Skip to main content

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

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

References

  1. 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.

    Article  PubMed  CAS  Google Scholar 

  2. 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.

    Article  PubMed  CAS  Google Scholar 

  3. 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.

    Article  PubMed  CAS  Google Scholar 

  4. Keefe, A.D., Szostak, J.W. Functional proteins from a random-sequence library. Nature 2001;410:715–718.

    Article  PubMed  CAS  Google Scholar 

  5. 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.

    Article  PubMed  CAS  Google Scholar 

  6. 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.

    Article  PubMed  CAS  Google Scholar 

  7. 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.

  8. 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.

    Article  PubMed  CAS  Google Scholar 

  9. Chen, B.P., Hai, T. Expression vectors for affinity purification and radiolabeling of proteins using Escherichia coli as host. Gene 1994;139:73–75.

    Article  PubMed  CAS  Google Scholar 

  10. 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.

    Article  PubMed  CAS  Google Scholar 

  11. Kozak, M. Initiation of translation in prokaryotes and eukaryotes. Gene 1999;234:187–208.

    Article  PubMed  CAS  Google Scholar 

  12. 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.

    Article  CAS  Google Scholar 

  13. Tabuchi, I., Soramoto, S., Nemoto, N., Husimi, Y. An in vitro DNA virus for in vitro protein evolution. FEBS Lett. 2001;508:309–312.

    Article  PubMed  CAS  Google Scholar 

  14. 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.

    Article  PubMed  CAS  Google Scholar 

  15. Lue, Q., Li, X, Sommer, S.S. pK-matched running buffers for gel electrophoresis. Anal.Biochem. 1999;270:112–122.

    Article  Google Scholar 

  16. 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.

    Article  PubMed  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Yuzuru Husimi.

Additional information

Published: October 28, 2002

Rights and permissions

Reprints and permissions

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

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1251/bpo33

Indexing terms