摘要
以大肠杆菌K12基因组DNA为模板,PCR扩增得到NAD+依赖型苹果酸酶(NAD-ME)的全长基因,并克隆到载体pET24b(+)中,得到表达质粒pET24b-ME。在IPTG诱导下,携带pET24b-ME的大肠杆菌BL21(DE3)高效表达分子量约为65 kDa的可溶性蛋白。重组NAD-ME经镍亲和层析纯化,比活达到100 U/mg以上。以上结果为深入研究苹果酸酶生物催化特性及其与辅酶的相互作用奠定了基础。
The gene coding NAD-linked malic enzyme （NAD-ME） from E. coli K12 was PCR amplified using genomic DNA as template, and cloned into vector pET24b（ ＋ ） to give an expression vector pET24b-ME. Upon IPTG induction, soluble NAD-ME was over-produced by E. coli BL21 （DE3） harboring the expression construct. Recombinant NAD-ME purified by Ni-NTA affinity chromatography showed a single band about 65 kDa on SDS-PAGE gel, and the specific activity was over 100 U per milligram protein. These results established a platform to further study biocatalytic properties of NAD-ME and interaction between cofactor and the enzyme.
出处
《生物加工过程》
CAS
CSCD
2006年第1期35-38,49,共5页
Chinese Journal of Bioprocess Engineering
基金
国家自然科学基金(编号:20472084)
关键词
苹果酸酶
表达
蛋白质纯化
辅酶
malic enzyme
expression
protein purification
coenzyme
