title: Highly stable and efficient mRNA templates for mRNA-protein fusions and C-terminally labeled proteins.
abstract: For high-throughput in vitro protein selection using genotype (mRNA)-phenotype (protein) fusion formation and C-terminal protein labeling as a post-selection analysis, it is important to improve the stability and efficiency of mRNA templates for both technologies. Here we describe an efficient single-strand ligation (90% of the input mRNAs) using a fluorescein-conjugated polyethylene glycol puromycin (Fluor-PEG Puro) spacer. This ligation provides a stable c-jun mRNA with a flexible Fluor-PEG Puro spacer for efficient fusion formation (70% of the input mRNA with the PEG spacer) in a cell-free wheat germ translation system. When using a 5' untranslated region including SP6 promoter and Omega29 enhancer (a part of tobacco mosaic virus Omega), an A(8) sequence (eight consecutive adenylate residues) at the 3' end is suitable for fusion formation, while an XA(8) sequence (XhoI and the A(8) sequence) is suitable for C-terminal protein labeling. Further, we report that Fluor-PEG N-t-butyloxycarbonylpuromycin [Puro(Boc)] spacer enhances the stability and efficiency of c-jun mRNA template for C-terminal protein labeling. These mRNA templates should be useful for puromycin-based technologies (fusion formation and C-terminal protein labeling) to facilitate high-throughput in vitro protein selection for not only evolutionary protein engineering, but also proteome exploration.
authors.affiliation: Department of Biosciences and Informatics, Faculty of Science and Technology, Keio University, 3-14-1, Hiyoshi, Kohoku-ku, Yokohama 223-8522, Japan.
authors.forename: Etsuko
authors.initials: E
authors.lastname: Miyamoto-Sato
authors.forename: Hideaki
authors.initials: H
authors.lastname: Takashima
authors.forename: Shinichiro
authors.initials: S
authors.lastname: Fuse
authors.forename: Kaori
authors.initials: K
authors.lastname: Sue
authors.forename: Masamichi
authors.initials: M
authors.lastname: Ishizaka
authors.forename: Seiji
authors.initials: S
authors.lastname: Tateyama
authors.forename: Kenichi
authors.initials: K
authors.lastname: Horisawa
authors.forename: Tatsuya
authors.initials: T
authors.lastname: Sawasaki
authors.forename: Yaeta
authors.initials: Y
authors.lastname: Endo
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Yanagawa
mesh_terms: D020413:3' Untranslated Regions; D001483:Base Sequence; D011088:DNA Ligases / Q000378:metabolism; D019793:Fluorescein / Q000737:chemistry; D005456:Fluorescent Dyes / Q000737:chemistry; D011092:Polyethylene Glycols / Q000737:chemistry; D014176:Protein Biosynthesis; D015202:Protein Engineering / Q000379:methods*; D011506:Proteins / Q000032:analysis* / Q000737:chemistry / Q000235:genetics; D016755:Proto-Oncogene Proteins c-jun / Q000235:genetics; D011691:Puromycin / Q000737:chemistry; D020871:RNA Stability; D012333:RNA, Messenger / Q000737:chemistry*; D013698:Templates, Genetic
chemical_list: D020413:3' Untranslated Regions; D005456:Fluorescent Dyes; D011506:Proteins; D016755:Proto-Oncogene Proteins c-jun; D012333:RNA, Messenger; D011092:Polyethylene Glycols; D011691:Puromycin; D011088:DNA Ligases; D019793:Fluorescein