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

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.

• Publication year

  2016

• Venue

  Scientific Reports

• Publication date

  2016-10-26

• Fields of study

  Biology, Medicine

• Identifiers
  DOI 10.1038/srep35932PMID 27782154PMCID 5080575
• External record

  Open on Semantic Scholar

• Source metadata

  Semantic Scholar, PubMed

• No claims are published for this paper.

• No concepts are published for this paper.

• Gγ recruitment systems specifically select PPI and affinity-enhanced candidate proteins that interact with membrane protein targets

  2015cited by this paper
• Rapid evaluation of tyrosine kinase activity of membrane-integrated human epidermal growth factor receptor using the yeast Gγ recruitment system.

  2015cited by this paper
• Promoter sequences direct cytoplasmic localization and translation of mRNAs during starvation in yeast

  2014cited by this paper
• Microbial fluorescence sensing for human neurotensin receptor type 1 using Gα-engineered yeast cells.

  2014cited by this paper
• A condition-specific codon optimization approach for improved heterologous gene expression in Saccharomyces cerevisiae

  2014cited by this paper
• Desired Alteration of Protein Affinities: Competitive Selection of Protein Variants Using Yeast Signal Transduction Machinery

  2014cited by this paper
• Three gene expression vector sets for concurrently expressing multiple genes in Saccharomyces cerevisiae.

  2014cited by this paper
• Improved Blue, Green, and Red Fluorescent Protein Tagging Vectors for S. cerevisiae

  2013cited by this paper
• Contribution of Bone Marrow-Derived Hematopoietic Stem/Progenitor Cells to the Generation of Donor-Marker+ Cardiomyocytes In Vivo

  2013cited by this paper
• A bright monomeric green fluorescent protein derived from Branchiostoma lanceolatum

  2013cited by this paper
• Bright Fluorescence Monitoring System Utilizing Zoanthus sp. Green Fluorescent Protein (ZsGreen) for Human G-Protein-Coupled Receptor Signaling in Microbial Yeast Cells

  2013cited by this paper
• Cell Wall Trapping of Autocrine Peptides for Human G-Protein-Coupled Receptors on the Yeast Cell Surface

  2012cited by this paper
• Gγ recruitment system incorporating a novel signal amplification circuit to screen transient protein‐protein interactions

  2011cited by this paper
• Amplification of agonist stimulation of human G-protein-coupled receptor signaling in yeast.

  2011cited by this paper
• Protein–protein interactions and selection: yeast‐based approaches that exploit guanine nucleotide‐binding protein signaling

  2010cited by this paper
• Very Bright Green Fluorescent Proteins from the Pontellid Copepod Pontella mimocerami

  2010cited by this paper
• A simple and immediate method for simultaneously evaluating expression level and plasmid maintenance in yeast.

  2009cited by this paper
• Construction of a novel detection system for protein–protein interactions using yeast G‐protein signaling

  2009cited by this paper
• Improving membrane voltage measurements using FRET with new fluorescent proteins

  2008cited by this paper
• Conversion of red fluorescent protein into a bright blue probe.

  2008cited by this paper
• A yEGFP‐based reporter system for high‐throughput yeast two‐hybrid assay by flow cytometry

  2008cited by this paper
• Hue-shifted monomeric variants of Clavularia cyan fluorescent protein: identification of the molecular determinants of color and applications in fluorescence imaging

  2008cited by this paper
• Cellular studies of binding, internalization and retention of a radiolabeled EGFR-binding affibody molecule.

  2007cited by this paper
• Optimized Protein Extraction for Quantitative Proteomics of Yeasts

  2007cited by this paper
• Endogenous Green Fluorescent Protein (GFP) in Amphioxus

  2007cited by this paper
• Single-cell proteomic analysis of S. cerevisiae reveals the architecture of biological noise

  2006cited by this paper
• The Fluorescent Toolbox for Assessing Protein Location and Function

  2006cited by this paper
• Post-transcriptional Regulation of the Yeast High Affinity Iron Transport System*

  2005cited by this paper
• Protein tagging and detection with engineered self-assembling fragments of green fluorescent protein

  2005cited by this paper
• Fluorescent proteins as a toolkit for in vivo imaging.

