engleski

In vitro mutagenesis of the gfp+ gene

The gfp gene from jellyfish Aequorea victoria encodes green fluorescent protein (Gfp). It is frequently used as a gene marker as well as a reporter gene. 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 of the gfp+ variant. Development of these forms, that would be strongly expressed and stable at 37°C in bacteria Escherichia coli, would allow combined use of different gfp variants in the experiments of double-labelling. Six point mutations resulting in replacement of 5 amino acids (K26R, Y66W, N146I, N164H and N212K) have been introduced into gfp+ gene in order to obtain cyan fluorescent protein. Seven point mutations resulting in replacement of 4 amino acids (T65G, V68L, S72A and T203Y) have been introduced in order to obtain yellow fluorescent protein. Optical densities and fluorescence signals of potential cfp+ and yfp+ transformants were measured. Relative fluorescence signals of those clones were put in relation to the signals of untransformed E. coli cells. The results confirmed the existence of cyan and yellow fluorescent proteins. Mutated gfp+ genes were sequenced. Sequences of the yfp+ mutants showed exceptional efficiency of mutagenesis. Mutation in the tripeptide active site of the cfp+ mutants proved to be necessary, while other mutations might have positive cumulative effect on solubility and brightness of the protein. The approach described in this work represents a good starting point for all future efforts to improve green fluorescent protein.

bioluminiscence; green fluorescent protein; gfp; mutagenesis

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