重组角蛋白单体外源表达、纯化及鉴定

Exogenous expression,purification and identification of recombinant keratin monomer

为了制备具有生物活性的可溶性重组角蛋白单体,构建能够外源表达重组角蛋白的重组菌株BL21(DE3)-pET32a(+)-Keratin。使用最适诱导条件(0.5 mmol/L IPTG,18℃培养15 h)诱导重组角蛋白基因表达,经Ni柱亲和层析法纯化得到蛋白产量约为40 mg/L,浓度约为2 mg/mL,Western Blot证实此蛋白是重组角蛋白。S.maltophilia DHHJ在重组角蛋白M9培养基中能够正常生长且不会产生絮状沉淀,与化学法制备的角蛋白相比,重组角蛋白溶解性更高。这更有利于S.maltophilia DHHJ降解角蛋白分子过程的研究。

In order to prepare soluble keratin monomer with biological activity,a recombinant strain BL21(DE3)-pET32a(+)-keratin capable of exogenously expressing recombinant keratin was constructed.Optimal induction conditions(0.5 mmol/L IPTG,cultured at 18℃for 15 h)were used to induce the expression of recombinant keratin genes.The yield of the protein purified by Ni column affinity chromatography was about 40 mg/L and the concentration was about 2 mg/mL.Western Blot confirmed that this protein was recombinant keratin.S.maltophilia DHHJ could grow normally in recombinant keratin M9 medium without flocculation.The solubility of recombinant keratin was more than that of the chemically prepared keratin.This was more conducive to studying the molecular process of keratin degradation by S.maltophilia DHHJ.