The site-directed mutagenesis and prokaryotic expression of midkine gene in Cynoglossus semilaevis

Abstract

Midkine is a kind of heparin-binding cytokine,and it promotes growth,survival,migration and other activities of target cells.The expression of fusion protein MK will facilitate further studies for its biological functions in half-smooth tongue sole(Cynoglossus semilaevis).Based on the plasmid containing full length of half-smooth tongue sole MK cDNA sequence,the site-directed mutagenesis of predicted mature MK sequence was successfully obtained by SOEing PCR method according to the E.coli codon bias.After being confirmed by sequencing,the resulting gene was cloned to the vector pET32a(+)to yield an identified recombinant plasmid pET32a(+)-MK,which was then transformed into E.coli host strain BL21(DE3)pLysS.SDS-PAGE analysis revealed that high level of fusion protein MK was induced by 1 mmol/L IPTG at 37 ℃ and was mainly detected in sonicated supernatant.The yielded fusion protein MK was approximately 33 ku which was in accordance with the theoretical molecular weight.This study establishes the basis for further study for its detailed functions in half-smooth tongue sole.