Isolation of receptor–ligand pairs by capture of long-lived multivalent interaction complexes

We have combined phage display and array screening for the rapid isolation of pairs of interacting polypeptides. Our strategy, named SAC (selection by avidity capture), is based on the avidity effect, the formation of highly stable complexes formed by multivalent interactions; in our case, between a receptor (multivalently displayed on phage) and a ligand (coexpressed as a multimeric fusion protein). Capture of the long-lived interaction complex allows the isolation of phage bearing cognate interaction pairs, as we demonstrate for a range of interactions, including Ab–antigen pairs and the rapamycin-dependent interaction of FKBP-12 and FRAP. Cognate phage are enriched by SAC up to 1000-fold and interacting pairs can be identified by array screening. Application of SAC to Ab–antigen interactions as a model system yielded over 140 specific Abs to a single antigen and 92 Abs to three different fetal human brain antigens in a single round of SAC each. Our results suggest that SAC should prove useful for the identification and study of receptor–ligand interactions in particular among extracellular proteins, as well as for the rapid generation of specific Abs to multiple antigens.

[1]  C. Borrebaeck.  Tapping the potential of molecular libraries in functional genomics. , 1998, Immunology today.

[2]  H. Lehrach,et al.  Protein microarrays for gene expression and antibody screening. , 1999, Analytical biochemistry.

[3]  T. Tudyka,et al.  Glutathione S‐transferase can be used as a C‐terminal, enzymatically active dimerization module for a recombinant protease inhibitor, and functionally secreted into the periplasm of Escherichia coli , 1997, Protein science : a publication of the Protein Society.

[4]  L. Serrano,et al.  Crystal structure of the abl-SH3 domain complexed with a designed high-affinity peptide ligand: implications for SH3-ligand interactions. , 1998, Journal of molecular biology.

[5]  K. J. Dorrington.  The structural basis for the functional versatility of immunoglobulin G1. , 1978, Canadian journal of biochemistry.

[6]  I. Tomlinson,et al.  Antibody arrays for high-throughput screening of antibody–antigen interactions , 2000, Nature Biotechnology.

[7]  S. Elledge,et al.  Isolation of an AP-1 repressor by a novel method for detecting protein-protein interactions , 1997, Molecular and cellular biology.

[8]  P G Schultz,et al.  Designing small-molecule switches for protein-protein interactions. , 2000, Science.

[9]  G. Whitesides,et al.  A miniaturized arrayed assay format for detecting small molecule-protein interactions in cells. , 1997, Chemistry & biology.

[10]  S. Michnick,et al.  Oligomerization domain-directed reassembly of active dihydrofolate reductase from rationally designed fragments. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[11]  G. Jovanovic,et al.  Filamentous phage infection-mediated gene expression: construction and propagation of the gIII deletion mutant helper phage R408d3. , 1997, Gene.

[12]  D M Crothers,et al.  The influence of polyvalency on the binding properties of antibodies. , 1972, Immunochemistry.

[13]  S. Schreiber,et al.  Identification of an 11-kDa FKBP12-rapamycin-binding domain within the 289-kDa FKBP12-rapamycin-associated protein and characterization of a critical serine residue. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[14]  H. Lehrach,et al.  A method for global protein expression and antibody screening on high-density filters of an arrayed cDNA library. , 1998, Nucleic acids research.

[15]  H. Blau,et al.  Gene expression and cell fusion analyzed by lacZ complementation in mammalian cells. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[16]  Dan S. Tawfik,et al.  Man-made cell-like compartments for molecular evolution , 1998, Nature Biotechnology.

[17]  J. Keith Joung,et al.  Activation of prokaryotic transcription through arbitrary protein–protein contacts , 1997, Nature.

[18]  C. Oker-Blom,et al.  Developments in the use of baculoviruses for the surface display of complex eukaryotic proteins. , 2001, Trends in biotechnology.

[19]  D. Ladant,et al.  A bacterial two-hybrid system based on a reconstituted signal transduction pathway. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[20]  I. Tomlinson,et al.  Methods for generating multivalent and bispecific antibody fragments. , 2000, Methods in enzymology.

[21]  K D Wittrup,et al.  Protein engineering by cell-surface display. , 2001, Current opinion in biotechnology.

[22]  G. Georgiou,et al.  Isolation of high-affinity ligand-binding proteins by periplasmic expression with cytometric screening (PECS) , 2001, Nature Biotechnology.

[23]  G. Winter,et al.  Retargeting serum immunoglobulin with bispecific diabodies , 1997, Nature Biotechnology.

[24]  F. Baneyx.  Recombinant protein expression in Escherichia coli. , 1999, Current opinion in biotechnology.

[25]  H. Bujard,et al.  Independent and tight regulation of transcriptional units in Escherichia coli via the LacR/O, the TetR/O and AraC/I1-I2 regulatory elements. , 1997, Nucleic acids research.

[26]  G. Winter,et al.  Making antibodies by phage display technology. , 1994, Annual review of immunology.

[27]  J W Szostak,et al.  RNA-peptide fusions for the in vitro selection of peptides and proteins. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[28]  D. Smith.  Generating fusions to glutathione S-transferase for protein studies. , 2000, Methods in enzymology.

[29]  P. Luisi,et al.  Protein expression in liposomes. , 1999, Biochemical and biophysical research communications.

[30]  A. Plückthun,et al.  In vitro selection and evolution of functional proteins by using ribosome display. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[31]  A. Skerra,et al.  Sequence analysis and bacterial production of the anti‐c‐myc antibody 9E10: the VH domain has an extended CDR‐H3 and exhibits unusual solubility , 1997, FEBS letters.

[32]  G. Winter,et al.  Mimicking somatic hypermutation: affinity maturation of antibodies displayed on bacteriophage using a bacterial mutator strain. , 1996, Journal of molecular biology.

[33]  Min Li.  Applications of display technology in protein analysis , 2000, Nature Biotechnology.

[34]  S. Fields,et al.  A novel genetic system to detect protein–protein interactions , 1989, Nature.

[35]  H R Hoogenboom,et al.  Multi-subunit proteins on the surface of filamentous phage: methodologies for displaying antibody (Fab) heavy and light chains. , 1991, Nucleic acids research.

[36]  G. Winter,et al.  Screening of phage antibody libraries. , 1996, Methods in enzymology.