Guo J, Wang H, Gu J
Institute of Hematology, CAMS and PUMC, Tianjin 300020.
Zhonghua Xue Ye Xue Za Zhi. 1998 Mar;19(3):146-8.
To express the recombinant hirudin variant 1 (rHV1) with biological activity in prokaryotic cells and then isolate and purify the expressed products.
The hirudin variant 1 gene in plasmid vector pBV220 was expressed in E. coli strain DH5 alpha. The biological activity of rHV1 was determined with chromogenic substrate method. The expressed product was purified by ultrafiltration, DEAE-Sephadex A-50 filtration and thrombin-Sepharose 4B affinity chromatography.
The expressed rHV1 accounted for approximately 16.9% of E. coli cell proteins with an activity of 20-30 ATU (antithrombin unit) per milliliter culture. The purified rHV1 showed a homogeneous band on SDS-PAGE.
This is the first report in China on successful expression of hirudin variant 1 gene in E. coli and purification of the expressed rHV1.
在原核细胞中表达具有生物活性的重组水蛭素变体1（rHV1），并对表达产物进行分离纯化。
将质粒载体pBV220中的水蛭素变体1基因在大肠杆菌DH5α菌株中表达。采用发色底物法测定rHV1的生物活性。通过超滤、DEAE-葡聚糖A-50过滤和凝血酶-琼脂糖4B亲和层析对表达产物进行纯化。
表达的rHV1约占大肠杆菌细胞蛋白的16.9%，每毫升培养物的活性为20 - 30抗凝血酶单位（ATU）。纯化后的rHV1在SDS-PAGE上呈现单一泳道。
这是国内首次关于水蛭素变体1基因在大肠杆菌中成功表达及对表达的rHV1进行纯化的报道。
