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Thermus caldophilus GK24 DNA Polymerase를 이용한 Polymerase Chain Reaction의 최적조건
권석태 성균관대학교 생명과학자원연구소 1994 生命資源科學硏究 Vol.1 No.1
A thermostable DNA polymerase was used in an in vitro amplification procedure, the polymerase chain reaction(PCR). Thermus caldophilus GK24(Tca) DNA polymerase greatly simplifies the procedure and, by enabling the amplificatioin reaction to be performed at higher temperature, significantly improves the specificity, yield and sensitivity. The optimal conditions for PCR were investigated in terms of pH range, MgCl_2, KCl and DTT concentration using the purified Tca DNA polymerase. The optimal pH for DNA amplification was found to be between 8.6, and 9.2 in 50mM Tris-HCl buffer. The optimal concentrations of MgCl_2 and KCl for PCR were 1-1.5mM and 10-50mM, respectively. The concentration of DTT above 0.5mM was sufficient to amplify template DNA by PCR. These conditions can be used to amplify a wide range of target sequences with excellent specificity.
Thermus aquaticus YT - 1 의 내열성 프로테아제 aqualysin Ⅰ 의 구조와 특징
권석태 한국농화학회 1988 Applied Biological Chemistry (Appl Biol Chem) Vol.31 No.3
Aqualysin I is an alkaline serine protease which is secretet into the culture medium by Thermos aquaticus YT-1, an extreme thermophile. Aqualysin I was purified, and its partial amino acid sequence was determined. The gene encoding aqualysin I was cloned into E. coli using synthetic oligodeoxyribonucleotides as hybridization probes. The nucleotide sequence of the cloned DNA was determined. The primary structure of aqualysin I, deduced from the nucleotide sequence, agreed with the determid amino acid sequences, including the NH₂- and COOH terminal sequence of the tryptides derived from aqualysin I. Aqualysin I comprised 281 amino acid residues and its molecular mass was determined to 1)e 28350. On alignment of the whole amino acid sequence, aqualysin I showed high sequence homology with the subtilisin type serine protease, and 43% identity with proteinase K, 37-30% with subtilisins and 34% with thermitase. Extremely high sequence identity was observed in the regions containing the active-site residues, corresponding to Asp32, His64 and Ser221 of subtilisin BPN'. Aqualysin I contains two disulfide bonds, Cys67-Cys99 and Cys163-Cysl94, and these disulfide bonds seem to contribute to the heat stability of the enzyme. The determined positions of the twe disulfide bonds of aqualysin I agreed with those predicted previously on the basis of computer graphics of the crystallographic data for subtilisin BPN'. Therefore, these findings sugests that the three-dimensional structure of aqualysin I is similar to that of subtilisin BPN' Aqualysin I is produced as a lage precursor, which contains NH₂- and COOH- terminal portions besides the mature protease sequence.
Thermus flavus AT-62 균주의 내열성 DNA Ligase 유전자의 Cloning 및 발현
권석태 성균관대학교 생명과학자원연구소 1994 生命資源科學硏究 Vol.1 No.1
The DNA fragment encoding DNA ligase from Thermus flovus AT-62 was cloned into Escherichia coli using polymerase chain reaction(PCR) method. Under tac promoter control, Thermus flavus AT-62(Tfl) DNA ligase was expressed in E colt. Tfl DNA ligase in E. coli was purified by simple methods like heat treating, DEAESephacel and Cellulose phosphate column chromatography. The purified enzyme had a molecular mass of about 77kDa, estimated by sodium dodecyl sulfate-polyacrylamide-gel electrophoresis. The purified Tfl DNA ligase was capable of catalyzing blunt-end ligation at 65℃.
대장균에서 Thermus caldophilus GK24의 DNA Polymerase유전자의 고발현
권석태,김현규,김중수,이대실 성균관대학교 생명과학자원연구소 1997 生命資源科學硏究 Vol.4 No.2
Thermus caldophilus GK24 (Tca) DNA polymerase is highly useful enzyme for amplifying DNA fragments by polymerase chain reaction (PCR). A plasmid, pTCA, is expression vector for Tca DNA polymerase gene in Escherichia coli under the control of tac promoter and its expression level is low. For the high expression, the DNA sequence coding for NH_2-amino acid sequence of Tca DNA polymerase was changed by PCR, and then an expression vector pTCAM was constructed. The activity of Tca DNA polymerase in E. coli harboring for pTCAM has enhanced nearly 6-fold/ml than that for E. coli harboring pTCA.