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

Abstract

G-protein-coupled receptors (GPCRs) are currently the most important pharmaceutical targets for drug discovery because they regulate a wide variety of physiological processes. Consequently, simple and convenient detection systems for ligands that regulate the function of GPCR have attracted attention as powerful tools for new drug development. We previously developed a yeast-based fluorescence reporter ligand detection system using flow cytometry. However, using this conventional detection system, fluorescence from a cell expressing GFP and responding to a ligand is weak, making detection of these cells by fluorescence microscopy difficult. We here report improvements to the conventional yeast fluorescence reporter assay system resulting in the development of a new highly-sensitive fluorescence reporter assay system with extremely bright fluorescence and high signal-to-noise (S/N) ratio. This new system allowed the easy detection of GPCR signaling in yeast using fluorescence microscopy. Somatostatin receptor and neurotensin receptor (implicated in Alzheimer’s disease and Parkinson’s disease, respectively) were chosen as human GPCR(s). The facile detection of binding to these receptors by cognate peptide ligands was demonstrated. In addition, we established a highly sensitive ligand detection system using yeast cell surface display technology that is applicable to peptide screening, and demonstrate that the display of various peptide analogs of neurotensin can activate signaling through the neurotensin receptor in yeast cells. Our system could be useful for identifying lead peptides with agonistic activity towards targeted human GPCR(s).

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

“…were used, unexpectedly resulting in a relatively similar codon usage to that in yeast (CAIs = 0.69~0.70 and GC contents = 44.9~45.0%) (Table 4). These support our previous result that ZsGreen (original codons) exhibited more intense fluorescence than EGFP (non-codon-optimized) when used as a fluorescent reporter protein34. Among the codon-optimized GFPs tested in this study, monomeric ymUkG1 and tetrameric yZsGreen exhibited the most intense green fluorescence in S. cerevisiae (Fig.…”

“…The plasmids used for the expression of the tested GFPs were constructed as follows: DNA fragments encoding the GFPs AcGFP1, TagGFP2 and mUkG1 were PCR-amplified from pAcGFP1 (Clontech Laboratories/Takara Bio, Shiga, Japan), pTagGFP2-tubulin (Evrogen, Moscow, Russia) and pmUkG1-S1 (Medical & Biological Laboratories, Nagoya, Japan) using primer pairs 1 and 2, 3 and 4, and 5 and 6; the fragments were then digested with SalI + BamHI and inserted into the same sites between the PGK1 promoter ( P PGK1 ) and the PGK1 terminator ( T PGK1 ) on pGK41633, yielding the plasmids pGK416-AcGFP1, pGK416-TagGFP2 and pGK416-mUkG1. For EGFP and ZsGreen, the previously constructed plasmids pGK416-EGFP33 and pGK416-ZsGreen34 were used. The expression plasmids used for codon-optimized GFPs (yEGFP, yAcGFP1, yTagGFP2, ymUkG1, yZsGreen, ymWasabi and ymNeonGreen) were constructed as follows: DNA fragments encoding the codon-optimized GFPs were PCR-amplified from the GeneArt ® Strings ™ DNA fragments using primer pairs 7 and 8, 9 and 10, 11 and 12, 13 and 14, 15 and 16, 17 and 18, and 19 and 20; the fragments were then digested with SalI + BamHI and inserted into the same sites between the P PGK1 and the T PGK1 on pGK41633, yielding the plasmids pGK416-yEGFP, pGK416-yAcGFP1, pGK416-yTagGFP2, pGK416-ymUkG1, pGK416-yZsGreen, pGK416-ymWasabi and pGK416-ymNeonGreen.…”

“…The plasmids used to integrate the GFP reporter genes at the FIG1 locus on the yeast chromosome were constructed as follows: A DNA fragment containing the homologous sequence of the FIG1 terminator (downstream of FIG1 gene; 200 bp) was PCR-amplified from pBlue-FIG1pt-ZsGreen34 using the primer pair 35 and 36. The amplified fragments were digested with XhoI + KpnI and inserted into the pBlueScript II KS(+) vector, yielding the plasmid pBlue-FIG1t.…”

“…The amplified fragments were digested with XhoI + KpnI and inserted into the pBlueScript II KS(+) vector, yielding the plasmid pBlue-FIG1t. A DNA fragment containing the URA3 selectable marker was PCR-amplified from pBlue-FIG1pt-ZsGreen34 using the primer pair 37 and 38. DNA fragments containing the GFPs were PCR-amplified from pGK416-AcGFP1, pGK416-TagGFP2, pGK416-mUkG1, pGK416-ZsGreen, pGK416-yEGFP, pGK416-yAcGFP1, pGK416-yTagGFP2, pGK416-ymUkG1, and pGK416-yZsGreen34 using primer pairs 39 and 40, 41 and 42, 43 and 44, 45 and 46, 47 and 48, 49 and 50, 51 and 52, 53 and 54, and 55 and 56, respectively.…”

Critical Role for CD103+/CD141+ Dendritic Cells Bearing CCR7 for Tumor Antigen Trafficking and Priming of T Cell Immunity in Melanoma

“…To provide a greater possibility to track antigens that might be degraded during hand-off or transit, we engineered another variant melanoma line, this time taking the parental B16 mela-noma cell line and introducing the bright and highly stable fluoro-phore ZsGreen (Nakamura et al, 2013), which is documented to resist both low pH and lysosomal degradation (Katayama et al, 2008), and which we have confirmed to be significantly less subject to degradation when ingested by phagocytes (data not shown). When we introduced these tumors into mice and removed dLN to analyze the myeloid populations, similarly to the previous assays we found that the fluorophore was again compartmentalized within CD103 + DC (Figure 3A).…”