Heparin Flavobacterium Heparanase Gene Expression In E. Coli

Heparanase (heparanase, Hpa) is a class of D glucuronidase enzyme (endo-D-glucuronidase) studied recently, both present in normal tissues such as placenta and lymphoid organs.It plays a normal physiological function on the formation of embryo and, nervous system development, angiogenesis, inflammation; also expressed in a variety of cancer cell surface, promotes cell invasion and metastasis, induces angiogenesis and reduces tumor survival. Heparin for the treatment and prevention of thrombosis has a long history, but studies have shown that heparin has some side effects, such as those affecting the stability of platelets which leading to post-operative bleeding. The molecular weight of4000to8000(11-14monosaccharides) of low molecular weight heparin (LMWH) can be effective in treatment of thrombosis, but no significant side effects. At present, the need of LMWH in China mainly relied on imports or produced by the joint venture production, so the production of LMWH study has important industrial applications. One of the most important uses of Heparanase is the production of LMWH.To solve the above problems, this research group builds a highly efficient expression of soluble heparin, recombinant in E. coli enzyme system, to obtain high purity, high activity, the nature of the stability of heparanase. It may significantly reduce the costs of production, making the heparanase be possible for the production of LMWH. The group has completed the cloning and expression of Flavobacterium heparanase gene in Escherichia coli expression system work, and to the gene as a starting material, with the determination of enzyme activity screening of high-activity strain, than optimize the fermentation medium. At last, expression and purification the recombinant protein, and study its enzymatic properties. The major findings are as follows:Extract the whole genome of Flavobacterium heparanase, design two appropriate primers (introduces two restriction sites, Hind Ⅲ and EcoR Ⅰ), heparin ORF was amplified by PCR; Than double digestion pET-28a vector and heparanase gene with those enzymes, in the role of T4ligase to connect the target gene and vector fragments. The cloning vector will be transformed into E. coli BL21strains, containing Kan on solid LB medium can be screened positive clones. By PCR, restriction enzyme digestion and sequenced to determine the recombinant expression vector was successfully constructed.With the appropriate concentration of IPTG induced the expression of heparanase, detected the heparanase expression by SDS-PAGE under different induction time, to determine the optimal induction time. Combination of enzyme activity determination methods, filter the highest activity of heparanase expression strains.FFD and BBD are used for optimizing fermentation medium. The former finds out three main fators:Tryptone, Yeast extract, Glucose. The later finds out an optimal fermentation medium composition (g/L): Tryptone11.95, Yeast extract6, Glucose2.74, Na2HPO4·12H2O10, NH4C13, MgSO4·7H2O0.5, KH2PO42. Comparing the specific activity before and after optimization, the later is1.95times the number of the former.Fermentation by nickel column affinity chromatography purified enzyme electrophoresis pure heparin, SDS-PAGE electrophoresis showed that purified by a single protein bands, analyze the enzyme's molecular weight that42.9kDa. Research on enzymatic properties indicate that the enzyme's optimum temperature is30℃, optimum pH is7.0. Poor thermal stability of the enzyme, when the temperature is higher than60℃, it's activity will be complete lossed in30min. When the pH is between the range of7-10,1h of enzyme activity not affected, deviation from this range is a greater impact on the activity. Comparing9different metal ions, we find that Ca2+and Cu2+has obvious activation on heparanase, while Mn2+and Pb2+has obvious inhibition effect on it.