噬菌体RB69 DNA聚合酶的表达、分离纯化和其聚合反应的稳态动力学研究

噬菌体ＲＢ６９外切酶活性缺失的ＤＮＡ 聚合酶突变体（Ｄ２２２Ａ／Ｄ３２７Ａ）在大肠杆菌细胞中表达，表达量达细胞蛋白总量的６９％ ．表达后经ＤＥＡＥ－Ｓｅｐｈａｒｏｓｅ ＦａｓｔＦｌｏｗ ， Ｓｏｕｒｃｅ ３０Ｑ 和ＨＴＰ三步分离纯化，纯度可达９９％ 以上．随后测定了该酶利用５种ｄＮＴＰ为底物进行聚合反应的酶促动力学常数（Ｋｍ 和Ｋｃａｔ），结果表明该酶利用ｄＵＴＰ的能力与利用ｄＴＴＰ的能力相近，Ｋｍ （ｄＴＴＰ）和Ｋｍ（ｄＵＴＰ）均较高于其它３种脱氧核苷酸的Ｋｍ （ｄＡＴＰ， ｄＣＴＰ， ｄＧＴＰ），推测其Ｋｍ 值的差异主要来源于Ｔ／Ｕ 碱基本身，而并非全部由ＧＣ碱基配对与ＡＴ碱基配对之间的氢键作用力的强弱差别所决定．

Expression and Purification of Bacteriophage RB69 DNA Polymerase and Characterization of its Kinetic Parameters for dNTP

The exo activity deficient polymerase mutant (D222A/D327)from bacteriophage RB69 was expressed in E. coli and purified to nearly homogenous. The kinetic parameters( K m & k cat ) for incorporation of 5 kinds of dNTP by this enzyme were determined. RB69 DNA polymerase could use dUTP for incorporation as well as dTTP. The fact that only K m (dTTP) & K m (dUTP), not K m (dATP) were higher than other nucleotides indicates that the K m difference comes from T/U bases themselves, not from the weaker strength of A/T(U) compared with G/C base pairing.