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Nm23-H1/NDPK-A基因在大肠杆菌中的高效表达及产物纯化的研究
引用本文:侯宇,赵利淦,张美英,何丽容,边六交,张玲,杨瑞仪,林剑. Nm23-H1/NDPK-A基因在大肠杆菌中的高效表达及产物纯化的研究[J]. 中国生物化学与分子生物学报, 1998, 14(6): 655-660
作者姓名:侯宇  赵利淦  张美英  何丽容  边六交  张玲  杨瑞仪  林剑
作者单位:暨南大学生物工程研究所
基金项目:国务院侨办重点学科科研基金
摘    要:利用聚合酶链反应(PCR)技术扩增人二磷酸核苷激酶A亚基(NDPK-A)基因,即nm23-H1/NDPK-K基因的编码序列,经序列分析后,定向克隆于表达质粒载体pBV220,在大肠杆菌DH5α中高效表达出重组人NDPK-A.表达产物为可溶性的非融合蛋白,占菌体总蛋白42%.斑点ELISA法鉴定表明表达产物与NDPK-A标准抗血清呈阳性反应.以DEAE纤维素弱阴离子交换层析、CibacronBlue染料亲和层析结合高效液相排阻色谱技术纯化rNDPK-A,得纯度为96.7%的目标蛋白.以反相高效液相色谱法进行酶活性分析,表明纯化的rNDPK-A能催化ATP+UDP=ADP+UTP的反应,比活性为800U/mg蛋白.

关 键 词:Nm23-H1/NDPK-A  基因表达  分离纯化  酶活性检测  
收稿时间:1998-12-20

Study on High level Expression and Purification of Nm23 H1/NDPK A in Escherichia coli
Hou Yu Zhao Li Gan Zhang Mei Ying He Li Rong Bian Liu Jiao Zhang Lin Yang Rui Yi Lin Jian. Study on High level Expression and Purification of Nm23 H1/NDPK A in Escherichia coli[J]. Chinese Journal of Biochemistry and Molecular Biology, 1998, 14(6): 655-660
Authors:Hou Yu Zhao Li Gan Zhang Mei Ying He Li Rong Bian Liu Jiao Zhang Lin Yang Rui Yi Lin Jian
Affiliation:(Bioengineering Institute of Jinan University,Guangzhou 510632
Abstract:Polymerase chain reaction (PCR) technique was used to amplify the whole coding sequence of Nm23 H1 gene while both EcoRⅠ and BamHⅠ recognition sites had been introduced to its 5′ and 3′ ends respectively.After sequcncing,the amplified gene was inserted into the same restriction enzyme sites downstream of the P RP L promoter of the expression vector pBV220 to form the recombinant expression plasmid pBVNMH1. E.coli DH5α strain transformed by the plasmid pBVNMH1 over expressed the product of the gene when it was induced at 42℃.Inclusion body of the rNDPK A did not form,and the amount of expressed product was 42% of the total bacterial proteins.A feasible method for obtaining the purified rNDPK A was established by using DEAE cellulose anion exchange chromatography,Cibacron Blue affinity chromatography and size exclusive high performace liquid chromatography (SE HPLC) techniques.The purity of purified rNDPK A was over 96 7%.For testing the kinase activity of the purified rNDPK A,reverse HPLC based assay was used,in which the substances (ATP and UDP) and products (ADP and UTP) of the enzyme reaction were seperated and UTP was quantitated.The specific activity of the purified rNDPK A is 800 U/mg protein.
Keywords:Nm23 H1/NDPK A  Expression  Purification  Assay  
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