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

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 1 Ab 4 Ab 3 Ab 2 Antibody - Mediated Protein Crystallization

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. ~Fold enrichment # distinct scFvs Enrichment of Library on Leischmania ORFs WB28L3223.250005 EQ370082210005 EQ81007965006 EQ111006181002 EQ13700388<10none EQ15300462100012 EQ16500076<10none EQ179002001003 EQ7850059210003 EQ17470065392000ND EQ196300687720002 EQ20050069515004 EQ21750072345002 EQ219700727610001 EQ221300730720003 EQ223900735220002 Protein IDORF ID

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-16EQ153/scFv-4

9. More EQ153 Complexes EQ153/scFv-B2 EQ153/scFv-B5

10. Immunoaffinity Purification of scFv/EQ37 EQ37/scFv-5EQ37/scFv-3

11. Immunoaffinity Purification of EQ2187 and EQ2005 complexes EQ2175/scFv-6 EQ2005/scFv-9

12. 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

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