Cao W, Lu J, Welch S G, Williams R A, Barany F
Department of Microbiology, Hearst Microbiology Research Center, Cornell University Medical College and Strang Cancer Prevention Center, New York, NY 10021, USA.
Biochem J. 1998 Jul 15;333 ( Pt 2)(Pt 2):425-31. doi: 10.1042/bj3330425.
Two TaqI endonuclease (hereafter referred to as TaqI) isoschizomer genes, tsp32IR from Thermus species SM32 of Azores and tfiTok6A1I from T. filiformis Tok6A1 of New Zealand, were cloned in Escherichia coli. The overexpressed enzymes were partly purified and their thermostability was determined. In the medium-salt buffer, Tsp32IR, TfiTok6A1I and one previously cloned TaqI isoschizomer (TthHB8I) were more thermostable than TaqI. Tsp32IR remained partly active up to 90 degreesC in the low-salt buffer. Six amino acid residues that are identical in the three high thermostability isoschizomers (Tsp32IR, TfiTok6A1I and TthHB8I) but differ in TaqI might provide added rigidity for thermostabilization. These include four proline residues located in or near loop regions, and one alanine and one arginine located at helix regions in the predicted TaqI endonuclease secondary structure. The possible role of these residues in thermostabilization was evaluated by mutagenizing the TaqI enzyme. Mutants generated at these six positions were less thermostable than wild-type TaqI. The results suggest that the surrounding sequence or structural context might be as important as the mutation itself.
克隆了两个TaqI核酸内切酶（以下简称TaqI）同裂酶基因，即来自亚速尔群岛嗜热栖热菌属SM32的tsp32IR和来自新西兰丝状嗜热栖热菌Tok6A1的tfiTok6A1I，并将其在大肠杆菌中进行表达。对过量表达的酶进行了部分纯化，并测定了它们的热稳定性。在中盐缓冲液中，Tsp32IR、TfiTok6A1I和一个先前克隆的TaqI同裂酶（TthHB8I）比TaqI更耐热。在低盐缓冲液中，Tsp32IR在高达90℃时仍保持部分活性。在三种高热稳定性同裂酶（Tsp32IR、TfiTok6A1I和TthHB8I）中相同但在TaqI中不同的六个氨基酸残基可能为热稳定性提供额外的刚性。这些残基包括位于环区或其附近的四个脯氨酸残基，以及预测的TaqI核酸内切酶二级结构中位于螺旋区的一个丙氨酸和一个精氨酸。通过对TaqI酶进行诱变评估了这些残基在热稳定性中的可能作用。在这六个位置产生的突变体比野生型TaqI的热稳定性更低。结果表明，周围的序列或结构环境可能与突变本身同样重要。
