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

Abstract:

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 pET-28a 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 mM 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.