title: Biosynthetic phage display: a novel protein engineering tool combining chemical and genetic diversity.
abstract: BACKGROUND
  Molecular diversity in nature is developed through a combination of genetic and chemical elements. We have developed a method that permits selective manipulation of both these elements in one protein engineering tool. It combines the ability to introduce non-natural amino acids into a protein using native chemical ligation with exhaustive targeted mutagenesis of the protein via phage-display mutagenesis.
  
  
  RESULTS
  A fully functional biosynthetic version of the protease inhibitor eglin c was constructed. The amino-terminal fragment (residues 8-40) was chemically synthesized with a non-natural amino acid at position 25. The remaining carboxy-terminal fragment was expressed as a 30-residue peptide extension of gIIIp or gVIIIp on filamentous phage in a phage-display mutagenesis format. Native chemical ligation was used to couple the two fragments and produced a protein that refolded to its active form. To facilitate the packing of the introduced non-natural amino acid, residues 52 and 54 in the carboxy-terminal fragment were fully randomized by phage-display mutagenesis. Although the majority of the observed solutions for residues 52 and 54 were hydrophobic - complementing the stereochemistry of the introduced non-natural amino acid - a significant number of residues (unexpected because of stereochemical and charge criteria) were observed in these positions.
  
  
  CONCLUSIONS
  Peptide synthesis and phage-display mutagenesis can be combined to produce a very powerful protein engineering tool. The physical properties of the environment surrounding the introduced non-natural residue can be selected for by evaluating all possible combinations of amino acid types at a targeted set of sites using phage-display mutagenesis.
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Chicago, IL 60637, USA.
authors.forename: M A
authors.initials: MA
authors.lastname: Dwyer
authors.forename: W
authors.initials: W
authors.lastname: Lu
authors.forename: J J
authors.initials: JJ
authors.lastname: Dwyer
authors.forename: A A
authors.initials: AA
authors.lastname: Kossiakoff
mesh_terms: D017104:Bacteriophage M13 / Q000378:metabolism; D007737:Kynurenine / Q000737:chemistry; D008958:Models, Molecular; D016296:Mutagenesis; D019151:Peptide Library; D010455:Peptides / Q000138:chemical synthesis; D015202:Protein Engineering / Q000379:methods*; D017510:Protein Folding; D011506:Proteins; D011994:Recombinant Proteins / Q000737:chemistry; D015843:Serpins / Q000096:biosynthesis* / Q000138:chemical synthesis / Q000235:genetics; D014633:Valine / Q000031:analogs & derivatives / Q000737:chemistry; D014764:Viral Proteins / Q000378:metabolism
chemical_list: D019151:Peptide Library; D010455:Peptides; D011506:Proteins; D011994:Recombinant Proteins; D015843:Serpins; D014764:Viral Proteins; C028656:eglin proteinase inhibitors; D007737:Kynurenine; C005313:norvaline; D014633:Valine