重组N-乙酰鸟氨酸脱乙酰基酶的高效表达与纯化  

The overexpression and purification of recombinant protein acetylornithine deacetylase

作　　者：陈悦[1] 李环[1] 姚忠[1] 韦萍[1] 

机构地区：[1]南京工业大学制药与生命科学学院,南京210009

出　　处：《生物加工过程》2006年第4期56-60,共5页Chinese Journal of Bioprocess Engineering

基　　金：国家973项目基金资助(编号2003CB7160004)

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200W，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43000。纯化倍数为139倍，回收率为11．1％。pET-22b（ ＋ ） was used as the expressing vector, the genetic engineering strain BL21-pET-22b（ ＋ ）- argE producing acetylomithine 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 colunm, 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%.

关 键 词：N-乙酰鸟氨酸脱乙酰基酶 基因工程菌 亲和层析 纯化 

分 类 号：Q55[生物学—生物化学]