Two regions of interest for further fertility vaccine development based on their immunogenicity and fertility trials and functional studies in other species were found to be immunogenic. These results suggest that immunocontraception based on targeting the ZP3 protein within the zona pellucida may be an effective strategy for fertility reduction in Eastern Grey kangaroos.

  19. Isolation of Escherichia coli synthesized recombinant eukaryotic proteins that contain epsilon-N-acetyllysine.

    PubMed Central

    Violand, B. N.; Schlittler, M. R.; Lawson, C. Q.; Kane, J. F.; Siegel, N. R.; Smith, C. E.; Kolodziej, E. W.; Duffin, K. L.

    1994-01-01

    Recombinant porcine (rpST) and bovine somatotropins (rbST) synthesized in Escherichia coli contain the amino acid, epsilon-N-acetyllysine. This amino acid was initially discovered in place of the normal lysine144 in a modified reversed-phase HPLC (RP-HPLC) species of rpST. Mass spectrometry and amino acid sequencing of a tryptic peptide isolated from this RP-HPLC purified protein were used to identify this altered residue as epsilon-N-acetyllysine. Ion-exchange chromatography was utilized to prepare low isoelectric point (pI) forms of rpST and rbST, which are enriched in epsilon-N-acetyllysine. Electrospray mass spectrometry demonstrated that the majority of the protein in these low pI fractions contained species 42 Da larger than normal. Immobilized pH gradient electrophoresis (IPG) of the ion-exchange purified low pI proteins was used to isolate several monoacetylated species of rpST and rbST. The location of the acetylated lysine in each IPG-purified protein was determined by tryptic peptide mapping and amino acid sequencing of the altered tryptic peptides. Amino acid analyses of enzymatic digests of rpST and rbST were also used to confirm the presence of epsilon-N-acetyllysine in these recombinant proteins. These data demonstrate that a significant portion of rpST and rbST produced in E. coli contain this unusual amino acid. PMID:7920255

  20. 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.

  1. Low Levels of Aflatoxin B1, Ricin, and Milk Enhance Recombinant Protein Production in Mammalian Cells

    PubMed Central

    Rasooly, Reuven; Hernlem, Bradley; Friedman, Mendel

    2013-01-01

    Gene expression in transduced mammalian cells correlates with virus titer, but high doses of vector for gene therapy leads to toxicity in humans and in animals. Changing the optimal tissue culture medium by adding low levels of environmental stressors, such as 1 µM of the fungal toxin aflatoxin B1 (AFB1), 1 ng of the castor bean protein toxin ricin, or 1% reconstituted milk, enhances transcription and increases production of proteins in transduced mammalian cells as demonstrated by production of the following three recombinant proteins: firefly luciferase, β-galactosidase, and green fluorescent protein (GFP). Higher concentrations of the stress-producing substances damage the cells beyond recovery, resulting in inhibited gene expression and cell death. We also evaluated the effect of the stressor substances on the enhanced infectivity of virus. The presented findings extend methods for large-scale transient recombinant protein production in mammalian cells and suggest that it may be possible to reduce the cytotoxicity of the adenovirus by reducing the virus titer without adversely affecting gene expression levels. PMID:23940780

  2. A Novel Recombinant DNA System for High Efficiency Affinity Purification of Proteins in Saccharomyces cerevisiae.

    PubMed

    Carrick, Brian H; Hao, Linxuan; Smaldino, Philip J; Engelke, David R

    2016-03-01

    Isolation of endogenous proteins from Saccharomyces cerevisiae has been facilitated by inserting encoding polypeptide affinity tags at the C-termini of chromosomal open reading frames (ORFs) using homologous recombination of DNA fragments. Tagged protein isolation is limited by a number of factors, including high cost of affinity resins for bulk isolation and low concentration of ligands on the resin surface, leading to low isolation efficiencies and trapping of contaminants. To address this, we have created a recombinant "CelTag" DNA construct from which PCR fragments can be created to easily tag C-termini of S. cerevisiae ORFs using selection for a nat1 marker. The tag has a C-terminal cellulose binding module to be used in the first affinity step. Microgranular cellulose is very inexpensive and has an effectively continuous ligand on its surface, allowing rapid, highly efficient purification with minimal background. Cellulose-bound proteins are released by specific cleavage of an included site for TEV protease, giving nearly pure product. The tag can be lifted from the recombinant DNA construct either with or without a 13x myc epitope tag between the target ORF and the TEV protease site. Binding of CelTag protein fusions to cellulose is stable to high salt, nonionic detergents, and 1 M urea, allowing stringent washing conditions to remove loosely associated components, as needed, before specific elution. It is anticipated that this reagent could allow isolation of protein complexes from large quantities of yeast extract, including soluble, membrane-bound, or nucleic acid-associated assemblies.

  3. Methods for efficient high-throughput screening of protein expression in recombinant Pichia pastoris strains.

    PubMed

    Camattari, Andrea; Weinhandl, Katrin; Gudiminchi, Rama K

    2014-01-01

    The methylotrophic yeast Pichia pastoris is becoming one of the favorite industrial workhorses for protein expression. Due to the widespread use of integration vectors, which generates significant clonal variability, screening methods allowing assaying hundreds of individual clones are of particular importance. Here we describe methods to detect and analyze protein expression, developed in a 96-well format for high-throughput screening of recombinant P. pastoris strains. The chapter covers essentially three common scenarios: (1) an enzymatic assay for proteins expressed in the cell cytoplasm, requiring cell lysis; (2) a whole-cell assay for a fungal cytochrome P450; and (3) a nonenzymatic assay for detection and quantification of tagged protein secreted into the supernatant. PMID:24744029

  4. Secretion of human interleukin-2 fused with green fluorescent protein in recombinant Pichia pastoris.

    PubMed

    Cha, Hyung Joon; Dalal, Nimish N; Bentley, William E

    2005-07-01

    Methylotrophic yeast Pichia pastoris is convenient for the expression of eukaryotic foreign proteins owing to its potential for posttranslational modifications, protein folding, and facile culturing. In this work, human interleukin (hIL)-2 was successfully produced as a secreted fusion form in recombinant P. pastoris. By employing green fluorescent protein (GFP) as a monitoring fusion partner, clear identification of fusion protein expression and quantification of intracellular hIL-2 were possible even though there was no correlation between culture supernatant fluorescence and secreted hIL-2 owing to high media interference. Importantly, by the addition of casamino acids in basal medium, we were able to enhance threefold amount of secreted hIL-2, which was present both as a fusion and as a clipped fragment. PMID:16014994

  5. Molecular responses of Escherichia coli caused by heat stress and recombinant protein production during temperature induction.

    PubMed

    Valdez-Cruz, Norma A; Ramírez, Octavio T; Trujillo-Roldán, Mauricio A

    2011-01-01

    In a recent review, we discussed the extensively used temperature-inducible expression system, based on the pL and/or pR phage lambda promoters that are finely regulated by the thermo-labile cI857 repressor. In this system, an increase in temperature induces the heterologous protein production and activates the heat shock response, as well as the stringent and SOS responses. The same responses are activated just by the overproduction of recombinant protein. All such responses result in a metabolic burden to the cells, a decrease in the specific growth rate, and alterations in the central carbon metabolism. Altogether, these effects can alter the quantity and quality of the produced foreign protein. Here, we compare and discuss the transcription of selected genes, and the concomitant synthesis of heat-shock proteins (hsp) soon after thermal induction, in relation to the responses that occur in other expression systems that also trigger the heat-shock response.

  6. N-Lauroylation during the Expression of Recombinant N-Myristoylated Proteins: Implications and Solutions.

    PubMed

    Flamm, Andrea Gabriele; Le Roux, Anabel-Lise; Mateos, Borja; Díaz-Lobo, Mireia; Storch, Barbara; Breuker, Kathrin; Konrat, Robert; Pons, Miquel; Coudevylle, Nicolas

    2016-01-01

    Incorporation of myristic acid onto the N terminus of a protein is a crucial modification that promotes membrane binding and correct localization of important components of signaling pathways. Recombinant expression of N-myristoylated proteins in Escherichia coli can be achieved by co-expressing yeast N-myristoyltransferase and supplementing the growth medium with myristic acid. However, undesired incorporation of the 12-carbon fatty acid lauric acid can also occur (leading to heterogeneous samples), especially when the available carbon sources are scarce, as it is the case in minimal medium for the expression of isotopically enriched samples. By applying this method to the brain acid soluble protein 1 and the 1-185 N-terminal region of c-Src, we show the significant, and protein-specific, differences in the membrane binding properties of lauroylated and myristoylated forms. We also present a robust strategy for obtaining lauryl-free samples of myristoylated proteins in both rich and minimal media.

  7. Emerging roles for centromere-associated proteins in DNA repair and genetic recombination.

    PubMed

    Osman, Fekret; Whitby, Matthew C

    2013-12-01

    Centromere proteins CENP-S and CENP-X are members of the constitutive centromere-associated network, which is a conserved group of proteins that are needed for the assembly and function of kinetochores at centromeres. Intriguingly CENP-S and CENP-X have alter egos going by the names of MHF1 (FANCM-associated histone-fold protein 1) and MHF2 respectively. In this guise they function with a DNA translocase called FANCM (Fanconi's anemia complementation group M) to promote DNA repair and homologous recombination. In the present review we discuss current knowledge of the biological roles of CENP-S and CENP-X and how their dual existence may be a common feature of CCAN (constitutive centromere-associated network) proteins.

  8. Quantitative GFP fluorescence as an indicator of recombinant protein synthesis in transgenic plants.

    PubMed

    Richards, H A; Halfhill, M D; Millwood, R J; Stewart, C N

    2003-09-01

    The utility of green fluorescent protein (GFP) for biological research is evident. A fluorescence-based method was developed to quantify GFP levels in transgenic plants and protein extracts. Fluorescence intensity was linear with increasing levels of GFP over a range that encompasses transgene expression in plants by the cauliflower mosaic virus 35S promoter. Standard curves were used to estimate GFP concentration in planta and in protein extracts. These values were consistent with ELISA measurements of GFP in protein extracts from transgenic plants, indicating that the technique is a reliable measure of recombinant GFP expression. The levels of in planta GFP expression in both homozygous and hemizygous plants was then estimated. Homozygous transgenic plants expressed twice the amount of GFP than hemizygous plants, suggesting additive transgene expression. This methodology may be useful to simplify the characterization of transgene expression in plants.

  9. Optimizing Production of Antigens and Fabs in the Context of Generating Recombinant Antibodies to Human Proteins.

    PubMed

    Zhong, Nan; Loppnau, Peter; Seitova, Alma; Ravichandran, Mani; Fenner, Maria; Jain, Harshika; Bhattacharya, Anandi; Hutchinson, Ashley; Paduch, Marcin; Lu, Vincent; Olszewski, Michal; Kossiakoff, Anthony A; Dowdell, Evan; Koide, Akiko; Koide, Shohei; Huang, Haiming; Nadeem, Vincent; Sidhu, Sachdev S; Greenblatt, Jack F; Marcon, Edyta; Arrowsmith, Cheryl H; Edwards, Aled M; Gräslund, Susanne

    2015-01-01

    We developed and optimized a high-throughput project workflow to generate renewable recombinant antibodies to human proteins involved in epigenetic signalling. Three different strategies to produce phage display compatible protein antigens in bacterial systems were compared, and we found that in vivo biotinylation through the use of an Avi tag was the most productive method. Phage display selections were performed on 265 in vivo biotinylated antigen domains. High-affinity Fabs (<20nM) were obtained for 196. We constructed and optimized a new expression vector to produce in vivo biotinylated Fabs in E. coli. This increased average yields up to 10-fold, with an average yield of 4 mg/L. For 118 antigens, we identified Fabs that could immunoprecipitate their full-length endogenous targets from mammalian cell lysates. One Fab for each antigen was converted to a recombinant IgG and produced in mammalian cells, with an average yield of 15 mg/L. In summary, we have optimized each step of the pipeline to produce recombinant antibodies, significantly increasing both efficiency and yield, and also showed that these Fabs and IgGs can be generally useful for chromatin immunoprecipitation (ChIP) protocols. PMID:26437229

  10. Rabbit Hemorrhagic Disease Virus Variant Recombinant VP60 Protein Induces Protective Immunogenicity.

    PubMed

    Yang, Dong-Kun; Kim, Ha-Hyun; Nah, Jin-Ju; Song, Jae-Young

    2015-11-01

    Rabbit hemorrhagic disease virus (RHDV) is highly contagious and often causes fatal disease that affects both wild and domestic rabbits of the species Oryctolagus cuniculus. A highly pathogenic RHDV variant (RHDVa) has been circulation in the Korean rabbit population since 2007 and has a devastating effect on the rabbit industry in Korea. A highly pathogenic RHDVa was isolated from naturally infected rabbits, and the gene encoding the VP60 protein was cloned into a baculovirus transfer vector and expressed in insect cells. The hemagglutination titer of the Sf-9 cell lysate infected with recombinant VP60 baculovirus was 131,072 units/50 μl and of the supernatant 4,096 units/50 μl. Guinea pigs immunized twice intramuscularly with a trial inactivated RHDVa vaccine containing recombinant VP60 contained 2,152 hemagglutination inhibition (HI) geometric mean titers. The 8-week-old white rabbits inoculated with one vaccine dose were challenged with a lethal RHDVa 21 days later and showed 100% survival rates. The recombinant VP60 protein expressed in a baculovirus system induced high HI titers in guinea pigs and rendered complete protection, which led to the development of a novel inactivated RHDVa vaccine. PMID:26198122

  11. Identification of recombinant baculoviruses using green fluorescent protein as a selectable marker.

    PubMed

    Wilson, L E; Wilkinson, N; Marlow, S A; Possee, R D; King, L A

    1997-04-01

    A rapid procedure for the production and identification of recombinant baculoviruses is described that uses the autofluorescent properties of the Aquorea victoria green fluorescent protein (GFP). Expression of the GFP cDNA (without signal peptide sequence) in Spodoptera frugiperda cells resulted in the synthesis of a 30-kDa protein, which was confirmed as GFP by Western blotting and by the emission of green fluorescence when illuminated with longwave UV light (495 or 365 nm). To use GFP as a marker for the selection of recombinant baculoviruses, we prepared a virus, BacGFP1, in which the GFP cDNA was inserted in lieu of lacZ in BacPAK6. Before the use of BacPAK6 or BacGFP1 in a cotransfection to prepare recombinant baculoviruses, the virus DNA was linearized with Bsu361 to improve the recovery of non-parental virus plaques. The use of BacGFP1 DNA resulted in the recovery of 79%-91% plaques with the non-parental phenotype. Plaques were rapidly identified by simply exposing them briefly to longwave UV light (365 nm) without the need for exogenous substrates or biological stains. PMID:9105619

  12. Optimizing Production of Antigens and Fabs in the Context of Generating Recombinant Antibodies to Human Proteins

    PubMed Central

    Zhong, Nan; Loppnau, Peter; Seitova, Alma; Ravichandran, Mani; Fenner, Maria; Jain, Harshika; Bhattacharya, Anandi; Hutchinson, Ashley; Paduch, Marcin; Lu, Vincent; Olszewski, Michal; Kossiakoff, Anthony A.; Dowdell, Evan; Koide, Akiko; Koide, Shohei; Huang, Haiming; Nadeem, Vincent; Sidhu, Sachdev S.; Greenblatt, Jack F.; Marcon, Edyta; Arrowsmith, Cheryl H.; Edwards, Aled M.; Gräslund, Susanne

    2015-01-01

    We developed and optimized a high-throughput project workflow to generate renewable recombinant antibodies to human proteins involved in epigenetic signalling. Three different strategies to produce phage display compatible protein antigens in bacterial systems were compared, and we found that in vivo biotinylation through the use of an Avi tag was the most productive method. Phage display selections were performed on 265 in vivo biotinylated antigen domains. High-affinity Fabs (<20nM) were obtained for 196. We constructed and optimized a new expression vector to produce in vivo biotinylated Fabs in E. coli. This increased average yields up to 10-fold, with an average yield of 4 mg/L. For 118 antigens, we identified Fabs that could immunoprecipitate their full-length endogenous targets from mammalian cell lysates. One Fab for each antigen was converted to a recombinant IgG and produced in mammalian cells, with an average yield of 15 mg/L. In summary, we have optimized each step of the pipeline to produce recombinant antibodies, significantly increasing both efficiency and yield, and also showed that these Fabs and IgGs can be generally useful for chromatin immunoprecipitation (ChIP) protocols. PMID:26437229

  13. Recombination-stable multimeric green fluorescent protein for characterization of weak promoter outputs in Saccharomyces cerevisiae.

