利用大腸桿菌胞外分泌蛋白質融合表現人類玻尿酸合成酶

Abstract

本研究利用大腸桿菌系統表現人類玻尿酸合成酶2(human_HAS2)，主要選擇其80-368的基因片段，此段為細胞質區的部位。建構在載體pET22b(+)作表現但無法獲得可溶性重組蛋白質，多在細胞殘體上。 為得到可溶性的重組蛋白質，以兩種策略進行，其一利用蛋白質復性的方法但仍無法得到具活性的重組蛋白質，其二為利用OsmY此蛋白質，藉由其本身蛋白質分泌至胞外的特性，跟HAS2結合建構於pET28a(+)作表現，可在可溶區得到重組蛋白質。經Ni2+ 管柱及DEAE 管柱純化可得到均質度極高的重組蛋白質。 在其合成的玻尿酸寡糖方面，目前僅偵測到其能合成雙醣，推測是因其在膜區的部位具有協助玻尿酸寡糖合成長鏈的功用。未來可利用此重組蛋白質進行反應機制的探討與抑制劑的發展，有助腫瘤細胞行為的研究。

This study is about the human hyaluronansynthase2 (human_HAS2) expressed by E. coil BL21(DE3) strain. We chose only cytoplasmic region of full human_HAS2 gene, it’s about 290 amino acids (80-368). This gene was constructed in pET22b(+) and expressed by E. coil. The recombinant protein could not get from cytosol but found in cell debris. To overcome this problem, we designed two methods. First, we tried to refold the recombinant protein, expected the recombinant protein to return to its native structure. But we could not recover the activity. Second, we chose the OsmY protein which could be secreted to extracellular by E. coil BL21 (DE3) strain as a carrier protein. We constructed HAS2 and OsmY as a fusion protein in pET28a(+) and found that protein in cytosol. The recombinant protein was purified by Ni2+ column and DEAE column. The homogeneity of purified protein was about 90%. We only could collect disaccharides of hyaluronan oligosaccharides which were synthesized by recombinant protein. We supposed there were still some functions of trans-membrane domain of full human_HAS2 could help to synthesize longer hyaluronan oligosaccharides. This recombinant protein can apply to study the mechanism and inhibitor in the future.