Riggs M G, Tudor S, Sivaram M, McDonough S H
Gen-Probe Incorporated, San Diego, CA 92121, USA.
Biochim Biophys Acta. 1996 Jun 7;1307(2):178-86. doi: 10.1016/0167-4781(96)00051-6.
Two individual amino acid substitutions were engineered at a selected site in the 5' --> 3' exonuclease domain of the cloned Bacillus stearothermophilus DNA polymerase I gene. These mutations resulted in the expression of enzymes lacking the 5' --> 3' exonuclease activity while maintaining normal polymerizing activity. The mutated and non-mutated enzymes were each constitutively expressed in an Escherichia coli host without the use of an exogenous or inducible promoter, and the mutated enzymes were demonstrated to be equivalent to the subtilisin large fragment of the native holoenzyme in sequencing reactions.
在克隆的嗜热脂肪芽孢杆菌DNA聚合酶I基因的5'→3'核酸外切酶结构域的选定位点进行了两个单独的氨基酸替换。这些突变导致了缺乏5'→3'核酸外切酶活性但保持正常聚合活性的酶的表达。突变型和非突变型酶均在大肠杆菌宿主中组成型表达，无需使用外源或诱导型启动子，并且在测序反应中证明突变型酶与天然全酶的枯草杆菌蛋白酶大片段相当。