    PubMed

    Rugbjerg, Peter; Knuf, Christoph; Förster, Jochen; Sommer, Morten O A

    2015-12-01

    Green fluorescent proteins (GFPs) are widely used for visualization of proteins to track localization and expression dynamics. However, phenotypically important processes can operate at too low expression levels for routine detection, i.e. be overshadowed by autofluorescence noise. While GFP functions well in translational fusions, the use of tandem GFPs to amplify fluorescence signals is currently avoided in Saccharomyces cerevisiae and many other microorganisms due to the risk of loop-out by direct-repeat recombination. We increased GFP fluorescence by translationally fusing three different GFP variants, yeast-enhanced GFP, GFP+ and superfolder GFP to yield a sequence-diverged triple GFP molecule 3vGFP with 74-84% internal repeat identity. Unlike a single GFP, the brightness of 3vGFP allowed characterization of a weak promoter in S. cerevisiae. Utilizing 3vGFP, we further engineered a less leaky Cu(2+)-inducible promoter based on CUP1. The basal expression level of the new promoter was approximately 61% below the wild-type CUP1 promoter, thus expanding the absolute range of Cu(2+)-based gene control. The stability of 3vGFP towards direct-repeat recombination was assayed in S. cerevisiae cultured for 25 generations under strong and slightly toxic expression after which only limited reduction in fluorescence was detectable. Such non-recombinogenic GFPs can help quantify intracellular responses operating a low copy number in recombination-prone organisms.

  14. Production of recombinant snakehead rhabdovirus: the NV protein is not required for viral replication.

    PubMed

    Johnson, M C; Simon, B E; Kim, C H; Leong, J A

    2000-03-01

    Snakehead rhabdovirus (SHRV) affects warm water fish in Southeast Asia and belongs to the genus Novirhabdovirus by virtue of its nonvirion gene (NV). Because SHRV grows best at temperatures between 28 and 31 degrees C, we were able to use the T7 expression system to produce viable recombinant SHRV from a cloned cDNA copy of the viral genome. Expression of a positive-strand RNA copy of the 11, 550-nucleotide SHRV genome along with the viral nucleocapsid (N), phosphoprotein (P), and polymerase (L) proteins resulted in the generation of infectious SHRV in cells preinfected with a vaccinia virus vector for T7 polymerase expression. Recombinant virus production was verified by detection of a unique restriction site engineered into the SHRV genome between the NV and L genes. Since we were now able to begin examining the function of the NV gene, we constructed a recombinant virus containing a nonsense mutation located 22 codons into the coding sequence of the NV protein. The NV knockout virus was produced at a concentration as high as that of wild-type virus in cultured fish cells, and the resulting virions appeared to be identical to the wild-type virions in electron micrographs. These initial studies suggest that NV has no critical function in SHRV replication in cultured fish cells.

  15. Effect of Chemical Chaperones in Improving the Solubility of Recombinant Proteins in Escherichia coli▿†

    PubMed Central

    Prasad, Shivcharan; Khadatare, Prashant B.; Roy, Ipsita

    2011-01-01

    The recovery of active proteins from inclusion bodies usually involves chaotrope-induced denaturation, followed by refolding of the unfolded protein. The efficiency of renaturation is low, leading to reduced yield of the final product. In this work, we report that recombinant proteins can be overexpressed in the soluble form in the host expression system by incorporating compatible solutes during protein expression. Green fluorescent protein (GFP), which was otherwise expressed as inclusion bodies, could be made to partition off into the soluble fraction when sorbitol and arginine, but not ethylene glycol, were present in the growth medium. Arginine and sorbitol increased the production of soluble protein, while ethylene glycol did not. Production of ATP increased in the presence of sorbitol and arginine, but not ethylene glycol. A control experiment with fructose addition indicated that protein solubilization was not due to a simple ATP increase. We have successfully reproduced these results with the N-terminal domain of HypF (HypF-N), a bacterial protein which forms inclusion bodies in Escherichia coli. Instead of forming inclusion bodies, HypF-N could be expressed as a soluble protein in the presence of sorbitol, arginine, and trehalose in the expression medium. PMID:21551288

  16. Signal peptide optimization tool for the secretion of recombinant protein from Saccharomyces cerevisiae.

    PubMed

    Mori, Akihiro; Hara, Shoichi; Sugahara, Tomohiro; Kojima, Takaaki; Iwasaki, Yugo; Kawarasaki, Yasuaki; Sahara, Takehiko; Ohgiya, Satoru; Nakano, Hideo

    2015-11-01

    The secretion efficiency of foreign proteins in recombinant microbes is strongly dependent on the combination of the signal peptides (SPs) used and the target proteins; therefore, identifying the optimal SP sequence for each target protein is a crucial step in maximizing the efficiency of protein secretion in both prokaryotes and eukaryotes. In this study, we developed a novel method, named the SP optimization tool (SPOT), for the generation and rapid screening of a library of SP-target gene fusion constructs to identify the optimal SP for maximizing target protein secretion. In contrast to libraries generated in previous studies, SPOT fusion constructs are generated without adding the intervening sequences associated with restriction enzyme digestion sites. Therefore, no extra amino acids are inserted at the N-terminus of the target protein that might affect its function or conformational stability. As a model system, β-galactosidase (LacA) from Aspergillus oryzae was used as a target protein for secretion from Saccharomyces cerevisiae. In total, 60 SPs were selected from S. cerevisiae secretory proteins and utilized to generate the SP library. While many of the SP-LacA fusions were not secreted, several of the SPs, AGA2, CRH1, PLB1, and MF(alpha)1, were found to enhance LacA secretion compared to the WT sequence. Our results indicate that SPOT is a valuable method for optimizing the bioproduction of any target protein, and could be adapted to many host strains.

  17. Production of recombinant proteins in Mycobacterium smegmatis for structural and functional studies

    PubMed Central

    Bashiri, Ghader; Baker, Edward N

    2015-01-01

    Protein production using recombinant DNA technology has a fundamental impact on our understanding of biology through providing proteins for structural and functional studies. Escherichia coli (E. coli) has been traditionally used as the default expression host to over-express and purify proteins from many different organisms. E. coli does, however, have known shortcomings for obtaining soluble, properly folded proteins suitable for downstream studies. These shortcomings are even more pronounced for the mycobacterial pathogen Mycobacterium tuberculosis, the bacterium that causes tuberculosis, with typically only one third of proteins expressed in E. coli produced as soluble proteins. Mycobacterium smegmatis (M. smegmatis) is a closely related and non-pathogenic species that has been successfully used as an expression host for production of proteins from various mycobacterial species. In this review, we describe the early attempts to produce mycobacterial proteins in alternative expression hosts and then focus on available expression systems in M. smegmatis. The advantages of using M. smegmatis as an expression host, its application in structural biology and some practical aspects of protein production are also discussed. M. smegmatis provides an effective expression platform for enhanced understanding of mycobacterial biology and pathogenesis and for developing novel and better therapeutics and diagnostics. PMID:25303009

  18. Tender coconut water an economical growth medium for the production of recombinant proteins in Escherichia coli

    PubMed Central

    2013-01-01

    Background Escherichia coli is most widely used prokaryotic expression system for the production of recombinant proteins. Several strategies have been employed for expressing recombinant proteins in E.coli. This includes the development of novel host systems, expression vectors and cost effective media. In this study, we exploit tender coconut water (TCW) as a natural and cheaper growth medium for E.coli and Pichia pastoris. Result E.coli and P.pastoris were cultivated in TCW and the growth rate was monitored by measuring optical density at 600 nm (OD600nm), where 1.55 for E.coli and 8.7 for P.pastoris was obtained after 12 and 60 hours, respectively. However, variation in growth rate was observed among TCW when collected from different localities (0.15-2.5 at OD600nm), which is attributed to the varying chemical profile among samples. In this regard, we attempted the supplementation of TCW with different carbon and nitrogen sources to attain consistency in growth rate. Here, supplementation of TCW with 25 mM ammonium sulphate (TCW-S) was noted efficient for the normalization of inconsistency, which further increased the biomass of E.coli by 2 to 10 folds, and 1.5 to 2 fold in P.pastoris. These results indicate that nitrogen source is the major limiting factor for growth. This was supported by total nitrogen and carbon estimation where, nitrogen varies from 20 to 60 mg/100 ml while carbohydrates showed no considerable variation (2.32 to 3.96 g/100 ml). In this study, we also employed TCW as an expression media for recombinant proteins by demonstrating successful expression of maltose binding protein (MBP), MBP-TEV protease fusion and a photo switchable fluorescent protein (mEos2) using TCW and the expression level was found to be equivalent to Luria Broth (LB). Conclusion This study highlights the possible application of TCW-S as a media for cultivation of a variety of microorganisms and recombinant protein expression. PMID:24004578

  19. Subdominant Outer Membrane Antigens in Anaplasma marginale: Conservation, Antigenicity, and Protective Capacity Using Recombinant Protein

    PubMed Central

    Ducken, Deirdre R.; Brown, Wendy C.; Alperin, Debra C.; Brayton, Kelly A.; Reif, Kathryn E.; Turse, Joshua E.; Palmer, Guy H.; Noh, Susan M.

    2015-01-01

    Anaplasma marginale is a tick-borne rickettsial pathogen of cattle with a worldwide distribution. Currently a safe and efficacious vaccine is unavailable. Outer membrane protein (OMP) extracts or a defined surface protein complex reproducibly induce protective immunity. However, there are several knowledge gaps limiting progress in vaccine development. First, are these OMPs conserved among the diversity of A. marginale strains circulating in endemic regions? Second, are the most highly conserved outer membrane proteins in the immunogens recognized by immunized and protected animals? Lastly, can this subset of OMPs recognized by antibody from protected vaccinates and conserved among strains recapitulate the protection of outer membrane vaccines? To address the first goal, genes encoding OMPs AM202, AM368, AM854, AM936, AM1041, and AM1096, major subdominant components of the outer membrane, were cloned and sequenced from geographically diverse strains and isolates. AM202, AM936, AM854, and AM1096 share 99.9 to 100% amino acid identity. AM1041 has 97.1 to 100% and AM368 has 98.3 to 99.9% amino acid identity. While all four of the most highly conserved OMPs were recognized by IgG from animals immunized with outer membranes, linked surface protein complexes, or unlinked surface protein complexes and shown to be protected from challenge, the highest titers and consistent recognition among vaccinates were to AM854 and AM936. Consequently, animals were immunized with recombinant AM854 and AM936 and challenged. Recombinant vaccinates and purified outer membrane vaccinates had similar IgG and IgG2 responses to both proteins. However, the recombinant vaccinates developed higher bacteremia after challenge as compared to adjuvant-only controls and outer membrane vaccinates. These results provide the first evidence that vaccination with specific antigens may exacerbate disease. Progressing from the protective capacity of outer membrane formulations to recombinant vaccines

  20. Holliday intermediates and reaction by-products in FLP protein-promoted site-specific recombination.

    PubMed Central

    Meyer-Leon, L; Huang, L C; Umlauf, S W; Cox, M M; Inman, R B

    1988-01-01

    Holliday structures are formed and resolved by FLP protein during site-specific recombination. These structures have been isolated and are visualized in both native and partially denatured states by electron microscopy. No single-strand breaks are found within the junction, indicating that the structure results from a reciprocal exchange of strands. These structures have properties consistent with being reaction intermediates. Double-strand cleavage products and "Y structures" are also detected and appear to be by-products of the reaction. The Y structures are three-armed branched molecules with a covalently closed junction located at the FLP recombination target site. Models are discussed, suggesting that both of these novel structures are made by aberrant cleavages during formation and resolution of the Holliday intermediate. Images PMID:3065624

  1. Canine Enteric Coronaviruses: Emerging Viral Pathogens with Distinct Recombinant Spike Proteins

    PubMed Central

    Licitra, Beth N.; Duhamel, Gerald E.; Whittaker, Gary R.

    2014-01-01

    Canine enteric coronavirus (CCoV) is an alphacoronavirus infecting dogs that is closely related to enteric coronaviruses of cats and pigs. While CCoV has traditionally caused mild gastro-intestinal clinical signs, there are increasing reports of lethal CCoV infections in dogs, with evidence of both gastrointestinal and systemic viral dissemination. Consequently, CCoV is now considered to be an emerging infectious disease of dogs. In addition to the two known serotypes of CCoV, novel recombinant variants of CCoV have been found containing spike protein N-terminal domains (NTDs) that are closely related to those of feline and porcine strains. The increase in disease severity in dogs and the emergence of novel CCoVs can be attributed to the high level of recombination within the spike gene that can occur during infection by more than one CCoV type in the same host. PMID:25153347

  2. Immunogenicity of recombinant Bacillus subtilis spores expressing Clonorchis sinensis tegumental protein.

    PubMed

    Zhou, Zhenwen; Xia, Huimin; Hu, Xuchu; Huang, Yan; Ma, Changling; Chen, Xiaoxiang; Hu, Fengyu; Xu, Jin; Lu, Fangli; Wu, Zhongdao; Yu, Xinbing

    2008-01-01

    Clonorchis sinensis, which causes clonorchiasis, is of major socioeconomic importance in China. In this study, we report the use of CotC, a major component of the Bacillus subtilis spore coat, as a fusion partner for the expression of C. sinensis TP20.8 (Tegumental Protein 20.8 kDa) on the spore coat. Western blotting was used to identify TP20.8 surface expression on spores. Recombinant spores displaying the TP20.8 antigen were used for oral immunization and were shown to generate mucosal response in rats. TP20.8-specific secretory IgA in feces reached significant levels 2 weeks after oral dosing. This report shows that surface display of recombinant C. sinensis TP20.8 on B. subtilis spores was immunogenic and B. subtilis spores can be used as a mucosal immunization vehicle for parasite prevention and control.

  3. HMG1-related DNA-binding protein isolated with V-(D)-J recombination signal probes.

    PubMed Central

    Shirakata, M; Hüppi, K; Usuda, S; Okazaki, K; Yoshida, K; Sakano, H

    1991-01-01

    In order to isolate cDNA clones for DNA-binding components of the V-(D)-J recombinase, phage libraries from a pre-B-cell line were screened with a radiolabeled probe containing recombination signal sequences (RSS). Among prospective clones, cDNA T160 was analyzed further. It produced a protein of 80.6 kDa which bound to DNA containing RSS but not to DNA in which the RSS had been mutated. A search of a data base revealed that the T160 protein has significant sequence homology (56%) to the nonhistone chromosomal protein HMG1 within the C-terminal region of 80 amino acids. DNA-binding analysis with truncated proteins showed that the HMG homology region is responsible for DNA binding. Using restriction fragment length polymorphisms, the T160 gene was mapped at the proximal end of mouse chromosome 2. Evidence was obtained for genetic linkage between the T160 gene and the recombination activator genes RAG-1 and RAG-2. Images PMID:1678855

  4. Antioxidant efficacy and adhesion rescue by a recombinant mussel foot protein-6.

    PubMed

    Nicklisch, Sascha C T; Das, Saurabh; Martinez Rodriguez, Nadine R; Waite, J Herbert; Israelachvili, Jacob N

    2013-01-01

    Mytilus foot protein type 6 (mfp-6) is crucial for maintaining the reducing conditions needed for optimal wet adhesion in marine mussels. In this report, we describe the expression and production of a recombinant Mytilus californianus foot protein type 6 variant 1 (rmfp-6.1) fused with a hexahistidine affinity tag in Escherichia coli and its purification by affinity chromatography. Recombinant mfp-6 showed high purification yields of 5-6 mg L(-1) cell culture and excellent solubility in low pH buffers that retard oxidation of its many thiol groups. Purified rmfp-6.1 protein showed high 2,2-diphenyl-1-picrylhydrazyl radical scavenging activity when compared with vitamin C. Using the highly sensitive surface forces apparatus (SFA) technique to measure interfacial surface forces in the nano-Newton range, we show that rmfp-6.1 is also able to rescue the oxidation-dependent adhesion loss of mussel foot protein 3 (mfp-3) at pH 3. The adhesion rescue is related to a reduction of dopaquinone back to 3,4-dihydroxyphenyl-l-alanine in mfp-3, which is the reverse reaction observed during the detrimental enzymatic browning process in fruits and vegetables. Broadly viewed, rmfp-6.1 has potential as a versatile antioxidant for applications ranging from personal products to antispoilants for perishable foods during processing and storage. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:1587-1593, 2013. PMID:24106182

  5. Residual endotoxin contaminations in recombinant proteins are sufficient to activate human CD1c+ dendritic cells.

    PubMed

    Schwarz, Harald; Schmittner, Maria; Duschl, Albert; Horejs-Hoeck, Jutta

    2014-01-01

    Many commercially available recombinant proteins are produced in Escherichia coli, and most suppliers guarantee contamination levels of less than 1 endotoxin unit (EU). When we analysed commercially available proteins for their endotoxin content, we found contamination levels in the same range as generally stated in the data sheets, but also some that were higher. To analyse whether these low levels of contamination have an effect on immune cells, we stimulated the monocytic cell line THP-1, primary human monocytes, in vitro differentiated human monocyte-derived dendritic cells, and primary human CD1c+ dendritic cells (DCs) with very low concentrations of lipopolysaccharide (LPS; ranging from 0.002-2 ng/ml). We show that CD1c+ DCs especially can be activated by minimal amounts of LPS, equivalent to the levels of endotoxin contamination we detected in some commercially available proteins. Notably, the enhanced endotoxin sensitivity of CD1c+ DCs was closely correlated with high CD14 expression levels observed in CD1c+ DCs that had been maintained in cell culture medium for 24 hours. When working with cells that are particularly sensitive to LPS, even low endotoxin contamination may generate erroneous data. We therefore recommend that recombinant proteins be thoroughly screened for endotoxin contamination using the limulus amebocyte lysate test, fluorescence-based assays, or a luciferase based NF-κB reporter assay involving highly LPS-sensitive cells overexpressing TLR4, MD-2 and CD14.

