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首页> 外文期刊>World Journal of Gastroenterology >Solubility of disulfide-bonded proteins in the cytoplasm of Escherichia coli and its 'oxidizing' mutant.
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Solubility of disulfide-bonded proteins in the cytoplasm of Escherichia coli and its 'oxidizing' mutant.

机译：二硫键结合蛋白在大肠杆菌及其“氧化”突变体细胞质中的溶解度。

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AIM: To study the influence of redox environment of Escherichia coli (E. coli) cytoplasm on disulfide bond formation of recombinant proteins. METHODS: Bovine 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. RESULTS: All 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. CONCLUSION: Modification of the redox environment of E. coli cytoplasm can significantly improve the folding of recombinant disulfide-bonded proteins produced in it.
机译：目的：研究大肠杆菌细胞质的氧化还原环境对重组蛋白二硫键形成的影响。方法：选择牛成纤维细胞生长因子（BbFGF）作为具有单二硫键和游离半胱氨酸的简单蛋白质的模型。抗人HBsAg单链Fv（HBscFv）是一种人工多域蛋白，被选作具有2个二硫键的复杂蛋白的模型分子。构建产生BbFGF的质粒pJN-BbFGF和产生HBscFv的质粒pQE-HBscFv，并将其分别转化到大肠杆菌菌株BL21（DE3）和M15（pREP4）中。同时，两个质粒均被转化为还原酶缺陷型宿主菌株大肠杆菌Origami（DE3）。培养4个重组大肠杆菌菌株，并纯化目标蛋白。分析并比较了在不同宿主菌株中产生的重组BbFGF和HBscFv的溶解度和生物活性。结果：所有重组大肠杆菌菌株均能有效产生靶蛋白。 BL21（DE3）中BbFGF的含量为总蛋白的15-23％，折纸（DE3）中为5-10％。另外，BL21（DE3）中产生的BbFGF的65％在细胞质中形成包涵体，并且所有靶蛋白都可溶于Origami（DE3）。从折纸（DE3）纯化的BbFGF的生物活性高于从BL21（DE3）提取的BbFGF。 Origami（DE3）和BL21（DE3）的BbFGF的ED（50）分别为1.6 microg / L和2.2 microg / L。 HBscFv在M15（pQE-HBscFv）或折纸（pQE-HBscFv）的细胞质中均形成包涵体。但是，Origami（pQE-HBscFv）裂解物的上清液显示出较弱的生物活性，而其与M15（pQE-HBscFv）的对应物则未显示任何生物活性。将折纸中的可溶性HBscFv（pQE-HBscFv）纯化为1-2 mg / L，测定其亲和常数为2.62 x 10（7）mol / L。从M15（pQE-HBscFv）中的包涵体重新折叠的天然HBscFv的产量为30-35 mg / L，亲和常数为1.98 x 10（7）mol / L。由不同宿主菌株产生的包涵体复性的HBscFvs的生物活性之间没有显着差异。结论：改变大肠杆菌细胞质的氧化还原环境可以显着改善其中产生的重组二硫键蛋白的折叠性。

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