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Gustav\'s Master\'s Degree Defense, 2004
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En Route To Peptide Based Vaccination
Elucidation Of Two HLA-B*1501 Structures And Establishment Of ANovel Mass Spectrometry MHC Disulfide Bond Configuration Assay
Gustav Roder, Dec. 2004
Project Corner StonesToday’s Agenda
MHC-I in Cellular ImmunologyCytotoxic T Cell Response
Apoptosis – Cell Death
MHC-I Protective Immunology
MHC-I in Cellular ImmunologyAntigen Processing
Immunology, Biochemistry and BioinformaticsMaking Vaccine Peptide Candidates
Cloning Expression Purification Active HCs
MHC-I Heavy Chain Manufacturing
Generation of Binding Data
Prediction of Peptide Binders
HC Refolding ELISA KD
KDQBC 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 specificitymatrices. Immunogenetics 55:797-810
Making the MHC-I Heavy ChainsGenetic Manipulation – An Overview
Making the MHC-I Heavy ChainsGenetic Manipulation – The Construct
Making the MHC-I Heavy ChainsGenetic Manipulation – QuikChange Mutagenesis
Making the MHC-I Heavy ChainsProduction – Expression and Purification
Purification by IMAC
Making the MHC-I Heavy ChainsProduction – Expression and Purification
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 ChainsProduction – Dose Response by ELISA
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*5801Why?
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*5801Making the Construct
2m-B*5801Expression and Purification
2m PCR HC-Vector PCR HC-Vector PCR
B2m-B*5801-HB Colony PCR
69oC 69oC (58-68)oC
2m-B*5801Expression and Purification
Fermentation IMAC Purification IEX Purification
2m-B*5801Testing the Functionality
Does 2m-B*5801 fold correctly and is it independent of external 2m
3M 2m1M pep
20nM 2m-B*5801HB2nM B*5801HB
Dependent on peptide and external 2m
MHC DSB MS AssayWhy?
Active? Active?
Why do we need ‘just another’ assay
MHC DSB MS AssayThe Basic Principle
MHC DSB MS AssayTrypsin Digestion Optimization
Assay Conditions: •trypsin/substrate ratio 1.5/100•2M urea, 25mM Tris-HCl, pH8.0•2hrs at 37C
% H
C L
oss
MHC DSB MS AssayHLA-A*0206-HB
MHC DSB MS AssaySumming Up
MHC DSB MS AssayFunctional DSB Isomer
MHC DSB MS AssayFunctional DSB Isomer
MHC DSB MS AssaySumming Up
MHC DSB MS AssayConclusion
Gorman, J. J. et al. 2002. Protein disulfide bond determination by mass spectrometry. Mass Spectrom. Rev. 21:183-216
• This assay is consistent with peptide binding data from RIA
• This assay does NOT need any addition of 2m or peptide
• This assay gets results faster than does ELISA and RIA
• This assay is capable of doing finger print analysis
HLA-B*1501 Crystal StructuresMaking the MHC Complexes
Denatured MHC Heavy ChainFolded ß2-microglobulin Peptide
Mixing Pot
Size Exclusion Chromatography
Concentration > 3 mg/mL
HLA-B*1501 Crystal StructuresMaking the Crystals
QuickTime™ and aTIFF (LZW) decompressor
are needed to see this picture.
Hanging Drops
MHC-I Complex
HLA-B*1501 Crystal StructuresFrom Diffraction to Model
Model Building
HLA-B*1501 Crystal StructuresProcess Flowchart
HLA-B*1501 Crystal StructuresOverall Structure
HLA-B*1501 Crystal StructuresPeptide Conformations
ILGPPGSVY
LEKARGSTY
Epstein-Barr virusnuclear protein 3EBNA3A (406-414)
Human ubiquitinConjugating enz.(83-91)
HLA-B*1501 Crystal StructuresILGPPGSVY Binding Pockets
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 LamberthLise-Lotte NielsenSune JustesenJeanette NielsenAnne SchmiegelowChristian LeisnerChristina Sylvester-HvidHenrik FerreKirstine Grandahl
…
And the rest!
Michael Gajhede
Thomas BlicherOle KristensenBritt Johannessen
Department Of Medical Anatomy
Mogens Cläesson
Thank You!