( Cytosolic expression of Green Fluoresc...)Cytosolic expression of Green Fluorescent Protein (GFP) and its derivatives ,,, in the yeast Saccharomyces cerevisiae: Detection in vivo using ,,, the Varian Cary Eclipse

Cytosolic expression of Green Fluorescent Protein (GFP) and its derivatives in the yeast Saccharomyces cerevisiae : Detection in vivo using the Varian Cary Eclipse

Paul Gavin# and Mark Prescott^#, Ph.D

Daren J. Fyfe, Ph.D^*

^#Department of Biochemistry and Molecular Biology, Monash University, Clayton campus Victoria 3800, Australia

* Technical assistance: Varian Australia Pty Ltd, Mulgrave, Victoria 3170, Australia Email: fluorescence@varianinc.com

Introduction

Since first isolated in 1994 by Chalfie and colleagues, green fluorescent protein (GFP) has proven to be an immensely popular and powerful tool for the non-invasive monitoring of cellular events in vivo ¹ . GFP is a 238 amino acid protein isolated from the fluorescent jellyfish, Aequoria victoria. The protein itself has an elegant -barrel structure measuring 30 x 40, which shields an internal tripeptide fluorophore (Figure 1).

Upon excitation with light of 395 nm (peak excitation) or 475 nm, the GFP fluorophore emits bright green fluorescence at 509 nm. This can be detected using a variety of instruments such as fluorescence microscopes, fluorescence-activated cell sorters and fluorometers.

GFP is an extremely attractive tool for todays life scientist, mainly because the protein matures without the need for any additional substrates or co-factors. This allows expression of GFP in an array of living organisms and cell systems, resulting in autocatalytic maturation and a fluorescent signal. In other words, GFP allows cells to produce their own fluorescent marker. This omits the need for conventiona
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