Cloning and gene expression of G protein competitive inhibitory polypeptide and its prophylactic effects on myocardial hypertrophy in vitro
Abstract
AIM: 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).
CONCLUSION: 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.
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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).
CONCLUSION: 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.