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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 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.



fluorescein isothicyanate


nitrilotriacetic acid


green fluorescent protein

OMe :

2’-O-methyl ribonucleotide


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Correspondence to Yuzuru Husimi.

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Published: October 28, 2002

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Tabuchi, I., Soramoto, S., Suzuki, M. et al. An efficient ligation method in the making of an in vitro virus for in vitro protein evolution. Biol Proced Online 4, 49–54 (2002).

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Indexing terms

  • ligation method
  • evolutionary protein engineering
  • in vitro selection
  • mRNA-protein fusion
  • mRNA display