An efficient ligation method in the making of an in vitro virus for in vitro protein evolutionReport as inadecuate

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Biological Procedures Online

, Volume 4, Issue 1, pp 49–54

Received: 03 July 2002Revised: 27 August 2002Accepted: 18 September 2002DOI: 10.1251-bpo33

Cite this article as: Tabuchi, I., Soramoto, S., Suzuki, M. et al. Biol Proced Online 2002 4: 49. doi:10.1251-bpo33


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.

Indexing termsligation method evolutionary protein engineering in vitro selection mRNA-protein fusion mRNA display AbbreviationsFITCfluorescein isothicyanate

NTAnitrilotriacetic acid

GFPgreen fluorescent protein

OMe2’-O-methyl ribonucleotide

Published: October 28, 2002

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Author: Ichiro Tabuchi - Sayaka Soramoto - Miho Suzuki - Koichi Nishigaki - Naoto Nemoto - Yuzuru Husimi


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