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Ribosome display: selecting and evolving proteins in vitro that specifically bind to a target

Abstract

Ribosome display is an in vitro selection and evolution technology for proteins and peptides from large libraries1. As it is performed entirely in vitro, there are two main advantages over other selection technologies2,3. First, the diversity of the library is not limited by the transformation efficiency of bacterial cells, but only by the number of ribosomes and different mRNA molecules present in the test tube. Second, random mutations can be introduced easily after each selection round, as no library must be transformed after any diversification step. This allows facile directed evolution of binding proteins over several generations (Box 1). A prerequisite for the selection of proteins from libraries is the coupling of genotype (RNA, DNA) and phenotype (protein). In ribosome display, this link is accomplished during in vitro translation by stabilizing the complex consisting of the ribosome, the mRNA and the nascent, correctly folded polypeptide (Fig. 1). The DNA library coding for a particular library of binding proteins is genetically fused to a spacer sequence lacking a stop codon. This spacer sequence, when translated, is still attached to the peptidyl tRNA and occupies the ribosomal tunnel, and thus allows the protein of interest to protrude out of the ribosome and fold. The ribosomal complexes are allowed to bind to surface-immobilized target. Whereas non-bound complexes are washed away, mRNA of the complexes displaying a binding polypeptide can be recovered, and thus, the genetic information of the binding polypeptides is available for analysis. Here we describe a step-by-step procedure to perform ribosome display selection using an Escherichia coli S30 extract for in vitro translation, based on the work originally described and further refined in our laboratory1. A protocol that makes use of eukaryotic in vitro translation systems for ribosome display4,6,7 is also included in this issue8.

A DNA library in the form of a PCR product ('library'; see Fig. 2 for construct details), coding for binding proteins, is ligated into the ribosome display vector pRDV, thereby genetically fusing it to a tolA spacer sequence in-frame, and providing a promoter and translation initiation region at the 5′ end. The final ribosome display construct is obtained by PCR amplification of both flanking regions and the library insert from the ligated vector. In vitro transcription of this PCR product yields mRNA that is used for in vitro translation. The ribosome stalls at the end of the mRNA and does not release the encoded and properly folded protein because of the absence of a stop codon. The mRNA-ribosome-protein ternary complexes are used for affinity selection on an immobilized target. mRNA of bound complexes is recovered after washing, reverse transcribed and amplified by PCR. Thereby the selected pools of binders can be used directly for the next cycle of ribosome display or analysis of single clones after cloning into expression vectors.

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Figure 2: The construct for ribosome display using E. coli ribosomes.

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Acknowledgements

We thank R. Skirgaila for originally suggesting and testing Phusion polymerase in ribosome display, as well as A. Batyuk, D. Ferrari, T. Huber, P. Martin Killias, P. Parizek, N. Sainz-Pastor and S.R. Wyss-Stoeckle for experimentally checking the protocol in detail and for many helpful suggestions and discussions, as well as former members of the Plückthun laboratory for establishing the protocol.

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Correspondence to Andreas Plückthun.

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A.P. is an inventor on a patent on ribosome display. Molecular Partners AG is using ribosome display commercially.

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Zahnd, C., Amstutz, P. & Plückthun, A. Ribosome display: selecting and evolving proteins in vitro that specifically bind to a target. Nat Methods 4, 269–279 (2007). https://doi.org/10.1038/nmeth1003

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