Expression of varied GFPs in Saccharomyces cerevisiae: codon optimization yields stronger than expected expression and fluorescence intensity

Abstract

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.

Modifying Expression Modes of Human Neurotensin Receptor Type 1 Alters Sensing Capabilities for Agonists in Yeast Signaling Biosensor

“…Instead of mNeptune far‐red protein, we used a monomeric green fluorescent protein (mUkG) that has been shown to be readily expressed in yeast cells . Multi‐copy autonomously replicating plasmids encoding mUkG‐fused NTSR1 protein were constructed and used to transform the IMFD‐72ZsD strain (Figure A; Table ).…”

“…Instead of mNeptune far-red protein, we used a monomeric green fluorescent protein (mUkG) that has been shown to be readily expressed in yeast cells. [30] Multi-copy autonomously replicating plasmids encoding mUkG-fused NTSR1 protein were constructed and used to transform the IMFD-72ZsD strain (Figure 4A; Table 1). When cultured in the absence of ligand (NTS) stimulation, the resulting strains showed higher fluorescence intensities than seen with far-red fluorescence obtained with mNeptune fusion proteins; however, the rankorder of green fluorescence intensities among the transformants was the same as that obtained with the mNeptune-fused NTSR1 constructs ( Figure 4B and Figure S2B, Supporting Information).…”

Secretory Overexpression of Bacillus thermocatenulatus Lipase in Saccharomyces cerevisiae Using Combinatorial Library Strategy

“…The transcription levels of the BTL2 gene were quantified by reverse‐transcription (RT) real‐time PCR . Total RNA was extracted from yeast cells cultivated in SDC medium for 72 h at 30 °C.…”

Consolidated bioprocessing of corn cob-derived hemicellulose: engineered industrialSaccharomyces cerevisiaeas efficient whole cell biocatalysts

“…Saccharomyces cerevisiae strains and main plasmids used in this work are listed in [42] for monitoring copy number integration. Plasmid assembling steps were performed with the In-Fusion HD Cloning Kit (Clontech, Mountain View, CA, USA).…”