[The effect of recombinant TSBP on protein kinase A activity].
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.
Su
Wenhui
W
Department of Biochemistry and Molecular Biology, China Medical University, Shenyang 110001, China.
Zhang
Zhe
Z
Zhang
Jie
J
Yu
Bingzhi
B
D020101:Acrosome Reaction / Q000187:drug effects*; D002460:Cell Line; D017868:Cyclic AMP-Dependent Protein Kinases / Q000187:drug effects*; D006801:Humans; D008297:Male; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000494:pharmacology; D029589:Seminal Plasma Proteins / Q000096:biosynthesis / Q000235:genetics / Q000494:pharmacology*; D013075:Sperm Capacitation / Q000187:drug effects*
D011994:Recombinant Proteins; D029589:Seminal Plasma Proteins; C425042:spermadhesin; D017868:Cyclic AMP-Dependent Protein Kinases