• Korean Journal of Veterinary Research
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• v.33 no.1
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• pp.81-85
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• 1993

Gel electrophoresis has proven to be one of the most useful of DNA separation and purification. The new technique of pulsed field gel electrophoresis (PFGE) is high resolution separation of large size DNA moleculs. Conventional continuous gel electrophresis can not be separation of large DNA fragments(20~50 k base). Field inversion gel electrophoresis(FIGE) is very useful for large DNA molecules. We have found that a pulse ratio ; 2 : 1, time ; 24hrs., volts ; $10^{volts}/_{cm}$, start ; 0.45sec, end ; 1sec, is most effectively resolves DNA fragment in the 6~50k base.

• Applied Biological Chemistry
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• v.31 no.3
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• pp.274-283
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• 1988

Aqualysin I is an alkaline serine protease which is secretet into the culture medium by Thermus 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 sequenc, agreed with the determid amino acid sequences, including the $NH_2-$ 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 be 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-Cys194, 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 $NH_2-$ and COOH- terminal portions besides the mature protease sequence.