  2005cited by this paper
• Optimized cassettes for fluorescent protein tagging in Saccharomyces cerevisiae

  2004cited by this paper
• Development and Use of Fluorescent Protein Markers in Living Cells

  2003cited by this paper
• Global analysis of protein localization in budding yeast

  2003cited by this paper
• Global analysis of protein expression in yeast

  2003cited by this paper
• A Green-emitting Fluorescent Protein from Galaxeidae Coral and Its Monomeric Version for Use in Fluorescent Labeling*

  2003cited by this paper
• A colourless green fluorescent protein homologue from the non-fluorescent hydromedusa Aequorea coerulescens and its fluorescent mutants.

  2003cited by this paper
• Visualization of maltose uptake in living yeast cells by fluorescent nanosensors

  2002cited by this paper
• Dual lipid modification motifs in G(alpha) and G(gamma) subunits are required for full activity of the pheromone response pathway in Saccharomyces cerevisiae.

  2000cited by this paper
• Plasma membrane compartmentalization in yeast by messenger RNA transport and a septin diffusion barrier.

  2000cited by this paper
• Dual Lipid Modification Motifs in Gα and Gγ Subunits Are Required for Full Activity of the Pheromone Response Pathway in Saccharomyces cerevisiae

  2000cited by this paper
• Fluorescent proteins from nonbioluminescent Anthozoa species

  1999cited by this paper
• The green fluorescent protein.

  1998cited by this paper
• Pheromone-regulated Genes Required for Yeast Mating Differentiation

  1998cited by this paper
• Subcellular distribution of proteasomes implicates a major location of protein degradation in the nuclear envelope–ER network in yeast

  1998cited by this paper
• Designer deletion strains derived from Saccharomyces cerevisiae S288C: A useful set of strains and plasmids for PCR‐mediated gene disruption and other applications

  1998cited by this paper
• Yeast-enhanced green fluorescent protein (yEGFP): a reporter of gene expression in Candida albicans.

  1997cited by this paper
• Use of the green fluorescent protein and its mutants in quantitative fluorescence microscopy.

  1997cited by this paper
• FACS-optimized mutants of the green fluorescent protein (GFP).

  1996cited by this paper
• Understanding, improving and using green fluorescent proteins.

  1995cited by this paper
• Improved green fluorescence

  1995cited by this paper
• GREEN FLUORESCENT PROTEIN

  1995cited by this paper
• Wavelength mutations and posttranslational autoxidation of green fluorescent protein.

  1994cited by this paper
• Green fluorescent protein as a marker for gene expression.

  1994cited by this paper
• FAR1 links the signal transduction pathway to the cell cycle machinery in yeast.

  1993cited by this paper
• Mutational analysis of the interaction between staphylococcal protein A and human IgG1.

  1993cited by this paper
• Improved method for high efficiency transformation of intact yeast cells.

  1992cited by this paper
• A synthetic IgG-binding domain based on staphylococcal protein A.

  1987cited by this paper
• Extraction, purification and properties of aequorin, a bioluminescent protein from the luminous hydromedusan, Aequorea.

  1962cited by this paper

• Chemical interactions in polyethylene glycol-induced condensates lead to an anomalous FRET response from a flexible linker-fluorescent protein crowding sensor

  2026cites this paper
• Combinatorial metabolic engineering of alkane biosynthesis in the osmotolerant yeast Debaryomyces hansenii CBS 767

  2025cites this paper
• QTL mapping, breeding, and debugging Saccharomyces cerevisiae strains through Reiterated Mass Selection and backcrosSing (ReMaSSing)

  2025cites this paper
• Promoters for high-efficiency expression and proinsulin secretion in Saccharomyces cerevisiae

  2025cites this paper
• Machine learning-guided engineering of T7 RNA polymerase and mRNA capping enzymes for enhanced gene expression in eukaryotic systems

  2025cites this paper
• Metabolic Engineering of Lachancea kluyveri with Enhanced Glucose and Xylose Co-utilization for Lignocellulosic Biofuels.

