Fluorescent protein engineering by in vivo site-directed mutagenesis

Melvys Valledor, Qinghua Hu, Paul Schiller, Richard S. Myers

Research output: Contribution to journalArticle

4 Scopus citations

Abstract

In vivo site-directed mutagenesis by single-stranded deoxyribonucleic acid recombineering is a facile method to change the color of fluorescent proteins (FPs) without cloning. Two different starting alleles of GFP were targeted for mutagenesis: gfpmut3* residing in the Escherichia coli genome and egfp carried by a bacterial/mammalian dual expression lentiviral plasmid vector. Fluorescent protein spectra were shifted by subtle modification of the chromophore region and residues interacting with the chromophore of the FP. Eight different FPs (Violeta, Azure, Aqua, Mar, Celeste, Amarillo, Mostaza, and Bronze) were isolated and shown to be useful in multicolor imaging and flow cytometry of bacteria and transgenic human stem cells. To make in vivo site-directed mutagenesis more efficient, the recombineering method was optimized using the fluorescence change as a sensitive quantitative assay for recombination. A set of rules to simplify mutant isolation by recombineering is provided.

Original languageEnglish (US)
Pages (from-to)684-689
Number of pages6
JournalIUBMB life
Volume64
Issue number8
DOIs
StatePublished - Aug 2012

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Keywords

  • fluorescent proteins
  • gene targeting
  • mutagenesis
  • oligonucleotides
  • recombination
  • recombineering

ASJC Scopus subject areas

  • Biochemistry
  • Cell Biology
  • Clinical Biochemistry
  • Molecular Biology
  • Genetics

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