  6. Macroscopic fibers self-assembled from recombinant miniature spider silk proteins.

    PubMed

    Stark, Margareta; Grip, Stefan; Rising, Anna; Hedhammar, My; Engström, Wilhelm; Hjälm, Göran; Johansson, Jan

    2007-05-01

    Strength, elasticity, and biocompatibility make spider silk an attractive resource for the production of artificial biomaterials. Spider silk proteins, spidroins, contain hundreds of repeated poly alanine/glycine-rich blocks and are difficult to produce recombinantly in soluble form. Most previous attempts to produce artificial spider silk fibers have included solubilization steps in nonphysiological solvents. It is here demonstrated that a miniature spidroin from a protein in dragline silk of Euprosthenops australis can be produced in a soluble form in Escherichia coli when fused to a highly soluble protein partner. Although this miniature spidroin contains only four poly alanine/glycine-rich blocks followed by a C-terminal non-repetitive domain, meter-long fibers are spontaneously formed after proteolytic release of the fusion partner. The structure of the fibers is similar to that of dragline silks, and although self-assembled from recombinant proteins they are as strong as fibers spun from redissolved silk. Moreover, the fibers appear to be biocompatible because human tissue culture cells can grow on and attach to the fibers. These findings enable controlled production of high-performance biofibers at large scale under physiological conditions. PMID:17402782

  7. Molecular characterization of recombinant mouse adenosine kinase and evaluation as a target for protein phosphorylation.

    PubMed

    Sahin, Bogachan; Kansy, Janice W; Nairn, Angus C; Spychala, Jozef; Ealick, Steven E; Fienberg, Allen A; Greene, Robert W; Bibb, James A

    2004-09-01

    The regulation of adenosine kinase (AK) activity has the potential to control intracellular and interstitial adenosine (Ado) concentrations. In an effort to study the role of AK in Ado homeostasis in the central nervous system, two isoforms of the enzyme were cloned from a mouse brain cDNA library. Following overexpression in bacterial cells, the corresponding proteins were purified to homogeneity. Both isoforms were enzymatically active and found to possess K(m) and V(max) values in agreement with kinetic parameters described for other forms of AK. The distribution of AK in discrete brain regions and various peripheral tissues was defined. To investigate the possibility that AK activity is regulated by protein phosphorylation, a panel of protein kinases was screened for ability to phosphorylate recombinant mouse AK. Data from these in vitro phosphorylation studies suggest that AK is most likely not an efficient substrate for PKA, PKG, CaMKII, CK1, CK2, MAPK, Cdk1, or Cdk5. PKC was found to phosphorylate recombinant AK efficiently in vitro. Further analysis revealed, however, that this PKC-dependent phosphorylation occurred at one or more serine residues associated with the N-terminal affinity tag used for protein purification.

  8. Recombinant Passenger Proteins Can Be Conveniently Purified by One-Step Affinity Chromatography.

    PubMed

    Wang, Hua-zhen; Chu, Zhi-zhan; Chen, Chang-chao; Cao, Ao-cheng; Tong, Xin; Ouyang, Can-bin; Yuan, Qi-hang; Wang, Mi-nan; Wu, Zhong-kun; Wang, Hai-hong; Wang, Sheng-bin

    2015-01-01

    Fusion tag is one of the best available tools to date for enhancement of the solubility or improvement of the expression level of recombinant proteins in Escherichia coli. Typically, two consecutive affinity purification steps are often necessitated for the purification of passenger proteins. As a fusion tag, acyl carrier protein (ACP) could greatly increase the soluble expression level of Glucokinase (GlcK), α-Amylase (Amy) and GFP. When fusion protein ACP-G2-GlcK-Histag and ACP-G2-Amy-Histag, in which a protease TEV recognition site was inserted between the fusion tag and passenger protein, were coexpressed with protease TEV respectively in E. coli, the efficient intracellular processing of fusion proteins was achieved. The resulting passenger protein GlcK-Histag and Amy-Histag accumulated predominantly in a soluble form, and could be conveniently purified by one-step Ni-chelating chromatography. However, the fusion protein ACP-GFP-Histag was processed incompletely by the protease TEV coexpressed in vivo, and a large portion of the resulting target protein GFP-Histag aggregated in insoluble form, indicating that the intracellular processing may affect the solubility of cleaved passenger protein. In this context, the soluble fusion protein ACP-GFP-Histag, contained in the supernatant of E. coli cell lysate, was directly subjected to cleavage in vitro by mixing it with the clarified cell lysate of E. coli overexpressing protease TEV. Consequently, the resulting target protein GFP-Histag could accumulate predominantly in a soluble form, and be purified conveniently by one-step Ni-chelating chromatography. The approaches presented here greatly simplify the purification process of passenger proteins, and eliminate the use of large amounts of pure site-specific proteases. PMID:26641240

  9. Recombinant Passenger Proteins Can Be Conveniently Purified by One-Step Affinity Chromatography

    PubMed Central

    Wang, Hua-zhen; Chu, Zhi-zhan; Chen, Chang-chao; Cao, Ao-cheng; Tong, Xin; Ouyang, Can-bin; Yuan, Qi-hang; Wang, Mi-nan; Wu, Zhong-kun; Wang, Hai-hong; Wang, Sheng-bin

    2015-01-01

    Fusion tag is one of the best available tools to date for enhancement of the solubility or improvement of the expression level of recombinant proteins in Escherichia coli. Typically, two consecutive affinity purification steps are often necessitated for the purification of passenger proteins. As a fusion tag, acyl carrier protein (ACP) could greatly increase the soluble expression level of Glucokinase (GlcK), α-Amylase (Amy) and GFP. When fusion protein ACP-G2-GlcK-Histag and ACP-G2-Amy-Histag, in which a protease TEV recognition site was inserted between the fusion tag and passenger protein, were coexpressed with protease TEV respectively in E. coli, the efficient intracellular processing of fusion proteins was achieved. The resulting passenger protein GlcK-Histag and Amy-Histag accumulated predominantly in a soluble form, and could be conveniently purified by one-step Ni-chelating chromatography. However, the fusion protein ACP-GFP-Histag was processed incompletely by the protease TEV coexpressed in vivo, and a large portion of the resulting target protein GFP-Histag aggregated in insoluble form, indicating that the intracellular processing may affect the solubility of cleaved passenger protein. In this context, the soluble fusion protein ACP-GFP-Histag, contained in the supernatant of E. coli cell lysate, was directly subjected to cleavage in vitro by mixing it with the clarified cell lysate of E. coli overexpressing protease TEV. Consequently, the resulting target protein GFP-Histag could accumulate predominantly in a soluble form, and be purified conveniently by one-step Ni-chelating chromatography. The approaches presented here greatly simplify the purification process of passenger proteins, and eliminate the use of large amounts of pure site-specific proteases. PMID:26641240

  10. Expression of biologically active recombinant rat IgE-binding protein in Escherichia coli.

    PubMed

    Frigeri, L G; Robertson, M W; Liu, F T

    1990-12-01

    IgE-binding protein (epsilon BP) is a protein which has affinity for IgE and was originally identified in rat basophilic leukemia (RBL) cells. Subsequently, it was found to be the rat homolog of CBP35, a murine beta-galactoside-specific lectin. This protein is also designated as L-34 and RL-29 and studied independently by several laboratories. More recently, CBP35 (epsilon BP) was found to be equivalent to Mac-2, a surface marker on activated macrophages. Using rat epsilon BP cDNA, we have succeeded in expressing recombinant epsilon BP in Escherichia coli. Milligram quantities of homogeneous epsilon BP could be obtained from bacterial lysate in a one-step affinity purification procedure utilizing lactosyl-Sepharose 4B and elution with a lactose gradient. The recombinant epsilon BP (r epsilon BP) binds mouse IgE and retains reactivity to anti-peptide antibodies specific for a sequence within rat epsilon BP. The purified r epsilon BP exhibits binding activity to various saccharides, with affinity for N-acetyllactosamine greater than thiodigalactoside greater than lactose much greater than D-galactose greater than L-arabinose, an order identical to that exhibited by native epsilon BP isolated from RBL cells. The recombinant lectin displayed hemagglutination activity when tested with rabbit erythrocytes. Although epsilon BP shares sequence homology to other lectins containing S-type (thiol-dependent) carbohydrate-recognition domains, r epsilon BP is resistant to air oxidation and does not require reducing agents for maintaining its activity. Furthermore, the single cysteine residue appears to be unexposed and can be alkylated only when the protein is denatured in 5.6 M guanidinium hydrochloride. The availability of a source for a large quantity of epsilon BP should facilitate the analysis of biological function(s) and structure-activity relationships of this lectin.

  11. Increasing pentose phosphate pathway flux enhances recombinant protein production in Pichia pastoris.

    PubMed

    Nocon, Justyna; Steiger, Matthias; Mairinger, Teresa; Hohlweg, Jonas; Rußmayer, Hannes; Hann, Stephan; Gasser, Brigitte; Mattanovich, Diethard

    2016-07-01

    Production of heterologous proteins in Pichia pastoris (syn. Komagataella sp.) has been shown to exert a metabolic burden on the host metabolism. This burden is associated with metabolite drain, which redirects nucleotides and amino acids from primary metabolism. On the other hand, recombinant protein production affects energy and redox homeostasis of the host cell. In a previous study, we have demonstrated that overexpression of single genes of the oxidative pentose phosphate pathway (PPP) had a positive influence on recombinant production of cytosolic human superoxide dismutase (hSOD). In this study, different combinations of these genes belonging to the oxidative PPP were generated and analyzed. Thereby, a 3.8-fold increase of hSOD production was detected when glucose-6-phosphate dehydrogenase (ZWF1) and 6-gluconolactonase (SOL3) were simultaneously overexpressed, while the combinations of other genes from PPP had no positive effect on protein production. By measuring isotopologue patterns of (13)C-labelled metabolites, we could detect an upshift in the flux ratio of PPP to glycolysis upon ZWF1 and SOL3 co-overexpression, as well as increased levels of 6-phosphogluconate. The substantial improvement of hSOD production by ZWF1 and SOL3 co-overexpression appeared to be connected to an increase in PPP flux. In conclusion, we show that overexpression of SOL3 together with ZWF1 enhanced both the PPP flux ratio and hSOD accumulation, providing evidence that in P. pastoris Sol3 limits the flux through PPP and recombinant protein production.

  12. Evaluation of the immunodiagnostic potential of a recombinant surface protein domain from Acanthamoeba castellanii.

    PubMed

    Sánchez, Alemao G Carpinteyro; Virginio, Veridiana Gomes; Maschio, Vinicius José; Ferreira, Henrique Bunselmeyer; Rott, Marilise Brittes

    2016-10-01

    Acanthamoeba spp. are free-living protists widely distributed in environment, able to cause keratitis, encephalitis and skin lesions in humans and animals. Acanthamoeba spp. exist in two forms: an infective trophozoite and a dormant cyst. Several factors contribute to the pathogenesis of Acanthamoeba spp. The parasite adhesion to the host cell is the primary step for infection and is mediated by a mannose binding-protein, expressed in the surface and considered the main pathogenicity factor in Acanthamoeba spp. So far, there was no evidence of another surface protein of Acanthamoeba spp. relevant for host invasion or infection by these organisms. The aims of this study were to identify and characterize an Acanthamoeba castellanii surface protein and to evaluate its diagnostic potential. In silico predictions of surface proteins allowed to identify the A. castellanii calreticulin as a possible surface antigen. The coding sequence of a predicted extracellular domain of A. castellanii calreticulin was cloned by in vivo homologous recombination and the recombinant polypeptide (AcCRT29-130) was produced. Its immunodiagnostic potential was assessed in a recombinant antigen-based ELISA with sera from experimentally infected rats that developed keratitis and encephalitis, and sera from patients with encephalitis. The AcCRT29-130 was significantly more recognized by sera from encephalitis infected rats in comparison with the non-infected controls. Human sera from encephalitis patients, however presented no significant response. These results showed the AcCRT29-130 potential for A. castellanii infection immunodiagnosis in animals, with further studies being required for assessment of its use for human infections.

  13. Rational Design of a Carrier Protein for the Production of Recombinant Toxic Peptides in Escherichia coli

    PubMed Central

    Pizzo, Elio; Varcamonti, Mario; Zanfardino, Anna; Sgambati, Valeria; Di Maro, Antimo; Carpentieri, Andrea; Izzo, Viviana; Di Donato, Alberto; Cafaro, Valeria; Notomista, Eugenio

    2016-01-01

    Commercial uses of bioactive peptides require low cost, effective methods for their production. We developed a new carrier protein for high yield production of recombinant peptides in Escherichia coli very well suited for the production of toxic peptides like antimicrobial peptides. GKY20, a short antimicrobial peptide derived from the C-terminus of human thrombin, was fused to the C-terminus of Onconase, a small ribonuclease (104 amino acids), which efficiently drove the peptide into inclusion bodies with very high expression levels (about 200–250 mg/L). After purification of the fusion protein by immobilized metal ion affinity chromatography, peptide was obtained by chemical cleavage in diluted acetic acid of an acid labile Asp-Pro sequence with more than 95% efficiency. To improve peptide purification, Onconase was mutated to eliminate all acid labile sequences thus reducing the release of unwanted peptides during the acid cleavage. Mutations were chosen to preserve the differential solubility of Onconase as function of pH, which allows its selective precipitation at neutral pH after the cleavage. The improved carrier allowed the production of 15–18 mg of recombinant peptide per liter of culture with 96–98% purity without the need of further chromatographic steps after the acid cleavage. The antimicrobial activity of the recombinant peptide, with an additional proline at the N-terminus, was tested on Gram-negative and Gram-positive strains and was found to be identical to that measured for synthetic GKY20. This finding suggests that N-terminal proline residue does not change the antimicrobial properties of recombinant (P)GKY20. The improved carrier, which does not contain cysteine and methionine residues, Asp-Pro and Asn-Gly sequences, is well suited for the production of peptides using any of the most popular chemical cleavage methods. PMID:26808536

  14. Rational Design of a Carrier Protein for the Production of Recombinant Toxic Peptides in Escherichia coli.

    PubMed

    Pane, Katia; Durante, Lorenzo; Pizzo, Elio; Varcamonti, Mario; Zanfardino, Anna; Sgambati, Valeria; Di Maro, Antimo; Carpentieri, Andrea; Izzo, Viviana; Di Donato, Alberto; Cafaro, Valeria; Notomista, Eugenio

    2016-01-01

    Commercial uses of bioactive peptides require low cost, effective methods for their production. We developed a new carrier protein for high yield production of recombinant peptides in Escherichia coli very well suited for the production of toxic peptides like antimicrobial peptides. GKY20, a short antimicrobial peptide derived from the C-terminus of human thrombin, was fused to the C-terminus of Onconase, a small ribonuclease (104 amino acids), which efficiently drove the peptide into inclusion bodies with very high expression levels (about 200-250 mg/L). After purification of the fusion protein by immobilized metal ion affinity chromatography, peptide was obtained by chemical cleavage in diluted acetic acid of an acid labile Asp-Pro sequence with more than 95% efficiency. To improve peptide purification, Onconase was mutated to eliminate all acid labile sequences thus reducing the release of unwanted peptides during the acid cleavage. Mutations were chosen to preserve the differential solubility of Onconase as function of pH, which allows its selective precipitation at neutral pH after the cleavage. The improved carrier allowed the production of 15-18 mg of recombinant peptide per liter of culture with 96-98% purity without the need of further chromatographic steps after the acid cleavage. The antimicrobial activity of the recombinant peptide, with an additional proline at the N-terminus, was tested on Gram-negative and Gram-positive strains and was found to be identical to that measured for synthetic GKY20. This finding suggests that N-terminal proline residue does not change the antimicrobial properties of recombinant (P)GKY20. The improved carrier, which does not contain cysteine and methionine residues, Asp-Pro and Asn-Gly sequences, is well suited for the production of peptides using any of the most popular chemical cleavage methods. PMID:26808536

  15. Frictional drag and electrical manipulation of recombinant proteins in polymer-supported membranes.

    PubMed

    Tanaka, Motomu; Hermann, Joachim; Haase, Ilka; Fischer, Markus; Boxer, Steven G

    2007-05-01

    We establish a lipid monolayer supported by a polymer interface that offers advantages over conventional solid-supported membranes for determining the frictional drag at the membrane-protein interface as well as for electric field manipulation of membrane-anchored proteins. Polymer-supported monolayers with functional lipid anchors allow for the specific docking of His-tagged green fluorescent protein variants (His-EGFP and His-DsRed tetramer) onto the membrane surface at a defined surface density. In the first part, we measure the lateral diffusion coefficients of lipids and proteins and calculate the frictional drag at the protein-membrane interface. The second part deals with the electric field-induced accumulation of recombinant proteins on a patterned surface. The mean drift velocity of proteins, which can be obtained analytically from the shape of the steady-state concentration gradient, can be controlled by tuning the interplay of electrophoresis and electroosmosis. The results demonstrate the potential of such molecular constructs for the local functionalization of solid substrates with membrane-associated proteins.

