Selection and evolution of enzymes from a partially randomized non-catalytic scaffold

Burckhard Seelig, Jack W. Szostak

Research output: Contribution to journalArticlepeer-review

157 Scopus citations

Abstract

Enzymes are exceptional catalysts that facilitate a wide variety of reactions under mild conditions, achieving high rate-enhancements with excellent chemo-, regio- and stereoselectivities. There is considerable interest in developing new enzymes for the synthesis of chemicals and pharmaceuticals and as tools for molecular biology. Methods have been developed for modifying and improving existing enzymes through screening, selection and directed evolution. However, the design and evolution of truly novel enzymes has relied on extensive knowledge of the mechanism of the reaction. Here we show that genuinely new enzymatic activities can be created de novo without the need for prior mechanistic information by selection from a naive protein library of very high diversity, with product formation as the sole selection criterion. We used messenger RNA display, in which proteins are covalently linked to their encoding mRNA, to select for functional proteins from an in vitro translated protein library of >10 independent sequences without the constraints imposed by any in vivo step. This technique has been used to evolve new peptides and proteins that can bind a specific ligand, from both random-sequence libraries and libraries based on a known protein fold. We now describe the isolation of novel RNA ligases from a library that is based on a zinc finger scaffold, followed by in vitro directed evolution to further optimize these enzymes. The resulting ligases exhibit multiple turnover with rate enhancements of more than two-million-fold.

Original languageEnglish (US)
Pages (from-to)828-831
Number of pages4
JournalNature
Volume448
Issue number7155
DOIs
StatePublished - Aug 16 2007

Bibliographical note

Funding Information:
Acknowledgements We thank G. S. Cho for providing the RXR library before its publication along with valuable advice; A. D. Keefe for critical input during the initiation of this project; C. Mueller-Vahl for help with protein expression and purification; G.-P. Zhou and J. J. Chou for NMR measurements; A. D. Keefe, G. S. Cho, G. F. Short, R. Larralde, J. M. Carothers, J. K. Ichida, F. P. Seebeck, S. S. Mansy, C. Del Bianco, D. A. Treco, D. S. Wilson, A. J. Bell, A. Luptak, R. Bruckner and Z. Sachs for helpful discussions. This work was supported by a grant from the NASA Astrobiology Institute. B.S. was supported in part by the Emmy Noether-Programm of the Deutsche Forschungsgemeinschaft. J.W.S. is an Investigator of the Howard Hughes Medical Institute.

Copyright:
Copyright 2015 Elsevier B.V., All rights reserved.

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