Improving proteolytic stability of Npu DnaE C-fragment in Escherichia coli expression system
10.11665/j.issn.1000-5048.20200312
- VernacularTitle:断裂内含肽C片段在大肠埃希菌表达系统中酶解稳定性的改良
- Author:
Hao CHEN
1
;
Jin CAO
;
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:
Npu DnaE;
C-terminal cleavage;
protein stability;
protein degradation
- From:
Journal of China Pharmaceutical University
2020;51(3):340-348
- CountryChina
- Language:Chinese
-
Abstract:
Naturally split Npu DnaE intein can mediate rapid trans-splicing and C-cleavage, which is of great use in many aspects of protein engineering. However, the degradation of NpuC during expression and purification reduces the yield and purity of recombinant protein. N2C, an extended NpuN2-containing N-terminal NpuC fragment, was constructed to improve NpuC stability. N2C was expressed in BL21(DE3) and purified by affinity chromatography. The degradation ratio was calculated by ImageJ, and the factors affecting the C-terminal cleavage reaction of intein, such as temperature, DTT concentration and N/C ratio, were also investigated. The results showed that N2C lowered the proportion of degradation to 2.7%-7.2% and the yield of C-terminal cleavage reached 90% in 30 min at 37 °C with an N/C ratio of 5∶1 catalyzed by 1 mmol/L DTT. N2C can not only improve the stability of NpuC in Escherichia coli expression system, but also retain the activity of C-terminal cleavage reaction, which is of great significance for its application in protein purification.
