Parasite vaccine development: large-scale recovery of immunogenic Taenia ovis fusion protein GST-45W(B/X) from Escherichia coli inclusion bodies

1996 ◽  
Vol 82 (4) ◽  
pp. 291-296 ◽  
Author(s):  
R. P. Dempster ◽  
C. M. Robinson ◽  
G. B. L. Harrison
Keyword(s):  
Escherichia Coli ◽  
Fusion Protein ◽  
Inclusion Bodies ◽  
Large Scale ◽  
Vaccine Development ◽  
Taenia Ovis

scholarly journals Refolding of ScFv-CBD fusion protein from Escherichia coli inclusion bodies

2008 ◽  
Vol 24 (1) ◽  
pp. 51-59 ◽  
Author(s):  
O. B. Gorbatuk ◽  
U. S. Nikolayev ◽  
D. M. Irodov ◽  
I. Ya. Dubey ◽  
P. V. Gilchuk
Keyword(s):  
Escherichia Coli ◽  
Fusion Protein ◽  
Inclusion Bodies

scholarly journals Expression in Bacteria of the Gene Encoding the gp43 Antigen of Paracoccidioides brasiliensis: Immunological Reactivity of the Recombinant Fusion Proteins

2002 ◽  
Vol 9 (6) ◽  
pp. 1200-1204 ◽  
Author(s):  
Susana N. Diniz ◽  
Kátia C. Carvalho ◽  
Patrícia S. Cisalpino ◽  
José F. Silveira ◽  
Luiz R. Travassos ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Amino Acids ◽  
Fusion Protein ◽  
Fusion Proteins ◽  
Inclusion Bodies ◽  
Paracoccidioides Brasiliensis ◽  
Immunological Reactivity ◽  
Gene Encoding ◽  
Recombinant Fusion Proteins ◽  
Gst Fusion Protein

ABSTRACT gp43 is the major diagnostic antigen of Paracoccidioides brasiliensis, the agent of paracoccidioidomycosis (PCM) in humans. In the present study, cDNA of the gp43 gene (PbGP43) was obtained by reverse transcriptase PCR, inserted into a pGEX vector in frame with the glutathione S-transferase (GST) gene, and expressed in Escherichia coli as inclusion bodies. Immunoblotting showed that all sera from patients with chronic pulmonary and acute lymphatic forms of PCM reacted with the recombinant fusion protein of the mature gp43 (381 amino acids). Reactivity with fusion proteins containing subfragments of the N-terminal, internal, or C-terminal regions occurred eventually, and the C-terminal region was the most antigenic. Lack of reactivity with the subfragments may be due to the conformational nature of the gp43 epitopes. Sera from patients with aspergillosis, candidiasis, and histoplasmosis did not react with the gp43-GST fusion protein. Our results suggest that recombinant gp43 corresponding to the processed antigen can be a useful tool in the diagnosis of PCM.

Matrix-assisted in vitro refolding of Pseudomonas aeruginosa class II polyhydroxyalkanoate synthase from inclusion bodies produced in recombinant Escherichia coli

2001 ◽  
Vol 358 (1) ◽  
pp. 263-268 ◽  
Author(s):  
Bernd H. A. REHM ◽  
Qingsheng QI ◽  
Br. Bernd BEERMANN ◽  
Hans-Jürgen HINZ ◽  
Alexander STEINBÜCHEL
Keyword(s):  
Escherichia Coli ◽  
Pseudomonas Aeruginosa ◽  
Fusion Protein ◽  
Molecular Mass ◽  
Inclusion Bodies ◽  
Specific Activity ◽  
Guanidine Hydrochloride ◽  
Class Ii ◽  
Random Coil ◽  
Pha Synthase

In order to facilitate the large-scale preparation of active class II polyhydroxyalkanoate (PHA) synthase, we constructed a vector pT7-7 derivative that contains a modified phaC1 gene encoding a PHA synthase from Pseudomonas aeruginosa possessing six N-terminally fused histidine residues. Overexpression of this phaC1 gene under control of the strong Ø10 promoter was achieved in Escherichia coli BL21(DE3). The fusion protein was deposited as inactive inclusion bodies in recombinant E. coli, and contributed approx. 30% of total protein. The inclusion bodies were purified by selective solubilization, resulting in approx. 70–80% pure PHA synthase, then dissolved and denatured by 6M guanidine hydrochloride. The denatured PHA synthase was reversibly immobilized on a Ni2+-nitrilotriacetate–agarose matrix. The matrix-bound fusion protein was refolded by gradual removal of the chaotropic reagent. This procedure avoided the aggregation of folding intermediates which often decreases the efficiency of refolding experiments. Finally, the refolded fusion protein was eluted with imidazole. The purified and refolded PHA synthase protein showed a specific enzyme activity of 10.8m-units/mg employing (R/S)-3-hydroxydecanoyl-CoA as substrate, which corresponds to 27% of the maximum specific activity of the native enzyme. The refolding of the enzyme was confirmed by CD spectroscopy. Deconvolution of the spectrum resulted in the following secondary structure prediction: 10% α-helix, 50% β-sheet and 40% random coil. Gel filtration chromatography indicated an apparent molecular mass of 69kDa for the refolded PHA synthase. However, light-scattering analysis of a 10-fold concentrated sample indicated a molecular mass of 128kDa. These data suggest that the class II PHA synthase is present in an equilibrium of monomer and dimer.

