Expanding the chemical diversity of M13 bacteriophage

Abstract

Bacteriophage M13 virions are very stable nanoparticles that can be modified by chemical and genetic methods. The capsid proteins can be functionalized in a variety of chemical reactions without loss of particle integrity. In addition, Genetic Code Expansion (GCE) permits the introduction of non-canonical amino acids (ncAAs) into displayed peptides and proteins. The incorporation of ncAAs into phage libraries has led to the discovery of high-affinity binders with low nanomolar dissociation constant (K _D) values that can potentially serve as inhibitors. This article reviews how bioconjugation and the incorporation of ncAAs during translation have expanded the chemistry of peptides and proteins displayed by M13 virions for a variety of purposes.

Keywords: antibody fragments; bioconjugation; combinatorial peptide libraries; cross-linking; cyclization; peptide; phage-display; stop codon suppression.

FIGURE 1

Cartoon of an M13 bacteriophage virion, three types of chemically reactive groups, and primary structure of the major capsid protein, pVIII. Each virus particle (A) is 900 nanometers long and 7 nanometers wide and contains one single-stranded, circular DNA molecule, and five copies of pIX (yellow), pVII (white), pIII (purple), and pVI (pink), and ∼2,700 copies of pVIII (blue). Chemical groups that are available for conjugation include the α-amino groups on the N-terminus and ε-amino groups of lysines (K) of pVIII, the carboxylate groups at the C-terminus and aspartic (D) and glutamic (E) acids, and the phenol groups of tyrosine (Y) are shown in violet, orange, and fushia, respectively. The primary structure for mature pVIII (B) is shown with the N-terminus, C-terminus, and residues theoretically capable of being chemically modified highlighted in color. It should be noted that the lysines in the anchoring region (KLFKKFTSKAS) of pVIII may not be sterically accessible for chemical modification.

FIGURE 2

Insertion of ncAAs into peptides or proteins by GCE. (A) An mRNA containing an engineered quadruplet or stop codon for insertion of an ncAA at a designated location in a protein. During translation, the ribosome (purple) decodes the engineered stop codon (B) or quadruplet codon (C) with a tRNA charged with the ncAA (blue). The tRNA anticodon is shown in bold and the growing polypeptide chain is shown in green. (D) Peptide or protein with an ncAA at the desired location.

FIGURE 3

Incorporation of ncAAs into phage-displayed constructs. (A) E. coli cell showing three genomes: phagemid with suppressible mutation (TAG, blue) in the coding region (green) of a protein fused to a truncated form of capsid protein pIII (purple), a plasmid encoding an orthogonal aaRS/tRNA_CUA pair (orange/red), and an M13 helper virus (i.e., K07) encoding full length pIII (black). Each of these genomes carry a different antibiotic resistance marker to allow for selective growth of bacterial cells containing all three. (B) Virions secreted from such cells contain both wild type (black) and recombinant (purple) pIII capsid proteins, the latter of which display the protein of interest (green) incorporating the desired ncAA (blue).

FIGURE 4

Current applications of GCE in phage display. (A) Conjugation of a ligand to an ncAA allows direct binding to the active site of a target enzyme. A ligand (orange) may be conjugated to an ncAA (blue) positioned within a peptide (green) and displayed on pIII of an M13 virion. After multiple rounds of phage-display affinity selections to the target protein, a consensus amino acid sequence flanking the ligand conjugated ncAA will be revealed. The final product is an optimized sequence with strong affinity for the target protein. (B) Macrocycle formation via spontaneous cyclization. The cysteine-reactive ncAA O2bey (O-(2-bromoethyl)-tyrosine, blue) spontaneously cyclizes with a cysteine residue (purple) to form a macrocycle that is displayed on pIII of an M13 virion. (C) Dual fluorophore labeling of a phage-displayed protein. Orthogonal incorporation of the ncAAs PrpF (p-propargyloxy-phenylalanine) and CypK (cyclopropene derivative of lysine) into the variable light chain (V_L, yellow) and variable heavy chain (V_H, orange) regions of a single-chain variable fragment (scFv) of a recombinant antibody. This allows for dual labeling with azide- and tetrazine-fluorophores, respectively, as depicted by the black and white stars.

FIGURE 5

Future applications of GCE in phage display. (A) Epitope steering. If the target protein (cyan) has incorporated a photoactivatable, crosslinking ncAA (pBpa, blue), recovery of virions can be biased toward those that bind to the labeled epitope. (B) Profiling the specificity of domains that recognize peptides that carry post-translational modifications. Phosphoamino acids such as phosphoserine (pS), phosphothreonine (pT), and phosphotyrosine (pY) can be incorporated into phage-displayed combinatorial peptide libraries or recombinant proteins. Affinity selections can be performed with these constructs to elucidate the specificity of protein interaction domains (top) as well as generate affinity reagents that recognize phosphoepitopes (bottom).