Sensitive and Convenient Yeast Reporter Assay for High-Throughput Analysis by Using a Secretory Luciferase from Cypridina noctiluca

Abstract

The yeast reporter assay has been widely used in various applications such as detection of endocrine disruptors and analysis of protein-protein interactions by the yeast two-hybrid system. The molecular characteristics of the reporter enzyme are critical determinants for this assay. We herein report the establishment of a novel yeast reporter assay using a secretory luciferase, Cypridina noctiluca luciferase (CLuc), as an alternative to the conventional beta-galactosidase. The CLuc reporter assay in yeast is more sensitive and convenient than the conventional assay. A yeast high-throughput reporter assay was established with a laboratory automation system, and the transcriptional activity of hundreds of yeast promoter fragments was comprehensively determined. Our results indicate that the yeast CLuc reporter assay is a promising tool for large-scale and sensitive analysis in the development of new drugs and in various fields of biotechnology research.

Rapid identification of mRNA processing defects with a novel single-cell yeast reporter

“…Another challenge with reporters, especially in high-throughput studies when using β-galactosidase activity, is the need for cell lysis. This can be alleviated using reporter genes/signals that do not require lysis, or other approaches such as a secreted luciferase (Tochigi et al 2010).…”

Construction of a cytosolic firefly luciferase reporter cassette for use in PCR‐mediated gene deletion and fusion in Saccharomyces cerevisiae

“…Most importantly, measurement of in vivo bioluminescence can be taken quickly, accurately and in one-step (Michelini et al, 2005), without the additional lysis step commonly required of other luciferase assays (McNabb et al, 2005;Liu et al, 2008). Additionally, recently discovered secretable luciferases show promise as nextgeneration reporters that remain compatible with current detection techniques (Tochigi et al, 2010;Ochi et al, 2011).…”

“…Current luciferase systems utilize one of two common methods of transformation: yeast integrative plasmids (YIps) (Leskinen et al, 2003) and yeast centromere plasmids (YCps) (Tochigi et al, 2010;Ochi et al, 2011), both of which present advantages and disadvantages. YIps directly integrate reporters into the genome at the promoter locus, which preserves transcriptional regulation and accurately reflects native expression levels.…”

A systems-level approach for metabolic engineering of yeast cell factories

“…For this, promoter libraries, which led to medium to large data sets on gene expression levels in yeast, have been created and screened using either native (Partow et al, 2010;Tochigi et al, 2010) or synthetic promoters (Jeppsson et al, 2003;Nevoigt et al, 2006;Hartner et al, 2008). In the first approach, the control of gene expression relies on the identification or design of optimal promoters.…”

“…Promoter library screening Yeast promoters were screened successfully using the novel yeast reporter system using a secretory Cypridina noctiluca luciferase (CLuc) Tochigi et al (2010) Promoter selection Expression vectors harboring bidirectional TEF1-PGK1 promoter, which was selected using LacZ reporter assay Partow et al (2010) Promoter engineering Mutation library of TEF1 promoter using error-prone PCR in S. cerevisiae Alper et al (2005) SPL (Synthetic Promoter Library) In Pichia pastoris, novel short synthetic promoter was developed from a synthetic promoter library, which was constructed by deletion and duplication of putative transcription factor-binding sites within the AOX1 promoter sequence Hartner et al (2008) Translation level control RNA regulators Using down-regulation of YBR1012 gene with the antisense RNA of YBR1012, S. cerevisiae was successfully arrested with its DNA unreplicated Nasr et al (1995) Ligand-dependent riboregulators by rational design showed to tightly regulate expression of the GFP reporter in S. cerevisiae dependent on the effector concentration Bayer & Smolke (2005) Using CAT reporter system, in vivo analysis of hammerhead ribozyme and antisense gene function in Saccharomyces cerevisiae were successfully performed Atkins & Gerlach (1994) Codon optimization Replacing a codon from major to synonymous minor at the 5′ end of the coding sequence caused a dramatic decline of the expression level 3′-UTRs of the target gene that are based on the hairpin substrate of the RNase III Rnt1. These recognition sites were modulated by either random mutagenesis (Babiskin & Smolke, 2011a) or introduction of RNA-aptamers (Babiskin & Smolke, 2011b).…”