  2025cites this paper
• A versatile genetic toolkit for engineering Wickerhamomyces ciferrii for tetraacetyl phytosphingosine production

  2025cites this paper
• SHIP identifies genomic safe harbors in eukaryotic organisms using genomic general feature annotation

  2025cites this paper
• Translation of the downstream ORF from bicistronic mRNAs by human cells: Impact of codon usage and splicing in the upstream ORF

  2025cites this paper
• Variability in Protein Expression and Fitness Under Stress: Distinct Modes of Early Adaptation of Populations

  2025cites this paper
• Enhanced eMAGE applied to identify genetic factors of nuclear hormone receptor dysfunction via combinatorial gene editing

  2024cites this paper
• Designing strong inducible synthetic promoters in yeasts

  2024cites this paper
• Structural and Functional Implications of Polyarginine Addition to Green Fluorescent Protein Expression in Saccharomyces cerevisiae INVSc1

  2024cites this paper
• A cryptic promoter in the exon of HKR1 drives expression of a truncated form of Hkr1 in Saccharomyces cerevisiae

  2024cites this paper
• Bioluminescent and fluorescent reporter-expressing recombinant Akabane virus (AKAV): an excellent tool to dissect viral replication

  2024influential citation
• Advances in recombinant protein production in microorganisms and functional peptide tags.

  2024cites this paper
• Screening of protein-based inhibitors for the intracellular domain of epidermal growth factor receptor by directed evolution using the yeast Gγ recruitment system.

  2024cites this paper
• Engineering strategies for enhanced heterologous protein production by Saccharomyces cerevisiae

  2024cites this paper
• The potency of mitochondria enlargement for mitochondria-mediated terpenoid production in yeast

  2024cites this paper
• Saccharomyces boulardii promoters for control of gene expression in vivo

  2024cites this paper
• Optimal trade-off between boosted tolerance and growth fitness during adaptive evolution of yeast to ethanol shocks

  2024cites this paper
• Engineering yeast with a light-driven proton pump system in the vacuolar membrane

  2024cites this paper
• Differential proteome diversification in yeast populations: modes of short-term adaptation and fitness outcomes

  2023cites this paper
• Expression of modified enhanced green fluorescent polyarginine protein in Saccharomyces cerevisiae INVSc1

  2023cites this paper
• Sterol interactions influence the function of Wsc sensors

  2023cites this paper
• Yeast Surface Dual Display Platform for Rapid Discovery of Shark VNAR from a Semi-Synthetic Library Followed by Next-Generation Sequencing

  2023cites this paper
• The punctate localisation of the yeast sterol transporter Ysp2p is determined by three dimerisation interfaces in its C-terminus

  2023cites this paper
• Evaluation of Long-Term Fermentation Performance with Engineered Saccharomyces cerevisiae Strains

  2023cites this paper
• Improving expression and assembly of difficult-to-express heterologous proteins in Saccharomyces cerevisiae by culturing at a sub-physiological temperature

  2023cites this paper
• Avoiding entry into intracellular protein degradation pathways by signal mutations increases protein secretion in Pichia pastoris

  2022cites this paper
• Emerging Mechanisms of Growth and Patterning Regulation by Dachsous and Fat Protocadherins

  2022cites this paper
• Development of a yeast whole-cell biocatalyst for MHET conversion into terephthalic acid and ethylene glycol

  2022cites this paper
• EasyGuide plasmids support in vivo assembly of gRNAs for CRISPR/Cas9 applications in Saccharomyces cerevisiae

  2022cites this paper
• Cross-kingdom expression of synthetic genetic elements promotes discovery of metabolites in the human microbiome.

  2022cites this paper
• Decorated networks of native proteins: nanomaterials with tunable mesoscopic domain size

  2021cites this paper
• Expression cassette and plasmid construction for Yeast Surface Display in Saccharomyces cerevisiae

  2021cites this paper
• Engineering oleaginous yeast Rhodotorula toruloides for overproduction of fatty acid ethyl esters

  2021cites this paper
• Bioinformatics analysis, codon optimization and expression of ovine pregnancy associated Glycoprotein-7 in HEK293 cells.

  2021cites this paper
• Protein-polymer mixtures in the colloid limit: Aggregation, sedimentation, and crystallization.

  2021cites this paper
• Enhancement for the fluorescent properties of new synthesized GFPs chromophore

  2021cites this paper
• Rapid inactivation of the yeast Sec complex selectively blocks transport of post-translationally translocated proteins

  2021cites this paper
• A Protein-Engineered, Enhanced Yeast Display Platform for Rapid Evolution of Challenging Targets

  2021cites this paper
• Expansion of the Yeast Modular Cloning Toolkit for CRISPR-Based Applications, Genomic Integrations and Combinatorial Libraries

  2021cites this paper
• Increased expression of the fluorescent reporter protein ymNeonGreen in Saccharomyces cerevisiae by reducing RNA secondary structure near the start codon.

