Pseudomonas putida S1海藻糖合成酶基因在大肠杆菌中的克隆表达

Cloning and expression of of trehalose synthase gene from Pseudomonas putida S1 in Escherichia coli

利用PCR和TA克隆方法扩增和克隆得到了恶臭假单胞菌Pseudomonas putidaS1的海藻糖合成酶基因treS。对其进行序列分析表明,其编码区含有2067bp,编码含688个氨基酸残基的蛋白质,其核苷酸序列和蛋白质序列与来源于其它假单胞菌属细菌的海藻糖合成酶的序列表现出了较高同源性。将该基因序列与表达载体pQE30T连接,构建重组质粒pQE30T-TS,并将其转化至E.coliM15菌株中。重组菌株经诱导表达后SDS-聚丙烯酰胺凝胶电泳结果显示有明显的分子量约77.5kD的特异蛋白条带出现。经测定酶活力达19U/mL,约是原始菌株P.putidaS1的50倍。

The treS gene encoding trehalose synthase was amplified and cloned by PCR and TA cloning from Pseudomonas putida S1. Sequence analysis revealed a 2067-bp trehalose synthase gene and a 688 residue amino acid sequence with high identities to those of trehalose synthase from other strains of Pseudomonas. The recombinant plasmid pQE30T-TS was constructed by inserting treS into vector pQE30T and then transformed into E. coli M15. The overexpression of treS gene in the IPTG-induced recombinant strain was confirmed by SDS-polyacrylamide gel electrophoresis with a distinct band corresponding to a molecular mass of about 77.5 kD. The recombinant trehalose synthase exhibited a high activity of 19U/mL, about 50-fold higher than the parent strain Pseudomonas putida S1.