Construction And Prokaryotic Expression Of The Heparinase â…  Plasmid Containing Gst-his Tags

Heparinases are a class of enzymes that are capable of specifically cleaving the(1â†'4)glycosidic likages in heparin-like glycosaminoglycans(GAG) such as heparin and heparansulfate. It is an usefull tool in producing low molecular weight heparin, understanding heparinstructure-function relationship and treating clear of the residual heparin in vitro circulation ofblood. The event of contaminated heparin that happened in2008puts forward higherrequirements on the safety of heparin-like drugs which demand for controlling the purity ofheparinase I in process for the preparation of LMWH. Furthermore, high purity heparinase isconvenient to the crystal structural analysis of heparinase I. Here, we report that the cloningand expression, in Escherichia coli, of heparinase I which contain GST-His double tags inupstream. This study provides a way for improving the expression and purity of heparinase Iwhich lays the foundation for preparing LMWH and studying the crystal struture ofheparinase I.The DNA sequence of heparinase I with6×His tag added in the gene upstream wasamplified with PCR from pET15b-Hep I, cloned into the cloning vector pMD-19T simplevector. The recombined cloning vector pMD-19T-His-Hep I was identified by PCR, doubledigestion and gene sequencing, then heparinase I with6×His tag cloned into the expressionvector pGEX-4T-1. The recombined expression vector pGEX-4T-1-His-Hep I was digested byBam H I and Sal I, then we could see that there were two bands consistent with the theoreticalvalue. The gene sequencing result coincided exactly with designde sequence.The recombined expression vector was transformed into E.coli BL21(DE3) and cultured.The optimization conditions of fermentation and induction were pH7.4,80ml of LB mediumcontaining in500ml flasks,2%the secondary seed inculum size, an optical density at OD600of1.6,0.8mmol/L IPTG,22℃, induced8h. Fermentation at the optimization conditions, thedry cell weight value and crude enzyme activity were0.973g/L and418.63IU/L, respectively.According to the flask results, high cell density cultivation of recombinant bacterium in5Lfermentor was investigated. The research results indicated that the recombinant bacteriumobtained highest cell density and crude enzyme activity with122.14g/L and1.697×104IU/L,respectively, when glycerol was used as the carbon source of R medium and induced atmid-logarithmic growth phase.The cell supernate was purified by GSTrap FF column followed by HisTrap HP column.SDS-PAGE results of the fusion protein showed specific bands at the size of66kDa and43kDa which were consistent with theoretical value of GST-His-Hep I and His-Hep I,respectively. The specific activity of His-Hep was measured as86.45IU/mg, and its puritywas calculated to be99%which was further improved compared with GST-His-Hep I. Thepreliminary enaymatic characteristics resuts of purified GST-His-Hep I and His-Hep Irevealed that the highest activity could be obtained at30℃and pH7.4. They also sharedsimilar stable at a broad pH range of2-10, and at a temperature of30℃. Moreover, we alsostudied the effect of different protective agent on GST-His-Hep I and His-Hep I anddegradation characteristic of two kind enzymes by GPC-MASS. The research results showed that BSA(0.1%), TW80(0.001mmol/L), CaCl2(1mmol/L) and DTT (0.5mmol/L) all havepositive effect on emzyme activity, and both of two kind enzyme can well cleavage of heparinprudcing about7000Da degradation product, with similar catalytic activity and substratespecificity.