The effect of recombinant TSBP on protein kinase A activity.
- Author:
Wenhui SU
1
;
Zhe ZHANG
;
Jie ZHANG
;
Bingzhi YU
Author Information
1. Department of Biochemistry and Molecular Biology, China Medical University, Shenyang 110001, China.
- Publication Type:Journal Article
- MeSH:
Acrosome Reaction;
drug effects;
Cell Line;
Cyclic AMP-Dependent Protein Kinases;
drug effects;
Humans;
Male;
Recombinant Proteins;
biosynthesis;
genetics;
pharmacology;
Seminal Plasma Proteins;
biosynthesis;
genetics;
pharmacology;
Sperm Capacitation;
drug effects
- From:
Journal of Biomedical Engineering
2007;24(6):1334-1342
- CountryChina
- Language:Chinese
-
Abstract:
To investigate the function of testis sperm binding protein (TSBP) in sperm capacitation and acrosome reaction, the effect of the recombinant TSBP on the activity of protein kinase A was detected in the transfected cell line. With the use of prokaryotic expressing plasmid pGEX-5X-1/tsbp as template, the novel gene tsbp was amplified by PCR and a eukaryotic expressing vector pcDNA3.1/myc-His(-)B/tsbp was constructed. DNA sequencing and restriction endonuclease digestion analysis indicated that the eukaryotic expression vector pcDNA3.1/myc-His(-)B/tsbp had been constructed successfully. After the recombinant plamid being transfected into HEK293 cells, Western blot verified the expression of tsbp. Fusion protein His6-TSBP was purified from the cell lysate by immobilized metal-ion affinity chromatography (IMAC). Radioautograph revealed a higher PKA activity in the transfected HEK293 cells than in the control group, which indicates that TSBP can increase the activity of PKA.
