title: Use of periplasmic target protein capture for phage display engineering of tight-binding protein-protein interactions.
abstract: Phage display is a powerful tool to study and engineer protein and peptide interactions. It is not without its limitations, however, such as the requirement for target protein purification and immobilization in a correctly folded state. A protein capture method is described here that allows enrichment of tight-binding protein variants in vivo thereby eliminating the need for target protein purification and immobilization. The linkage of genotype to phenotype is achieved by placing both receptor and ligand encoding genes on the same plasmid. This allows the isolation of the tight-binding ligand-receptor pair complexes after their association in the bacterial periplasm. The interaction between the TEM-1-β-lactamase fused to the gene 3 coat protein displayed on the surface of M13 bacteriophage and the β-lactamse inhibitory protein (BLIP) expressed in soluble form with a signal sequence to export it to the periplasm was used as a model system to test the method. The system was experimentally validated using a previously characterized collection of BLIP alanine mutants with a range of binding affinities for TEM-1 β-lactamase and by isolating tight-binding variants from a library of mutants randomized at residue position Tyr50 in BLIP which contacts β-lactamase.
authors: Department of Biochemistry and Molecular Biology, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030,  USA.
authors: Bartlomiej G
authors: BG
authors: Fryszczyn
authors: Nicholas G
authors: NG
authors: Brown
authors: Wanzhi
authors: W
authors: Huang
authors: Miriam A
authors: MA
authors: Balderas
authors: Timothy
authors: T
authors: Palzkill
mesh_terms: D000595:Amino Acid Sequence; D017104:Bacteriophage M13 / Q000235:genetics; D004791:Enzyme Inhibitors / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D019151:Peptide Library; D033903:Periplasmic Proteins / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D015202:Protein Engineering / Q000379:methods*; D065093:beta-Lactamase Inhibitors; D001618:beta-Lactamases / Q000235:genetics / Q000378:metabolism*
chemical_list: D004791:Enzyme Inhibitors; D029968:Escherichia coli Proteins; D019151:Peptide Library; D033903:Periplasmic Proteins; D065093:beta-Lactamase Inhibitors; D001618:beta-Lactamases; C034413:beta-lactamase TEM-1