Naslov
The change of spectroscopic properties of the green fluorescent protein Gfp+ by in vitro mutagenesis

Autori
Marcelić, Tea ; Lindner, Ariel ; Radman, Miroslav

Vrsta, podvrsta i kategorija rada
Sažeci sa skupova, sažetak, znanstveni

Izvornik
8th Croatian Biological Congress with International Participation, Proceeding of Abstracts / Besendorfer, Višnja ; Kopjar, Nevenka - Zagreb : Hrvatsko biološko društvo, 2003, 105-106

Skup
8th Croatian Biological Congress with International Participation

Mjesto i datum
Zagreb, Hrvatska, 27.09.2003. - 02.10.2003

Vrsta sudjelovanja
Poster

Vrsta recenzije
Nije recenziran

Ključne riječi
bioluminiscence; green fluorescent protein; gfp; mutagenesis

Sažetak
The gfp gene that encodes green fluorescent protein (Gfp), is frequently used as a gene marker as well as a reporter gene. Numerous forms of Gfp that show greater brightness, better folding at 37 C and enhanced fluorescence properties have been developed until now. The aim of this research was to produce (i) cyan (Cfp+) and (ii) yellow (Yfp+) fluorescent protein using the method of multiple site-directed mutagenesis. Aminoacids that could be significant for the spectroscopic properties of the proteins have been chosen on the basis of sequences of the ECFP and EYFP genes expressed in human cells. In order to gain cyan fluorescent protein, 6 mutations that change 5 aminoacids have been introduced: K26R, Y66W, N146I, N164H and N212K. For the purpose of producing yellow fluorescent protein, 7 mutations that change 4 aminoacids have been introduced: T65G, V68L, S72A and T203Y. Optical density and fluorescence signals of potential cfp+ and yfp+ transformants were measured. Relative fluorescence signals of these clones were calculated using carefully conceived mathematical form and the signals were compared with signals of untransformed cells of E. coli. It was confirmed that analyzed mutants possess cyan and yellow fluorescing properties. Mutated gfp+ genes of the clones that showed the best results were sequenced and exceptional efficiency of mutagenesis was found. Furthermore, sequencing analysis confirmed the necessity of the mutations in the tripeptide active site while the other mutations might have cumulative effect on solubility and brightness of proteins. The approach described above represents a good starting point for all subsequent experiments of improving green fluorescent proteins.

Izvorni jezik
Engleski

Znanstvena područja
Biologija

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