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Mark A. Sullivan Structural Genomics of Pathogenic Protozoa Center for Human Genetics and

Application of Phage Antibody Technology to Structural Genomics: Antibody Selection and Complex Formation. Mark A. Sullivan Structural Genomics of Pathogenic Protozoa Center for Human Genetics and Molecular Pediatric Disease University of Rochester.

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Mark A. Sullivan Structural Genomics of Pathogenic Protozoa Center for Human Genetics and

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  1. Application of Phage Antibody Technology to Structural Genomics: Antibody Selection and Complex Formation Mark A. Sullivan Structural Genomics of Pathogenic Protozoa Center for Human Genetics and Molecular Pediatric Disease University of Rochester

  2. Antibody - Mediated Protein Crystallization Ab 1 Ab 2 Goal: Develop a panel of scFvs for each protein produced for evaluation in crystallization trials. The scFvs may also be valuable in defining the role of these proteins in future studies of parasite biology. Ab 4 Ab 3

  3. Antibody Facilitated Crystallization • Majority of purified ORF proteins fail to crystallize • Complexing the targets with an scFv or Fab can improve the chances of crystallization • Reducing protein flexibility • Providing different surface contacts • Known structure of antibodies can facilitate solving of structures • Many antibody variable regions contain multiple methionine residues for selenium incorporation • May be possible to re-engineer the linker to provide additional methionine residues

  4. Requirements for Demonstrating Feasibility of Phage Display Technology • Phage display must yield useful antibodies • Capable of high throughput isolation • Single-chain Fvs must function equivalent to Fabs in crystallization • Direct purification of antibody-target complexes desirable

  5. Essential Elements for Effective Antibody Isolation • Good library diversity • Stable vector system for efficient selection • Minimize deletion of scFvs during enrichment • Good expression and purification of scFvs • Assays for complex formation and effective purification

  6. Enrichment of Library on Leischmania ORFs ~Fold enrichment Protein ID ORF ID # distinct scFvs

  7. Immunoaffinity Purification of scFv/Target Complexes on an Anti-Flag Column - Ca+2 + Ca+2

  8. The First Complexes - Immunoaffinity Purification of EQ153-scFv EQ153/scFv-16 EQ153/scFv-4

  9. Immunoaffinity Purification of scFv/EQ37 EQ37/scFv-5 EQ37/scFv-3 Load FT Elute conc. complex scFv Target Load FT Elute

  10. Immunoaffinity Purification of EQ2187 and EQ2005 complexes EQ2175/scFv-6 EQ2005/scFv-9 scFv Target Load FT Elute scFv Target FT Elute

  11. Unresolved Issues for Complex Preparation • Effect of reducing agents on scFv stability • Concentration of complex • Optimum method • Solubility limits of complexes • Flag epitope accessibility in scFv/target complex • scFv multimers • scFv suitability vs. Fab

  12. Antibody Lab Colleen Shea Laura Bloedorn Qian Zhao SGPP Rochester Solubles Erin Quartley Christina DeVries Julie Babulski Danielle DeRosa Eric Phizicky Acknowledgements

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