scholarly journals Recombinant soluble human tissue factor secreted by Saccharomyces cerevisiae and refolded from Escherichia coli inclusion bodies: glycosylation of mutants, activity and physical characterization

1995 ◽  
Vol 310 (2) ◽  
pp. 605-614 ◽  
Author(s):  
M J Stone ◽  
W Ruf ◽  
D J Miles ◽  
T S Edgington ◽  
P E Wright
Keyword(s):  
Escherichia Coli ◽  
Saccharomyces Cerevisiae ◽  
Fusion Protein ◽  
Tissue Factor ◽  
Inclusion Bodies ◽  
Factor Viia ◽  
Factor X ◽  
Wild Type ◽  
Proteolytic Activation ◽  
E Coli

Tissue factor (TF) is the cell-surface transmembrane receptor that initiates both the extrinsic and intrinsic blood coagulation cascades. The abilities of TF to associate with Factor VIIa and Factor X in a ternary complex and to enable proteolytic activation of Factor X by Factor VIIa reside in the extracellular domain of TF. We describe the expression of the surface domain of TF (truncated TF, tTF) in both Saccharomyces cerevisiae and Escherichia coli and the biochemical and physical characterization of the recombinant proteins. Wild-type tTF and several glycosylation-site mutants were secreted efficiently by S. cerevisiae under the control of the yeast prepro-alpha-signal sequence; the T13A,N137D double mutant was the most homogeneous variant expressed in milligram quantities. Wild-type tTF was expressed in a non-native state in E. coli inclusion bodies as a fusion protein with a poly(His) leader. The fusion protein could be fully renatured and the leader removed by proteolysis with thrombin; the correct molecular mass (24,729 Da) of the purified protein was confirmed by electrospray mass spectrometry. Recombinant tTFs from yeast, E. coli and Chinese hamster ovary cells were identical in their abilities to bind Factor VIIa, to enhance the catalytic activity of Factor VIIa and to enhance the proteolytic activation of Factor X by Factor VIIa. Furthermore, CD, fluorescence emission and NMR spectra of the yeast and E. coli proteins indicated that these proteins are essentially identical structurally.

Chaperones Promote Remarkable Solubilization of Salmonella enterica serovar Enteritidis Flagellin Expressed in Escherichia coli

2020 ◽  
Vol 27 (3) ◽  
pp. 210-218
Author(s):  
Bahador Bakhtiarvand ◽  
Zahra Sadeghi ◽  
Shirin Tarahomjoo ◽  
Soheila Yaghmaie
Keyword(s):  
Escherichia Coli ◽  
Molecular Chaperones ◽  
Salmonella Enterica ◽  
Inclusion Bodies ◽  
Vaccine Development ◽  
Limited Proteolysis ◽  
Soluble Form ◽  
Salmonella Enterica Serovar Enteritidis ◽  
Native Page ◽  
Chemical Chaperones

Background: Flagellin of Salmonella enterica serovar Enteritidis (SEF) stimulates immune responses to both itself and coapplied antigens. It is therefore used in vaccine development and immunotherapy. Removal of pathogenic S. enterica ser. Enteritidis from SEF production process is advantageous due to the process safety improvement. The protein solubility analysis using SDS-PAGE indicated that 53.49% of SEF expressed in Escherichia coli formed inclusion bodies. However, the protein recovery from inclusion bodies requires a complex process with a low yield. Objective: We thus aim to study possibility of enhancing SEF expression in E. coli in soluble form using chemical and molecular chaperones. Methods: Chemical chaperones including arginine, sorbitol, trehalose, sodium chloride and benzyl alcohol were used as cultivation medium additives during SEF expression. SEF solubilization by coexpression of molecular chaperones DnaK, DnaJ, and GrpE was also investigated. Results: All of the chemical chaperones were effective in improving SEF solubility. However, sorbitol showed the most profound effect. SEF solubilization by molecular chaperones was slightly better than that using sorbitol and this approach enhanced noticeably SEF soluble concentration and SEF solubility percentage to almost two folds and 96.37% respectively. Results of limited proteolysis assay and native PAGE indicated similar conformational states and proper folding for SEF obtained without using chaperones and for those obtained using sorbitol and the molecular chaperones. However, the molecular chaperones based system was less costly than the sorbitol based system. Conclusion: The coexpression of molecular chaperones was then considered as the most appropriate approach for soluble SEF production. Therefore, SEF production for medical purposes is expected to be facilitated.

