突变型(α99,β82位Lys-Cys)人重组血红蛋白的纯化

Purification of α99,β82 Recombinant Human Mutant (α99,β82 Lys-Cys)Hemoglobin

将基因突变型 (α99,β82位Lys Cys)血红蛋白在 pBV2 2 0载体中诱导表达 ,表达产物达细菌总蛋白的 2 0 %左右。该表达产物以包涵体形式存在 ,包涵体经洗涤后 ,用 8mol/L尿素溶解 ,先用Q SepharoseFastFlow阴离子交换纯化 ,再经Sephacryl 10 0凝胶过滤纯化。纯化产物经复性 。

Recombinant human α99,β82 mutant genes were introduced into the expression vector pBV220 The expression product reached about 20% of total bacterial protein They were expressed as inclusion bodies and must be dissolved in denatured condition and then renatured After the washing by 4 mol/L urea,purification was performed on a column of Q sepharose Fast Flow Further purification was performed on a column of Sephacryl 100 gel filtration which resulted in the elimination of most of the contaminated proteins Then a refolding reaction was established in the presence of glutathione oxide shuffling system The purified and renatured product had biological activity