  2021cites this paper
• An enhanced yeast display platform demonstrates the binding plasticity under various selection pressures

  2020cites this paper
• Directly imaging the localisation and photosensitization properties of the pan-mTOR inhibitor, AZD2014, in living cancer cells☆

  2020cites this paper
• Comparative analyses of site‐directed mutagenesis of human melatonin MTNR1A and MTNR1B receptors using a yeast fluorescent biosensor

  2020cites this paper
• Distance-Based Phylogenetic Methods

  2020cites this paper
• Nucleotide Substitution Models and Evolutionary Distances

  2020cites this paper
• G protein-coupled receptors expressed and studied in yeast. The adenosine receptor as a prime example.

  2020cites this paper
• Advanced eMAGE for highly efficient combinatorial editing of a stable genome

  2020cites this paper
• Novel xylose transporter Cs4130 expands the sugar uptake repertoire in recombinant Saccharomyces cerevisiae strains at high xylose concentrations

  2020cites this paper
• Real-time detection of changes in yeast plasma membrane potential using genetically encoded voltage indicator proteins.

  2020cites this paper
• Consolidated bioprocessing of corn cob-derived hemicellulose: engineered industrial Saccharomyces cerevisiae as efficient whole cell biocatalysts

  2020cites this paper
• Engineering Oleaginous Yeast Rhodosporidium Toruloides for Overproduction of Fatty Acid Ethyl Esters

  2020cites this paper
• Broadening the spectrum of fluorescent protein tools for use in the encapsulated human fungal pathogen Cryptococcus neoformans.

  2020cites this paper
• The in vivo detection and measurement of the unfolded protein response in recombinant cellulase producing Saccharomyces cerevisiae strains

  2019cites this paper
• De novo production of aromatic m-cresol in Saccharomyces cerevisiae mediated by heterologous polyketide synthases combined with a 6-methylsalicylic acid decarboxylase

  2019cites this paper
• Mechanism of Long-Range Chromosome Motion Triggered by Gene Activation.

  2019cites this paper
• Sec16 function in ER export and autophagy is independent of its phosphorylation in Saccharomyces cerevisiae

  2019cites this paper
• Engineering G protein-coupled receptor signalling in yeast for biotechnological and medical purposes

  2019cites this paper
• Decorated Protein Networks: Functional Nanomaterials with Tunable Domain Size

  2019cites this paper
• Engineering two species of yeast as cell factories for 2'-fucosyllactose.

  2019cites this paper
• Quantifying bacterial cell lysis using GFP based fluorimetric assay.

  2019cites this paper
• Modifying Expression Modes of Human Neurotensin Receptor Type 1 Alters Sensing Capabilities for Agonists in Yeast Signaling Biosensor.

  2018cites this paper
• Modular 5′-UTR hexamers for context-independent tuning of protein expression in eukaryotes

  2018cites this paper
• Determining S6K1 localisation and interactions with mTORC1 in live cells using fluorescence lifetime imaging microscopy

  2018cites this paper
• Artificial Mating-Type Conversion and Repetitive Mating for Polyploid Generation.

  2018cites this paper
• Protein Isoelectric Point and Helicobacter pylori

  2018cites this paper
• Biosensing Techniques in Yeast: G-Protein Signaling and Protein-Protein Interaction Assays for Monitoring Ligand Stimulation and Oligomer Formation of Heterologous GPCRs

  2018cites this paper
• Bioinformatics and Translation Initiation

  2018cites this paper
• Bioinformatics and Translation Elongation

  2018cites this paper
• Fundamentals of Proteomics

  2018cites this paper
• Optimization of mNeonGreen for Homo sapiens increases its fluorescent intensity in mammalian cells

  2018cites this paper
• Secretory Overexpression of Bacillus thermocatenulatus Lipase in Saccharomyces cerevisiae Using Combinatorial Library Strategy.

  2018cites this paper
• In vivo selection of sfGFP variants with improved and reliable functionality in industrially important thermophilic bacteria

  2018cites this paper
• CRISPR-UnLOCK: Multipurpose Cas9-Based Strategies for Conversion of Yeast Libraries and Strains

  2017cites this paper
• Positive Detection of GPCR Antagonists Using a System for Inverted Expression of a Fluorescent Reporter Gene.

  2017cites this paper
• Enhancing the performance of brewing yeasts.

  2017cites this paper
• Localization of Proteins in the Secretory Pathway of Pichia pastoris Using Fluorescence Microscopy.

  year unknowncites this paper