色褐链霉菌磷脂酶D基因pld的克隆与表达

利用表达载体pET-22b( ),实现了色褐链霉菌磷脂酶D基因在大肠杆菌BL21(DE3)中的高效表达.利用镍亲和柱对表达产物进行纯化,将纯化后的重组磷脂酶D作用于底物卵磷脂和丝氨酸定向合成磷脂酰丝氨酸,并用HPLC法检测酶活力.结果表明,目的蛋白可在短时间内进行大量表达.转酯反应6 h后卵磷脂的转化率达到31%,重组磷脂酶D活力达到39 U·(mg蛋白)-1.

Cloning, Expression of pld Gene from Streptomyces Chromofuscus

In this research,the gene encoding phospholipase D from Streptomyces chromofuscus was cloned into expressed vector pET-22b( ) constituting recombinant plasmid pET-pld which was tranformed into E.coli BL21(DE3).Positive transformant was cultivated and IPTG was added into culture to induce expression of recombinant phospholipase D.Detected by SDS-PAGE,the aim protein was expressed in E.coli successfully,the relative yield of the phospholipase D was higher than that of the initial strain.Purified step was carried out by use Ni column.The purified phospholipase D was used to catalyze the synthesis of phosphatidylserine by substrates lecithin and serine.The transformation rate of lecithin or the activity of the recombinant phospholipase D was detected by HPLC.With transphosphatidylation reaction time 6 h,the transformation rate was 31%,and the enzymatic activity was 39 U per mg recombinant protein.