Efficient Phage Display with Multiple Distinct Non-Canonical Amino Acids Using Orthogonal Ribosome-Mediated Genetic Code Expansion.

Benjamí Oller‐Salvia,Jason W Chin

doi:10.1002/anie.201902658

Abstract

Phage display is a powerful approach for evolving proteins and peptides with new functions, but the properties of the molecules that can be evolved are limited by the chemical diversity encoded. Herein, we report a system for incorporating non‐canonical amino acids (ncAAs) into proteins displayed on phage using the pyrrolysyl‐tRNA synthetase/tRNA pair. We improve the efficiency of ncAA incorporation using an evolved orthogonal ribosome (riboQ1), and encode a cyclopropene‐containing ncAA (CypK) at diverse sites on a displayed single‐chain antibody variable fragment (ScFv), in response to amber and quadruplet codons. CypK and an alkyne‐containing ncAA are incorporated at distinct sites, enabling the double labeling of ScFv with distinct probes, through mutually orthogonal reactions, in a one‐pot procedure. These advances expand the number of functionalities that can be encoded on phage‐displayed proteins and provide a foundation to further expand the scope of phage display applications.

Highlights

We report a system for incorporating non-canonical amino acids into proteins displayed on phage using the pyrrolysyl-tRNA synthetase/tRNA pair
The MjTyrRS/tRNACUA pair has been exclusively used to incorporate amino acids derived from phenylalanine; this precludes the genetic encoding of diverse aliphatic non-canonical amino acids (ncAAs)
Each phage generated using this approach only incorporates a single type of ncAA in response to a single amber codon in the gene of interest; this precludes the incorporation of multiple distinct ncAAs on a single phage, which may facilitate a range of applications, including the selective double labeling of displayed proteins

Summary

Introduction

We improve the efficiency of ncAA incorporation using an evolved orthogonal ribosome (riboQ1), and encode a cyclopropenecontaining ncAA (CypK) at diverse sites on a displayed singlechain antibody variable fragment (ScFv), in response to amber and quadruplet codons. We demonstrate the site-specific incorporation of a cyclopropene-containing ncAA (CypK, Ne-[((2-methylcycloprop2-en-1-yl)methoxy)carbonyl]-l-lysine) into proteins displayed on phage using the PylRS/tRNA pair.

Results

Conclusion