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Selection of proteins with desired properties from natural proteome libraries using mRNA display

Nature Protocols volume 6, pages 1163–1182 (2011)Cite this article

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Abstract

mRNA display is a powerful yet challenging in vitro selection technique that can be used to identify proteins with desired properties from both natural proteome and combinatorial polypeptide libraries. The physical conjugation between a protein and its own RNA presents unique challenges in manipulating the displayed proteins at a low nanomolar scale in an RNase-free environment. The following protocol outlines the generation of cDNA libraries derived from natural organisms as well as the steps required for generation of mRNA-protein fusion molecules, in vitro functional selection and regeneration of the selected cDNA library. The selection procedures for the identification of protease substrates and Ca2+-dependent calmodulin-binding proteins from natural cDNA libraries are presented as examples. The method can be generally applied to the identification of protein sequences with desired properties from various natural proteome libraries. One round of mRNA display–based selection can be accomplished in ∼7 d.

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Figure 1: Flowchart and timing of the experimental steps required for one round of mRNA display–based selection.
Figure 2: Scheme for the selection of conditional (Ca2+-dependent) target-binding partners from an mRNA-displayed proteome library.
Figure 3: Scheme for the selection of the downstream substrates of a protease of interest from an mRNA-displayed proteome library.
Figure 4: cDNA library distribution and fusion formation.
Figure 5: Representative oligo(dT) purification of mRNA-protein fusion molecules from counting radioactivity present in the collected fractions.
Figure 6: Representative anti-FLAG purification of mRNA-protein fusion molecules from radioactivity present in the collected fractions.
Figure 7: Enrichment data for two different mRNA-display-based selections.
Figure 8: Functional validation of proteins identified from mRNA display–based selections.

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Acknowledgements

This work was supported by American Cancer Society Research Scholar Grant RSG-06-073 (to R.L.) and National Institutes of Health Grants NS047650, CA119343 and DA025702 (to R.L.).

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Authors and Affiliations

  1. Eshelman School of Pharmacy and Carolina Center for Genome Sciences, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA

    Steven W Cotten, Jianwei Zou, C Alexander Valencia & Rihe Liu

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  1. Steven W Cotten
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Contributions

R.L. designed the experiments and supervised the projects. S.W.C., J.Z., C.A.V. and R.L. conducted the experiments and analyzed the data. S.W.C., J.Z., C.A.V. and R.L. wrote the paper.

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Correspondence to Rihe Liu.

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Cotten, S., Zou, J., Valencia, C. et al. Selection of proteins with desired properties from natural proteome libraries using mRNA display. Nat Protoc 6, 1163–1182 (2011). https://doi.org/10.1038/nprot.2011.354

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