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An efficient ligation method in the making of an in vitro virus for in vitro protein evolution
Biological Procedures Online volume 4, pages 49–54 (2002)
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.
Abbreviations
- FITC :
-
fluorescein isothicyanate
- NTA :
-
nitrilotriacetic acid
- GFP :
-
green fluorescent protein
- OMe :
-
2’-O-methyl ribonucleotide
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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). https://doi.org/10.1251/bpo33
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DOI: https://doi.org/10.1251/bpo33