ArticleWorld journal of gastroenterology2005
Solubility of disulfide-bonded proteins in the cytoplasm of Escherichia coli and its "oxidizing" mutant.
Article in World journal of gastroenterology, 2005. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 11 papers.
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Who cites it
11 citing papers in PubMed, 33 citations in OpenAlex.
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- Article
- High throughput screening identifies disulfide isomerase DsbC as a very efficient partner for recombinant expression of small disulfide-rich proteins in E. coli.Microbial cell factories · 2013Article
- Functional recombinant extra membrane loop of human CD20, an alternative of the full length CD20 antigen.Iranian biomedical journal · 2012Article
- Toxins from cone snails: properties, applications and biotechnological production.Applied microbiology and biotechnology · 2008Review
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Authors and funding
9 authors at 1 institution in 1 country.
Funding
No grant is acknowledged in the PubMed record.
Abstract
aimTo study the influence of redox environment of Escherichia coli (E. coli) cytoplasm on disulfide bond formation of recombinant proteins.
methodsBovine fibroblast growth factor (BbFGF) was selected as a model of simple proteins with a single disulfide bond and free cysteines. Anti-HBsAg single-chain Fv (HBscFv), an artificial multidomain protein, was selected as the model molecule of complex protein with 2 disulfide bonds. A BbFGF-producing plasmid, pJN-BbFGF, and a HBscFv producing-plasmid, pQE-HBscFv, were constructed and transformed into E. coli strains BL21(DE3) and M15(pREP4) respectively. At the same time, both plasmids were transformed into a reductase-deficient host strain, E. coli Origami(DE3). The 4 recombinant E. coli strains were cultured and the target proteins were purified. Solubility and bioactivity of recombinant BbFGF and HBscFv produced in different host strains were analyzed and compared respectively.
resultsAll recombinant E. coli strains could efficiently produce target proteins. The level of BbFGF in BL21(DE3) was 15-23% of the total protein, and was 5-10% in Origami (DE3). In addition, 65% of the BbFGF produced in BL21(DE3) formed into inclusion body in the cytoplasm, and all the target proteins became soluble in Origami(DE3). The bioactivity of BbFGF purified from Origami(DE3) was higher than its counterpart from BL21(DE3). The ED(50) of BbFGF from Origami(DE3) and BL21(DE3) was 1.6 microg/L and 2.2 microg/L, respectively. Both HBscFv formed into inclusion body in the cytoplasm of M15(pQE-HBscFv) or Origami(pQE-HBscFv). But the supernatant of Origami(pQE-HBscFv) lysate displayed weak bioactivity and its counterpart from M15(pQE-HBscFv) did not display any bioactivity. The soluble HBscFv in Origami(pQE-HBscFv) was purified to be 1-2 mg/L and its affinity constant was determined to be 2.62 x 10(7) mol/L. The yield of native HBscFv refolded from inclusion body in M15(pQE-HBscFv) was 30-35 mg/L and the affinity constant was 1.98 x 10(7) mol/L. There was no significant difference between the bioactivity of HBscFvs refolded from the inclusion bodies produced in different host strains.
conclusionModification of the redox environment of E. coli cytoplasm can significantly improve the folding of recombinant disulfide-bonded proteins produced in it.
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