Expression And Application Of Aryl Sulfotranferase ? And ?4,5-unsaturated Glycuronidase

Heparin has been used widely as an anticoagulant and antithrombotic drug in clinical application.About 100 tons of pharmaceutical grade heparin products are produced and used annually worldwide.The biological safety of heparin produced from animal source has received scrutiny since the crisis of heparin contamination in 2008.The preparation of a bioengineered heparin from a microbially produced heparosan offers a potentially safer alternative than animal sourced heparin.Heparosan can be used as precursor for heparin and heparan sulfate biosynthesis due to a polysaccharide backbone similar to heparin.Heparosan need sulfated modification and depolymerization for preparation of heparin or low molecular heparin.Aryl sulfotransferase IV with PAPS regeneration system was required to provid sulfate groups for sulfated modification of heparosan.Meanwile,?4,5 unsaturated glycuronidase was also required to recover the unsaturated A4,5 uronic acid at the nonreducing end of oligosaccharides resulting from heparinase eliminative cleavage.In the present study,Aryl Sulfotransferase IV was expressed and purified.A4,5 unsaturated glycuronidase gene of Pedobacter heparinus was cloned and expressed in E.coli.Heparinase I,Heparinase II,and Heparinase? were used to depolymerize heparosan,heparin sulfate,and heparin soldium.Then,the recover effect of A4,5 unsaturated glycuronidase on the depolymerization products was evaluated.Aryl Sulfotransferase ? was successfully expressed in engineering bacteria which contains AST-? gene via IPTG induction.The molecular weight of this protein is 34.08 kDa.Soluble fusion Aryl Sulfotransferase ?was obtained after low temperature culturing.The soluble fraction was purified by Ni-NTA affinity chromatography.The specific activity of the purified enzyme was 40.95 U/mg,purification fold was 3.38.Two pairs of specific primers were designed to amplify ?4,5 and ?4,5?20 gene.The DNA fragment had 100%identity with glycosyl hydrolase family 88 from Pedobacter heparinus DSM 2366 by BLAST alignment in NCBI.The gene fragment of ?4,5 and ?4,5?20 encode 402 and 382 amino acids with a molecular weight of 46 kDa and 44 kDa.The ?4,5 and ?4,5?20 fragments was successfully cloned into expression vector pMAL-c2X.The resultant plasmid pMAL-c2X-A4,5 and pMAL-c2X-?4,5?20 was transformed into E.coli TB1.Fusion protein MBP-?4,5 and MBP-?4,5A20 was expressed with IPTG inducing.After purifying by amylose resin affinity chromatography and analysis of UN-Scan-it gel 6.1,the expressed protein was the target protein with a molecular weight of 76 kDa.The specific activity of the purified enzyme was 325.47 U/mg,purification fold was 8.09.Heparinase ?,Heparinase ?,and Heparinase ? were expressed and purified.And then these enzymes were utilized to depolymerize heparosan,heparin sulfate,and heparin soldium.Finally,the recover effect of ?4,5 unsaturated glycuronidase on unsaturated ?4,5 uronic acid of these depolymerization products was evaluated.