Green fluorescent protein (GFP), which was originally isolated from jellyfish, is a widely used tool in biological research, and homologs from other organisms are available. However, researchers must determine which GFP is the most suitable for a specific host. Here, we expressed GFPs from several sources in codon-optimized and non-codon-optimized forms in the yeast Saccharomyces cerevisiae, which represents an ideal eukaryotic model. Surprisingly, codon-optimized mWasabi and mNeonGreen, which are typically the brightest GFPs, emitted less green fluorescence than did the other five codon-optimized GFPs tested in S. cerevisiae. Further, commercially available GFPs that have been optimized for mammalian codon usage (e.g., EGFP, AcGFP1 and TagGFP2) unexpectedly exhibited extremely low expression levels in S. cerevisiae. In contrast, codon-optimization of the GFPs for S. cerevisiae markedly increased their expression levels, and the fluorescence intensity of the cells increased by a maximum of 101-fold. Among the tested GFPs, the codon-optimized monomeric mUkG1 from soft coral showed the highest levels of both expression and fluorescence. Finally, the expression of this protein as a fusion-tagged protein successfully improved the reporting system’s ability to sense signal transduction and protein–protein interactions in S. cerevisiae and increased the detection rates of target cells using flow cytometry.

Optical density at 600 nm (OD₆₀₀) measurements are routinely and quickly taken to estimate cell density in cultivation and to track cell growth. The yeast Saccharomyces cerevisiae is one of the microorganisms most used in industry, and the OD₆₀₀ values are frequently adopted as the indicator of yeast cell density, according to the Beer–Lambert law. Because the OD₆₀₀ value is based on turbidity measurement, the Beer–Lambert law can be applied only for microbial cultivation with low cell densities. The proportionality constants strongly depend on several parameters such as cell size. Typically, yeast strains are categorized into haploids and diploids. It is well known that cell size of diploid yeasts is larger than haploid cells. Additionally, polyploid (especially triploid and tetraploid) yeast cells are also employed in several human-activities such as bread-making and lager-brewing. As a matter of fact, there is almost no attention paid to the difference in the proportionality constants depending on the yeast ploidy. This study presents information for cell size of haploid, diploid, triploid, and tetraploid yeasts with isogenic background, and describes their proportionality constants (k) corresponding to the molar extinction coefficient (ε) in the Beer–Lambert law. Importantly, it was found that the constants are inversely proportional to apparent cell diameters estimated by flow cytometric analysis. Although each cell property highly depends on genetic and environmental factors, a set of results obtained from yeast strains with different ploidy in the current study would serve as a major reference source for researchers and technical experts.

For elucidating protein–protein interactions, many methodologies have been developed during the past two decades. For investigation of interactions inside cells under physiological conditions, yeast is an attractive organism with which to quickly screen for hopeful candidates using versatile genetic technologies, and various types of approaches are now available. Among them, a variety of unique systems using the guanine nucleotide‐binding protein (G‐protein) signaling pathway in yeast have been established to investigate the interactions of proteins for biological study and pharmaceutical research. G‐proteins involved in various cellular processes are mainly divided into two groups: small monomeric G‐proteins, and heterotrimeric G‐proteins. In this minireview, we summarize the basic principles and applications of yeast‐based screening systems, using these two types of G‐protein, which are typically used for elucidating biological protein interactions but are differentiated from traditional yeast two‐hybrid systems.