海洋细菌Pseudoalteromonas sp.NJ631胶原蛋白酶的克隆与表达

Molecular Cloning and Expression of Collagenase Produce by Marine Bacteria Pseudoalteromonas sp. NJ631

对交替假单胞菌Pseudoalteromonas sp.NJ631基因组中潜在的胶原蛋白酶基因进行克隆与表达,并鉴定重组蛋白PNJC的酶活力。根据NJ631基因组序列草图,利用基因组信息发掘方法获得胶原蛋白酶功能提示的编码基因,进而利用高效表达质粒p ET28a构建重组表达系统。经IPTG诱导表达、亲和层析纯化获得重组蛋白纯化产物。以不溶性Ⅰ型胶原蛋白为底物,测定胶原蛋白酶酶活力。对Pseudoalteromonas sp.NJ631基因组信息挖掘,从中发现一条疑似编码胶原蛋白酶的基因序列,命名为Pnjc。该基因全长1 359 bp,GC含量45.62%,编码由452个氨基酸残基组成的约51 000大小的胶原蛋白酶。SDS-PAGE检测表明,胶原蛋白酶PNJC在0.2 mmol/L的IPTG诱导下得到高效表达。经过亲和层析获得纯化重组蛋白PNJC,蛋白质质量浓度约为1 mg/m L。酶活检测表明,PNJC的酶活力为160.34 U/mg,为标准品的70.48%。PNJC高酶活力表明,Pseudoalteromonas sp.NJ631来源的胶原蛋白酶具有重要的研究价值与工业开发潜力。

The gene encoding collagenase from Pseudoalteromonas sp.NJ631 was cloned and expressed in E.coli cells. Subsequently,the bioactivity of recombinant protein,named PNJC,was assessed. The gene sequence encoding a putative collagenase,designed Pnjc,was obtained from the genome of Pseudoalteromonas sp. NJ631 using a genome mining approach. The Pnjc coding region was amplified and cloned into p ET-28 a Vector. The plasmids harboring the assembled full-length sequence encoding PNJC were transformed into the E.coli cells for the expression of the PNJC protein. The expression and purification of the recombinant protein were carried on according to manufacturer＇s instruction. Finally,the enzymatic activity of collagenase was assessed by using recombinant protein decomposing the type I collagen as substrate. The 1359-bp open reading frame with 45.62% GC was obtained from the genome of Pseudoalteromonas sp. NJ631,which was encoded of a protein of 452 amino acids and shared significant homology with the collagenase from the other genera. The PNJC was highly expressed by adding 0.2 m M IPTG. The assay of enzyme activity showed that the enzyme activity of PNJC was 160.34 U/mg,70.48% of the standard,confirming that the Pnjc gene encoded a functional collagenase. This is the first report of the cloning the expression of collagenase from marine bacteria Pseudoalteromonas sp. The PNJC encoded by Pnjc gene from NJ631 showed high bioactivity on decomposing the type I collagen,which are of potential value for the commercial exploitation.