Highly stable and efficient mRNA templates for mRNA-protein fusions and C-terminally labeled proteins.

Etsuko Miyamoto-Sato, Hideaki Takashima, Shinichiro Fuse, Kaori Sue, Masamichi Ishizaka, Seiji Tateyama, Kenichi Horisawa, Tatsuya Sawasaki, Yaeta Endo, Hiroshi Yanagawa

Research output: Contribution to journalArticle

55 Citations (Scopus)

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.

Original languageEnglish
JournalNucleic acids research
Volume31
Issue number15
Publication statusPublished - Jan 1 2003
Externally publishedYes

Fingerprint

Protein C
Puromycin
Messenger RNA
Proteins
Fluorescein
Ligation
Technology
Tobacco Mosaic Virus
Protein Engineering
5' Untranslated Regions
RNA Stability
Proteome
Triticum
Genotype
Phenotype

All Science Journal Classification (ASJC) codes

  • Genetics

Cite this

Miyamoto-Sato, E., Takashima, H., Fuse, S., Sue, K., Ishizaka, M., Tateyama, S., ... Yanagawa, H. (2003). Highly stable and efficient mRNA templates for mRNA-protein fusions and C-terminally labeled proteins. Nucleic acids research, 31(15).

Highly stable and efficient mRNA templates for mRNA-protein fusions and C-terminally labeled proteins. / Miyamoto-Sato, Etsuko; Takashima, Hideaki; Fuse, Shinichiro; Sue, Kaori; Ishizaka, Masamichi; Tateyama, Seiji; Horisawa, Kenichi; Sawasaki, Tatsuya; Endo, Yaeta; Yanagawa, Hiroshi.

In: Nucleic acids research, Vol. 31, No. 15, 01.01.2003.

Research output: Contribution to journalArticle

Miyamoto-Sato, E, Takashima, H, Fuse, S, Sue, K, Ishizaka, M, Tateyama, S, Horisawa, K, Sawasaki, T, Endo, Y & Yanagawa, H 2003, 'Highly stable and efficient mRNA templates for mRNA-protein fusions and C-terminally labeled proteins.', Nucleic acids research, vol. 31, no. 15.
Miyamoto-Sato E, Takashima H, Fuse S, Sue K, Ishizaka M, Tateyama S et al. Highly stable and efficient mRNA templates for mRNA-protein fusions and C-terminally labeled proteins. Nucleic acids research. 2003 Jan 1;31(15).
Miyamoto-Sato, Etsuko ; Takashima, Hideaki ; Fuse, Shinichiro ; Sue, Kaori ; Ishizaka, Masamichi ; Tateyama, Seiji ; Horisawa, Kenichi ; Sawasaki, Tatsuya ; Endo, Yaeta ; Yanagawa, Hiroshi. / Highly stable and efficient mRNA templates for mRNA-protein fusions and C-terminally labeled proteins. In: Nucleic acids research. 2003 ; Vol. 31, No. 15.
@article{9cb84c567ff04e0ab7c80bcc0ee9da9c,
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.",
author = "Etsuko Miyamoto-Sato and Hideaki Takashima and Shinichiro Fuse and Kaori Sue and Masamichi Ishizaka and Seiji Tateyama and Kenichi Horisawa and Tatsuya Sawasaki and Yaeta Endo and Hiroshi Yanagawa",
year = "2003",
month = "1",
day = "1",
language = "English",
volume = "31",
journal = "Nucleic Acids Research",
issn = "0305-1048",
publisher = "Oxford University Press",
number = "15",

}

TY - JOUR

T1 - Highly stable and efficient mRNA templates for mRNA-protein fusions and C-terminally labeled proteins.

AU - Miyamoto-Sato, Etsuko

AU - Takashima, Hideaki

AU - Fuse, Shinichiro

AU - Sue, Kaori

AU - Ishizaka, Masamichi

AU - Tateyama, Seiji

AU - Horisawa, Kenichi

AU - Sawasaki, Tatsuya

AU - Endo, Yaeta

AU - Yanagawa, Hiroshi

PY - 2003/1/1

Y1 - 2003/1/1

N2 - 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.

AB - 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.

UR - http://www.scopus.com/inward/record.url?scp=0043163705&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0043163705&partnerID=8YFLogxK

M3 - Article

C2 - 12888530

AN - SCOPUS:0043163705

VL - 31

JO - Nucleic Acids Research

JF - Nucleic Acids Research

SN - 0305-1048

IS - 15

ER -