还原酶缺陷型大肠杆菌对重组蛋白溶解性的影响

探讨大肠杆菌细胞质氧化还原环境对重组蛋白溶解性的影响。选择含有1对二硫键的牛碱性成纤维细胞生长因子（BbFGF）作为简单蛋白的模式分子，选择含有2对二硫键的人抗HBsAg单链抗体（HBscFv）作为复杂蛋白的模式分子，分别构建表达质粒并转化普通宿主菌和还原酶缺陷型宿主菌E. coli Origami(DE3)，比较表达产物的溶解性和纯化产物的活性。结果发现，BbFGF在普通宿主菌中大部分形成包涵体，在Origami(DE3)中为可溶性表达，但表达量降低。两种工程菌的表达产物经离子交换和肝素亲和层析两步纯化后，MTT法测定活性，发现来自还原酶缺陷型宿主菌的BbFGF活性高于普通宿主菌表达产物，二者的ED₅₀分别是1.6 ng/mL和2.2 ng/mL；HBscFv在两种宿主菌中均形成包涵体，包涵体以6 mol/L盐酸胍缓冲液溶解后，镍离子螯合亲和层析纯化并透析复性，间接ELISA测定抗原结合活性，发现二者活性无明显差异，但在Origami(DE3)菌体破碎后的的上清中可检测到HBscFv活性，纯化后产量为1～2 mg/L，而在普通宿主菌破碎后的上清中检测不到HBscFv活性。上述结果说明，改变宿主菌细胞质氧化还原环境对于含有1～2对二硫键的重组蛋白的可溶性表达具有明显促进作用。

Influence of the Reductase Deficient Escherichia coli on the Solubility of Recombinant Proteins Produced in It

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.