A novel strategy for constructing N‐terminal chromosomal fusions to green fluorescent protein in the yeast Saccharomyces cerevisiae

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Abstract
A novel rapid polymerase chain reaction (PCR)‐based technique for N‐terminal attachment of green fluorescent protein (GFP) to a yeast protein is described. Genomic integration of a PCR‐generated loxPkanMX4loxP‐yEGFP fusion cassette immediately upstream of the open reading frame (ORF) allows for selection of G418 resistant transformants carrying GFP fused N‐terminally to the protein of interest. In a subsequent step, the loxPkanMX4loxP selection marker that is inserted between the tagged ORF and the endogenous promoter is excised upon site‐specific recombination between the loxP sites by Cre recombinase, leaving behind in the promoter one loxP site, immediately upstream of the GFP start codon. The essential protein Ydl193wp of unknown function and the oleate‐inducible fatty acid activation protein, encoded by FAA2, were N‐terminally tagged using the novel technique. Both experiments yielded viable haploid strains with growth phenotypes indistinguishable from the wild type strain. The subcellular localization pattern for the chromosomally expressed GFP‐Ydl193wp to the endoplasmic reticulum and lipid particles was identical to the pattern observed for a plasmid‐borne GFP construct expressed under control of the MET25p promoter, albeit at a lower level and with a more homogeneous distribution among the cell population. GFP‐FAA2 was inducible by oleate, as is the wild type gene, demonstrating that specific expression patterns are not grossly affected by the promoter manipulation. In agreement with previous reports, GFP‐Faa2p was found to localize to peroxisomes.