title: [Influence of the reductase deficient Escherichia coli on the solubility of recombinant proteins produced in it].
abstract: The cytoplasm of E. coli is a reducing environment where cysteines do not engage in disulfide bonds. Any disulfide bonds that do appear are rapidly reduced through the action of disulfide reducing enzymes such as thioredoxin and glutaredoxin. To study the influence of E. coli cytoplasm on the solubility of recombinant proteins produced in it, bovine fibroblast growth factor (BbFGF), with single disulfide bond, and anti-HBsAg single-chain Fv (HBscFv), with two disulfide bonds, were selected as the pattern molecules of simple protein and complex protein, respectively. pJN98-BbFGF, a BbFGF expressing plasmid based on the vector pET3c, was constructed and transformed into normal host BL21(DE3) and a reductase deficient strain, E. coli Origami(DE3). At the same time, pQE-HBscFv, a HBscFv expressing plasmid was constructed and transformed into M15 [pREP4] and Origami(DE3). The recombinant BbFGF and HBscFv were produced in 2 types of bacteria and their solubilities and bioactivities were determined, respectively. It was found that the majority of BbFGF had formed inclusion body in the cytoplasm of BL21 (DE3) and all of them turned into soluble protein in Origami(DE3). It was also found the productivity of BbFGF in Origami (DE3) was 5% - 10% of the total protein and the value was 15% - 23% in BL21(DE3). BbFGFs produced in 2 recombinant bacteria were purified by cation exchange and heparin affinity chromatography. MTT assay revealed that the bioactivity of BbFGF purified from Origami(DE3) was higher than its counterpart from BL21(DE3). The ED50 of BbFGFs from different bacteria was 1.6ng/mL and 2.2ng/mL, respectively. As far as HBscFvs, both of them formed inclusion body in the cytoplasm of M15 [pQE-HBscFv] and Origami [pQE-HBscFv]. The inclusion body was solubilized in 6mol/L GuHCl, purified with a His-Trap column and then refolded by dialysis step-by-step against buffers containing downtrend concentration of GuHCl. Indirect ELISA was applied to determine the HBsAg binding activity of HBscFvs. It was found there was no obvious difference between the bioactivity of refolded HBscFvs produced from 2 recombinant bacteria. On the other hand, the supernatant of Origami [pQE-HBscFv] lysate displayed weak bioactivity and its counterpart from M15 [pQE-HBscFv] displayed without any bioactivity. The soluble HBsFv in the cytoplasm of Origami [pQE-HBscFv] was purified by cation exchange and immobilized metal affinity chromatography (IMAC) and the yield was 1 - 2mg/L. Those results suggested that modification of the redox environment of E. coli cytoplasm greatly improved the solubility of recombinant disulfide-bonded proteins produced in it. In the next step, we had like to co-express of molecular chaperones or refoldase to raise the yield of soluble recombinant proteins, as well as optimizing the culture condition of the "oxidizing" E. coli.
authors.affiliation: Biomedical Research & Development Center, Jinan University, Guangzhou 510640, China.
authors.forename: Sheng
authors.initials: S
authors.lastname: Xiong
authors.forename: Mei-Ying
authors.initials: MY
authors.lastname: Zhang
authors.forename: Chui-Wen
authors.initials: CW
authors.lastname: Qian
authors.forename: Yan-Chao
authors.initials: YC
authors.lastname: Ran
authors.forename: Yi-Fei
authors.initials: YF
authors.lastname: Wang
authors.forename: Xiang-Rong
authors.initials: XR
authors.lastname: Ren
authors.forename: Kuan-Yuan
authors.initials: KY
authors.lastname: Su
authors.forename: Zhou-Yao
authors.initials: ZY
authors.lastname: Yu
mesh_terms: D000818:Animals; D000906:Antibodies / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D002417:Cattle; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000235:genetics*; D005346:Fibroblast Growth Factors / Q000235:genetics / Q000378:metabolism; D005822:Genetic Vectors / Q000235:genetics; D006514:Hepatitis B Surface Antigens / Q000276:immunology; D002479:Inclusion Bodies / Q000737:chemistry / Q000378:metabolism; D010088:Oxidoreductases / Q000235:genetics*; D010957:Plasmids / Q000235:genetics; D015202:Protein Engineering; D011994:Recombinant Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D012995:Solubility
chemical_list: D000906:Antibodies; D029968:Escherichia coli Proteins; D006514:Hepatitis B Surface Antigens; D011994:Recombinant Proteins; D005346:Fibroblast Growth Factors; D010088:Oxidoreductases