Modification of streptokinase using protein engineering technique and its effects on the activity = 단백질공학 기법을 이용한 스트렙토카이네즈의 변형과 그 효과

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The chromosomal DNA from Streptococcus equisimilis ATCC 9542 was cloned in E, coli HB101 by using the plasmid vector pBR 322. A casein/plasminogen overla technique was used to screen the gene bank for recombinants carring the streptokinase gene. The gene was present with a frequency of 1 in 460 recombinants and 26 independent clones containing streptokinase gene were isolated and phsicall characterized. One recombinant clone (pSK II) was used to subclone in M13 phage and site-directed mutagenesis was carried out to replace Gly 24 residue with histidine, glutamic acid, and alanine. The mutated streptokinase genes were expressed in E. coli C600 using the tacpromoter and the streptokinases were purified by ammonium sulfate fractionation DEAE-cellulose, and Sephade G-150 chromatography. The active fractions were detected using dot immunoblot assay of eluate. The plasminogen activator activit of the purified streptokinases was determined. Wild-tpe streptokinase and alanine mutant streptokinase were found to be active as plasminogen activators. The activity of alanine mutant streptokinase was somewhat less than that of wild-type and histidine mutant streptokinase and glutamic acid mutant streptokinase showed activities onl 4\% and 3\%, respectively of the activit of wild-tpe. A modified radioimmunoassa method was used to detect streptokinase bound to the human plasminogen coated on the well.s The radioactivity increased and saturated with increasing streptokinase concentration, indicating normal binding of the streptokinases with human uncharged alkyl group side-chain on the 24th amino acid residue of streptokinase is indidspensable for the activity of the human plasminogen-streptokinase compelex.
Byun, Si-Myung변시명
한국과학기술원 : 생물공학과,
Issue Date
61473/325007 / 000825223

학위논문(박사) - 한국과학기술원 : 생물공학과, 1990.2, [ xiii, 178 p. ]


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