Splicing reaction of split inteins between non-natural exteins containing amino acid sequences of antibody hinge area
10.11665/j.issn.1000-5048.20190515
- VernacularTitle:断裂内含肽在含有抗体铰链区氨基酸序列的非天然外显肽中的剪接
- Author:
Tonglu DOU
1
;
Hao CHEN
;
Jin CAO
;
Junsheng CHEN
;
Jianwei ZHU
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:
split intein;
extein;
bi-specific antibody drug;
protein splicing;
exogenous amino acids
- From:
Journal of China Pharmaceutical University
2019;50(5):606-613
- CountryChina
- Language:Chinese
-
Abstract:
Intein is a functional protein that mediates self-cleavage from precursor protein and simultaneously connects the exteins on both sides of the intein via peptide bonds. Among all kinds of inteins, split intein has a wide range of applications in the field of antibody ligation. However, since the naturally split intein specifically recognizes the first three amino acid sequences “cysteine, phenylalanine, and asparagine”(CFN)of the extein, exogenous amino acids are inevitably introduced after the protein splicing reaction. In this study, the amino acid sequence “cysteine, aspartic acid, and lysine”(CDK)of the antibody hinge region was substituted for “CFN” as the recognition site for the intein and the split intein Npu DnaE was mutated into Npu*GEP DnaE. The results showed that the mutant could recognize “CDK” and the intein splicing reaction could successfully take place. The factors affecting the intein splicing reaction platform, such as pH, temperature and the concentration of NaCl and DTT were investigated in this study. The results showed that the splicing reaction of the mutant performed well, which indicated its potential usefulness in bispecific antibody assembly. In conclusion, the problem of introducing foreign amino acids was alleviated, the broadness of intein substrate was further expanded, and further technical support for the application of intein to the antibody assembly was provided.
