定点突变人α_(99)、β_(82)位的血红蛋白及在大肠杆菌中的表达

Site-directed Mutagenesis of Human α_(99) 、β_(82) Hemoglobin and its Expression in Escherichia coli

模拟分析发现血红蛋白两条α珠蛋白链上 99位的 Lys突变为 Cys后 ,它们之间可以形成二硫键 ,两条β珠蛋白链上 82位的 Lys突变为 Cys后可以增加血红蛋白四聚体间氢键的作用 ,分别起到稳定四聚体的作用 .利用寡核苷酸介导的定点突变技术将α99、β82 位的 Lys突变为 Cys.将突变后的血红蛋白插入 p BV2 2 0载体 ,在大肠杆菌中获得了高效表达 ,其表达产物达细菌总蛋白的2 0 %左右 ,并经

In order to increase the stability of hemoglobin tetramer,with computer modeling predication two α 99 lysines of α globin mutated to cysteines can form a disulfide bond,and two β 82 lysines of β globin mutated to cysteines can form a strong hydrogen bond so that they can stabilize the hemoglobin tetramer.With site directed mutagenesis technique,α 99 and β 82 lysines were mutated to cysteines.Then mutant α and β genes were introduced into the expression vector pBV220 and exhibited a high expression level after induction.The expression product was as inclusion body and the yield reached about 20% of total bacterial protein.The products were confirmed by Western blotting.