title: 5´-UTR introns enhance protein expression in the yeast Saccharomyces cerevisiae.
abstract: Saccharomyces cerevisiae is one of the most suitable microorganisms for recombinant protein production. To enhance protein production, various expression systems have been intensively studied. However, the effect of introns on protein expression has not been examined deeply in S. cerevisiae. In this study, we analyzed the effect of some introns on protein expression. RPS25A, RPS26A, and RPS26B contain single introns within the 5´-untranslated regions (5´-UTRs), and RPS24A has an intron just downstream of the initiation codon. Expression activity of the promoter regions containing introns (intron promoters) were analyzed by luciferase reporter assays. These intron promoters showed higher expression than the TDH3 promoter (TDH3p), which is one of the strongest promoters in S. cerevisiae. Deletion of the introns from these promoters decreased luciferase expression, indicating that introns have a role in enhancing protein expression. To develop artificial strong intron promoters, several chimeric promoters were constructed using the TDH3p and the RPS25A intron promoter. A construct containing the entire TDH3p followed by the RPS25A intron showed about 50-fold higher expression than the TDH3p alone. Inducible expressions driven by the GAL10 promoter and the CUP1 promoter were also enhanced by the RPS25A intron. However, enhancement of mRNA accumulation by the TDH3p and the GAL10 promoter with the RPS25A intron was lower than the effect on luciferase activity, suggesting that the intron affects post-transcriptionally. The chimeric promoter, TDH3p-RPS25A-intron, enhanced expressions of some, but not all proteins examined, indicating that 5'-UTR introns increase production of a certain type of recombinant proteins in S. cerevisiae.
authors.affiliation: Department of Applied Chemistry, Graduate School of Sciences and Technology for Innovation, Yamaguchi University, Tokiwadai 2-16-1, Ube, 755-8611, Japan. hoshida@yamaguchi-u.ac.jp.|Research Center for Thermotolerant Microbial Resources, Yamaguchi University, Yoshida 1677-1, Yamaguchi, 753-8315, Japan. hoshida@yamaguchi-u.ac.jp.|Yamaguchi University Biomedical Engineering Center, Tokiwadai 2-16-1, Ube, 755-8611, Japan. hoshida@yamaguchi-u.ac.jp.
authors.forename: Hisashi
authors.identifier: http://orcid.org/0000-0003-2613-232X
authors.initials: H
authors.lastname: Hoshida
authors.affiliation: Department of Applied Chemistry, Graduate School of Sciences and Technology for Innovation, Yamaguchi University, Tokiwadai 2-16-1, Ube, 755-8611, Japan.
authors.forename: Masaki
authors.initials: M
authors.lastname: Kondo
authors.affiliation: Department of Applied Chemistry, Graduate School of Sciences and Technology for Innovation, Yamaguchi University, Tokiwadai 2-16-1, Ube, 755-8611, Japan.
authors.forename: Takafumi
authors.initials: T
authors.lastname: Kobayashi
authors.affiliation: Department of Applied Chemistry, Graduate School of Sciences and Technology for Innovation, Yamaguchi University, Tokiwadai 2-16-1, Ube, 755-8611, Japan.|Environmental Biofunction Division, National Institute for Agro-Environmental Sciences, 3-1-3 Kan-nondai, Tsukuba, Ibaraki, 305-8604, Japan.
authors.forename: Tohru
authors.initials: T
authors.lastname: Yarimizu
authors.affiliation: Department of Applied Chemistry, Graduate School of Sciences and Technology for Innovation, Yamaguchi University, Tokiwadai 2-16-1, Ube, 755-8611, Japan.|Research Center for Thermotolerant Microbial Resources, Yamaguchi University, Yoshida 1677-1, Yamaguchi, 753-8315, Japan.|Yamaguchi University Biomedical Engineering Center, Tokiwadai 2-16-1, Ube, 755-8611, Japan.
authors.forename: Rinji
authors.initials: R
authors.lastname: Akada
mesh_terms: D020121:5' Untranslated Regions; D019677:Artificial Gene Fusion; D020869:Gene Expression Profiling; D015966:Gene Expression Regulation, Fungal; D017930:Genes, Reporter; D007438:Introns; D008156:Luciferases / Q000032:analysis / Q000235:genetics; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000235:genetics*; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000096:biosynthesis*; D014158:Transcription, Genetic
chemical_list: D020121:5' Untranslated Regions; D012333:RNA, Messenger; D029701:Saccharomyces cerevisiae Proteins; D008156:Luciferases
keywords: 5´-UTR; Intron; Protein expression; Saccharomyces cerevisiae; Secretory luciferase