Expression of soluble cloned porcine pepsinogen A in Escherichia coli.
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ABSTRACT:
SUBMITTER: Tanaka T
PROVIDER: S-EPMC1217215 | biostudies-other | 1996 Apr
REPOSITORIES: biostudies-other
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Project description:BackgroundPorcine epidemic diarrhea virus (PEDV) is a highly contagious enteric pathogen of swine. The spike glycoprotein (S) of PEDV is the major immunogenic determinant that plays a pivotal role in the induction of neutralizing antibodies against PEDV, which therefore is an ideal target for the development of subunit vaccine. In an attempt to develop a subunit vaccine for PEDV, we cloned two different fragments of S protein and expressed as glutathione S-transferase (GST)-tagged fusion proteins, namely rGST-COE and rGST-S1D, in E.coli. However, the expression of these recombinant protein antigens using a variety of expression vectors, strains, and induction conditions invariably resulted in inclusion bodies. To achieve the soluble expression of recombinant proteins, several chaperone co-expression systems were tested in this study.ResultsWe firstly tested various chaperone co-expression systems and found that co-expression of trigger factor (TF) with recombinant proteins at 15 °C was most useful in soluble production of rGST-COE and rGST-S1D compared to GroEL-ES and DnaK-DnaJ-GrpE/GroEL-ES systems. The soluble rGST-COE and rGST-S1D were purified using glutathione Sepharose 4B with a yield of 7.5 mg/l and 5 mg/l, respectively. Purified proteins were detected by western blot using mouse anti-GST mAb and pig anti-PEDV immune sera. In an indirect ELISA, purified proteins showed immune reactivity with pig anti-PEDV immune sera. Finally, immunization of mice with 10 μg of purified proteins elicited highly potent serum IgG and serum neutralizing antibody titers.ConclusionsIn this study, soluble production of recombinant spike protein of PEDV, rGST-COE and rGST-S1D, were achieved by using TF chaperone co-expression system. Our results suggest that soluble rGST-COE and rGST-S1D produced by co-expressing chaperones may have the potential to be used as subunit vaccine antigens.
| S-EPMC4855837 | biostudies-literature
Project description:As a vital tumor suppressor, PTEN (Phosphatase and tension homolog deleted on chromosome 10) is involved in inherited syndromes, and is among the most frequently inactivated tumor suppressor gene in sporadic cancers. PTEN loss-of-function widely occurs in human cancers via a variety of mechanisms, including genetic alterations and posttranslational modification. These suggest PTEN has a role of functional importance in a variety of cancers. In the present study, we constructed a prokaryotic expression vector that efficiently expresses GST-PTEN (the target protein in which PTEN is fused with glutathione S-transferase tag) in E. coli. We found that the target protein was partially soluble although major portions of the protein remained in the inclusion bodies. Furthermore, we explored the optimal induction temperature, isopropyl β-D-1-thiogalactopyranoside (IPTG) concentration and induction time in a series of experiments. Expression level analysis indicated that PTEN reached its peak level at 36(○)C for 8 h with 1.5625mM IPTG, while solubility analysis revealed the optimal induction temperature was at 20(○)C, the optimal IPTG concentration was 0.1µM and the optimal induction time was up to 8 h. Taken together, we provide an optimal induction condition for expressing soluble fusion protein of PTEN in E. coli, facilitating further analysis of PTEN's biological function in vitro.
| S-EPMC4598374 | biostudies-literature
Project description:MotivationPoor protein solubility hinders the production of many therapeutic and industrially useful proteins. Experimental efforts to increase solubility are plagued by low success rates and often reduce biological activity. Computational prediction of protein expressibility and solubility in Escherichia coli using only sequence information could reduce the cost of experimental studies by enabling prioritization of highly soluble proteins.ResultsA new tool for sequence-based prediction of soluble protein expression in E.coli, SoluProt, was created using the gradient boosting machine technique with the TargetTrack database as a training set. When evaluated against a balanced independent test set derived from the NESG database, SoluProt's accuracy of 58.5% and AUC of 0.62 exceeded those of a suite of alternative solubility prediction tools. There is also evidence that it could significantly increase the success rate of experimental protein studies. SoluProt is freely available as a standalone program and a user-friendly webserver at https://loschmidt.chemi.muni.cz/soluprot/.Availability and implementationhttps://loschmidt.chemi.muni.cz/soluprot/.Supplementary informationSupplementary data are available at Bioinformatics online.
