An efficient ligation method in the making of an in vitro virus for in vitro protein evolution
Open Access
- 1 June 2002
- journal article
- Published by Springer Nature in Biological Procedures Online
- Vol. 4 (1) , 49-54
- https://doi.org/10.1251/bpo33
Abstract
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.Keywords
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