| Sea cucumber, a kind of nourishing food and Chinese medicine medicinal food, is deeply loved by people. However, sea cucumbers have a strong ability of autolysis, which causes huge losses in fishing, transportation and processing procedures. Previous researches have shown that endogenous proteinases of sea cucumber are involved in the autolysis process. However, limited researches have been found to focus on the genetic information and structural characteristics of endogenous proteinases. In this study, genes of serine proteinase(SP) and matrix metalloproteinase-2(MMP-2) were cloned from sea cucumber, respectively. Moreover, active recombinant proteinases were acquired by using genetic engineering technology, and the nature of which was analysed. This study provides a solid theoretical basis for understanding sea cucumber endogenous enzyme gene.In the present study, we designed primers according to the part of amino acid fragments of SP from sea cucumber viscera. And the gene encoding SP was cloned from sea cucumber(Stichopus japonicus) by RT-PCR and RACE. The full-length c DNA of SP was 1367 bp, including 44 bp 5’ non-coding regions and 192 bp 3’ non-coding regions. The complete open reading frame was 1131 bp, encoding a protein composed of 377 amino acid residues. Asp138、His176 and Ser325 consist the catalytic domain, which are the active sites responsible for enzymatic activity of SP. The SP is part of the family of Subtilisin serine protease, its tertiary structure contains six parallel β folded piece, surrounded by α screw on both sides, which forms a sandwich structure. In addition, two β folded structure involved in protein folding process correctly in the C-terminal domain of SP. Furtherly, using in vitro expression of E.coli expression system, the recombinant SP was effectively expressed as inclusion bodies in BL21(DE3). Then a large amount of highly purified recombinant SP was obtained by His Trap affinity column chromatography, and it is used to prepare specific polyclonal antibody. This antibody showed good titer and specificity. Further,we coupled the specificity antibody and CNBr-activated Sepharose 4B affinity column to purify the natural SP in sea cucumber viscera. At the same time, the SP gene from sea cucumber was inserted into p PIC9 K and expressed in pichia to acquire active recombinant SP. SDS-PAGE and Western-blot analysis indicated that the molecular mass of active recombinant SP was about 70 k Da.According to the MMPs conservative sequence, the primers were designed. We had expanded sea cucumber MMP-2 enzyme gene sequence in the first time. The deduced MMP-2 protein contains a propeptide domain, three repeats of fibronectin-type II domain inserted in the catalytic domain and a C-terminal hemopexin-like domain. The 3D structure of MMP-2 showed that catalytic domain and hemopexin-like domain are completely independent, and connected by hinge area. The catalytic domain of MMP-2 gene was cloned and inserted into E.coli expression vector p ET-28 a, and was effectively expressed as inclusion bodies in BL21(DE3). SDS-PAGE analysis indicated that the molecular mass of target protein was about 40 k Da, which was consistent with the predicted value. After dissolving inclusion bodies, the recombinant MMP-2 was furtherly purified by His Trap agarose affinity column and subsequently followed by renaturation. Obtained recombinant MMP-2 showed high activity at 35 °C~40 °C. High proteolytic activity was exhibited in the p Hs range from 7.5 to 9.0, and the highest activity was emerged at the p H of 8.0, suggesting that recombinant MMP-2 is a kind of weak alkaline protease. Divalent metal ion Ca2+ was essential for the gelatinolytic activity, indicating that the recombinant MMP-2 was similar to natural MMP-2, and both of them are calcium-dependent metalloproteinases. |
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