| S-EPMC8034534 | biostudies-literature
Project description:We have cloned the cDNA of rat hsc70 (clathrin-uncoating ATPase) into a T7 expression system and have expressed this enzyme in Escherichia coli. The recombinant clathrin-uncoating ATPase is in the cytosolic fraction of the bacterium and is soluble. It was purified to homogeneity by DEAE-cellulose and ATP-agarose column chromatography. From 1 litre of bacterial culture (0.3-0.4 g of proteins), 5-20 mg of pure recombinant clathrin-uncoating ATPase was routinely obtained. The cloned enzyme is capable of dissociating clathrin from bovine coated vesicle. Furthermore, it is not methylated on basic amino acid residues and is not blocked at the N-terminus, indicating that these modifications on hsc70 are not essential for uncoating of clathrin. Binding of [alpha-32P]ATP by purified recombinant hsc70 was analysed by Scatchard plot. The results indicate that there one high-affinity binding component with a Kd (dissociation constant) of 0.2-0.3 microM. The peptide-stimulated ATPase activities of recombinant hsc70 at 37 degrees C with respect to S-peptide peptides P3a and GT4 at a concentration of 1.2 mM are 142 +/- 6, 214 +/- 8 and 362 +/- 5 pmol/h per micrograms of hsc70 protein respectively. The EC50 values of hsc70 ATPase for S-peptide, peptides P3a and GT4 are 2, 0.67 and 0.17 mM respectively. On the other hand, the dissociation constants of S-peptide, peptides P3a and GT4 for recombinant hsc70 are 7.6, 13 and 100 microM respectively. Thus peptide GT4 is the only peptide examined for which the binding constant is comparable with the EC50 for stimulation ATPase activity, albeit it has the lowest affinity for hsc70.
| S-EPMC1134567 | biostudies-other
Project description:Marine luciferases are regularly employed as useful reporter molecules across a range of various applications. However, attempts to transition expression from their native eukaryotic environment into a more economical prokaryotic, i.e. bacterial, expression system often presents several challenges. Specifically, bacterial protein expression inherently lacks chaperone proteins to aid in the folding process, while Escherichia coli presents a reducing cytoplasmic environment in. These conditions contribute to the inhibition of proper folding of cysteine-rich proteins, leading to incorrect tertiary structure and ultimately inactive and potentially insoluble protein. Vargula luciferase (Vluc) is a cysteine-rich marine luciferase that exhibits glow-type bioluminescence through a reaction between its unique native substrate and molecular oxygen. Because most other commonly used bioluminescent proteins exhibit flash-type emission kinetics, this emission characteristic of Vluc is desirable for high-throughput applications where stability of emission is required for the duration of data collection. A truncated form of Vluc that retains considerable bioluminescence activity (55%) compared to the native full-length protein has been reported in the literature. However, expression and purification of this luciferase from bacterial systems has proven difficult. Herein, we demonstrate the expression and purification of a truncated form of Vluc from E. coli. This truncated Vluc (tVluc) was subsequently characterized in terms of both its biophysical and bioluminescence properties.
| S-EPMC5772761 | biostudies-literature
Project description:BACKGROUND:Cyclodextrin glycosyltransferases (CGTases) catalyze the synthesis of cyclodextrins, which are circular ?-(1,4)-linked glucans used in many applications in the industries related to food, pharmaceuticals, cosmetics, chemicals, and agriculture, among others. Economic use of these CGTases, particularly ?-CGTase, requires their efficient production. In this study, the effects of chemical chaperones, temperature and inducers on cell growth and the production of soluble ?-CGTase by Escherichia coli were investigated. RESULTS:The yield of soluble ?-CGTase in shake-flask culture approximately doubled when ?-cyclodextrin was added to the culture medium as a chemical chaperone. When a modified two-stage feeding strategy incorporating 7.5 mM ?-cyclodextrin was used in a 3-L fermenter, a dry cell weight of 70.3 g·L-?1 was achieved. Using this cultivation approach, the total yield of ?-CGTase activity (50.29 U·mL-?1) was 1.71-fold greater than that observed in the absence of ?-cyclodextrin (29.33 U·mL-?1). CONCLUSIONS:Since ?-cyclodextrin is inexpensive and nontoxic to microbes, these results suggest its universal application during recombinant protein production. The higher expression of soluble ?-CGTase in a semi-synthetic medium showed the potential of the proposed process for the economical production of many enzymes on an industrial scale.
