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

Study on High level Expression and Purification of Nm23 H1/NDPK A in Escherichia coli

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．

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.