【摘要】 利用质粒pET-22b(+)为表达载体,成功构建了高效表达N-乙酰鸟氨酸脱乙酰基酶的基因工程菌BL21-pET-22b(+)-argE。研究了该菌的最适超声破壁条件及硫酸铵分级沉淀的最适范围,并以金属螯合亲和层析法纯化含有6-His-Tag的目的蛋白。结果表明:26℃诱导表达的菌体,最佳超声破碎条件为功率200 W,超声时间为15min;目的蛋白主要存在于40%~50%硫酸铵沉淀中。通过Ni-NTA亲和层析柱纯化目的蛋白,可以达到SDS-PAGE电泳纯,并显示单亚基相对分子质量为43 000。纯化倍数为139倍,回收率为11.1%。更多还原

【Abstract】 pET-22b（+） was used as the expressing vector,the genetic engineering strain BL21-pET-22b（+）-argE producing acetylornithine deacetylase was successfully constructed.In this report,the optimal condition of sonication and the suitable saturation of(NH₄)₂SO₄ precipitation were studied.And the recombinant protein with 6-His-Tag was purified by the immobilized metal affinity chromatography.The results showed,when the strain was induced at 26 ℃,the optimal condition of sonication was that the sample was sonicated under 200 W for 15 min.Most of the recombinant protein was present in 40%-50% saturation of(NH₄)₂SO₄ precipitation.NAO was purified by the Ni-NTA immobilized metal affinity column,purified enzyme presented as a single protein band on SDS-PAGE with a molecular weight of 4.3×10⁴.The Purification fold of NAO was 139,the yield was 11.1%.更多还原