Construction of single ammo acid substitution mutants of cloned Bacillus stearothermophilus DNA polymerase I which lack 5'→3' exonuclease activity

摘要

Two individual ami no acid substitutions were engineered at a selected site in the 5' - 3' exonuclease domain of the cloned Bacillus stedt'othermophiius 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 constilutively 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.