Fine tuning of functionally salvaged green fluorescent protein variant by directed evolution = 기능적으로 복원된 변이 녹색 형광 단백질의 방향적 진화를 이용한 미세 조정

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Functional salvage screen (FSS) method was invented as a novel method that could create new protein lineage with different sequence space. Functionally salvaged GFP variants with randomly inserted fragments were generated by FSS. However, these mutants showed little fluorescence intensity. Among these variants, GFP-I5 was selected and directly evolved to optimize the spectral property. As a result, best mutant, E-2S4 was obtained from the library generated by ep-PCR and DNA shuffling. It possessed the mutations in F64L, E111V, K166Q and Kins-8N residues. The fluorescence intensity of purified protein of E-2S4 was ~28-fold improved over that of GFP-I5, but other spectral properties were unchanged. This improvement of fluorescence was also observed in the whole cell of E.coli. Purified protein of E-2S4 has different conformation with other GFP mutants. In order to investigate the effect of each mutated residues on fluorescence intensity, site-directed mutagenesis was carried out. Site-specific mutations in other templates showed the different effect of fluorescence intensity. These results suggest that salvaged variant, GFP-I5 had new sequence space by the incorporation of 12 amino acids and it was subjected to new evolution pathway in further artificial evolution.
Advisors
Kim, Hak-Sungresearcher김학성researcher
Description
한국과학기술원 : 생물과학과,
Publisher
한국과학기술원
Issue Date
2002
Identifier
173557/325007 / 020003165
Language
eng
Description

학위논문(석사) - 한국과학기술원 : 생물과학과, 2002.2, [ iv, 43 p. ]

Keywords

green fluorescent protein; functional salvage screen; 방향적 진화; 녹색형광단백질; directed evolution

URI
http://hdl.handle.net/10203/27955
Link
http://library.kaist.ac.kr/search/detail/view.do?bibCtrlNo=173557&flag=dissertation
Appears in Collection
BS-Theses_Master(석사논문)
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