Construction and application of N-terminal Strep-tagged protein expression vector
10.3969/j.issn.1001-1978.2016.01.021
- VernacularTitle:N 末端 Strep 蛋白标签表达载体的构建和应用
- Author:
Yu SHI
;
Hongzhang JI
;
Xiaofeng BAO
- Publication Type:Journal Article
- Keywords:
Strep-tag;
expression vector;
fusion pro-tein;
cloning;
RNA polymerase;
chlamydia
- From:
Chinese Pharmacological Bulletin
2016;(1):98-102
- CountryChina
- Language:Chinese
-
Abstract:
Aims To construct the N-terminal Strep-tagged ( NS-tagged) fusion protein expression vector, and to apply the vector to express NS-tagged fusion proteins of Chlamydia RNA polymerase subunit. Meth-ods By using PCR method, NS fusion protein tag and a new multiple cloning sites (MCS) were inserted into pET21c-DH plasmid by primers to replace the original T7 protein tag and MCS. The newly introduced Not I cutting site was chosen for self-ligation of PCR prod-uct. Then, the cyclized PCR product was transformed into DH-5α competent cells. The positive clones were selected by PCR and sequencing. To get NS-tagged fu-sion proteins of chlamydial RNA polymerase subunits, the α, β and β′ subunits were inserted between BamH I and Sal I cutting sites of the newly constructed ex-pression vector. Then, the NS-α, NS-β and NS-β′ ex-pression vectors were transformed into Arctic Express expression cells. The fusion protein expression statuses of transformed cells were identified by Commassie blue staining and Western blot. Results The NS-tagged fusion protein expression vector pET21c-NS-MCS was successfully constructed, and NS-α, NS-β and NS-β′fusion proteins were obtained by using this newly con-structed expression vector. Conclusions In this pro-ject, we constructed an NS-tagged fusion protein ex-pression vector and applied it to express NS-α, NS-βand NS-β′ fusion proteins. Our study can lay a solid foundation for the study of transcriptional regulation of Chlamydia genes.
