磷脂酰丝氨酸合成酶基因pss的克隆与表达

磷脂酰丝氨酸合成酶能催化转酯反应,是定向合成特定磷脂类物质特别是磷脂酰丝氨酸的工具酶,但出发菌株产量低,很大程度上限制了酶法合成磷脂酰丝氨酸的工业化应用。利用表达载体pET-22b,实现了大肠杆菌磷脂酰丝氨酸合成酶基因在大肠杆菌BL21(DE3)中的同源高效表达。利用镍亲和柱对表达产物进行纯化,并用HPLC法对纯化后的重组酶的活力进行检测。结果表明,目的蛋白可在短时间内进行大量表达,蛋白含量是出发菌株的100倍,同时经6h的转酯反应转化率达到33%,重组磷脂酰丝氨酸合成酶活力达到69U/mg蛋白。

Cloning,Expression of pss gene from Escherichia coli K12

Phosphatidylserine synthase could catalyze the transphosphatidylation reaction to synthesize lipid compounds that what people need.But the low yield of initial strain limited industrial-scale of enzymatic synthesis phosphatidylserine.In this research,the gene encoding phosphatidylserine synthase from Escherichia coli K12Sgal-was cloned into expression vector pET-22b( )constituting recombinant plasmid pET-pss which was tranformed into E.coli BL21(DE3).Positive transformant was cultivated and IPTG was added into culture to induce expression of recombinant phosphatidylserine synthase.Detected by SDS-PAGE,the aim prtein was expression in E.coli successfully,the relative yield of the phosphatidylserine synthase was 100-fold higher than the initial strain.Purified step was carried out by use Ni column.The transformation rate of the transphosphatidylation reaction or the activity of the recombinant phosphatidylserine synthase was detected by HPLC.After 6h transphosphatidylation reaction,the transformation rate was 33%,and enzymatic activity was 69U/mg recombinant protein.