  16. Production of cocktail of polyclonal antibodies using bacterial expressed recombinant protein for multiple virus detection.

    PubMed

    Kapoor, Reetika; Mandal, Bikash; Paul, Prabir Kumar; Chigurupati, Phaneendra; Jain, Rakesh Kumar

    2014-02-01

    Cocktail of polyclonal antibodies (PAb) were produced that will help in multiple virus detection and overcome the limitation of individual virus purification, protein expression and purification as well as immunization in multiple rabbits. A dual fusion construct was developed using conserved coat protein (CP) sequences of Cucumber mosaic virus (CMV) and Papaya ringspot virus (PRSV) in an expression vector, pET-28a(+). The fusion protein (∼40kDa) was expressed in Escherichia coli and purified. Likewise, a triple fusion construct was developed by fusing conserved CP sequences of CMV and PRSV with conserved nucleocapsid protein (N) sequence of Groundnut bud necrosis virus (GBNV) and expressed as a fusion protein (∼50kDa) in pET-28a(+). PAb made separately to each of these three viruses recognized the double and triple fusion proteins in Western blot indicating retention of desired epitopes for binding with target antibodies. The fusion proteins (∼40kDa and ∼50kDa) were used to produce cocktail of PAb by immunizing rabbits, which simultaneously detected natural infection of CMV and PRSV or CMV, PRSV and GBNV in Cucurbitaceous, Solanaceous and other hosts in DAC-ELISA. This is the first report on production of a cocktail of PAb to recombinant fusion protein of two or three distinct viruses.

  17. Protection of Mice from Fatal Measles Encephalitis by Vaccination with Vaccinia Virus Recombinants Encoding Either the Hemagglutinin or the Fusion Protein

    NASA Astrophysics Data System (ADS)

    Drillien, Robert; Spehner, Daniele; Kirn, Andre; Giraudon, Pascale; Buckland, Robin; Wild, Fabian; Lecocq, Jean-Pierre

    1988-02-01

    Vaccinia virus recombinants encoding the hemagglutinin or fusion protein of measles virus have been constructed. Infection of cell cultures with the recombinants led to the synthesis of authentic measles proteins as judged by their electrophoretic mobility, recognition by antibodies, glycosylation, proteolytic cleavage, and presentation on the cell surface. Mice vaccinated with a single dose of the recombinant encoding the hemagglutinin protein developed antibodies capable of both inhibiting hemagglutination activity and neutralizing measles virus, whereas animals vaccinated with the recombinant encoding the fusion protein developed measles neutralizing antibodies. Mice vaccinated with either of the recombinants resisted a normally lethal intracerebral inoculation of a cell-associated measles virus subacute sclerosing panencephalitis strain.

  18. Expression of the structural proteins of dengue 2 virus and yellow fever virus by recombinant vaccinia viruses.

    PubMed

    Hahn, Y S; Lenches, E M; Galler, R; Rice, C M; Dalrymple, J; Strauss, J H

    1990-01-01

    Vaccinia virus recombinants were constructed which contained cDNA sequences encoding the structural region of dengue 2 virus (PR159/S1 strain) or yellow fever virus (17D strain). The flavivirus cDNA sequences were expressed under the control of the vaccinia 7.5k early/late promotor. Cultured cells infected with these recombinants expressed immunologically reactive flavivirus structural proteins, precursor prM and E. These proteins appeared to be cleaved and glycosylated properly since they comigrated with the authentic proteins from dengue 2 virus- and yellow fever virus-infected cells. Mice immunized with the dengue/vaccinia recombinant showed a dengue-specific immune response that included low levels of neutralizing antibodies. Immunization of mice with the yellow fever/vaccinia recombinant was less effective at inducing an immune response to yellow fever virus and in only some of the mice were low titers of neutralizing antibodies produced.

  19. Immunogenicity of a novel tetravalent vaccine formulation with four recombinant lipidated dengue envelope protein domain IIIs in mice

    PubMed Central

    Chiang, Chen-Yi; Pan, Chien-Hsiung; Chen, Mei-Yu; Hsieh, Chun-Hsiang; Tsai, Jy-Ping; Liu, Hsueh-Hung; Liu, Shih-Jen; Chong, Pele; Leng, Chih-Hsiang; Chen, Hsin-Wei

    2016-01-01

    We developed a novel platform to express high levels of recombinant lipoproteins with intrinsic adjuvant properties. Based on this technology, our group developed recombinant lipidated dengue envelope protein domain IIIs as vaccine candidates against dengue virus. This work aims to evaluate the immune responses in mice to the tetravalent formulation. We demonstrate that 4 serotypes of recombinant lipidated dengue envelope protein domain III induced both humoral and cellular immunity against all 4 serotypes of dengue virus on the mixture that formed the tetravalent formulation. Importantly, the immune responses induced by the tetravalent formulation in the absence of the exogenous adjuvant were functional in clearing the 4 serotypes of dengue virus in vivo. We affirm that the tetravalent formulation of recombinant lipidated dengue envelope protein domain III is a potential vaccine candidate against dengue virus and suggest further detailed studies of this formulation in nonhuman primates. PMID:27470096

  20. Immunogenicity of a novel tetravalent vaccine formulation with four recombinant lipidated dengue envelope protein domain IIIs in mice.

    PubMed

    Chiang, Chen-Yi; Pan, Chien-Hsiung; Chen, Mei-Yu; Hsieh, Chun-Hsiang; Tsai, Jy-Ping; Liu, Hsueh-Hung; Liu, Shih-Jen; Chong, Pele; Leng, Chih-Hsiang; Chen, Hsin-Wei

    2016-01-01

    We developed a novel platform to express high levels of recombinant lipoproteins with intrinsic adjuvant properties. Based on this technology, our group developed recombinant lipidated dengue envelope protein domain IIIs as vaccine candidates against dengue virus. This work aims to evaluate the immune responses in mice to the tetravalent formulation. We demonstrate that 4 serotypes of recombinant lipidated dengue envelope protein domain III induced both humoral and cellular immunity against all 4 serotypes of dengue virus on the mixture that formed the tetravalent formulation. Importantly, the immune responses induced by the tetravalent formulation in the absence of the exogenous adjuvant were functional in clearing the 4 serotypes of dengue virus in vivo. We affirm that the tetravalent formulation of recombinant lipidated dengue envelope protein domain III is a potential vaccine candidate against dengue virus and suggest further detailed studies of this formulation in nonhuman primates. PMID:27470096

  1. Disulfide bonds in a recombinant protein modeled after a core repeat in an aquatic insect's silk protein.

    PubMed Central

    Smith, S. V.; Correia, J. J.; Case, S. T.

    1995-01-01

    We constructed a gene encoding rCAS, recombinant constant and subrepeat protein, modeled after tandem repeats found in the major silk proteins synthesized by aquatic larvae of the midge, Chironomus tentans. Bacterially synthesized rCAS was purified to near homogeneity and characterized by several biochemical and biophysical methods including amino-terminal sequencing, amino acid compositional analysis, sedimentation equilibrium ultracentrifugation, and mass spectrometry. Complementing these techniques with quantitative sulfhydryl assays, we discovered that the four cysteines present in rCAS form two intramolecular disulfide bonds. Mapping studies revealed that the disulfide bonds are heterogeneous. When reduced and denatured rCAS was allowed to refold and its disulfide bonding state monitored, it again adopted a conformation with two intramolecular disulfide bonds. The inherent ability of rCAS to quantitatively form two intramolecular disulfide bonds may reflect a previously unknown feature of the in vivo silk proteins from which it is derived. PMID:7663350

  2. Triatoma Virus Recombinant VP4 Protein Induces Membrane Permeability through Dynamic Pores

    PubMed Central

    Sánchez-Eugenia, Rubén; Goikolea, Julen; Gil-Cartón, David; Sánchez-Magraner, Lissete

    2015-01-01

    ABSTRACT In naked viruses, membrane breaching is a key step that must be performed for genome transfer into the target cells. Despite its importance, the mechanisms behind this process remain poorly understood. The small protein VP4, encoded by the genomes of most viruses of the order Picornavirales, has been shown to be involved in membrane alterations. Here we analyzed the permeabilization activity of the natively nonmyristoylated VP4 protein from triatoma virus (TrV), a virus belonging to the Dicistroviridae family within the Picornavirales order. The VP4 protein was produced as a C-terminal maltose binding protein (MBP) fusion to achieve its successful expression. This recombinant VP4 protein is able to produce membrane permeabilization in model membranes in a membrane composition-dependent manner. The induced permeability was also influenced by the pH, being greater at higher pH values. We demonstrate that the permeabilization activity elicited by the protein occurs through discrete pores that are inserted on the membrane. Sizing experiments using fluorescent dextrans, cryo-electron microscopy imaging, and other, additional techniques showed that recombinant VP4 forms heterogeneous proteolipidic pores rather than common proteinaceous channels. These results suggest that the VP4 protein may be involved in the membrane alterations required for genome transfer or cell entry steps during dicistrovirus infection. IMPORTANCE During viral infection, viruses need to overcome the membrane barrier in order to enter the cell and replicate their genome. In nonenveloped viruses membrane fusion is not possible, and hence, other mechanisms are implemented. Among other proteins, like the capsid-forming proteins and the proteins required for viral replication, several viruses of the order Picornaviridae contain a small protein called VP4 that has been shown to be involved in membrane alterations. Here we show that the triatoma virus VP4 protein is able to produce membrane

  3. Serological Analysis of Immunogenic Properties of Recombinant Meningococcus IgA1 Protease-Based Proteins.

    PubMed

    Kotelnikova, O V; Zinchenko, A A; Vikhrov, A A; Alliluev, A P; Serova, O V; Gordeeva, E A; Zhigis, L S; Zueva, V S; Razgulyaeva, O A; Melikhova, T D; Nokel, E A; Drozhzhina, E Yu; Rumsh, L D

    2016-07-01

    Using the genome sequence of IgA1 protease of N. meningitidis of serogroup B, four recombinant proteins of different structure and molecular weight were constructed. These proteins were equal in inducing the formation of specific antibodies to IgA1 protease and had protective properties against meningococci. In the sera of immunized mice, anti-IgA1 protease antibodies were detected by whole-cell ELISA, which indicated the presence of IgA1 protease on the surface of these bacteria. We hypothesized that the protective properties of IgA1 protease-based antigens and IgA1 protease analogs could be realized not only via impairment of bacterium adhesion to the mucosa, but also via suppression of this pathogen in the organism. The presented findings seem promising for using these proteins as the basis for anti-meningococcus vaccine.

  4. Immunogenicity of recombinant class 1 protein from Neisseria meningitidis refolded into phospholipid vesicles and detergent.

    PubMed

    Niebla, O; Alvarez, A; Martín, A; Rodríguez, A; Delgado, M; Falcón, V; Guillén, G

    2001-05-14

    The possibility of eliciting bactericidal antibodies against a recombinant class 1 protein (P1) from Neisseria meningitidis, joined to the first 45 amino acids of the neisserial LpdA protein (PM82), was examined. P1 was produced in Escherichia coli as intracellular inclusion bodies, from which it was purified and reconstituted by (a) inclusion into phospholipid vesicles and detergent and (b) refolding in 0.1% SDS. When Balb/c mice were immunised, high titres of subtype-specific bactericidal antibodies against P1 were obtained in both cases. These results suggest that in spite of being a denaturing agent, it is possible to use SDS to reconstitute the P1 protein in a conformation that exposes the immunodominat regions.

  5. [Characterization of a panel of monoclonal antibodies to hepatitis C NS3 recombinant protein ].

    PubMed

    Abdulmedzhidova, A G; Masalova, O V; Atanadze, S N; Ulanova, T I; Burkov, A N; Khudiakov, Iu E; Fields, H; Kushch, A A

    2002-01-01

    Recombinant protein rNS3 imitating helicase region (1356-1459 amino acid residues) of hepatitis C virus (HCV) was expressed in E. coli cells and used for BALB/c mice immunization. Seven hybrydoma clones producing monoclonal antibodies (MAbs) to rHS3 were obtained. All MAbs reacted in ELISA with NS3 protein from Murex anti-HCV Version III and in immunoblotting from RIBA 3. These MAbs detect 5 individual epitopes, 4 of which were conformational and 1 discontinuous. All MAbs could compete for rNS3 binding with serum antibodies from patients with chronic hepatitis C, which suggests that these MAbs can recognize the natural HCV NS3 protein.

  6. Homologous recombination and human health: the roles of BRCA1, BRCA2, and associated proteins.

    PubMed

    Prakash, Rohit; Zhang, Yu; Feng, Weiran; Jasin, Maria

    2015-04-01

    Homologous recombination (HR) is a major pathway for the repair of DNA double-strand breaks in mammalian cells, the defining step of which is homologous strand exchange directed by the RAD51 protein. The physiological importance of HR is underscored by the observation of genomic instability in HR-deficient cells and, importantly, the association of cancer predisposition and developmental defects with mutations in HR genes. The tumor suppressors BRCA1 and BRCA2, key players at different stages of HR, are frequently mutated in familial breast and ovarian cancers. Other HR proteins, including PALB2 and RAD51 paralogs, have also been identified as tumor suppressors. This review summarizes recent findings on BRCA1, BRCA2, and associated proteins involved in human disease with an emphasis on their molecular roles and interactions. PMID:25833843

  7. Selective Blockade of Trypanosomatid Protein Synthesis by a Recombinant Antibody Anti-Trypanosoma cruzi P2β Protein

    PubMed Central

    Simonetti, Leandro; Duffy, Tomas; Longhi, Silvia A.; Gómez, Karina A.; Hoebeke, Johan; Levin, Mariano J.; Smulski, Cristian R.

    2012-01-01

    The ribosomal P proteins are located on the stalk of the ribosomal large subunit and play a critical role during the elongation step of protein synthesis. The single chain recombinant antibody C5 (scFv C5) directed against the C-terminal region of the Trypanosoma cruzi P2β protein (TcP2β) recognizes the conserved C-terminal end of all T. cruzi ribosomal P proteins. Although this region is highly conserved among different species, surface plasmon resonance analysis showed that the scFv C5 possesses very low affinity for the corresponding mammalian epitope, despite having only one single amino-acid change. Crystallographic analysis, in silico modelization and NMR assays support the analysis, increasing our understanding on the structural basis of epitope specificity. In vitro protein synthesis experiments showed that scFv C5 was able to specifically block translation by T. cruzi and Crithidia fasciculata ribosomes, but virtually had no effect on Rattus norvegicus ribosomes. Therefore, we used the scFv C5 coding sequence to make inducible intrabodies in Trypanosoma brucei. Transgenic parasites showed a strong decrease in their growth rate after induction. These results strengthen the importance of the P protein C terminal regions for ribosomal translation activity and suggest that trypanosomatid ribosomal P proteins could be a possible target for selective therapeutic agents that could be derived from structural analysis of the scFv C5 antibody paratope. PMID:22570698

  8. Display of recombinant proteins at the surface of lactic acid bacteria: strategies and applications.

    PubMed

    Michon, C; Langella, P; Eijsink, V G H; Mathiesen, G; Chatel, J M

    2016-01-01

    Lactic acid bacteria (LAB) are promising vectors of choice to deliver active molecules to mucosal tissues. They are recognized as safe by the World Health Organization and some strains have probiotic properties. The wide range of potential applications of LAB-driven mucosal delivery includes control of inflammatory bowel disease, vaccine delivery, and management of auto-immune diseases. Because of this potential, strategies for the display of proteins at the surface of LAB are gaining interest. To display a protein at the surface of LAB, a signal peptide and an anchor domain are necessary. The recombinant protein can be attached to the membrane layer, using a transmembrane anchor or a lipoprotein-anchor, or to the cell wall, by a covalent link using sortase mediated anchoring via the LPXTG motif, or by non-covalent liaisons employing binding domains such as LysM or WxL. Both the stability and functionality of the displayed proteins will be affected by the kind of anchor used. The most commonly surfaced exposed recombinant proteins produced in LAB are antigens and antibodies and the most commonly used LAB are lactococci and lactobacilli. Although it is not necessarily so that surface-display is the preferred localization in all cases, it has been shown that for certain applications, such as delivery of the human papillomavirus E7 antigen, surface-display elicits better biological responses, compared to cytosolic expression or secretion. Recent developments include the display of peptides and proteins targeting host cell receptors, for the purpose of enhancing the interactions between LAB and host. Surface-display technologies have other potential applications, such as degradation of biomass, which is of importance for some potential industrial applications of LAB. PMID:27142045

