title: Mutational analysis of the major coat protein of M13 identifies residues that control protein display.
abstract: We have reported variants of the M13 bacteriophage major coat protein (P8) that enable high copy display of monomeric and oligomeric proteins, such as human growth hormone and steptavidin, on the surface of phage particles (Sidhu SS, Weiss GA, Wells JA. 2000. High copy display of large proteins on phage for functional selections. J Mol Biol 296:487-495). Here, we explore how an optimized P8 variant (opti-P8) could evolve the ability to efficiently display a protein fused to its N-terminus. Reversion of individual opti-P8 residues back to the wild-type P8 residue identifies a limited set of hydrophobic residues responsible for the high copy protein display. These hydrophobic amino acids bracket a conserved hydrophobic face on the P8 alpha helix thought to be in contact with the phage coat. Mutations additively combine to promote high copy protein display, which was further enhanced by optimization of the linker between the phage coat and the fusion protein. These data are consistent with a model in which protein display-enhancing mutations allow for better packing of the fusion protein into the phage coat. The high tolerance for phage coat protein mutations observed here suggests that filamentous phage coat proteins could readily evolve new capabilities.
authors.affiliation: Department of Protein Engineering, Genentech Inc., South San Francisco, California 94080, USA.
authors.forename: G A
authors.initials: GA
authors.lastname: Weiss
authors.forename: J A
authors.initials: JA
authors.lastname: Wells
authors.forename: S S
authors.initials: SS
authors.lastname: Sidhu
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D002213:Capsid / Q000737:chemistry* / Q000235:genetics; D036022:Capsid Proteins; D004274:DNA, Recombinant; D013006:Growth Hormone / Q000737:chemistry; D006801:Humans; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed
chemical_list: D036022:Capsid Proteins; D004274:DNA, Recombinant; D008565:Membrane Proteins; C025668:coat protein, Bacteriophage M13; D013006:Growth Hormone