Expression of a Fusion Protein of Human Proinsulin with Glutathione-S-Transferase in Escherichia coli

2014 ◽  
Vol 998-999 ◽  
pp. 248-251
Author(s):  
Zhi Xin Di ◽  
Jian Zhong Ma ◽  
Yong Gang Wang
Keyword(s):  
Escherichia Coli ◽  
Fusion Protein ◽  
Fusion Proteins ◽  
Inclusion Bodies ◽  
Glutathione S Transferase ◽  
E Coli ◽  
Human Proinsulin ◽  
Sds Page ◽  
Dna Fragment ◽  
Sequence Encoding

A DNA sequence encoding for the human proinsulin was designed according to the codon bias of Escherichia coli and then chemically synthesized. The synthesized DNA fragment was subcloned into pGEX-3X for expression in E. coli BL21 (DE3) and E. coli BL21 Star (DE3), respectively. Conditions for the highest expression of the GST-proinsulin fusion proteins were optimized. These conditions are that cells of E. coli BL21 star (DE3) are incubated in 100mL of the LB medium with 2 mmol/L IPTG and 60μ?g/mL ampicillin at 26oCfor 4h. After disrupted E. coli cells with ultrasonication, inclusion bodies were precipitated from cell lysis and washed. Fusion proteins from the inclusion bodies were redissolved in 8mmol/L of urea. After dialysed in purified water, fusion proteins were analysed by SDS-PAGE. The purity of the fusion protein is about 80.5% in total. The fusion protein from SDS-PAGE was further identified by mass/mass spectrum. GST in the dyad protein is confirmed by the 9 matched sequences. However, the left part is proved a polypeptide of which is completely different from the human proinsulin.

The use of inclusion bodies, isolated from Escherichia coli expressing corticotrophin-releasing hormone precursor, to raise specific antibodies against the neuropeptide moiety

1990 ◽  
Vol 5 (3) ◽  
pp. 221-230 ◽  
Author(s):  
M. G. Castro ◽  
P. R. Lowenstein ◽  
P. W. Saphier ◽  
E. A. Linton ◽  
P. J. Lowry
Keyword(s):  
Escherichia Coli ◽  
Fusion Protein ◽  
Inclusion Bodies ◽  
Rat Tissues ◽  
Skin Extract ◽  
Parallel Displacement ◽  
Releasing Hormone ◽  
Tissue Extracts ◽  
Immunocytochemical Techniques

ABSTRACT We have expressed human pre-procorticotrophin-releasing hormone (pre-proCRH) as a fusion protein to β-galactosidase in Escherichia coli. The chimeric fusion protein was found in insoluble bacterial inclusion bodies. The inclusion bodies were isolated, purified and solubilized, and used as imunogens in rabbits to raise antibodies against the neuropeptide moiety. The antibodies generated were characterized by immunoassays and immunocytochemical techniques. The immunoassay results showed that the recombinant pre-proCRH antibodies cross-reacted with the full-length CRH precursor and several cleavage products derived from it, i.e. CRH(1–41) and CRH(36–41). They did not cross-react with the CRH antagonist CRH(9–41). Extracts of stalk median eminence from various species were also studied. The antibodies cross-reacted with extracts from ovine, bovine, human and rat tissues, exhibiting parallel displacement curves to that of synthetic rat/human CRH(1–41) used as standard. They also cross-reacted with a skin extract of the frog, a species known to contain a CRH-related peptide, i.e. sauvagine, in this tissue. The immunocytochemical studies demonstrated that the antibodies generated against recombinant human preproCRH labelled neurones in the rat paraventricular nucleus of the hypothalamus. They exhibited the same pattern of staining as that obtained with an antibody generated against synthetic CRH(1–41). The results indicate that these antibodies can recognize CRH(1–41) or CRH-related molecules in the hypothalamus in situ as well as in tissue extracts from several species. Hence, they will be useful tools in the study of the CRH biosynthetic pathway and its intracellular compartmentalization.

Large-scale preparation of recombinant human calcitonin from a multimeric fusion protein produced in Escherichia coli

1999 ◽  
Vol 87 (3) ◽  
pp. 296-301 ◽  
Author(s):  
Hirokazu Ishikawa ◽  
Junko Kawaguchi ◽  
Yoshio Yao ◽  
Hidetsune Tamaoki ◽  
Takeshi Ono ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Fusion Protein ◽  
Large Scale ◽  
Human Calcitonin ◽  
Large Scale Preparation ◽  
Scale Preparation

Mammalian G-protein-coupled receptor expression in Escherichia coli: I. High-throughput large-scale production as inclusion bodies

2009 ◽  
Vol 386 (2) ◽  
pp. 147-155 ◽  
Author(s):  
Kerstin Michalke ◽  
Marie-Eve Gravière ◽  
Céline Huyghe ◽  
Renaud Vincentelli ◽  
Renaud Wagner ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
G Protein ◽  
High Throughput ◽  
Inclusion Bodies ◽  
Large Scale ◽  
Receptor Expression ◽  
Scale Production ◽  
Expression In Escherichia Coli ◽  
Large Scale Production ◽  
G Protein Coupled
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