  9. Recombinant Streptococcus equi Proteins Protect Mice in Challenge Experiments and Induce Immune Response in Horses

    PubMed Central

    Flock, Margareta; Jacobsson, Karin; Frykberg, Lars; Hirst, Timothy R.; Franklin, Anders; Guss, Bengt; Flock, Jan-Ingmar

    2004-01-01

    Horses that have undergone infection caused by Streptococcus equi subspecies equi (strangles) were found to have significantly increased serum antibody titers against three previously characterized proteins, FNZ (cell surface-bound fibronectin binding protein), SFS (secreted fibronectin binding protein), and EAG (α2-macroglobulin, albumin, and immunoglobulin G [IgG] binding protein) from S. equi. To assess the protective efficacy of vaccination with these three proteins, a mouse model of equine strangles was utilized. Parts of the three recombinant proteins were used to immunize mice, either subcutaneously or intranasally, prior to nasal challenge with S. equi subsp. equi. The adjuvant used was EtxB, a recombinant form of the B subunit of Escherichia coli heat-labile enterotoxin. It was shown that nasal colonization of S. equi subsp. equi and weight loss due to infection were significantly reduced after vaccination compared with a mock-vaccinated control group. This effect was more pronounced after intranasal vaccination than after subcutaneous vaccination; nearly complete eradication of nasal colonization was obtained after intranasal vaccination (P < 0.001). When the same antigens were administered both intranasally and subcutaneously to healthy horses, significant mucosal IgA and serum IgG antibody responses against FNZ and EAG were obtained. The antibody response was enhanced when EtxB was used as an adjuvant. No adverse effects of the antigens or EtxB were observed. Thus, FNZ and EAG in conjunction with EtxB are promising candidates for an efficacious and safe vaccine against strangles. PMID:15155624

  10. Imidazole-free purification of His3-tagged recombinant proteins using ssDNA aptamer-based affinity chromatography.

    PubMed

    Bartnicki, Filip; Kowalska, Ewa; Pels, Katarzyna; Strzalka, Wojciech

    2015-10-30

    Immobilized metal ion affinity chromatography (IMAC) is widely used for the purification of many different His6-tagged recombinant proteins. On the one hand, it is a powerful technique but on the other hand it has its disadvantages. In this report, we present the development of a unique ssDNA aptamer for the purification of His3-tagged recombinant proteins. Our study shows that stability of the His3-tag/H3T aptamer complex can be controlled by the sodium ion concentration. Based on this feature, we demonstrate that H3T aptamer resin was successfully employed for the purification of three out of four tested His3-tagged recombinant proteins from an E. coli total protein extract using imidazole-free buffers. Finally, we show that the purity of His3-tagged proteins is superior when purified with the help of the H3T aptamer in comparison with Ni-NTA resin. PMID:26427325

  11. Cell culture media supplementation of uncommonly used sugars sucrose and tagatose for the targeted shifting of protein glycosylation profiles of recombinant protein therapeutics.

    PubMed

    Hossler, Patrick; McDermott, Sean; Racicot, Christopher; Chumsae, Christopher; Raharimampionona, Haly; Zhou, Yu; Ouellette, David; Matuck, Joseph; Correia, Ivan; Fann, John; Li, Jianmin

    2014-01-01

    Protein glycosylation is an important post-translational modification toward the structure and function of recombinant therapeutics. The addition of oligosaccharides to recombinant proteins has been shown to greatly influence the overall physiochemical attributes of many proteins. It is for this reason that protein glycosylation is monitored by the developer of a recombinant protein therapeutic, and why protein glycosylation is typically considered a critical quality attribute. In this work, we highlight a systematic study toward the supplementation of sucrose and tagatose into cell culture media for the targeted modulation of protein glycosylation profiles on recombinant proteins. Both sugars were found to affect oligosaccharide maturation resulting in an increase in the percentage of high mannose N-glycan species, as well as a concomitant reduction in fucosylation. The latter effect was demonstrated to increase antibody-dependent cell-mediated cytotoxicity for a recombinant antibody. These aforementioned results were found to be reproducible at different scales, and across different Chinese hamster ovary cell lines. Through the selective supplementation of these described sugars, the targeted modulation of protein glycosylation profiles is demonstrated, as well as yet another tool in the cell culture toolbox for ensuring product comparability.

  12. Physical and kinetic characterization of recombinant human cholesteryl ester transfer protein.

    PubMed Central

    Connolly, D T; McIntyre, J; Heuvelman, D; Remsen, E E; McKinnie, R E; Vu, L; Melton, M; Monsell, R; Krul, E S; Glenn, K

    1996-01-01

    Cholesteryl ester transfer protein (CETP) mediates the exchange of triglycerides (TGs), cholesteryl esters (CEs) and phospholipids (PLs) between lipoproteins in the plasma. In order to better understand the lipid transfer process, we have used recombinant human CETP expressed in cultured mammalian cells, purified to homogeneity by immunoaffinity chromatography. Purified recombinant CETP had a weight-average relative molecular mass (MW) of 69561, determined by sedimentation equilibrium, and a specific absorption coefficient of 0.83 litre.g-1.cm-1. The corresponding hydrodynamic diameter (Dh) of the protein, determined by dynamic light scattering, was 14 nm, which is nearly twice the expected value for a spheroidal protein of this molecular mass. These data suggest that CETP has a non-spheroidal shape in solution. The secondary structure of CETP was estimated by CD to contain 32% alpha-helix, 35% beta-sheet, 17% turn and 16% random coil. Like the natural protein from plasma, the recombinant protein consisted of several glycoforms that could be only partially deglycosylated using N-glycosidase F. Organic extraction of CETP followed by TLC showed that CE, unesterified cholesterol (UC), PL, TG and fatty acids (FA) were associated with the pure protein. Quantitative analyses verified that each mol of CETP contained 1.0 mol of cholesterol, 0.5 mol of TG and 1.3 mol of PL. CETP mediated the transfer of CE, TG, PL, and UC between lipoproteins, or between protein-free liposomes. In dual-label transfer experiments, the transfer rates for CE or TG from HDL to LDL were found to be proportional to the initial concentrations of the respective ligands in the donor HDL particles. Kinetic analysis of CE transfer was consistent with a carrier mechanism, having a Km of 700 nM for LDL particles and of 2000 nM for HDL particles, and a kcat of 2 s-1. The Km values were thus in the low range of the normal physiological concentration for each substrate. The carrier mechanism was verified

  13. Recombinant VSV G proteins reveal a novel raft-dependent endocytic pathway in resorbing osteoclasts

    SciTech Connect

    Mulari, Mika T.K. Nars, Martin; Laitala-Leinonen, Tiina; Kaisto, Tuula; Metsikkoe, Kalervo; Sun Yi; Vaeaenaenen, H. Kalervo

    2008-05-01

    Transcytotic membrane flow delivers degraded bone fragments from the ruffled border to the functional secretory domain, FSD, in bone resorbing osteoclasts. Here we show that there is also a FSD-to-ruffled border trafficking pathway that compensates for the membrane loss during the matrix uptake process and that rafts are essential for this ruffled border-targeted endosomal pathway. Replacing the cytoplasmic tail of the vesicular stomatitis virus G protein with that of CD4 resulted in partial insolubility in Triton X-100 and retargeting from the peripheral non-bone facing plasma membrane to the FSD. Recombinant G proteins were subsequently endosytosed and delivered from the FSD to the peripheral fusion zone of the ruffled border, which were both rich in lipid rafts as suggested by viral protein transport analysis and visualizing the rafts with fluorescent recombinant cholera toxin. Cholesterol depletion by methyl-{beta}-cyclodextrin impaired the ruffled border-targeted vesicle trafficking pathway and inhibited bone resorption dose-dependently as quantified by measuring the CTX and TRACP 5b secreted to the culture medium and by measuring the resorbed area visualized with a bi-phasic labeling method using sulpho-NHS-biotin and WGA-lectin. Thus, rafts are vital for membrane recycling from the FSD to the late endosomal/lysosomal ruffled border and bone resorption.

  14. Meiotic recombination protein Rec12: functional conservation, crossover homeostasis and early crossover/non-crossover decision.

    PubMed

    Kan, Fengling; Davidson, Mari K; Wahls, Wayne P

    2011-03-01

    In fission yeast and other eukaryotes, Rec12 (Spo11) is thought to catalyze the formation of dsDNA breaks (DSBs) that initiate homologous recombination in meiosis. Rec12 is orthologous to the catalytic subunit of topoisomerase VI (Top6A). Guided by the crystal structure of Top6A, we engineered the rec12 locus to encode Rec12 proteins each with a single amino acid substitution in a conserved residue. Of 21 substitutions, 10 significantly reduced or abolished meiotic DSBs, gene conversion, crossover recombination and the faithful segregation of chromosomes. Critical residues map within the metal ion-binding pocket toprim (E179A, D229A, D231A), catalytic region 5Y-CAP (R94A, D95A, Y98F) and the DNA-binding interface (K201A, G202E, R209A, K242A). A subset of substitutions reduced DSBs but maintained crossovers, demonstrating crossover homeostasis. Furthermore, a strong separation of function mutation (R304A) suggests that the crossover/non-crossover decision is established early by a protein-protein interaction surface of Rec12. Fission yeast has multiple crossovers per bivalent, and chromosome segregation was robust above a threshold of about one crossover per bivalent, below which non-disjunction occurred. These results support structural and functional conservation among Rec12/Spo11/Top6A family members for the catalysis of DSBs, and they reveal how Rec12 regulates other features of meiotic chromosome dynamics. PMID:21030440

  15. Structure and stability of recombinant bovine odorant-binding protein: II. Unfolding of the monomeric forms.

    PubMed

    Stepanenko, Olga V; Roginskii, Denis O; Stepanenko, Olesya V; Kuznetsova, Irina M; Uversky, Vladimir N; Turoverov, Konstantin K

    2016-01-01

    In a family of monomeric odorant-binding proteins (OBPs), bovine OBP (bOBP), that lacks conserved disulfide bond found in other OBPs, occupies unique niche because of its ability to form domain-swapped dimers. In this study, we analyzed conformational stabilities of the recombinant bOBP and its monomeric variants, the bOBP-Gly121+ mutant containing an additional glycine residue after the residue 121 of the bOBP, and the GCC-bOBP mutant obtained from the bOBP-Gly121+ form by introduction of the Trp64Cys/His155Cys double mutation to restore the canonical disulfide bond. We also analyzed the effect of the natural ligand binding on the conformational stabilities of these bOBP variants. Our data are consistent with the conclusion that the unfolding-refolding pathways of the recombinant bOBP and its mutant monomeric forms bOBP-Gly121+ and GCC-bOBP are similar and do not depend on the oligomeric status of the protein. This clearly shows that the information on the unfolding-refolding mechanism is encoded in the structure of the bOBP monomers. However, the process of the bOBP unfolding is significantly complicated by the formation of the domain-swapped dimer, and the rates of the unfolding-refolding reactions essentially depend on the conditions in which the protein is located. PMID:27114857

  16. Structure and stability of recombinant bovine odorant-binding protein: II. Unfolding of the monomeric forms

    PubMed Central

    Stepanenko, Olga V.; Roginskii, Denis O.; Stepanenko, Olesya V.; Kuznetsova, Irina M.

    2016-01-01

    In a family of monomeric odorant-binding proteins (OBPs), bovine OBP (bOBP), that lacks conserved disulfide bond found in other OBPs, occupies unique niche because of its ability to form domain-swapped dimers. In this study, we analyzed conformational stabilities of the recombinant bOBP and its monomeric variants, the bOBP-Gly121+ mutant containing an additional glycine residue after the residue 121 of the bOBP, and the GCC-bOBP mutant obtained from the bOBP-Gly121+ form by introduction of the Trp64Cys/His155Cys double mutation to restore the canonical disulfide bond. We also analyzed the effect of the natural ligand binding on the conformational stabilities of these bOBP variants. Our data are consistent with the conclusion that the unfolding-refolding pathways of the recombinant bOBP and its mutant monomeric forms bOBP-Gly121+ and GCC-bOBP are similar and do not depend on the oligomeric status of the protein. This clearly shows that the information on the unfolding-refolding mechanism is encoded in the structure of the bOBP monomers. However, the process of the bOBP unfolding is significantly complicated by the formation of the domain-swapped dimer, and the rates of the unfolding-refolding reactions essentially depend on the conditions in which the protein is located. PMID:27114857

  17. Solution behavior of synthetic silk peptides and modified recombinant silk proteins

    NASA Astrophysics Data System (ADS)

    Foo, C. Wong Po; Bini, E.; Huang, J.; Lee, S. Y.; Kaplan, D. L.

    2006-02-01

    Spider dragline silk from Nephila clavipes possesses impressive mechanical properties derived in part from repetitive primary sequence containing polyalanine regions that self-assemble into crystalline β-sheets. In the present study, we have sought to understand more details of redox responses related to conformational transitions of modified silk peptides and a recombinant protein containing encoded methionine triggers. Regardless of the position of the methionine trigger relative to the polyalanine domain, chemical oxidation was rapid and slight increases in the α-helical structure and decreases in the β-sheet and random coil content were observed by CD and FTIR in the assembled silk-like peptides and the recombinant protein. CD results indicated that the decrease in β-sheet and random coil conformations, coupled with the increase in helical content during oxidation, occurred during the first 30 min of the reaction. No further conformational changes occurred after this time and the response was independent of methionine trigger location relative to the penta-alanine domain. These results were confirmed with fluorescence studies. The design, processing and utility of these modified redox triggered silk-like peptides and proteins suggest a range of potential utility, from biomaterials to engineered surface coatings with chemically alterable secondary structure and, thus, properties.

  18. Inhibition of apoptosis by expression of antiapoptotic proteins in recombinant human keratinocytes.

    PubMed

    Choi, Claudia Y U; Reimers, Kerstin; Allmeling, Christina; Kall, Susanne; Choi, Yeong-Hoon; Vogt, Peter M

    2007-01-01

    The Fas ligand/Fas interaction plays an important role in the regulation of immune responses. Allografted cells undergo Fas-mediated apoptosis induced by CD8+ T cells. Our objective was to prevent human keratinocytes from immunologically induced apoptosis. We focused on three proteins with inhibitory function on Fas-mediated apoptosis. Human keratinocytes were transfected with either Flip, Faim, or Lifeguard (LFG). The treatment proved to be practicable and efficient. The recombinant keratinocytes with expression of our target proteins were cocultured with CD8+ T cells and the apoptotic activity was then evaluated. Activation of caspase-8 was detectable in control but not in the recombinant cells. Quantitative analysis revealed significant induction of T-cell-induced apoptosis in nontransfected keratinocytes (p = 0.04, n = 12) but not in Flip (p = 0.66), Faim (p = 0.42), or LFG (p = 0.44) expressing cells. Our results suggest that heterotopic expression of antiapoptotic proteins can induce the resistance of keratinocytes to a major mechanism of rejection.

  19. Recombinant Reg3β protein protects against streptozotocin-induced β-cell damage and diabetes

    PubMed Central

    Luo, Chen; Yu, Lu-Ting; Yang, Meng-Qi; Li, Xiang; Zhang, Zhi-Yuan; Alfred, Martin O; Liu, Jun-Li; Wang, Min

    2016-01-01

    Regenerating genes (Reg) have been found during the search for factors involved in pancreatic islet regeneration. Our recent study discovered that pancreatic β-cell-specific overexpression of Reg3β protects against streptozotocin (Stz) -induced diabetes in mice. To investigate its potential roles in the treatment of diabetes, we produced a recombinant Reg3β protein and provided evidence that it is active in promoting islet β-cell survival against Stz- triggered cell death. Though ineffective in alleviating preexisting diabetes, pretreatment of recombinant Reg3β was capable of minimizing the Stz-induced hyperglycemia and weight loss, by preserving serum and pancreatic insulin levels, and islet β-cell mass. No obvious changes were observed in the rate of cell proliferation and hypertrophy in α- or acinar-cells after treatment with recombinant Reg3β. The underlying mechanism of Reg3β-mediated protection seems to involve Akt activation which upregulates Bcl-2 and Bcl-xL levels and consequently promotes cell survival. PMID:27767186

  20. Genetic diversity and recombination analysis in the coat protein gene of Banana bract mosaic virus.

    PubMed

    Balasubramanian, V; Selvarajan, R

    2014-06-01

    Banana bract mosaic virus (BBrMV), a member of the genus Potyvirus, family Potyviridae, is the causal agent of the bract mosaic disease (BBrMD) that causes serious yield losses in banana and plantain in India and the Philippines. In this study, global genetic diversity and molecular evolution of BBrMV based on the capsid protein (CP) gene were investigated. Multiple alignments of CP gene of 49 BBrMV isolates showed nucleotide (nt) and amino acid (aa) identity of 79-100 and 80-100 %, respectively. Phylogenetic analysis revealed that except two Indians isolates (TN14 and TN16), all isolates clustered together. Eleven recombination events were detected using Recombination Detection Program. Codon-based maximum-likelihood methods revealed that most of the codons in the CP gene were under negative or neutral selection except for codons 28, 43, and 92 which were under positive selection. Gene flow between BBrMV populations of banana and cardamom was relatively frequent but not between two different populations of banana infecting isolates identified in this study. This is the first report on genetic diversity, and evolution of BBrMV isolates based on recombination and phylogenetic analysis in India. PMID:24691817

  1. Genetic diversity and recombination analysis in the coat protein gene of Banana bract mosaic virus.

    PubMed

    Balasubramanian, V; Selvarajan, R

    2014-06-01

    Banana bract mosaic virus (BBrMV), a member of the genus Potyvirus, family Potyviridae, is the causal agent of the bract mosaic disease (BBrMD) that causes serious yield losses in banana and plantain in India and the Philippines. In this study, global genetic diversity and molecular evolution of BBrMV based on the capsid protein (CP) gene were investigated. Multiple alignments of CP gene of 49 BBrMV isolates showed nucleotide (nt) and amino acid (aa) identity of 79-100 and 80-100 %, respectively. Phylogenetic analysis revealed that except two Indians isolates (TN14 and TN16), all isolates clustered together. Eleven recombination events were detected using Recombination Detection Program. Codon-based maximum-likelihood methods revealed that most of the codons in the CP gene were under negative or neutral selection except for codons 28, 43, and 92 which were under positive selection. Gene flow between BBrMV populations of banana and cardamom was relatively frequent but not between two different populations of banana infecting isolates identified in this study. This is the first report on genetic diversity, and evolution of BBrMV isolates based on recombination and phylogenetic analysis in India.

