Enhancement of streptokinase activity by directed evolution = 인위적 분자 진화에 의한 스트렙토키나제 활성 증진

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A bacterial plasminogen activator, streptokinase is widely used as a thrombolytic agent in the treatment of myocardial infarction. In this study, streptokinase was modified by directed evolution. Through six rounds of random mutagenesis and three rounds of error-prone PCR, over 50 thousands of clones were screened by skim milk-plasminogen overlay test and plasminogen activation assay in 96-well plates. As a result, seven mutants with amino acid substitution were selected. Among them, S221F was purified and its amidolytic activity and plasminogen activation activity were analyzed at pH 7.4 and 37℃ with a chromogenic substrate, $NH_{2-D}-Val-Leu-Lys-p-nitroanilide. The S221F showed the 7.7-fold increased specific activity than wild-type SK owing to the decreased Michaelis constant for the substrate plasminogen. The evolved F221 may contribute to a hydrophobic core of SK β domain for substrate plasminogen binding.
Advisors
Byun, Si-Myung변시명
Description
한국과학기술원 : 생물과학과,
Publisher
한국과학기술원
Issue Date
2002
Identifier
177026/325007 / 020003247
Language
eng
Description

학위논문(석사) - 한국과학기술원 : 생물과학과, 2002.8, [ iv, 47 p. ]

Keywords

plasminogen; streptokinase; plasminogen activation; 방향성 있는 인위적 분자 진화; 플라스미노젠; 스트렙토키나제; directed evolution

URI
http://hdl.handle.net/10203/27978
Link
http://library.kaist.ac.kr/search/detail/view.do?bibCtrlNo=177026&flag=dissertation
Appears in Collection
BS-Theses_Master(석사논문)
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