- Open Access
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)
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.
- FITC :
- NTA :
- GFP :
green fluorescent protein
- OMe :
Nemoto, N., Miyamoto-Sato, E., Husimi, Y., Yanagawa, H. In vitro virus: bonding of mRNA bearing puromycin at the 3′-terminal end to the C-terminal end of its encoded protein on the ribosome in vitro. FEBS Lett. 1997;414:405–408.
Roberts, R.W., Szostak, J.W. RNA-peptide fusions for the in vitro selection of peptides and proteins. Proc.Natl.Acad.Sci. USA 1997;94:12297–12302.
Wilson, D.S., Keefe, A.D., Szostak, J.W. The use of mRNA display to select high-affinity protein-binding peptides. Proc.Natl.Acad.Sci.USA 2001;98:3750–3755.
Keefe, A.D., Szostak, J.W. Functional proteins from a random-sequence library. Nature 2001;410:715–718.
Hammond, P.W., Alpin, J., Rise, C.E., Wright, M., Kreider, B.L. In vitro selection and characterization of Bcl-X(L)-binding proteins from a mix of tissue-specific Mrna display libraries. J Biol Chem. 2001;276:20898–20906.
Liu, R., Barrick, J.E, Szostak, J.W., Roberts, R.W. Optimized synthesis of RNA-protein fusions for in vitro protein selection. Methods Enzymol. 2000;318:268–293.
Kurz, M., Gu, K., Lohse, P.A. Psoralen photo-crosslinked mRNA-puromycin conjugates: a novel template for the rapid and facile preparation of mRNA-protein fusions. Nucleic Acids Res. 2000;28:e83.
Nishigaki, K., Taguchi, K., Kinoshita, Y., Aita, T., Husimi, Y. Y-ligation: an efficient method for ligating single-stranded DNAs and RNAs with T4 RNA ligase. Mol Divers. 1998;4:187–190.
Chen, B.P., Hai, T. Expression vectors for affinity purification and radiolabeling of proteins using Escherichia coli as host. Gene 1994;139:73–75.
Gallie, D.R., Watts, J.W. Identification of the motifs within the tobacco mosaic virus 5′-leader responsible for enhancing translation. Nucleic Acids Res. 1992;20:4631–4638.
Kozak, M. Initiation of translation in prokaryotes and eukaryotes. Gene 1999;234:187–208.
Hochuli, E., Bannwarth, W., Döbeli, H., Gentz, R. Stüber, D. Genetic approach to facilitate purification of recombinant proteins with a novel metal chelate adsorbent. Biotechnology 1988;6:1321–1325.
Tabuchi, I., Soramoto, S., Nemoto, N., Husimi, Y. An in vitro DNA virus for in vitro protein evolution. FEBS Lett. 2001;508:309–312.
Schagger, H., Jagow, G.V. Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal.Biochem. 1987;166:368–379.
Lue, Q., Li, X, Sommer, S.S. pK-matched running buffers for gel electrophoresis. Anal.Biochem. 1999;270:112–122.
Barrick, J.E., Takahashi, T.T., Balakin, A., Roberts, R.W. Selection of RNA-binding peptides using mRNA-peptide fusions. Methods 2001;23:287–293.
Published: October 28, 2002
About this article
Cite this article
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
- ligation method
- evolutionary protein engineering
- in vitro selection
- mRNA-protein fusion
- mRNA display