Expression and purification of soluble recombinant antibody fragment and immunotoxin in Escherichia coli using Npu DnaE C-cleavage mutant
10.11665/j.issn.1000-5048.20200213
- VernacularTitle: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
- Author:
Jin CAO
1
;
Hao CHEN
;
Jing ZHANG
;
Jianwei ZHU
;
Junsheng CHEN
Author Information
1. 上海交通大学药学院细胞工程及抗体药物教育部工程研究中心
- Publication Type:Journal Article
- Keywords:
immunotoxin;
solubilizing label;
split intein
- From:
Journal of China Pharmaceutical University
2020;51(2):213-222
- CountryChina
- Language:Chinese
-
Abstract:
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.
