重组N-乙酰鸟氨酸脱乙酰基酶的表达、纯化和复性研究

Studies on the Expression, Purification and Renaturation of Recombinant N_acety_L_ornithine Deacetylase

摘要:

报道重组N-乙酰鸟氨酸脱乙酰基酶（NAOase）的研究进展。重组NAOase由大肠杆菌argE基因编码，在重组菌BL21(DE3)-pET22b -argE中的表达量为32.5％，大多以无活性的包涵体存在。低温诱导可增大有活性的可溶表达部分的比例。可溶性NAOase经Ni-NTA凝胶亲和纯化后得到SDS-PAGE电泳纯的酶，比酶活为1193.2u/mg蛋白。诱导条件影响整菌蛋白的成分及比例。37℃诱导生成的包涵体经尿素梯度洗涤后纯度较22℃高。低的蛋白浓度和合适的氧化还原体系是影响复性的关键因素。稀释法和透析法皆可使包涵体部分复性。在合适的条件下以稀释法复性时，约有17.78%包涵体可顺利复活。包涵体经尿素洗涤、溶解、Ni-NTA凝胶柱亲和纯化后，获得了高纯度的NAOase。

Abstract:

The argE gene from Escherichia coli coding for N-acety-L-ornithine deacetylase（NAOase）, the key enzyme involved in the L-arginine biosynthesis, had been cloned in pET22b and transformed into BL21(DE3). With 32.5% expression level in the optimal fermentation medium at 37℃, most NAOase was expressed as inclusion bodies. The soluble and active proportion could be slightly increased when expressed at low temperature. The specific activity of soluble NAOase purified by Ni-NTA resin chromatography was 1193.2u/mg. The species and proportions of whole cell proteins varied with induction conditions. The inclusion bodies expressed at 37℃ was more pure than 22℃after gradient wash with urea. Inclusion bodies could be partly refolding and reactivated by dilution and dialysis. Low protein concentration and suitable rate of oxidant/reducing agents were important to renaturation. In the optimal conditions 17.78% of Urea-denatured NAOase could be refolding and reactivated by dilution. The purified fusion protein was obtained after wash, solubilization and Ni-NTA resin affinity chromatography purification of inclusion bodies.