Fusion to Mistic improved fatty acid desaturase Fat-1 overexpression in E .coli / 重庆医学

ABSTRACT

Objective To construct a highly efficient expression plasmid of eukaryotic nuclear membrane protein Omega 3 fatty acid desaturase gene Fat‐1 in E .coli .Methods Using molecular cloning technology to construct the recombinant prokaryotic expression plasmid pET32a Fat‐1 and pET32a‐Mistic‐Fat‐1 fused with Membrane proteins expression chaperon mistic ;the two re‐combinant plasmids were transformed into E .coli strain BL21 (DE3) ,the expression of Fat‐1 protein and M110 Fat‐1 protein in‐duced by IPTG were identified by SDS‐PAGE and gray degree analysed the amount of expression ,further identified by Western blot .Results The results of enzyme digestion and sequencing demonstrated that we successfully constructed the prokaryotic ex‐pression vectors pET32a Fat‐1 and pET32a‐Mistic‐Fat‐1;SDS‐PAGE and Western blot showed that Fat‐1 fatty acid desaturase wasn′t significantly induced ,but the overexpression of M110 Fat‐1 fusion protein was obtained in E .coli ,accounting for 15% of the total amount of whole cell proteins .Conclusion The fusion with Mistic proteins to express the Fat‐1 gene has realized the overex‐pression of eukaryotic nuclear membrane integrated protein Omega 3 fatty acid desaturase in prokaryotic host .