Cloning And Expression Of Collagenase From Marine Bacteria Pseudoalteromonas Sp.Nj631
Keywords
Pseudoalteromonas,Collagenase,Genome Mining,Enzymatic Activity Detectionpseudoalteromonas,Collagenase,Genome Mining,Enzymatic Activity
Abstract
The potential collagenase gene in the genome of Pseudoalteromonas sp.NJ631 was cloned and expressed, and the enzyme activity of the recombinant protein PNJC was identified. According to the draft genome sequence of NJ631, the genome information mining method was used to obtain the coding gene suggesting the function of collagenase, and then the high-efficiency expression plasmid pET28a was used to construct a recombinant expression system. The purified recombinant protein product was obtained through IPTG-induced expression and affinity chromatography purification. Insoluble type I collagen was used as the substrate to determine collagenase enzyme activity. Mining the genome information of Pseudoalteromonas sp.NJ631, a gene sequence suspected to encode collagenase was found, named Pnjc. The gene has a full length of 1 359 bp, a GC content of 45.62%, and encodes a collagenase of approximately 51 000 pieces consisting of 452 amino acid residues. SDS-PAGE detection showed that collagenase PNJC was highly expressed under the induction of 0.2 mmol/L IPTG. Purified recombinant protein PNJC was obtained through affinity chromatography, and the protein mass concentration was approximately 1 mg/mL. The enzyme activity test showed that the enzyme activity of PNJC was 160.34 U/mg, which was 70.48% of the standard substance. The high enzyme activity of PNJC shows that collagenase derived from Pseudoalteromonas sp.NJ631 has important research value and industrial development potential. 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
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Original research was done by Xue Chunxu, Chen Wei, Zhou Yufang, Zhu Peng
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