PHOTO CONVERSION OF GREEN FLUORESCENT PROTEIN

University Of Colorado At Boulder, Boulder CO

Investigators

Linked publications & trials

Abstract

The green fluorescent protein (GFP) from jelly fish of the genus Aequoreus has been isolated and characterized by several workers, and the gene that encodes it has now been cloned and expressed in numerous organisms to serve as a marker for aspects of cell physiology. We are interested in devising a way to convert the fluorescence of GFP to an electron scattering stain so that fine structural studies can be use to complement and extend the work ongoing in many labs that uses GFP as a reporter for protein position. We have had limited success in photoconverting GFP by fixing cells that express it, bleaching their fluorescence with either an argon ion laser or a mercury vapor lamp in the presence of diamino benzadine (DAB), followed by further fixation with OsO4, then embedding, sectioning and electron microscopy. Conditions that might improve the photoconversion of GFP have been explored, using first cultured mammalian cells, expressing a chimera of microtubule-associated protein 4 with GFP, then bacteria expressing either of two alleles of GFP at high levels. Exposure of such cells to fixation then bleaching irradiation in the presence of various buffers has shown that ionic chaotrops, like KI increase the rate of photobleaching, but we have not found conditions that enhance the rate or extent of GFP photoconversion. We are now beginning to select a mutant allele of the GFP that photoconverts better than either the wild type GFP or the mutants alleles that are additionally fluorescent. Robert West, an experienced molecular biologist will carry out this mutagenesis by PCR and the selection for alleles with the desired phenotype.