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Biological Procedures Online

Open Access

An efficient ligation method in the making of an in vitro virus for in vitro protein evolution

  • Ichiro Tabuchi1, 2, 3,
  • Sayaka Soramoto1,
  • Miho Suzuki1,
  • Koichi Nishigaki1,
  • Naoto Nemoto2 and
  • Yuzuru Husimi1Email author
Biological Procedures Online4:41049

Received: 3 July 2002

Accepted: 18 September 2002


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 terms

ligation methodevolutionary protein engineering in vitro selectionmRNA-protein fusionmRNA display