Cloning And Expression Of Heparan Sulfate 2-O-Sulfotransferase From Mouse Liver

Heparin and heparan sulfate have been widely used as anticoagulant drugs in clinical application, and may play an important role in anti-cancer and anti-virus. Pharmaceutical heparin is isolated from porcine, however, heparin, contaminated by oversulfated chondroitin sulfate, has a potential risk and unstable resources. Traditional chemical synthesis of heparin is costly and complicated, although it has successfully manufactured heparin. Therefore, synthesis of heparin and its derivatives from microorganism becomes a hot topic. Aiming to synthesize heparin by enzymatic method, heparan sulfate 2-O-sulfotransferase (Hs2st) was cloned, expressed and purified. Optimization of the expression conditions was also studied.An Hs2st fragment was cloned from the total RNA of mouse liver via RT-PCR technology. Then the fragment was inserted into the cloning vector pMD-19-T. After PCR identification, sequencing and Blast analysis of the resultant plasmid pMD-19T-Hs2st were carried out. The Hs2st fragment on the vector was proved 100% similar to the target gene, and it encoded 356 amino acids with a molecular weight of 33 kDa.The Hs2st fragment was successfully cloned into expression vector pMAL-c2X. The resultant plasmid pMAL-c2X-Hs2st was transformed into E. coli TB1. Fusion protein MBP-Hs2st was expressed with IPTG inducing. After purifed by amylose resin affinity chromatography and analysis of UN-Scan-it gel 6.1, it proved that the expressed protein was the target protein with a molecular weight of 75 kDa. And the yield of one-step purification was 47%.In order to achieve high production of Hs2st, the recombinant plasmid pMAL-c2X-Hs2st was constructed so that Hs2st was fused to the C-terminal of maltose binding protein (MBP) to realize its soluble expression in E. coli. Based on the constructed vector, optimization of the cultivation conditions using LB medium was carried out, including the host bacteria, the induction time, the IPTG concentration, the additions of glucose and ethanol. The results showed the optimal cultivation conditions were as follows:E. coli TB1 as the host,0.4 mmol/L IPTG inducing when OD6oo reached 0.6; the additional 0.5-1.5g/L glucose and 1%-3% ethanol.