Expression of human hyaluronan synthase by fusing E.coli. secretory protein
|關鍵字:||人類玻尿酸合成酶;胞外分泌蛋白質;human hyaluronan synthase;HAS2;OSMY|
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。
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.
|Appears in Collections:||Thesis|