Functional primordial proteins presumably originated from random sequences, but it is not known how frequently functional, or even folded, proteins occur in collections of random sequences. Here we have used in vitro selection of messenger RNA displayed proteins, in which each protein is covalently linked through its carboxy terminus to the 3′ end of its encoding mRNA1, to sample a large number of distinct random sequences. Starting from a library of 6 × 1012 proteins each containing 80 contiguous random amino acids, we selected functional proteins by enriching for those that bind to ATP. This selection yielded four new ATP-binding proteins that appear to be unrelated to each other or to anything found in the current databases of biological proteins. The frequency of occurrence of functional proteins in random-sequence libraries appears to be similar to that observed for equivalent RNA libraries2,3.
Subscribe to Journal
Get full journal access for 1 year
only $3.83 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Rent or Buy article
Get time limited or full article access on ReadCube.
All prices are NET prices.
Roberts, R. W. & Szostak, J. W. RNA-peptide fusions for the in vitro selection of peptides and proteins. Proc. Natl Acad. Sci. USA 94, 12297– 12302 (1997).
Sassanfar, M. & Szostak, J. W. An RNA motif that binds ATP. Nature 364, 550– 553 (1993).
Wilson, D. S. & Szostak, J. W. In vitro selection of functional nucleic acids. Annu. Rev. Biochem. 68, 611– 647 (1999).
Cho, G., Keefe, A. D., Liu, R. L., Wilson, D. S. & Szostak, J. W. Constructing high complexity synthetic libraries of long ORFs using in vitro selection. J. Mol. Biol. 297, 309– 319 (2000).
Wilson, D. S., Keefe, A. D. & Szostak, J. W. In vitro selection of high affinity protein-binding peptides using mRNA display. Proc. Natl Acad. Sci. USA (in the press).
Freedman, L. P. et al. The function and structure of the metal coordination sites within the glucocorticoid receptor DNA binding domain. Nature 334, 543– 546 (1988).
Altschul, S. F. et al. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 25, 3389– 3402 (1997).
Considine, D. M. (ed.) in Van Nostrand's Scientific Encyclopedia 7th edn, 3067 (Van Nostrand Reinhold, New York, 1989).
Liu, R., Barrick, J., Szostak, J. W. & Roberts, R. W. Optimized synthesis of RNA–protein fusions for in vitro protein selection. Methods Enzymol. 318, 268– 293 (2000).
Keefe, A. D. in Current Protocols in Molecular Biology (eds Ausubel, F. M. et al.) Unit 24.5 (Wiley, New York, 2001).
Cadwell, R. C. & Joyce, G. F. Randomization of genes by PCR mutagenesis. PCR Methods Appl. 2, 28– 33 (1992).
Wilson, D. S. & Keefe, A. D. in Current Protocols in Molecular Biology (eds Ausubel, F. M. et al.) Unit 8.3 (Wiley, New York, 2000).
McCafferty, D. G., Lessard, I. A. D. & Walsh, C. T. Mutational analysis of potential zinc-binding residues in the active site of the enterococcal d-Ala-d-Ala dipeptidase VanX. Biochemistry 36, 10498– 10505 (1997).
Jenison, R. D., Gill, S. C., Pardi, A. & Polisky, B. High resolution molecular discrimination by RNA. Science 263, 1425– 1429 (1994).
Wang, Y. & von Hippel, P. H. Escherichia coli transcription termination factor Rho. J. Biol. Chem. 268, 13947– 13955 (1993).
We thank members of the Szostak laboratory and especially D. Wilson, G. Cho, G. Short, J. Pollard, G. Zimmermann, R. Liu, J. Urbach and R. Larralde-Ridaura for their helpful advice. This work was supported in part by the NASA Astrobiology Institute and the NIH. J.W.S. is an investigator at the Howard Hughes Medical Institute.
About this article
Cite this article
Keefe, A., Szostak, J. Functional proteins from a random-sequence library. Nature 410, 715–718 (2001) doi:10.1038/35070613
Nucleic Acid-Barcoding Technologies: Converting DNA Sequencing into a Broad-Spectrum Molecular Counter
Angewandte Chemie International Edition (2019)
Methods and Protocols (2019)
Trends in Genetics (2019)
Superfunneled Energy Landscape of Protein Evolution Unifies the Principles of Protein Evolution, Folding, and Design
Physical Review Letters (2019)