肝素硫酸转移酶优化表达及其在动物源肝素硫酸化中的应用

Optimized expression of heparin sulfotransferases and their application in sulfation of animal derived heparin

摘要:

肝素是一种重要的凝血药物，目前主要依赖于动物小肠粘膜的提取。动物源肝素含有的抗凝血活性五糖单位GlcNS6S-GlcA-GlcNS6S3S-Ido2S-GlcNS6S少，抗凝血活性低下。文中提出并验证了一种基于酶法催化动物源肝素，提高其硫酸化程度和抗凝血活性的方法。通过比较3种硫酸转移酶肝素-2-硫酸转移酶 (Heparan sulfate-2-O-sulfotransferase，HS2ST)、肝素-6-硫酸转移酶 (Heparan sulfate-6-O- sulfotransferase，HS6ST)、肝素-3-硫酸转移酶 (Heparan sulfate-3-O-sulfotransferase，HS3ST) 在重组大肠杆菌及重组毕赤酵母中表达，确定了毕赤酵母作为3种硫酸转移酶的表达宿主；进一步通过N端融合麦芽糖融合蛋白MBP和硫氧还蛋白TrxA，HS2ST和HS6ST的酶表达水平分别提高至 (839±14) U/L和 (792±23) U/L。通过3种硫酸转移酶HS2ST、HS6ST和HS3ST共同催化动物源肝素，其抗凝血活性由 (76±2) IU/mg提高至 (189±17) IU/mg。

Abstract:

Heparin is a very important anticoagulant drug. Currently, heparin is mainly extracted from porcine mucosa. However, animal-derived heparin shows low anticoagulant activity due to the low proportion of the anticoagulant active unit, the GlcNS6S-GlcA-GlcNS6S3S-Ido2S-GlcNS6S pentasaccharide. In this study we proposed an enzymatic strategy to sulfate the animal-sourced heparin to increase the proportion of anticoagulant pentasaccharide and the anticoagulant activity. First, three sulfotransferases HS2ST, HS6ST, and HS3ST were expressed tentatively in Escherichia coli and Pichia pastoris. After measuring the sulfotransferase activity, we confirmed P. pastoris GS115 is the better host for sulfotransferases production. Then, the maltose binding protein (MBP) and thioredoxin (TrxA) were fused separately to the N-terminal of sulfotransferases to increase enzyme solubility. As a result, the yields of HS2ST and HS6ST were increased to (839±14) U/L and (792±23) U/L, respectively. Subsequent sulfation of the animal-sourced heparin with the recombinant HS2ST, HS6ST and HS3ST increased the anticoagulant activity from (76±2) IU/mg to (189±17) IU/mg.