Aiping M Zhao

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

M A Dwyer; W Lu; J J Dwyer; A A Kossiakoff (Profiled Author: Wuyuan Lu)

Department of Biochemistry and Molecular Biology, University of Chicago, IL 60637, USA.
Chemistry & biology 2000;7(4):263-74.

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.

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