MSc Defense, 2004

En Route To Peptide Based Vaccination Elucidation Of Two HLA-B*1501 Structures And Establishment Of A Novel Mass Spectrometry MHC Disulfide Bond Configuration Assay Gustav Roder, Dec. 2004

Project Corner Stones ,[object Object]

MHC-I in Cellular Immunology ,[object Object],Apoptosis – Cell Death MHC-I Protective Immunology

MHC-I in Cellular Immunology ,[object Object]

Immunology, Biochemistry and Bioinformatics ,[object Object],Cloning Expression Purification Active HCs MHC-I Heavy Chain Manufacturing Generation of Binding Data Prediction of Peptide Binders HC Refolding ELISA K D K D QBC ANN Novel Peptides Some Cooperation Results Sylvester-Hvid C , Nielsen M, Lamberth K , Roder G , Justesen S , Lundegaard C, Worning P, Thomadsen H, Lund O, Brunak S, Buus S . 2004. SARS CTL vaccine candidates; HLA supertype-, genome-wide scanning and biochemical validation. Tissue Antigens 63 :395-400 Lund O, Nielsen M, Kesmir C, Petersen AG, Lundegaard C, Worning P, Sylvester-Hvid C, Lamberth K, Roder G, Justesen S, Buus S, Brunak S. 2004. Definition of supertypes for HLA molecules using clustering of specificity matrices. Immunogenetics 55 :797-810

Making the MHC-I Heavy Chains ,[object Object]

Making the MHC-I Heavy Chains ,[object Object],Purification by IMAC

Making the MHC-I Heavy Chains ,[object Object],Ferre et al . Protein Sci. 2003, 12:551 Melander et al . J.Chromatogr. 1984, 317:67 Purification by HIC Fractions tested in RIA or ELISA

Making the MHC-I Heavy Chains ,[object Object],Testing of Peptide Binding Capability HC Dose-Response in Quantitative ELISA Sylvester-Hvid, C. et al . 2002. Establishment of a quantitative ELISA capable of determining peptide - MHC class I interaction. Tissue Antigens 59 :251-258

 2m-B*5801 ,[object Object],Potential Advantages : Previously : Sylvester-Hvid, C. et al . 1999. A Single-Chain Fusion Molecule Consisting of Peptide, Major Histocompatibility Gene Complex Class I Heavy Chain and b2-Microglobulin Can Fold Partially Correctly, but Binds Peptide Inefficiently. Tissue Antigens 59 :251-258 On-site  2m mediated refolding of HC No need for  2m production

 2m-B*5801 ,[object Object]

 2m-B*5801 ,[object Object], 2m PCR HC-Vector PCR HC-Vector PCR B2m-B*5801-HB Colony PCR 69 o C 69 o C (58-68) o C

 2m-B*5801 ,[object Object],Fermentation IMAC Purification IEX Purification

 2m-B*5801 ,[object Object],Does  2m-B*5801 fold correctly and is it independent of external  2m 3  M  2m 1  M pep 20nM  2m-B*5801HB 2nM B*5801HB Dependent on peptide and external  2m

MHC DSB MS Assay ,[object Object],Active? Active? Why do we need ‘just another’ assay

MHC DSB MS Assay ,[object Object]

MHC DSB MS Assay ,[object Object],[object Object],[object Object],[object Object],[object Object],% HC Loss

MHC DSB MS Assay ,[object Object],Gorman, J. J. et al . 2002. Protein disulfide bond determination by mass spectrometry. Mass Spectrom. Rev. 21 :183-216 ,[object Object],[object Object],[object Object],[object Object]

HLA-B*1501 Crystal Structures ,[object Object],Denatured MHC Heavy Chain Folded ß2-microglobulin Peptide Mixing Pot Size Exclusion Chromatography Concentration > 3 mg/mL

HLA-B*1501 Crystal Structures ,[object Object],Hanging Drops MHC-I Complex

HLA-B*1501 Crystal Structures ,[object Object],Model Building

HLA-B*1501 Crystal Structures ,[object Object]

HLA-B*1501 Crystal Structures ,[object Object],ILGPPGSVY LEKARGSTY Epstein-Barr virus nuclear protein 3 EBNA3A (406-414) Human ubiquitin Conjugating enz. (83-91)

HLA-B*1501 Crystal Structures ,[object Object],Matsumura, M. et al . 1992. Emerging principles for the recognition of peptide antigens by MHC class I molecules. Science. 257 :927-934 A pocket

Acknowlegdements Department Of Medical Microbiology And Immunology Department Of Medicinal Chemistry Søren Buus Kasper Lamberth Lise-Lotte Nielsen Sune Justesen Jeanette Nielsen Anne Schmiegelow Christian Leisner Christina Sylvester-Hvid Henrik Ferre Kirstine Grandahl … And the rest! Michael Gajhede Thomas Blicher Ole Kristensen Britt Johannessen Department Of Medical Anatomy Mogens Cläesson Thank You!

MSc Defense, 2004

Recommended

Recommended

More Related Content

What's hot

What's hot (20)

Similar to MSc Defense, 2004

Similar to MSc Defense, 2004 (20)

More from groder

More from groder (7)

MSc Defense, 2004