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

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

<p>The <it>&ldquo;in vitro</it> virus&rdquo; is a molecular construct to perform evolutionary protein engineering. The &ldquo;virion (=viral particle)&rdquo; (mRNA-peptide fusion), is made by bonding a nascent protein with its coding mRNA via puromycin in a test tub...

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Main Authors: Tabuchi Ichiro, Soramoto Sayaka, Suzuki Miho, Nishigaki Koichi, Nemoto Naoto, Husimi Yuzuru
Format: Article
Language:English
Published: BMC 2002-01-01
Series:Biological Procedures Online
Subjects:
methods
protein engineering
Online Access:http://www.biologicalprocedures.com/bpo/arts/1/33/m33.htm
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spelling doaj-ac994b21aade4d9f99da54dc8aeb183a2020-11-25T00:16:48ZengBMCBiological Procedures Online1480-92222002-01-0141495410.1251/bpo33An Efficient Ligation Method in the Making of an in vitro Virus for in vitro Protein Evolution Tabuchi IchiroSoramoto SayakaSuzuki MihoNishigaki KoichiNemoto NaotoHusimi Yuzuru<p>The <it>&ldquo;in vitro</it> virus&rdquo; is a molecular construct to perform evolutionary protein engineering. The &ldquo;virion (=viral particle)&rdquo; (mRNA-peptide fusion), is made by bonding a nascent protein with its coding mRNA via puromycin in a test tube for <it>in vitro</it> 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 <it>in vitro</it> virus with this &ldquo;viral genome&rdquo; was demonstrated.http://www.biologicalprocedures.com/bpo/arts/1/33/m33.htmmethodsprotein engineering
collection DOAJ
language English
format Article
sources DOAJ
author Tabuchi Ichiro
Soramoto Sayaka
Suzuki Miho
Nishigaki Koichi
Nemoto Naoto
Husimi Yuzuru
spellingShingle 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
Biological Procedures Online
methods
protein engineering
author_facet Tabuchi Ichiro
Soramoto Sayaka
Suzuki Miho
Nishigaki Koichi
Nemoto Naoto
Husimi Yuzuru
author_sort Tabuchi Ichiro
title 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_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_sort efficient ligation method in the making of an in vitro virus for in vitro protein evolution
publisher BMC
series Biological Procedures Online
issn 1480-9222
publishDate 2002-01-01
description <p>The <it>&ldquo;in vitro</it> virus&rdquo; is a molecular construct to perform evolutionary protein engineering. The &ldquo;virion (=viral particle)&rdquo; (mRNA-peptide fusion), is made by bonding a nascent protein with its coding mRNA via puromycin in a test tube for <it>in vitro</it> 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 <it>in vitro</it> virus with this &ldquo;viral genome&rdquo; was demonstrated.
topic methods
protein engineering
url http://www.biologicalprocedures.com/bpo/arts/1/33/m33.htm
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