  2. Maxillary anterior ridge augmentation with recombinant human bone morphogenetic protein 2.

    PubMed

    Edmunds, Ryan K; Mealey, Brian L; Mills, Michael P; Thoma, Daniel S; Schoolfield, John; Cochran, David L; Mellonig, Jim

    2014-01-01

    No human studies exist on the use of recombinant human bone morphogenetic protein 2 (rhBMP-2) on an absorbable collagen sponge (ACS) as a sole graft material for lateral ridge augmentation in large ridge defect sites. This series evaluates the treatment outcome of maxillary anterior lateral ridge augmentation with rhBMP-2/ACS. Twenty patients were treated with rhBMP-2/ACS and fixation screws for space maintenance. Cone beam volumetric tomography measurements were used to determine gain in ridge width, and a bone core biopsy was obtained. The mean horizontal ridge gain was 1.2 mm across sites, and every site gained width. PMID:25006772

  3. Molecular cloning, purification and immunogenicity of recombinant Brucella abortus 544 malate dehydrogenase protein.

    PubMed

    Reyes, Alisha Wehdnesday Bernardo; Simborio, Hannah Leah Tadeja; Hop, Huynh Tan; Arayan, Lauren Togonon; Kim, Suk

    2016-03-01

    The Brucella mdh gene was successfully cloned and expressed in E. coli. The purified recombinant malate dehydrogenase protein (rMDH) was reactive to Brucella-positive bovine serum in the early stage, but not reactive in the middle or late stage, and was reactive to Brucella-positive mouse serum in the late stage, but not in the early or middle stage of infection. In addition, rMDH did not react with Brucella-negative bovine or mouse sera. These results suggest that rMDH has the potential for use as a specific antigen in serological diagnosis for early detection of bovine brucellosis.

  4. Molecular cloning, purification and immunogenicity of recombinant Brucella abortus 544 malate dehydrogenase protein

    PubMed Central

    Reyes, Alisha Wehdnesday Bernardo; Simborio, Hannah Leah Tadeja; Hop, Huynh Tan; Arayan, Lauren Togonon

    2016-01-01

    The Brucella mdh gene was successfully cloned and expressed in E. coli. The purified recombinant malate dehydrogenase protein (rMDH) was reactive to Brucella-positive bovine serum in the early stage, but not reactive in the middle or late stage, and was reactive to Brucella-positive mouse serum in the late stage, but not in the early or middle stage of infection. In addition, rMDH did not react with Brucella-negative bovine or mouse sera. These results suggest that rMDH has the potential for use as a specific antigen in serological diagnosis for early detection of bovine brucellosis. PMID:27051349

  5. Autoclaving as a chemical-free process to stabilize recombinant silk-elastinlike protein polymer nanofibers

    NASA Astrophysics Data System (ADS)

    Qiu, Weiguo; Cappello, Joseph; Wu, Xiaoyi

    2011-06-01

    We report here that autoclaving is a chemical-free, physical crosslinking strategy capable of stabilizing electrospun recombinant silk-elastinlike protein (SELP) polymer nanofibers. Fourier transform infrared spectroscopy showed that the autoclaving of SELP nanofibers induced a conformational conversion of β-turns and unordered structures to ordered β-sheets. Tensile stress-strain analysis of the autoclaved SELP nanofibrous scaffolds in phosphate buffered saline at 37 °C revealed a Young's modulus of 1.02 ± 0.28 MPa, an ultimate tensile strength of 0.34 ± 0.04 MPa, and a strain at failure of 29% ± 3%.

  6. Recombinant protein expression in Pichia pastoris strains with an engineered methanol utilization pathway

    PubMed Central

    2012-01-01

    Βackground The methylotrophic yeast Pichia pastoris has become an important host organism for recombinant protein production and is able to use methanol as a sole carbon source. The methanol utilization pathway describes all the catalytic reactions, which happen during methanol metabolism. Despite the importance of certain key enzymes in this pathway, so far very little is known about possible effects of overexpressing either of these key enzymes on the overall energetic behavior, the productivity and the substrate uptake rate in P. pastoris strains. Results A fast and easy-to-do approach based on batch cultivations with methanol pulses was used to characterize different P. pastoris strains. A strain with MutS phenotype was found to be superior over a strain with Mut+ phenotype in both the volumetric productivity and the efficiency in expressing recombinant horseradish peroxidase C1A. Consequently, either of the enzymes dihydroxyacetone synthase, transketolase or formaldehyde dehydrogenase, which play key roles in the methanol utilization pathway, was co-overexpressed in MutS strains harboring either of the reporter enzymes horseradish peroxidase or Candida antarctica lipase B. Although the co-overexpression of these enzymes did not change the stoichiometric yields of the recombinant MutS strains, significant changes in the specific growth rate, the specific substrate uptake rate and the specific productivity were observed. Co-overexpression of dihydroxyacetone synthase yielded a 2- to 3-fold more efficient conversion of the substrate methanol into product, but also resulted in a reduced volumetric productivity. Co-overexpression of formaldehyde dehydrogenase resulted in a 2-fold more efficient conversion of the substrate into product and at least similar volumetric productivities compared to strains without an engineered methanol utilization pathway, and thus turned out to be a valuable strategy to improve recombinant protein production. Conclusions Co

  7. Serum antibody response to recombinant major inner capsid protein following human infection with group B rotavirus.

    PubMed Central

    Eiden, J J; Mouzinho, A; Lindsay, D A; Glass, R I; Fang, Z Y; Taylor, J L

    1994-01-01

    Recombinant major inner capsid protein (VP6) of the IDIR strain of group B rotavirus (GBR) was incorporated in a solid-phase immunoassay to access antibody response to infection in humans. Expression of VP6 in insect cells permitted design of a highly sensitive assay that avoided the contaminants present in GBR antigens obtained from fecal specimens. Among patients infected with the ADRV strain of GBR in China, increased reactivity with recombinant VP6 was observed in convalescent-phase sera in comparison with sera obtained shortly after infection (P = 0.0084). Anti-VP6 antibodies were detectable as soon as 7 days after onset of gastrointestinal symptoms, and serum reactivity persisted in specimens drawn more than 1 year after infection. Solid-phase immunoassay with recombinant VP6 was next employed in order to assess anti-GBR antibody in 513 serum specimens obtained from 423 Maryland residents (ages, 7 months to 96 years; median age, 42 years). Four individuals (< 1%) exhibited serum antibodies directed against the recombinant VP6 (ages, 54 to 95 years; mean age, 77 years). Examination of 129 additional serum specimens including some from other geographic regions of the United States failed to reveal the presence of anti-GBR antibody. Anti-GBR antibody was also not detected in any of 131 serum specimens from 60 staff and residents of a nursing home in Switzerland. While infection of humans with GBR has been uncommon in these locations outside of China, the detection of serum antibodies in older individuals in the United States either indicated an unknown, age-related risk factor or may have indicated infection in the more distant past. The availability of these reagents should allow surveys for GBR infection among additional populations that have not previously been investigated. PMID:8077413

  8. Efficient expression and purification of recombinant therapeutic protein candidates, human midkine and pleiotrophin.

    PubMed

    Murasugi, Akira

    2013-01-01

    Midkine is a heparin-binding growth factor that promotes cell growth, survival, and migration. Externally added midkine prevents ventricular remodeling and improves long-term survival after myocardial infarction in the mouse. Preclinical testing of this protein is in progress. Externally added pleiotrophin, a member of the midkine protein family, promotes functional recovery after neural transplantation in rats. Thus, pleiotrophin is also a candidate therapeutic protein. Large amounts of these proteins were obtained by using the heterologous protein expression system of Pichia pastoris, and the recombinant P. pastoris clones were cultured in a controlled fermentor. Intracellular expression yielded about 300 mg/L recombinant human (rh)-midkine, which was extracted, renatured, and purified. From 1 L of the culture, 64 mg of rh-midkine was purified. Secretory expression induced by the midkine secretion signal resulted in about 100 mg of rhmidkine in 1 L of the culture supernatant, but over 70% of the rh-midkine had yeast-specific glycosylation. Three threonyl residues that are targets for glycosylation were substituted with alanyl residues, and nonglycosylated, active rh-midkine was obtained. In secretory expression using α-mating factor prepro-sequence, about 640 mg/L rh-midkine was obtained, but it was partially truncated. Therefore, a protease-deficient host was used, and about 360 mg/L intact rh-midkine was then obtained. The rh-midkine was recovered and purified, with 70% final yield. All purified rh-midkine, regardless of expression method, was able to promote mammalian cell proliferation. In secretory expression of rh-pleiotrophin using α- mating factor prepro-sequence, 260 mg/L rh-pleiotrophin could be secreted. The rh-pleiotrophin was recovered and efficiently purified with 72% final yield. PMID:24372230

  9. Bio-assisted potentiometric multisensor system for purity evaluation of recombinant protein A.

    PubMed

    Voitechovič, Edita; Korepanov, Anton; Kirsanov, Dmitry; Jahatspanian, Igor; Legin, Andrey

    2016-08-15

    Recombinant proteins became essential components of drug manufacturing. Quality control of such proteins is routine task, which usually requires a lot of time, expensive reagents, specialized equipment and highly educated personnel. In this study we propose a new concept for protein purity evaluation that is based on application of bio-assisted potentiometric multisensor system. The model object for analysis was recombinant protein A from Staphylococcus aureus (SpA), which is commonly used for monoclonal antibody purification. SpA solutions with different amount of host cell related impurities (Escherichia coli, bacterial lysate) were analyzed. Two different bio-transducers were employed: proteinase K from Tritirachium album and baker's yeast Saccharomyces cerevisiae. It was shown that both bio-transducers are able to induce changes in pure and lysate-contaminated SpA samples. Different products of yeast digestion and proteolysis with proteinase of pure SpA and lysate were detected with size exclusion high-performance liquid chromatography (SE-HPLC). The induced changes of chemical composition are detectible with potentiometric multisensor system and can be related to SpA purity through projection on latent structures (PLS) regression technique. The proposed method allows for estimation of the impurity content with 12% accuracy using proteinase K and 16% accuracy using baker's yeast. The suggested approach could be useful for early contamination warning at initial protein purification steps. The analysis requires no expensive materials and equipment, no bio-material immobilization, and its duration time is comparable with other commonly used methods like chromatography or electrophoresis though the main part of this time is related to the sample preparation. PMID:27260439

  10. Capture of a recombinant protein from unclarified canola extract using streamline expanded bed anion exchange.

    PubMed

    Bai, Yun; Glatz, Charles E

    2003-03-30

    The feasibility of applying expanded bed adsorption technology to recombinant protein recovery from extracts of transgenic canola (rapeseed) was assessed. The extraction step results in a suspension of high solids content that is difficult to clarify. The coarse portion of the solids can be removed easily, and our aim was to operate the expanded bed in the presence of the recalcitrant particulates. Recombinant beta-glucuronidase (rGUS) produced in transgenic canola seed was the model system. Diethylaminoethyl (DEAE) and Streamline DEAE resin exhibited similar binding and elution properties for both rGUS and native canola proteins. More than 95% of native canola proteins did not bind to DEAE resins at pH 7.5, whereas the bound proteins were fractionated by two-step salt elution into two groups with the first peak, containing 70% of total bound proteins, at 20 mS/cm, followed by elution of rGUS at 50 mS/cm. The adsorption isotherm was only slightly influenced by the presence of up to 14 mg solids/mL extract; C(m) and K(d) changed by -1% and +39%, respectively. Bed expansion was semiquantitatively predictable from physical properties of the fluid together with Stokes's law and the Richardson-Zaki correlation for both clarified and partially clarified extracts. The presence of 1.4% solids did not change rGUS breakthrough behavior of the expanded bed; however, a small difference between expanded bed and packed bed was observed early in the sample loading stage, during which bed expansion adjusts. Canola solids moved through the column in approximately plug flow with no detriment to bed stability. Seventy-two percent recovery of 34-fold purified rGUS was obtained after initial loading of 1.4% (w/w) solids extract to 25% breakthrough.

  11. 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

  12. Efficient expression and purification of recombinant therapeutic protein candidates, human midkine and pleiotrophin.

    PubMed

    Murasugi, Akira

    2013-01-01

    Midkine is a heparin-binding growth factor that promotes cell growth, survival, and migration. Externally added midkine prevents ventricular remodeling and improves long-term survival after myocardial infarction in the mouse. Preclinical testing of this protein is in progress. Externally added pleiotrophin, a member of the midkine protein family, promotes functional recovery after neural transplantation in rats. Thus, pleiotrophin is also a candidate therapeutic protein. Large amounts of these proteins were obtained by using the heterologous protein expression system of Pichia pastoris, and the recombinant P. pastoris clones were cultured in a controlled fermentor. Intracellular expression yielded about 300 mg/L recombinant human (rh)-midkine, which was extracted, renatured, and purified. From 1 L of the culture, 64 mg of rh-midkine was purified. Secretory expression induced by the midkine secretion signal resulted in about 100 mg of rhmidkine in 1 L of the culture supernatant, but over 70% of the rh-midkine had yeast-specific glycosylation. Three threonyl residues that are targets for glycosylation were substituted with alanyl residues, and nonglycosylated, active rh-midkine was obtained. In secretory expression using α-mating factor prepro-sequence, about 640 mg/L rh-midkine was obtained, but it was partially truncated. Therefore, a protease-deficient host was used, and about 360 mg/L intact rh-midkine was then obtained. The rh-midkine was recovered and purified, with 70% final yield. All purified rh-midkine, regardless of expression method, was able to promote mammalian cell proliferation. In secretory expression of rh-pleiotrophin using α- mating factor prepro-sequence, 260 mg/L rh-pleiotrophin could be secreted. The rh-pleiotrophin was recovered and efficiently purified with 72% final yield.

  13. Recombinant production and solution structure of lipid transfer protein from lentil Lens culinaris

    SciTech Connect

    Gizatullina, Albina K.; Finkina, Ekaterina I.; Mineev, Konstantin S.; Melnikova, Daria N.; Bogdanov, Ivan V.; Shenkarev, Zakhar O.; Ovchinnikova, Tatiana V.

    2013-10-04

    Highlights: •Lipid transfer protein from lentil seeds (Lc-LTP2) was overexpressed in E. coli. •Antimicrobial activity and spatial structure of the recombinant Lc-LTP2 were examined. •Internal tunnel-like lipid-binding cavity occupies ∼7% of the total Lc-LTP2 volume. •Binding of DMPG lipid induces moderate rearrangements in the Lc-LTP2 structure. •Lc-LTP2/DMPG complex has limited lifetime and dissociates within tens of hours. -- Abstract: Lipid transfer protein, designated as Lc-LTP2, was isolated from seeds of the lentil Lens culinaris. The protein has molecular mass 9282.7 Da, consists of 93 amino acid residues including 8 cysteines forming 4 disulfide bonds. Lc-LTP2 and its stable isotope labeled analogues were overexpressed in Escherichia coli and purified. Antimicrobial activity of the recombinant protein was examined, and its spatial structure was studied by NMR spectroscopy. The polypeptide chain of Lc-LTP2 forms four α-helices (Cys4-Leu18, Pro26-Ala37, Thr42-Ala56, Thr64-Lys73) and a long C-terminal tail without regular secondary structure. Side chains of the hydrophobic residues form a relatively large internal tunnel-like lipid-binding cavity (van der Waals volume comes up to ∼600 Å{sup 3}). The side-chains of Arg45, Pro79, and Tyr80 are located near an assumed mouth of the cavity. Titration with dimyristoyl phosphatidylglycerol (DMPG) revealed formation of the Lc-LTP2/lipid non-covalent complex accompanied by rearrangements in the protein spatial structure and expansion of the internal cavity. The resultant Lc-LTP2/DMPG complex demonstrates limited lifetime and dissociates within tens of hours.

