Cloning, Expression And Purification Of A Heparinase Ⅰ Mutant In E.Coli Rosetta

Background and Purpose-Heparinase is one of the polysaccharide lyases that are capable of acting on linkages present in heparin and heparan sulfate.It has important application in the preparation of low molecular weight heparin and the deheparinization of heparin-treated blood.It is also used as a tool in the biochemical studies of the fine structure and physiological activity of heparin and heparin sulfate.Heparinaseâ… purified from Flavobacterium heparinum,is much more useful because of its specificity to degrade the linkage of heparin,but the high cost due to its low productivity,complexity of purification procedures and poor stability has hampered both analytical and commercial applications of the enzyme.So,doing researches on heparinaseâ… ,especially on the methods to increase its purity and productivity,has significance in expanding its applications in medical and pharmaceutical industry.In our study,a heparinaseâ… mutant from Flavobacterium heparinum was found and a sequencing vector was constructed to determine the precise positions of the mutational amino acid residues.In order to acquire a recombinant heparinaseâ… protein with higher productivity and purity,the mutational heparinaseâ… gene was cloned and expressed in engineering strain E.coli Rosetta(DE3).Methods-Firstly,Heparinseâ… gene(HepA) was obtained by PCR using Flavobacterium heparinum NBRC 12017 chromosome DNA as template and cloned to pGEM-T Easy vector to determine the precise positions of the mutational bases in HepA.The enzymatic activity was determined by Azure A assay to investigate the effects resulted by the mutaions on heparinaseâ… .Secondly,the mutational HepA was cloned into the vectors pET28a and pET22b to construct three expression vectors(pET28a/H1, pET28a/H2,pET22b/H1),then the recombinant plasmids were transformed into E.coli Rosetta(DE3) and the expression products were analyzed by SDS-PAGE,the highest heparinaseâ… -producing transformant with optimization expression conditions was selected out and cultivated to express recombinant protein.Finally,the heparinaseâ… existing as inclusion bodys obtained from bacterial broth by sonication and centrifugation was refolded and purified by combining the urea concentration and pH gradient changes in ion exchange chromatography.Results-1.A mutated heparinaseâ… from F.heparinum with four variations of amino acid residue was found.It was proved that the mutations did not affect the enzymatic activity and these four amino acid residues were not essential for the enzyme activity of heparinaseâ… .2.Three expression vectors pET22b/H1,pET28a/H1 and pET28a/H2 were constructed and transformed into E.coli Rosetta.Positive clones were screened and the heparinaseâ… protein was expressed successfully from the engineering bacteria.In this expression system,target gene was the heparinaseâ… mutant from Flavobacterium heparinum (NBRC 12017),the host strain was E.coli Rosetta(DE3) which can enhance the expression of the proteins that contain codons rarely used in E.coli.The results also showed that the yield of heparinaseâ… was up to 128mg/L,which was approximately twice higher than that obtained in E.coli BL21(DE3) as reported.3.In this research,the active recombinant heparinaseâ… was refolded and purified by urea concentration and pH combined gradients ion exchange chromatography.The total enzyme activity of the recombinant protein could reach about 3000U/L,but the protein folding efficiency was only 40%,which need to be improved by further study.