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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．
The DNA polymerase mutant (D222A / D327A) with deletion of phage RB69 exonuclease activity was expressed in E. coli cells in an amount of 69% of the total cellular protein. After the expression DEAE-Sepharose FastFlow, Source 30Q and HTP three-step separation and purification, purity up to 99%. The enzymatic kinetic constants (Km and Kcat) of the enzyme using five dNTPs as substrates were determined. The results showed that the enzyme’s ability to utilize dUTP was similar to that of dTTP. Km (dTTP) and Km (dUTP ) Were higher than the other three kinds of deoxynucleotides Km (dATP, dCTP, dGTP), speculated that the difference in Km value mainly from the T / U base itself, but not all of the GC base pairing and AT base Pairing between the hydrogen bond strength of the difference between the strength of the decision.