Kim J S, Koh S, Kim J J, Kwon S T, Lee D S
Molecular Glycobiology Research Unit, Korea Research Institute of Bioscience and Biotechnology, KIST, Taejon, Korea.
Mol Cells. 1998 Apr 30;8(2):157-61.
A gene, top encoding Thermus thermophilus HB27 (Top) DNA polymerase, was cloned in E. coli and its nucleotide sequence was determined. Based on its deduced amino acid sequence, Top DNA polymerase is a 93.8 kDa protein comprising 834 amino acid residues. Top DNA polymerase showed high amino acid homology with those of other DNA polymerases from the Thermus sp., for example, 87.3% identity with Taq DNA polymerase. Codon usage in the top gene was similar to those of the proteins from other Thermus strains. The G + C content in the third position of the codons was as high as 93%. The top gene under the control of the tac promoter was expressed in E. coli [plasmid pTOP9]. DNA amplification using the recombinant Top DNA polymerase performed the same as other thermostable DNA polymerases from Thermus strains. The optimum temperature for its reaction was 76 degrees C. An interesting observation was that the recombinant Top DNA polymerase was slowly cleaved into two fragments of about 60 kDa and 35 kDa at 4 degrees C and -20 degrees C. The larger fragment possessed polymerase activity like the Klenow fragment of E. coli DNA polymerase I. To prevent the cleavage of the Top DNA polymerase, a variety of protecting agents were examined. Among those examined, (NH4)2SO4 (100 mM) solution demonstrated an outstanding ability to block its cleavage for a prolonged period.
编码嗜热栖热菌HB27（Top）DNA聚合酶的基因在大肠杆菌中克隆，并测定了其核苷酸序列。根据推导的氨基酸序列，Top DNA聚合酶是一种93.8 kDa的蛋白质，由834个氨基酸残基组成。Top DNA聚合酶与来自栖热菌属的其他DNA聚合酶具有高度的氨基酸同源性，例如，与Taq DNA聚合酶的同一性为87.3%。top基因的密码子使用与其他栖热菌菌株的蛋白质相似。密码子第三位的G + C含量高达93%。在tac启动子控制下的top基因在大肠杆菌[质粒pTOP9]中表达。使用重组Top DNA聚合酶进行的DNA扩增与来自栖热菌菌株的其他耐热DNA聚合酶相同。其反应的最佳温度为76℃。一个有趣的观察结果是，重组Top DNA聚合酶在4℃和-20℃下会缓慢切割成两个片段，分别约为60 kDa和35 kDa。较大的片段具有类似于大肠杆菌DNA聚合酶I的Klenow片段的聚合酶活性。为了防止Top DNA聚合酶的切割，研究了多种保护剂。在所研究的保护剂中，（NH4）2SO4（100 mM）溶液表现出在长时间内阻止其切割的出色能力。
