Study On Cloning And Expressing Of Mouse GAcrp30 Gene And Biological Activities Of Expression Products

Adipose tissue is the largest reservoir of fuel, storing energy in the rapidly utilizable triglycerides. Adipocyte complement-related protein (Acrp30) is a secreted protein exclusively in differentiated adipocytes. gAcrp30 is the protease-generated globular head domain of gAcrp30 which has not the signal sequence of peptides. The molecular weight of mice gAcrp30 is 25 KDa. The effect of gAcrp30 was caused, at least in part, by an acute increase in fatty acid oxidation by muscle.Mice gAcrp30 cDNA from adipocytes was synthesized by RT-PCR from mRNA extracted from adipose tissue of mice. The mice gAcrp30 cDNA from adipose tissue of mice was then subcloned into pMD18-T vector. The sequence of cloned mice gAcrp30 cDNA was determined. The length of the mice gAcrp30 gene was 744 bp, encoding a peptide of 255 amino acids whose molecular weight is 25 KDa. The signal sequence of gAcrp30 peptides is not cloned in our study. By blasting the homologous sequences in GenBank databases, the sequence of mice gAcrp30 cDNA from adipose tissue of mice is homologous to the previously cloned mice gAcrp30 cDNA.The prokaryotic expression plasmid pET-21a/gAcrp30 was constructed. pET-21a/gAcrp30 was transformed into E.coli BL21. competent cells and the clones expressing the gAcrp30 protein was identified by SDS-PAGE. The mRNA expression of pET-21a/gAcrp30 was detected by RT-PCR. A new protein band was found with molecular mass of about 25KDa in SDS-PAGE. This result was consistent with a 25 KDa protein deduced from gAcrp30 gene sequence.