Biosynthetic phage display: a novel protein engineering tool combining chemical and genetic diversity

Background: Molecular diversity in nature is developed through a combinatio n of genetic and chemical elements. We have developed a method that permits selective manipulation of both these elements in one protein engineering t ool. It combines the ability to introduce non-natural amino acids into a pr otein using native chemical ligation with exhaustive targeted mutagenesis o f 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 ch emically synthesized with a non-natural amino acid at position 25, The rema ining carboxy-terminal fragment was expressed as a 30-residue peptide exten sion 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 pack ing of the introduced non-natural amino acid, residues 52 and 54 in the car boxy-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-natura l amino acid a significant number of residues (unexpected because of stereo chemical and charge criteria) were observed in these positions. Conclusions: Peptide synthesis and phage-display mutagenesis can be combine d to produce a very powerful protein engineering tool. The physical propert ies of the environment surrounding the introduced non-natural residue can b e selected for by evaluating all possible combinations of amino acid types at a targeted set of sites using phage-display mutagenesis.