This is a proposal to establish a proof-of-principle for a vector system to isolate high-affinty antibodies through selective rounds in both yeast and Escherichia coli. Antibodies have quickly become extremely useful as therapeutic medicines to treat a wide-variety of disorders. We propose to develop a means of making the discovery of immunotherapeutics faster and cheaper. In this proposal we describe a system to isolate antigen-specific antibodies in yeast two-hybrid and then quickly evolve them for both higher affinity and specificity using phage display. This new vector system will eliminate the need for any subcloning needed for protein expression, Y2H and phage display. The option for expressing and purifying antibodies directly from E. coli or yeast is a direct application of the described system. The described system will allow for simultaneous testing of cDNA and antibody-antigen interactions in both yeast and E.coi-based systems. Completion of this project will form the basis of a scalable system for a rapid and inexpensive means of affinity-maturation of recombinant antibodies screened in Y2H and increasing our understanding of protein- protein interactions.
Public Health Relevance: Researchers will benefit from an increased understanding of the function of human proteins. Methods are needed that can facilitate this understanding. The immediate objective of our research is to generate a method that will enable researchers to multiplex the screening of affinity reagents. We anticipate using our high-throughput pipeline for producing recombinant antibodies as a means to generate the reagents needed to make this method possible.
Thesaurus Terms: Affinity; Antibodies; Antibodies, Bispecific; Antibody Affinity; Antigenic Determinants; Bifunctional Antibodies; Binding; Binding (Molecular Function); Binding Determinants; Bispecific Antibodies; Cdr; Cell Communication And Signaling; Cell Nucleus; Cell Signaling; Chimera Protein; Chimeric Proteins; Cloning; Cloning Vectors; Complementarity Determining Regions; Complementary Dna; Complimentarity Determining Region; Dna Binding Domain; Dna Rearrangement; Dna, Complementary; Detection; Disease; Disorder; E Coli; Epitopes; Escherichia Coli; Fusion Protein; Gene Rearrangement; Genes; Human; Human, General; Hypervariable Loop; Hypervariable Regions; Immune Globulins; Immunoglobulin Hypervariable Region; Immunoglobulin V; Immunoglobulin Variable Region; Immunoglobulins; Immunoglobulins / Antibodies; Immunotherapeutic Agent; In Vitro; Intracellular Communication And Signaling; Investigators; Libraries; Light; Maintenance; Maintenances; Man (Taxonomy); Man, Modern; Medicine; Messenger Rna; Methods; Moab, Clinical Treatment; Molecular Interaction; Monoclonal Antibodies; Nature; Nuclear; Nucleus; Phage Display; Photoradiation; Plasmids; Polyomavirus Macacae; Polyomavirus Maccacae 1; Promoter; Promoters (Genetics); Promotor; Promotor (Genetics); Protein Motifs, Dna-Binding; Proteins; Proteomics; Rna, Messenger; Reagent; Recombinant Antibody; Reporter Genes; Research; Research Personnel; Researchers; Sv 40; Sv 40 Virus; Sv40; Sv40 Virus; Saccharomyces; Science Of Medicine; Screening Procedure; Signal Transduction; Signal Transduction Systems; Signaling; Simian Vacuolating Virus 40; Simian Virus 40; Single-Stranded Dna; Site; Specificity; System; System, Loinc Axis 4; Testing; Therapeutic; Time; Transcriptional Activation Domain; Two Hybrid; Vacuolating Agent; Variable Region; Variable Region, Ig; Yeast One Hybrid System; Yeast One/Two-Hybrid System; Yeasts; Antigen Antibody Affinity; Base; Biological Signal Transduction; Bsab; Cdna; Combinatorial; Cost; Design; Designing; Direct Application; Disease/Disorder; Gene Cloning; Gene Product; Immunologic Preparation; Immunotherapeutics; Mrna; Protein Expression; Protein Protein Interaction; Public Health Relevance; Screening; Screenings; Transcription Termination; Vacuolating Virus; Vector; Yeast Two Hybrid System
