Presentation at Immunologisk Selskab, 2006

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A presentation at the Immunologisk Selskab meeting in 2006 about Tapasin and MHC-I antigen presentation

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Presentation at Immunologisk Selskab, 2006

  1. 1. Agenda – IS 2006.05.18 <ul><li>Advertisement: My PhD project </li></ul><ul><li>Why All this? - The Human MHC Project </li></ul><ul><li>Production of recombinant HLA complexes </li></ul><ul><li>Crystallization and structure determination </li></ul><ul><li>3D modeling and structure based affinity prediction </li></ul>
  2. 2. Advertisement: My PhD project Gustav Røder Panum Institute (by Søren Buus) University of Copenhagen Tapasin mediated receptor editing and quality control of major histocompatibility complex class I molecules CONTACT ME: [email_address] OR http://www.tapasin.com
  3. 3. The Human MHC Project <ul><li>More than 1100 MHC-I alleles are currently discovered and sequenced </li></ul><ul><li>Determine peptide specificity for representative MHC-I alleles </li></ul><ul><li>Divide MHC-I molecules into representative supertypes covering the world population </li></ul><ul><li>Knowing some of the peptide binding specificity; being able to predict the rest </li></ul>Buus, S. et al., Tissue Antigens (2003) 62:378
  4. 4. The Human MHC Project Cloning Expression Purification Active HCs MHC-I Heavy Chain Manufacturing
  5. 5. Production of recombinant HLA complexes
  6. 6. Production of recombinant HLA complexes
  7. 7. Production of recombinant HLA complexes Jumping from one MHC-I allele to another by introducing mutations: These constructs are then ‘ codon optimized’ for efficient Expression in E.coli
  8. 8. Production of recombinant HLA complexes Purification by IMAC
  9. 9. Production of recombinant HLA complexes Ferre et al . Protein Sci. 2003, 12:551 Purification by HIC Fractions tested in RIA or ELISA
  10. 10. Production of recombinant HLA complexes Denatured MHC Heavy Chain Folded ß2-microglobulin Peptide Mixing Pot Size Exclusion Chromatography Concentration > 3 mg/mL
  11. 11. Crystallization and structure determination Hanging Drops MHC-I Complex
  12. 12. Crystallization and structure determination Model Building
  13. 13. Crystallization and structure determination
  14. 14. Crystallization and structure determination
  15. 15. Crystallization and structure determination ILGPPGSVY LEKARGSTY Epstein-Barr virus nuclear protein 3 EBNA3A (406-414) Human ubiquitin Conjugating enz. (83-91)
  16. 16. Modeling and structure based affinity prediction
  17. 17. Acknowledgements IMMI: Lotte Nielsen, Sune Justesen, Kasper Lamberth, Søren Buus DFH: Thomas Blicher, Ole Kristensen, Jette Kastrup, Michael Gajhede

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