  14. 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

  15. Biochemical analysis of the human ENA/VASP-family proteins, MENA, VASP and EVL, in homologous recombination.

    PubMed

    Takaku, Motoki; Ueno, Hiroyuki; Kurumizaka, Hitoshi

    2011-06-01

    MENA, VASP and EVL are members of the ENA/VASP family of proteins and are involved in cytoplasmic actin remodeling. Previously, we found that EVL directly interacts with RAD51, an essential protein in the homologous recombinational repair of double-strand breaks (DSBs) and stimulates the RAD51-mediated recombination reactions in vitro. The EVL-knockdown MCF7 cells exhibited a clear reduction in RAD51-foci formation, suggesting that EVL may function in the DSB repair pathway through RAD51-mediated homologous recombination. However, the DSB repair defects were less significant in the EVL-knockdown cells, implying that two EVL paralogues, MENA and VASP, may complement the EVL function in human cells. Therefore, in the present study, we purified human MENA, VASP and EVL as recombinant proteins, and compared their biochemical activities in vitro. We found that all three proteins commonly exhibited the RAD51 binding, DNA binding and DNA-annealing activities. Stimulation of the RAD51-mediated homologous pairing was also observed with all three proteins. In addition, surface plasmon resonance analyses revealed that MENA, VASP and EVL mutually interacted. These results support the ideas that the ENA/VASP-family proteins are functionally redundant in homologous recombination, and that all three may be involved in the DSB repair pathway in humans.

  16. BTag: a novel six-residue epitope tag for surveillance and purification of recombinant proteins.

    PubMed

    Wang, L F; Yu, M; White, J R; Eaton, B T

    1996-02-22

    Epitope tagging (Eta) is becoming an increasingly useful technique in molecular biology and biotechnology for the detection, characterisation and purification of recombinant proteins (re-proteins). Here we describe a novel Eta system composed of two different monoclonal antibodies (mAb; D11 and F10) and a 6-amino-acid Eta (Gln-Tyr-Pro-Ala-Leu-Thr or QYPALT). This Eta was derived from a highly conserved region of the major core protein, VP7, of bluetongue (BT) viruses, hence the name BTag. BTag is unique among current tagging systems in its lack of charge and the fact the tag sequence can be placed and detected in any region of a re-protein. Other useful features of BTag include its small size and its recognition by two different mAb. Using the BTag system, more than 30 re-proteins have been produced from a variety of host organisms, and the antigenicity of the tag sequence was maintained in all of the proteins tested to date. Our result demonstrated that BTag could be superior to other existing Eta systems for certain applications.

  17. Genetic selection system for improving recombinant membrane protein expression in E. coli

    PubMed Central

    Massey-Gendel, Elizabeth; Zhao, Anni; Boulting, Gabriella; Kim, Hye-Yeon; Balamotis, Michael A; Seligman, Len M; Nakamoto, Robert K; Bowie, James U

    2009-01-01

    A major barrier to the physical characterization and structure determination of membrane proteins is low yield in recombinant expression. To address this problem, we have designed a selection strategy to isolate mutant strains of Escherichia coli that improve the expression of a targeted membrane protein. In this method, the coding sequence of the membrane protein of interest is fused to a C-terminal selectable marker, so that the production of the selectable marker and survival on selective media is linked to expression of the targeted membrane protein. Thus, mutant strains with improved expression properties can be directly selected. We also introduce a rapid method for curing isolated strains of the plasmids used during the selection process, in which the plasmids are removed by in vivo digestion with the homing endonuclease I-CreI. We tested this selection system on a rhomboid family protein from Mycobacterium tuberculosis (Rv1337) and were able to isolate mutants, which we call EXP strains, with up to 75-fold increased expression. The EXP strains also improve the expression of other membrane proteins that were not the target of selection, in one case roughly 90-fold. PMID:19165721

  18. Recombinant human bone morphogenetic protein-2 binding and incorporation in PLGA microsphere delivery systems.

    PubMed

    Schrier, J A; DeLuca, P P

    1999-01-01

    The objective of this research was to determine the binding capacity and kinetics, and total incorporation of recombinant human bone morphogenetic protein-2 (rhBMP-2) in microspheres made from hydrophilic and hydrophobic poly(lactide-co-glycolide) (PLGA). Polymers were characterized by molecular weight, polydispersity, and acid number. Microspheres were produced via a water-in-oil-in-water double emulsion system and characterized for bulk density, size, specific surface area, and porosity. Protein concentrations were determined by reversed phase HPLC. Protein was loaded by soaking microspheres in a buffered solution, pH 4.5, of rhBMP-2, decanting excess liquid, and vacuum drying the wetted particles. Total loading and binding were determined by comparing protein concentration remaining to non-microsphere containing samples. Polymer acid number was the dominant polymer feature affecting the binding. Higher acid values correlated with increased rhBMP-2 binding. The amount of non-bound incorporated rhBMP-2 linearly correlated with the concentration of protein used in binding. High rhBMP-2 concentrations inhibit binding to PLGA microspheres. Binding was also inhibited by increased lactide content in the PLGA polymer. The polymer characteristics controlling rhBMP-2 binding to PLGA microspheres are acid value foremost followed by molecular weight and lactide/glycolide ratio. The total amount of rhBMP-2 incorporated depends on the bound amount and on the amount of free protein present.

  19. Recombinant Preparation, Biochemical Analysis, and Structure Determination of Sirtuin Family Histone/Protein Deacylases.

    PubMed

    Suenkel, B; Steegborn, C

    2016-01-01

    Lysine acetylation is long known as a regulatory posttranslational modification of histone proteins and is emerging as a ubiquitous intracellular protein modification. Additional lysine acylations such as succinylation and glutarylation have also been found on histones and other proteins. Acylations are reversibly attached through nonenzymatic acylation mechanisms and the action of protein acyl transferases and protein deacylases (PDACs). Sirtuins are an evolutionary defined class of PDACs and act as metabolic sensors by catalyzing a unique deacylation reaction that requires the cosubstrate NAD(+). Sirtuins are found in all domains of life, and the mammalian sirtuin family comprises seven isoforms in different cellular compartments. They regulate a wide range of cellular targets and functions, such as energy metabolism and stress responses, and they have been implicated in aging processes and aging-related diseases. A large body of functional, biochemical, biophysical, and structural work on isolated sirtuins has provided many important insights that complement the many physiological studies on this enzyme family. They enabled the comprehensive structural and biochemical analysis of sirtuin catalysis, substrate selectivity, and regulation. Here, we describe the recombinant production of sirtuin proteins, with an emphasis on the mammalian isoforms. We then describe their application in activity and binding assays and for crystal structure analysis. We provide protocols for these procedures, and we discuss typical pitfalls in studying this enzyme family and how to avoid them. This information will support further molecular studies on sirtuin mechanisms and functions. PMID:27372754

  20. Cell Penetrating Peptide POD Mediates Delivery of Recombinant Proteins to Retina, Cornea and Skin

    PubMed Central

    Johnson, Leslie N.; Cashman, Siobhan M.; Read, Sarah Parker; Kumar-Singh, Rajendra

    2009-01-01

    Recently we described a novel cell penetrating peptide, POD (peptide for ocular delivery) that could deliver small molecules including fluorescent dyes into retinal cells. The objective of the current study was to examine whether biologically relevant macromolecules such as proteins, genetically fused with POD could also be delivered into retinal tissues in vivo. We generated a POD-GFP fusion protein and examined its cell and tissue penetrating properties. We found that endogenously expressed POD-GFP fusion protein localized to the nucleus, suggesting that POD acts as a nuclear localization signal. Adenovirus (Ad) vectors expressing POD-GFP fusion protein were constructed and the recombinant protein was purified from Ad-infected human embryonic retinoblasts (HER). Exogenously supplied POD-GFP fusion protein rapidly transduced A549 and HER cells and colocalized in part with markers of late endosomes, from which it could escape. Following subretinal delivery, POD-GFP localized to the retinal pigment epithelium and the photoreceptor cell bodies. When injected into the vitreous, POD-GFP localized to the ganglion cells and the inner nuclear layer of the retina as well as the lens capsule. Topical application of POD-GFP to ocular surfaces resulted in uptake by the corneal epithelium. POD-GFP also transduced non-ocular tissues, including the epidermis of the skin following topical application. PMID:19733192

  1. Immunogenicity of recombinant VP2 proteins of all nine serotypes of African horse sickness virus

    PubMed Central

    Kanai, Yuta; van Rijn, Piet A.; Maris-Veldhuis, Mieke; Kaname, Yuki; Athmaram, T.N.; Roy, Polly

    2014-01-01

    African horse sickness (AHS) is an equine disease with a mortality of up to 90% for susceptible horses. The causative agent AHS virus (AHSV) is transmitted by species of Culicoides. AHSV serogroup within the genus Orbivirus of the Reoviridae family consists of nine serotypes that show no or very limited cross-neutralization. Of the seven structural proteins (VP1-VP7) of AHSV, VP2 is the serotype specific protein, and the major target for neutralizing antibodies. In this report, recombinant VP2 proteins of all nine serotypes were expressed individually by the baculovirus expression system and the immunogenicity of each was studied by immunization of guinea pigs with single VP2 as well as with cocktails of VP2 proteins. Homologous neutralizing antibodies measured by 50% plaque reduction assay showed varying degrees (from 37 to 1365) of titers for different VP2 proteins. A low cross-neutralizing antibody titer was found for genetically related AHSV serotypes. Immunization with VP2 cocktails containing equal amounts of each of the VP2 proteins also triggered neutralizing antibodies albeit to lower titers (4-117) to each of the serotypes in the cocktail. This study is a first step to develop a VP2 subunit vaccine for AHS and our results indicate that VP2 subunit vaccines are feasible individually or in a multi-serotype cocktail. PMID:25045805

  2. Isolation of Camelid Single-Domain Antibodies Against Native Proteins Using Recombinant Multivalent Peptide Ligands.

    PubMed

    Alturki, Norah A; Henry, Kevin A; MacKenzie, C Roger; Arbabi-Ghahroudi, Mehdi

    2015-01-01

    Generation of antibodies against desired epitopes on folded proteins may be hampered by various characteristics of the target protein, including antigenic and immunogenic dominance of irrelevant epitopes and/or steric occlusion of the desired epitope. In such cases, peptides encompassing linear epitopes of the native protein represent attractive alternative reagents for immunization and screening. Peptide antigens are typically prepared by fusing or conjugating the peptide of interest to a carrier protein. The utility of such antigens depends on many factors including the peptide's amino acid sequence, display valency, display format (synthetic conjugate vs. recombinant fusion) and characteristics of the carrier. Here we provide detailed protocols for: (1) preparation of DNA constructs encoding peptides fused to verotoxin (VT) multimerization domain; (2) expression, purification, and characterization of the multivalent peptide-VT ligands; (3) concurrent panning of a non-immune phage-displayed camelid VHH library against the peptide-VT ligands and native protein; and (4) identification of VHHs enriched via panning using next-generation sequencing techniques. These methods are simple, rapid and can be easily adapted to yield custom peptide-VT ligands that appear to maintain the antigenic structures of the peptide. However, we caution that peptide sequences should be chosen with great care, taking into account structural, immunological, and biophysical information on the protein of interest.

  3. Better and faster: improvements and optimization for mammalian recombinant protein production

    PubMed Central

    Almo, Steven C.; Love, James D.

    2014-01-01

    Thanks to numerous technological advances, the production of recombinant proteins in mammalian cell lines has become an increasingly routine task that is no longer viewed as a heroic enterprise. While production in prokaryotic or lower eukaryotic systems may be more rapid and economical, the advantages of producing large amounts of protein that closely resembles the native form is often advantageous and may be essential for the realization of functionally active material for biological studies or biopharmaceuticals. The correct folding, processing and post-translational modifications conferred by expression in a mammalian cell is relevant to all classes of proteins, including cytoplasmic, secreted or integral membrane proteins. Therefore considerable efforts have focused on the development of growth media, cell lines, transformation methods and selection techniques that enable the production of grams of functional protein in weeks, rather than months. This review will focus on a plethora of methods that are broadly applicable to the high yield production of any class of protein (cytoplasmic, secreted or integral membrane) from mammalian cells. PMID:24721463

  4. Strategies for successful recombinant expression of disulfide bond-dependent proteins in Escherichia coli

    PubMed Central

    de Marco, Ario

    2009-01-01

    Bacteria are simple and cost effective hosts for producing recombinant proteins. However, their physiological features may limit their use for obtaining in native form proteins of some specific structural classes, such as for instance polypeptides that undergo extensive post-translational modifications. To some extent, also the production of proteins that depending on disulfide bridges for their stability has been considered difficult in E. coli. Both eukaryotic and prokaryotic organisms keep their cytoplasm reduced and, consequently, disulfide bond formation is impaired in this subcellular compartment. Disulfide bridges can stabilize protein structure and are often present in high abundance in secreted proteins. In eukaryotic cells such bonds are formed in the oxidizing environment of endoplasmic reticulum during the export process. Bacteria do not possess a similar specialized subcellular compartment, but they have both export systems and enzymatic activities aimed at the formation and at the quality control of disulfide bonds in the oxidizing periplasm. This article reviews the available strategies for exploiting the physiological mechanisms of bactera to produce properly folded disulfide-bonded proteins. PMID:19442264

  5. Development of rhizosecretion as a production system for recombinant proteins from hydroponic cultivated tobacco.

    PubMed

    Drake, Pascal M W; Barbi, Tommaso; Sexton, Amy; McGowan, Edward; Stadlmann, Johannes; Navarre, Catherine; Paul, Matthew J; Ma, Julian K-C

    2009-10-01

    Rhizosecretion is an attractive technology for the production of recombinant proteins from transgenic plants. However, to date, yields of plant-derived recombinant pharmaceuticals by this method have been too low for commercial viability. Studies conducted focused on three transgenic plant lines grown in hydroponic culture medium, two expressing monoclonal antibodies Guy's 13 and 4E10 and one expressing a small microbicide polypeptide cyanovirin-N. Rhizosecretion rates increased significantly by the addition of the plant growth regulator alpha-naphthalene acetic acid. The maximum rhizosecretion rates achieved were 58 microg/g root dry weight/24 h for Guy's 13, 10.43 microg/g root dry weight/24 h for 4E10, and 766 microg/g root dry weight/24 h for cyanovirin-N, the highest figures so far reported for a full-length antibody and a recombinant protein, respectively. The plant growth regulators indole-butyric acid, 6-benzylaminopurine, and kinetin were also demonstrated to increase rhizosecretion of Guy's 13. The effect of the growth regulators differed, as alpha-naphthalene acetic acid and indole-butyric acid increased the root dry weight of hydroponic plants, whereas the cytokinins benzylaminopurine and kinetin increased rhizosecretion without affecting root mass. A comparative glycosylation analysis between MAb Guy's 13 purified from either hydroponic culture medium or from leaf extracts demonstrated a similar pattern of glycosylation comprising high mannose to complex glycoforms. Analysis of the hydroponic culture medium at harvest revealed significantly lower and less complex levels of proteolytic enzymes, in comparison with leaf extracts, which translated to a higher proportion of intact Guy's 13 IgG in relation to other IgG products. Hydroponic medium could be added directly to a chromatography column for affinity purification, allowing simple and rapid production of high purity Guy's 13 antibody. In addition to the attractiveness of controlled cultivation within

  6. Diversity and Recombination of Dispersed Ribosomal DNA and Protein Coding Genes in Microsporidia

    PubMed Central

    Ironside, Joseph Edward

    2013-01-01

    Microsporidian strains are usually classified on the basis of their ribosomal DNA (rDNA) sequences. Although rDNA occurs as multiple copies, in most non-microsporidian species copies within a genome occur as tandem arrays and are homogenised by concerted evolution. In contrast, microsporidian rDNA units are dispersed throughout the genome in some species, and on this basis are predicted to undergo reduced concerted evolution. Furthermore many microsporidian species appear to be asexual and should therefore exhibit reduced genetic diversity due to a lack of recombination. Here, DNA sequences are compared between microsporidia with different life cycles in order to determine the effects of concerted evolution and sexual reproduction upon the diversity of rDNA and protein coding genes. Comparisons of cloned rDNA sequences between microsporidia of the genus Nosema with different life cycles provide evidence of intragenomic variability coupled with strong purifying selection. This suggests a birth and death process of evolution. However, some concerted evolution is suggested by clustering of rDNA sequences within species. Variability of protein-coding sequences indicates that considerable intergenomic variation also occurs between microsporidian cells within a single host. Patterns of variation in microsporidian DNA sequences indicate that additional diversity is generated by intragenomic and/or intergenomic recombination between sequence variants. The discovery of intragenomic variability coupled with strong purifying selection in microsporidian rRNA sequences supports the hypothesis that concerted evolution is reduced when copies of a gene are dispersed rather than repeated tandemly. The presence of intragenomic variability also renders the use of rDNA sequences for barcoding microsporidia questionable. Evidence of recombination in the single-copy genes of putatively asexual microsporidia suggests that these species may undergo cryptic sexual reproduction, a

  7. Evaluation of immobilized metal affinity chromatography kits for the purification of histidine-tagged recombinant CagA protein.

