mRNA display for the selection and evolution of enzymes from in vitro-translated protein libraries

Abstract

The mRNA display technology enables the in vitro selection and directed evolution of functional proteins from libraries of more than 1012 different mutants in a single test tube. The size of these libraries is well beyond the limit of screening technologies and of most in vivo and in vitro selection methods. The mRNA display technology has been used to select peptides and proteins that bind to a specific ligand, as well as novel enzymes. This protocol details the procedure to produce mRNA-displayed proteins (3 d) and to subject them to a selection and evolution of enzymes for bond-forming reactions (4–10 weeks). This method is demonstrated by the generation of new RNA ligase enzymes.

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Figure 1: Overview scheme of the selection of enzymes for bond-forming reactions by mRNA display.
Figure 2: DNA library design for an mRNA display selection.
Figure 3: Selection of enzymes catalyzing a bond-forming reaction of two substrates, A and B.
Figure 4: Autoradiogram of an electrophoresis gel of radiolabeled mRNA-displayed proteins.
Figure 5: Selection progress.

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Acknowledgements

The author thanks J.W. Szostak and members of the Szostak lab for their advice and help. The author is supported in part by the National Aeronautics and Space Administration under Agreement No. NNX09AH70A issued through the NASA Astrobiology Institute/Ames Research Center.

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Correspondence to Burckhard Seelig.

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Seelig, B. mRNA display for the selection and evolution of enzymes from in vitro-translated protein libraries. Nat Protoc 6, 540–552 (2011). https://doi.org/10.1038/nprot.2011.312

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