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 language | English |
|---|---|
| Journal | Nucleic acids research |
| Volume | 31 |
| Issue number | 15 |
| Publication status | Published - Jan 1 2003 |
| Externally published | Yes |
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All Science Journal Classification (ASJC) codes
- Genetics
Cite this
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 journal › Article
}
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 -