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Project description:Leptin, a 16 kDa nonglycosylated hormone, is produced by mature adipocytes and functions primarily in the hypothalamus to reduce food intake and body weight. To explore a new approach for high-level expression of human Leptin in Escherichia coli, the human Leptin gene, synthesized according to the published sequence, was cloned into the vector pET32a to construct a fusion expression plasmid: Trx-Leptin/pET32a. Our data showed that more than 40% of the fusion protein Trx-Leptin was expressed in soluble form. After purified by Ni-IDA affinity chromatography, cleaved by enterokinase and applied Ni-IDA affinity chromatography again, purified Leptin with homogeneity over 96% was achieved. The bio-functional experiments of purified Leptin showed a significant reduction in food intake and body weight of female mice treated with Leptin by comparing with control mice, and it indicated that the purified Leptin has full biological activity. In addition, our expression system was a very low-cost and efficient prokaryotic expression system. So taken together, our results demonstrated that our expression system of bio-active Leptin provided a new method for producing Leptin in big scale and would be widely applied in commercial Leptin producing industries.
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Project description:The emergence of highly pathogenic variant porcine epidemic diarrhea virus (PEDV) strains, from 2013 to 2014, in North American and Asian countries have greatly threatened global swine industry. Therefore, development of effective vaccines against PEDV variant strains is urgently needed. Recently, it has been reported that the N-terminal domain (NTD) of S1 domain of PEDV spike protein is responsible for binding to the 5-N-acetylneuraminic acid (Neu5Ac), a possible sugar co-receptor. Therefore, the NTD of S1 domain could be an attractive target for the development of subunit vaccines. In this study, the NTD spanning amino acid residues 25-229 (S25-229) of S1 domain of PEDV variant strain was expressed in Escherichia coli BL21 (DE3) in the form of inclusion bodies (IBs). S25-229 IBs were solubilized in 20 mM sodium acetate (pH 4.5) buffer containing 8 M urea and 1 mM dithiothreitol with 95% yield. Solubilized S25-229 IBs were refolded by 10-fold flash dilution and purified by one-step cation exchange chromatography with >95% purity and 20% yield. The CD spectrum of S25-229 showed the characteristic pattern of alpha helical structure. In an indirect ELISA, purified S25-229 showed strong reactivity with mouse anti-PEDV sera. In addition, immunization of mice with 20 ?g of purified S25-229 elicited highly potent serum IgG titers. Finally, mouse antisera against S25-229 showed immune reactivity with native PEDV S protein in an immunofluorescence assay. These results suggest that purified S25-229 may have potential to be used as a subunit vaccine against PEDV variant strains.
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Project description:Genetically engineered variants of human lysozyme represent promising leads in the battle against drug-resistant bacterial pathogens, but early stage development and testing of novel lysozyme variants is constrained by the lack of a robust, scalable and facile expression system. While wild type human lysozyme is reportedly produced at 50–80 kg per hectare of land in recombinant rice, this plant-based system is not readily scaled down to bench top production, and it is therefore not suitable for development and characterization of novel lysozyme variants. Here, we describe a novel and efficient expression system capable of producing folded, soluble and functional human lysozyme in Escherichia coli cells. To achieve this goal, we simultaneously co-express multiple protein folding chaperones as well as harness the lysozyme inhibitory protein, Ivy. Our strategy exploits E. coli's ease of culture, short doubling time, and facile genetics to yield upwards of 30 mg/l of soluble lysozyme in a bioreactor system, a 3000-fold improvement over prior efforts in E. coli. Additionally, molecular interactions between lysozyme and a his-tagged Ivy allows for one-step purification by IMAC, yielding as much as 21 mg/l of purified enzyme. We anticipate that our expression and purification platform will facilitate further development of engineered lysozymes having utility in disease treatment and other practical applications.
| S-EPMC3594478 | biostudies-literature
Project description:The UDP-glucosyltransferase UGT76G1 from Stevia rebaudiana converts stevioside to rebaudioside A via a one-step glycosylation reaction, which increases the amount of sweet-tasting rebaudioside A and decreases the amount of stevioside that has a bitter aftertaste. This enzyme could, therefore, conceivably be used to improve the organoleptic properties of steviol glycosides and offer a cost-effective preparation of high-purity rebaudioside A. Producing soluble enzymes by overexpression is a prerequisite for large-scale biocatalysis. However, most of the UGT76G1 overexpressed in Escherichia coli is in inclusion bodies. In this study, three N-terminal fusion partners, 3'-phosphoadenosine-5'-phosphatase (CysQ), 2-keto-3-deoxy-6-phosphogluconate aldolase (EDA) and N-utilisation substance A (NusA), were tested to improve UGT76G1 expression and solubility in E. coli. Compared with the fusion-free protein, the solubility of UGT76G1 was increased 40% by fusion with CysQ, and the glucosyltransferase activity of the crude extract was increased 82%. This successful CysQ fusion strategy could be applied to enhance the expression and solubility of other plant-derived glucosyltransferases and presumably other unrelated proteins in the popular, convenient and cost-effective E. coli host.
| S-EPMC5624807 | biostudies-literature