An Efficient Ligation Method in the Making of an in vitro Virus for in vitro Protein Evolution

The “in vitro virus” is a molecular construct to perform evolutionary protein engineering. The “virion (=viral particle)” (mRNA-peptide fusion), is made by bonding a nascent protein with its coding mRNA via puromycin in a test tube for in vitro translation. In this work, the puromycin-linker was att...

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Detalles Bibliográficos
Autores principales: Tabuchi, Ichiro, Soramoto, Sayaka, Suzuki, Miho, Nishigaki, Koichi, Nemoto, Naoto, Husimi, Yuzuru
Formato: Texto
Lenguaje:English
Publicado: Biological Procedures Online 2002
Materias:
Research Article
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC145556/
https://www.ncbi.nlm.nih.gov/pubmed/12734569
http://dx.doi.org/10.1251/bpo33
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author Tabuchi, Ichiro
Soramoto, Sayaka
Suzuki, Miho
Nishigaki, Koichi
Nemoto, Naoto
Husimi, Yuzuru
author_facet Tabuchi, Ichiro
Soramoto, Sayaka
Suzuki, Miho
Nishigaki, Koichi
Nemoto, Naoto
Husimi, Yuzuru
author_sort Tabuchi, Ichiro
collection PubMed
description The “in vitro virus” is a molecular construct to perform evolutionary protein engineering. The “virion (=viral particle)” (mRNA-peptide fusion), is made by bonding a nascent protein with its coding mRNA via puromycin in a test tube for in vitro translation. In this work, the puromycin-linker was attached to mRNA using the Y-ligation, which was a method of two single-strands ligation at the end of a double-stranded stem to make a stem-loop structure. This reaction gave a yield of about 95%. We compared the Y-ligation with two other ligation reactions and showed that the Y-ligation gave the best productivity. An efficient amplification of the in vitro virus with this “viral genome” was demonstrated.
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spelling pubmed-1455562003-04-15 An Efficient Ligation Method in the Making of an in vitro Virus for in vitro Protein Evolution Tabuchi, Ichiro Soramoto, Sayaka Suzuki, Miho Nishigaki, Koichi Nemoto, Naoto Husimi, Yuzuru Biol Proced Online Research Article The “in vitro virus” is a molecular construct to perform evolutionary protein engineering. The “virion (=viral particle)” (mRNA-peptide fusion), is made by bonding a nascent protein with its coding mRNA via puromycin in a test tube for in vitro translation. In this work, the puromycin-linker was attached to mRNA using the Y-ligation, which was a method of two single-strands ligation at the end of a double-stranded stem to make a stem-loop structure. This reaction gave a yield of about 95%. We compared the Y-ligation with two other ligation reactions and showed that the Y-ligation gave the best productivity. An efficient amplification of the in vitro virus with this “viral genome” was demonstrated. Biological Procedures Online 2002-10-28 /pmc/articles/PMC145556/ /pubmed/12734569 http://dx.doi.org/10.1251/bpo33 Text en Copyright © October 10, 2002, I Tabuchi et al. Published in Biological Procedures Online under license from the authors. Copying, printing, redistribution and storage permitted.
spellingShingle Research Article
Tabuchi, Ichiro
Soramoto, Sayaka
Suzuki, Miho
Nishigaki, Koichi
Nemoto, Naoto
Husimi, Yuzuru
An Efficient Ligation Method in the Making of an in vitro Virus for in vitro Protein Evolution
title An Efficient Ligation Method in the Making of an in vitro Virus for in vitro Protein Evolution
title_full An Efficient Ligation Method in the Making of an in vitro Virus for in vitro Protein Evolution
title_fullStr An Efficient Ligation Method in the Making of an in vitro Virus for in vitro Protein Evolution
title_full_unstemmed An Efficient Ligation Method in the Making of an in vitro Virus for in vitro Protein Evolution
title_short An Efficient Ligation Method in the Making of an in vitro Virus for in vitro Protein Evolution
title_sort efficient ligation method in the making of an in vitro virus for in vitro protein evolution
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC145556/
https://www.ncbi.nlm.nih.gov/pubmed/12734569
http://dx.doi.org/10.1251/bpo33
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