presentation at immunologisk selskab, 2006
DESCRIPTION
A presentation at the Immunologisk Selskab meeting in 2006 about Tapasin and MHC-I antigen presentationTRANSCRIPT
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Maj 2006 IS Årsmøde Århus
Agenda – IS 2006.05.18
• Advertisement: My PhD project
• Why All this? - The Human MHC Project
• Production of recombinant HLA complexes
• Crystallization and structure determination
• 3D modeling and structure based affinity prediction
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Maj 2006 IS Årsmøde Århus
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 protected] OR http://www.tapasin.com
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Maj 2006 IS Årsmøde Århus
The Human MHC Project
• More than 1100 MHC-I alleles are currently discovered and sequenced
• Determine peptide specificity for representative MHC-I alleles
• Divide MHC-I molecules into representative supertypes covering the world population
• Knowing some of the peptide binding specificity; being able to predict the rest
Buus, S. et al., Tissue Antigens (2003) 62:378
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Maj 2006 IS Årsmøde Århus
The Human MHC Project
Cloning Expression Purification Active HCs
MHC-I Heavy Chain Manufacturing
![Page 5: Presentation at Immunologisk Selskab, 2006](https://reader034.vdocuments.site/reader034/viewer/2022052621/557d2aaed8b42a90748b4aaa/html5/thumbnails/5.jpg)
Maj 2006 IS Årsmøde Århus
Production of recombinant HLA complexes
![Page 6: Presentation at Immunologisk Selskab, 2006](https://reader034.vdocuments.site/reader034/viewer/2022052621/557d2aaed8b42a90748b4aaa/html5/thumbnails/6.jpg)
Maj 2006 IS Årsmøde Århus
Production of recombinant HLA complexes
![Page 7: Presentation at Immunologisk Selskab, 2006](https://reader034.vdocuments.site/reader034/viewer/2022052621/557d2aaed8b42a90748b4aaa/html5/thumbnails/7.jpg)
Maj 2006 IS Årsmøde Århus
Production of recombinant HLA complexes
Jumping from one MHC-I allele to another by introducing mutations:
These constructs are then ‘codon optimized’ for efficientExpression in E.coli
![Page 8: Presentation at Immunologisk Selskab, 2006](https://reader034.vdocuments.site/reader034/viewer/2022052621/557d2aaed8b42a90748b4aaa/html5/thumbnails/8.jpg)
Maj 2006 IS Årsmøde Århus
Production of recombinant HLA complexes
Purification by IMAC
![Page 9: Presentation at Immunologisk Selskab, 2006](https://reader034.vdocuments.site/reader034/viewer/2022052621/557d2aaed8b42a90748b4aaa/html5/thumbnails/9.jpg)
Maj 2006 IS Årsmøde Århus
Production of recombinant HLA complexes
Ferre et al.Protein Sci. 2003, 12:551
Purification by HIC
Fractions tested in RIA or ELISA
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Maj 2006 IS Årsmøde Århus
Production of recombinant HLA complexes
Denatured MHC Heavy ChainFolded ß2-microglobulin Peptide
Mixing Pot
Size Exclusion Chromatography
Concentration > 3 mg/mL
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Maj 2006 IS Årsmøde Århus
Crystallization and structure determination
Hanging Drops
MHC-I Complex
![Page 12: Presentation at Immunologisk Selskab, 2006](https://reader034.vdocuments.site/reader034/viewer/2022052621/557d2aaed8b42a90748b4aaa/html5/thumbnails/12.jpg)
Maj 2006 IS Årsmøde Århus
Crystallization and structure determination
Model Building
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Maj 2006 IS Årsmøde Århus
Crystallization and structure determination
![Page 14: Presentation at Immunologisk Selskab, 2006](https://reader034.vdocuments.site/reader034/viewer/2022052621/557d2aaed8b42a90748b4aaa/html5/thumbnails/14.jpg)
Maj 2006 IS Årsmøde Århus
Crystallization and structure determination
![Page 15: Presentation at Immunologisk Selskab, 2006](https://reader034.vdocuments.site/reader034/viewer/2022052621/557d2aaed8b42a90748b4aaa/html5/thumbnails/15.jpg)
Maj 2006 IS Årsmøde Århus
Crystallization and structure determination
ILGPPGSVY
LEKARGSTY
Epstein-Barr virusnuclear protein 3EBNA3A (406-414)
Human ubiquitinConjugating enz.(83-91)
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Maj 2006 IS Årsmøde Århus
Modeling and structure based affinity prediction
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Maj 2006 IS Årsmøde Århus
Acknowledgements
IMMI:Lotte Nielsen,Sune Justesen,Kasper Lamberth,Søren Buus
DFH:Thomas Blicher,Ole Kristensen,Jette Kastrup,Michael Gajhede