Enhancement of streptokinase activity by site directed mutagenesis = 특정위치 돌연변이에 의한 스트렙토키나아제 활성 증진

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A bacterial plasminogen activator, streptokinase (SK) is widely used as a thrombolytic agent in the treatment of myocardicac infarction. In this study, streptokinase gene was modified by using the technique of directed evolution. Through six-rounds of random mutagenesis and three rounds of error-prone PCR, over 50 thousands of clones showing larger hollow than wild type SK were screened by skim-milk plasminogen overlay test. Among them, SK-S221F which showed the highest activity was purified and characterized. SK-S221F shows the lower Km value than wild type SK and showed 2.5 folds higher overall second order rate constant than wild type complex. It suggests that the SK-221F mutant could efficiently recruit substrate plasminogen. To improve the better activity of SK, the amino acid sequences in β4 strand were changed by site directed mutagenesis. Among them, SK-R219L was purified and characterized. It shows 3 folds higher activation activity. To confirm the mechanism, SK-R219L and SK-E219F were made and these mutants show 1.3 and 3.9 times higher activity than wild type SK. It suggests that these mutants efficiently recruited substrate plasminogen. The possible usefulness of SK-221F in developing thrombolytic agent was discussed
Advisors
Byung, Si-Myung변시명
Description
한국과학기술원 : 생명과학과,
Publisher
한국과학기술원
Issue Date
2005
Identifier
243520/325007  / 020023266
Language
eng
Description

학위논문(석사) - 한국과학기술원 : 생명과학과, 2005.2, [ 44 p. ]

Keywords

R219Lne; Site Directed Mutagenesis; Streptokinase; Directed evolutiontep-in mode of AFM; R219Lne 방향적 진화 분석; 특정위치 돌연변이; 스트렙토키나아제; frequency analysis

URI
http://hdl.handle.net/10203/28046
Link
http://library.kaist.ac.kr/search/detail/view.do?bibCtrlNo=243520&flag=dissertation
Appears in Collection
BS-Theses_Master(석사논문)
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