Cloning and gene expression of G protein competitive inhibitory polypeptide and its prophylactic effects on myocardial hypertrophy in vitro.
OBJECTIVE
To clone and express G protein competitive inhibitory polypeptide (GCIP) gene and investigate the prophylactic effects of GCIP on myocardial hypertrophy in vitro.


METHODS
The pIVEX2.3MCS-GCIP plasmid expressing GCIP was constructed by inserting double-stranded oligonucleotide into pIVEX2.3-MCS plasmid vector. Recombinant plasmid was expressed in Rapid Translation System 500 (RTS500). The expression of GCIP was identified by SDS-PAGE and Western blotting. The purification of GCIP with 6xHis tag was carried out on a Ni-NTA agarose column. The prophylactic effects of GCIP was observed on cardiomyocytes isolated from newborn Wistar rats. Protein synthesis rate were assessed by [3H]Leu incorporation. Protein contents were measured by Lowry method.


RESULTS
The plasmid pIVEX2.3MCS-GCIP was successfully constructed. The expression of GCIP was about 2.43 % of total protein. GCIP was purified on Ni-NTA agarose column. The purity of the purified GCIP peptide was about 98 %. The contents of total protein and the rate of [3H]Leu incorporation were significantly decreased in (100 microg/L, 1 mg/L, and 10 mg/L) GCIP-treated groups in myocardial cellular hypertrophy model (P<0.01).


CONCLUSIONS
The pIVEX2.3MCS-GCIP expression vector has been constructed successfully. The GCIP was expressed in RTS500 system and was purified with Ni-NTA agarose. GCIP was able to inhibit myocardial hypertrophy concentration-dependently in vitro.
Zhou
Jian-Zhi
JZ
Department of Pharmacology, Third Military Medical University, Chongqing 400038, China.
Li
Xiao-Hui
XH
Zhang
Hai-Gang
HG
Tang
Yuan
Y
Wang
Xiao-Qin
XQ
D000818:Animals; D000831:Animals, Newborn; D002478:Cells, Cultured; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000737:chemistry / Q000235:genetics* / Q000494:pharmacology; D015870:Gene Expression; D005822:Genetic Vectors; D007930:Leucine / Q000378:metabolism*; D008970:Molecular Weight; D032383:Myocytes, Cardiac / Q000166:cytology / Q000378:metabolism*; D010957:Plasmids; D051381:Rats; D017208:Rats, Wistar; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000494:pharmacology
C488275:G protein competitive inhibitory polypeptide, rat; D011994:Recombinant Proteins; D019204:GTP-Binding Proteins; D007930:Leucine