血小板生成素突变体融合蛋白表达及纯化研究

Studies on expression and purification of human thrombopoietin mutant fusion protein

目的：用大肠杆菌融合蛋白表达载体pMAL—c2构建人血小板生成素突变体融合蛋白表达质粒pMAL-TPOM。方法：采用基因重组和表达、SD-聚丙烯酰胺凝胶电泳和蛋白纯化技术。结果：SDS-PAGE分析表明，IPTG诱导5h后大肠杆菌JM109表达到最高水平，约占大肠杆菌菌体总体蛋白的36．6％。除了大肠杆菌RR1不表达以外，大肠杆菌DH5a、H101均有较高水平的表达，表达产物血小板生成素突变体融合蛋白经SephacyIS-200和DEAE-SepharoseFF层析纯化后，蛋白纯度约为87．6％。结论：人血小板生成素突变体融合蛋白在大肠杆菌JM109、DH5a和H101中均获得高效表达。

Objective: The plasmid pMAl-TPOM for expression human thrombopoietin mutant fusion protein (MBP-TPOM) was constructed by using and E. coli fusion protein vector pMAL-c2. MBP-TPOM expressed by E. coli was purified by means of chromatography,which will provide sufficient material for biological activity study. Methods: Gene recombination and expression, SDS-PAGE and protein purification techniques were employed. Results:SDS-PAGE analysis indicated that MBP-TPOM expressed by E. coli JM109 strain enhanced the highest level after IPTG induced for 5 hours and accounted up to 36. 6 % of total E. colt protein. There was higher level expression in E. colt DH5a and Hb101 except RR1 strain. After purification of MBP -TPOM by the Sephacryl S-200 chromatoraphy and DEAE-Sepharose FF chromatography, the purity of MBP- TPOM was about 87. 6 %. Conclusion: MBP - TPOM was expressed in E. coli JM109, DH5a, Hb101 strains in high level. After purification of the protein,the purity was about 87. 6%.