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PubMed · 10739955

A new approach to gene mutation analysis using "GFP-Display".

T Aoki·R Ami·H Onagi·H Fujino·H Watabe
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Abstract

The unique behavior of green fluorescent protein (GFP) on SDS-PAGE was applied to the detection of a single amino acid substitution in GFP-tagged polypeptides. This simple detection method using SDS/urea gels was designated GFP-display. The N-terminal 18 or 37 amino acids of K-Ras was used as a model GFP-tagged polypeptide. K-ras exon 1 was fused to a gfp cDNA at each end and expressed in Escherichia coli. Amino acid number 12 of K-Ras (wild type; Gly) was changed to Ser, Arg, Cys, Asp, Ala, or Val, and the mobility shift of the greenish fluorescent bands in the SDS/urea gel was analyzed. These mutants were easily detected by GFP-display; however, detection depended strongly on the urea concentration and electrophoresis temperature. Subsequently, GFP-display was applied to the 36 amino acids encoding human p53 exon 7. Amino acid number 248 (wild type; Arg) was changed to Gly, Trp, Gln, Pro, or Leu, and similar mobility shifts were observed. GFP-display could be coupled with an in vitro translation system. Fluorescent active GFP and GFP-Ras fusion proteins were synthesized within a few hours. GFP-display shows potential as a modern approach to gene mutation analysis at the protein level, and is a useful method for protein engineering studies.

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DNA Mutational AnalysisElectrophoresis, Polyacrylamide GelEscherichia coliGenes, rasGreen Fluorescent ProteinsHumansLuminescent ProteinsPoint MutationRecombinant Fusion ProteinsUreaJournal ArticleResearch Support, Non-U.S. Gov't

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T Aoki, R Ami, H Onagi, H Fujino, H Watabe. 2000. A new approach to gene mutation analysis using "GFP-Display".. https://doi.org/10.1093/oxfordjournals.jbchem.a022650

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