    PubMed

    Karakus, Cebrail; Uslu, Merve; Yazici, Duygu; Salih, Barik A

    2016-05-15

    Immobilized metal affinity chromatography (IMAC) technique is used for fast and reliable purification of histidine(His)-tagged recombinant proteins. The technique provides purification under native and denaturing conditions. The aim of this study is to evaluate three commercially available IMAC kits (Thermo Scientific, GE Healthcare and Qiagen) for the purification of a 6xHis-tagged recombinant CagA (cytotoxin-associated gene A) protein from IPTG-induced Escherichia coli BL21(DE3) culture. The kits were tested according to the manufacturer instructions and the protein was purified with only GE Healthcare and Qiagen kits under denaturing conditions. 1% (w/v) SDS was used as denaturing agent in PBS instead of extraction reagent of Thermo Scientific kit to lyse bacterial cells from 100ml culture. The 6xHis-tagged recombinant protein was purified by the three kits equally. PMID:26657801

  8. A heteromeric protein that binds to a meiotic homologous recombination hot spot: correlation of binding and hot spot activity.

    PubMed

    Wahls, W P; Smith, G R

    1994-07-15

    Homologous recombination hot spots are DNA sites that increase the frequency of recombination in their vicinity. The M26 allele of the ade6 gene in Schizosaccharomyces pombe is the first meiotic hot spot with an identified unique nucleotide sequence. We have purified 40,000-fold a heteromeric protein, containing polypeptides Mts1 (70 kD) and Mts2 (28 kD), that binds to the M26 site. Binding in vitro strictly correlates with hot spot activity in vivo for numerous single base pair substitutions in the vicinity of the M26 site, indicating that Mts1/Mts2 activates the M26 site and promotes a rate-limiting step of meiotic recombination. These and other data suggest that homologous recombination may be regulated primarily by discrete DNA sites and proteins that interact with those sites. PMID:7958849

  9. Production of Polyclonal Antibodies to the Recombinant Coat Protein of Citrus tristeza virus and Their Effectiveness for Virus Detection

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The p25 coat protein gene of three Citrus tristeza virus (CTV) isolates, two from Mexico and one from India, was amplified by RT-PCR and further cloned and expressed in Escherichia coli cells. The recombinant coat protein (rCP) of the three CTV isolates was injected into rabbits and goats for antibo...

  10. "Recombinant Protein of the Day": Using Daily Student Presentations to Add Real-World Aspects to a Biotechnology Course

    ERIC Educational Resources Information Center

    Shaffer, Justin F.

    2013-01-01

    To provide a realistic view of the biotechnology industry for students, a novel course focusing on recombinant proteins and their importance in medicine, pharmaceuticals, industry, scientific research, and agriculture was developed. ''Designer Proteins and Society,'' an upper-division elective, was taught in the Fall 2012…

  11. Immobilized palladium(II) ion affinity chromatography for recovery of recombinant proteins with peptide tags containing histidine and cysteine.

    PubMed

    Kikot, Pamela; Polat, Aise; Achilli, Estefania; Fernandez Lahore, Marcelo; Grasselli, Mariano

    2014-11-01

    Fusion of peptide-based tags to recombinant proteins is currently one of the most used tools for protein production. Also, immobilized metal ion affinity chromatography (IMAC) has a huge application in protein purification, especially in research labs. The combination of expression systems of recombinant tagged proteins with this robust chromatographic system has become an efficient and rapid tool to produce milligram-range amounts of proteins. IMAC-Ni(II) columns have become the natural partners of 6xHis-tagged proteins. The Ni(II) ion is considered as the best compromise of selectivity and affinity for purification of a recombinant His-tagged protein. The palladium(II) ion is also able to bind to side chains of amino acids and form ternary complexes with iminodiacetic acid and free amino acids and other sulfur-containing molecules. In this work, we evaluated two different cysteine- and histidine-containing six amino acid tags linked to the N-terminal group of green fluorescent protein (GFP) and studied the adsorption and elution conditions using novel eluents. Both cysteine-containing tagged GFPs were able to bind to IMAC-Pd(II) matrices and eluted successfully using a low concentration of thiourea solution. The IMAC-Ni(II) system reaches less than 20% recovery of the cysteine-containing tagged GFP from a crude homogenate of recombinant Escherichia coli, meanwhile the IMAC-Pd(II) yields a recovery of 45% with a purification factor of 13.

  12. Immobilized palladium(II) ion affinity chromatography for recovery of recombinant proteins with peptide tags containing histidine and cysteine.

    PubMed

    Kikot, Pamela; Polat, Aise; Achilli, Estefania; Fernandez Lahore, Marcelo; Grasselli, Mariano

    2014-11-01

    Fusion of peptide-based tags to recombinant proteins is currently one of the most used tools for protein production. Also, immobilized metal ion affinity chromatography (IMAC) has a huge application in protein purification, especially in research labs. The combination of expression systems of recombinant tagged proteins with this robust chromatographic system has become an efficient and rapid tool to produce milligram-range amounts of proteins. IMAC-Ni(II) columns have become the natural partners of 6xHis-tagged proteins. The Ni(II) ion is considered as the best compromise of selectivity and affinity for purification of a recombinant His-tagged protein. The palladium(II) ion is also able to bind to side chains of amino acids and form ternary complexes with iminodiacetic acid and free amino acids and other sulfur-containing molecules. In this work, we evaluated two different cysteine- and histidine-containing six amino acid tags linked to the N-terminal group of green fluorescent protein (GFP) and studied the adsorption and elution conditions using novel eluents. Both cysteine-containing tagged GFPs were able to bind to IMAC-Pd(II) matrices and eluted successfully using a low concentration of thiourea solution. The IMAC-Ni(II) system reaches less than 20% recovery of the cysteine-containing tagged GFP from a crude homogenate of recombinant Escherichia coli, meanwhile the IMAC-Pd(II) yields a recovery of 45% with a purification factor of 13. PMID:25277090

  13. Chromatographic separation of low-molecular-mass recombinant proteins and peptides on Superdex 30 prep grade.

    PubMed

    Joyce, J G; Cook, J C; Przysiecki, C T; Lehman, E D

    1994-12-01

    The chromatographic properties of Superdex 30 prep grade medium have been investigated in non-denaturing and denaturing mobile phases using commercially available proteins and peptides as well as low-molecular-mass (M(r)) recombinant polypeptides. The medium is a macroreticular gel composed of crosslinked agarose beads to which dextran has been covalently bound. The mean particle size is approximately 34 microns. Experimental results show a linear relation between the distribution coefficient (KD) and the log10 M(r) in the fractionation range 24,000-3000. The relationships between resolution and flow-rate or load volume were investigated and shown to be comparable with those of Superdex 75 and 200 prep grade media. Minimal loss of resolution occurred in the flow-range from 30-60 cm/h. Load volumes of up to 5% total column volume could be applied while maintaining baseline resolution of polypeptide mixtures. Non-specific interactions between the matrix and certain samples were characterized. The predominant interactions with the resin appear to be hydrophobic in nature rather than ionic. Hydrogen bonding may also play a role in the retardation of certain small molecules. The applicability of the resin for separating dimeric and oligomeric forms of low-molecular-mass recombinant proteins was shown.

  14. Meiotic recombination protein Rec12: functional conservation, crossover homeostasis and early crossover/non-crossover decision

    PubMed Central

    Kan, Fengling; Davidson, Mari K.; Wahls, Wayne P.

    2011-01-01

    In fission yeast and other eukaryotes, Rec12 (Spo11) is thought to catalyze the formation of dsDNA breaks (DSBs) that initiate homologous recombination in meiosis. Rec12 is orthologous to the catalytic subunit of topoisomerase VI (Top6A). Guided by the crystal structure of Top6A, we engineered the rec12 locus to encode Rec12 proteins each with a single amino acid substitution in a conserved residue. Of 21 substitutions, 10 significantly reduced or abolished meiotic DSBs, gene conversion, crossover recombination and the faithful segregation of chromosomes. Critical residues map within the metal ion-binding pocket toprim (E179A, D229A, D231A), catalytic region 5Y-CAP (R94A, D95A, Y98F) and the DNA-binding interface (K201A, G202E, R209A, K242A). A subset of substitutions reduced DSBs but maintained crossovers, demonstrating crossover homeostasis. Furthermore, a strong separation of function mutation (R304A) suggests that the crossover/non-crossover decision is established early by a protein–protein interaction surface of Rec12. Fission yeast has multiple crossovers per bivalent, and chromosome segregation was robust above a threshold of about one crossover per bivalent, below which non-disjunction occurred. These results support structural and functional conservation among Rec12/Spo11/Top6A family members for the catalysis of DSBs, and they reveal how Rec12 regulates other features of meiotic chromosome dynamics. PMID:21030440

  15. Protease-Triggered, Integrin-Targeted Cellular Uptake of Recombinant Protein Micelles.

    PubMed

    Gao, Chen; Vargo, Kevin B; Hammer, Daniel A

    2016-09-01

    Targeting nanoparticles for drug delivery has great potential for improving efficacy and reducing side effects from systemic toxicity. New developments in the assembly of materials afford the opportunity to expose cryptic targeting domains in tissue-specific microenvironments in which certain proteases are expressed. Here, recombinant proteins are designed to combine the responsiveness to environmental proteases with specific targeting. Materials made recombinantly allow complete control over amino acid sequence, in which each molecule is identically functionalized. Previously, oleosin, a naturally occurring plant protein that acts as a surfactant, has been engineered to self-assemble into spherical micelles-a useful structure for drug delivery. To make oleosins that are locally activated to bind receptors, oleosin is genetically modified to incorporate the integrin-binding motif RGDS just behind a domain cleavable by thrombin. The resulting modified oleosin self-assembles into spherical micelles in aqueous environments, with the RGDS motif protected by the thrombin-cleavable domain. Upon the addition of thrombin, the RGDS is exposed and the binding of the spherical micelles to breast cancer cells is increased fourfold. PMID:27284959

  16. Expression and Characterization of Recombinant Human Secretory Leukocyte Protease Inhibitor (SLPI) Protein from Pichia pastoris

    PubMed Central

    Li, Zhiguo; Moy, Allison; Sohal, Kirti; Dam, Carolyn; Kuo, Peter; Ulrich, Beau; Whittaker, James; Whittaker, Mei; Düzgünes, Nejat; Konopka, Kryatyna; Franz, Andreas H.; Lin-Cereghino, Joan; Lin-Cereghino, Geoff P.

    2009-01-01

    The human secretory leukocyte protease inhibitor (SLPI) has been shown to possess anti-protease, anti-inflammatory and antimicrobial properties. Its presence in saliva is believed to be a major deterrent to oral transmission of human immunodeficiency virus-1. The 11.7 kD peptide is a secreted, nonglycosylated protein rich in disulfide bonds. Currently, recombinant SLPI is only available as an expensive bacterial expression product. We have investigated the utility of the methylotrophic yeast Pichia pastoris to produce and secrete SLPI with C-terminal c-myc and polyhistidine tags. The posttransformational vector amplification protocol was used to isolate strains with increased copy number, and culturing parameters were varied to optimize SLPI expression. Modification of the purification procedure allowed the secreted, recombinant protein to be isolated from the cell-free fermentation medium with cobalt affinity chromatography. This yeast-derived SLPI was shown to have an anti-protease activity comparable to the commercially available bacterial product. Thus, P. pastoris provides an efficient, cost-effective system for producing SLPI for structure function analysis studies as well as a wide array of potential therapeutic applications. PMID:19505578

  17. Recombinant Fv-Hsp70 protein mediates neuroprotection after focal cerebral ischemia in rats

    PubMed Central

    Zhan, Xinhua; Ander, Bradley P; Liao, Isaac H; Hansen, James E; Kim, Chester; Clements, Douglas; Weisbart, Richard H; Nishimura, Robert N; Sharp, Frank R

    2010-01-01

    Background and Purpose This study investigated the effects of intravenous recombinant Fv-Hsp70 protein on infarction volume and behavior following experimental ischemic stroke. Methods Focal cerebral ischemia was produced by occluding the middle cerebral artery (MCA) using the intraluminal suture technique. Rats subjected to 2 hours of focal ischemia were allowed to survive 24 h. At 2 ¼ h and 3 h after onset of ischemia, Fv-Hsp70 recombinant protein (0.5 mg / kg) or saline was injected via the tail vein. Sensory-motor function and infarction volume were assessed at 24 h following ischemia. Results Administration of Fv-Hsp70 following focal cerebral ischemia significantly decreased infarct volume by 68% and significantly improved sensory-motor function compared to the saline-treated control group. Western blots showed Fv-Hsp70 in ischemic but not in control brain; and Fv-Hsp70 suppressed endogenous Hsp70. Conclusion Fv-Hsp70 protects ischemic brain in this experimental stroke model. PMID:20075343

  18. Recombinant human epidermal growth factor precursor is a glycosylated membrane protein with biological activity.

    PubMed Central

    Mroczkowski, B; Reich, M; Chen, K; Bell, G I; Cohen, S

    1989-01-01

    NIH 3T3 cells were transfected with cDNA corresponding to human kidney prepro-epidermal growth factor (preproEGF) under control of the inducible mouse metallothionein promoter. The synthesis of recombinant human EGF precursor by these cells has provided us with a model system for analysis of the structure and activity of this precursor. In transfected cells, the precursor was present as an intrinsic 170-kilodalton membrane protein as well as a soluble protein in the extracellular medium; both forms were N glycosylated. Glycosylation of the EGF precursor was determined by (i) the direct incorporation of [3H]mannose and [3H]glucosamine, (ii) metabolic labeling in the presence or absence of glycosylation inhibitors, (iii) enzymatic cleavage of the precursor by N-glycanase or endoglycosidase II, and (iv) lectin chromatography. Recombinant human preproEGF was purified by affinity chromatography, using wheat germ lectin and antibodies to human EGF. The intact precursor was biologically active. Purified preparations of preproEGF (i) competed with 125I-labeled EGF for binding to the EGF receptor in intact fibroblast cells, (ii) activated the intrinsic tyrosine kinase activity of the EGF receptor in membrane preparations, and (iii) sustained the growth of a mouse keratinocyte cell line that is dependent on EGF for growth. These results suggest that proteolytic processing of the precursor may not be essential for its biological function. Images PMID:2789334

  19. De novo generation of infectious prions with bacterially expressed recombinant prion protein.

    PubMed

    Zhang, Zhihong; Zhang, Yi; Wang, Fei; Wang, Xinhe; Xu, Yuanyuan; Yang, Huaiyi; Yu, Guohua; Yuan, Chonggang; Ma, Jiyan

    2013-12-01

    The prion hypothesis is strongly supported by the fact that prion infectivity and the pathogenic conformer of prion protein (PrP) are simultaneously propagated in vitro by the serial protein misfolding cyclic amplification (sPMCA). However, due to sPMCA's enormous amplification power, whether an infectious prion can be formed de novo with bacterially expressed recombinant PrP (rPrP) remains to be satisfactorily resolved. To address this question, we performed unseeded sPMCA with rPrP in a laboratory that has never been exposed to any native prions. Two types of proteinase K (PK)-resistant and self-perpetuating recombinant PrP conformers (rPrP-res) with PK-resistant cores of 17 or 14 kDa were generated. A bioassay revealed that rPrP-res(17kDa) was highly infectious, causing prion disease in wild-type mice with an average survival time of about 172 d. In contrast, rPrP-res(14kDa) completely failed to induce any disease. Our findings reveal that sPMCA is sufficient to initiate various self-perpetuating PK-resistant rPrP conformers, but not all of them possess in vivo infectivity. Moreover, generating an infectious prion in a prion-free environment establishes that an infectious prion can be formed de novo with bacterially expressed rPrP.

  20. Recombinant lipidated dengue-3 envelope protein domain III stimulates broad immune responses in mice.

    PubMed

    Chiang, Chen-Yi; Liu, Shih-Jen; Hsieh, Chun-Hsiang; Chen, Mei-Yu; Tsai, Jy-Ping; Liu, Hsueh-Hung; Chen, I-Hua; Chong, Pele; Leng, Chih-Hsiang; Chen, Hsin-Wei

    2016-02-17

    The linkage of an immunogen with a toll-like receptor ligand has great potential to induce highly potent immune responses with the initial features of antigen-presenting cell activation. In the current study, we expressed recombinant dengue-3 envelope protein domain III (D3ED III) in lipidated form using an Escherichia coli-based system. The recombinant lipidated dengue-3 envelope