C端断裂型内含肽Npu DnaE介导的抗体片段及免疫毒素在大肠埃希菌中的可溶性表达与纯化

Expression and purification of soluble recombinant antibody fragment and immunotoxin in Escherichia coli using Npu DnaE C-cleavage mutant

为实现免疫毒素在大肠杆菌内的可溶性表达,避免包涵体的形成带来的繁琐操作,本实验选用抗间皮素单克隆抗体SS1,以及其与毒素PE38KDEL连接的产物SS1P为实验对象;采用促溶标签结合低温诱导方式实现重组蛋白在大肠埃希菌(SHuffle T7)胞质内可溶性表达,其中促溶标签连接在NpuCD118G突变体的N端,SS1/SS1P连接在NpuCD118G突变体的C端;采用Dextrin Beads 6FF柱亲和色谱和镍柱亲和色谱方法实现目的蛋白纯化;采用分别表达纯化后混合的方法实现断裂内含肽Npu DnaE的自我断裂;采用镍柱亲和色谱除去未剪切的前体和断裂后促溶标签,其中,SS1进一步用Capto L捕获富集;采用Fortebio检测蛋白亲和力。本实验通过内含肽与促溶标签和纯化标签连用,实现了免疫毒素在大肠埃希菌中的可溶性表达,简化了纯化方式。该方法为开发基于大肠埃希菌表达系统的可溶性表达、纯化免疫毒素类药用重组蛋白方法提供了理论参考。

The purpose of this experiment is to achieve the soluble expression of immunotoxins in Escherichia coli and avoid the complicated operation caused by the formation of inclusion bodies.An anti-mesothelin monoclonal antibody SS1 and it′s derivative SS1P which composing of SS1 and a truncated pseudomonas exotoxin PE38KDEL were used as the passenger protein.We took advantages of solubility promoting fusion tags and the self-cleaving split intein in recombinant antibody fragment and immunotoxin expression and purification.We constructed solubilizing tags-NpuCD118G fusion tags,and recombinant SS1/SS1P were fused at the C-terminal of the fusion tags.The constructs were expressed in E.coli(SHuffle T7)cytoplasm in soluble form at low temperature.Dextrin Beads 6FF column and Nickel column was used to purify the fusion protein.The self-cleavage of the fusion protein was achieved by adding the NpuN fragment.The released solubilizing tag and unreacted precursor were removed by Nickel column and the cleaved antibody fragment and immunotoxinwere further captured by Capto L.The dissociation constant of the obtained Fv and immunotoxin were determined by Fortebio.In summary,the current method could enhance the solubility of antibody fragment and immunotoxin in E.coli,and could improve the purification process.This method provides a reference for the development of a method for soluble expression and purification of immunotoxin-type pharmaceutical recombinant proteins based on the Escherichia coli expression system.