Presentation at Immunologisk Selskab, 2006

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


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


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


The Human MHC Project

Cloning Expression Purification Active HCs

MHC-I Heavy Chain Manufacturing


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



Purification by IMAC



Ferre et al.Protein Sci. 2003, 12:551

Purification by HIC

Fractions tested in RIA or ELISA



Denatured MHC Heavy ChainFolded ß2-microglobulin Peptide

Mixing Pot

Size Exclusion Chromatography

Concentration > 3 mg/mL


Crystallization and structure determination

Hanging Drops

MHC-I Complex



Model Building



ILGPPGSVY

LEKARGSTY

Epstein-Barr virusnuclear protein 3EBNA3A (406-414)

Human ubiquitinConjugating enz.(83-91)


Modeling and structure based affinity prediction


Acknowledgements

IMMI:Lotte Nielsen,Sune Justesen,Kasper Lamberth,Søren Buus

DFH:Thomas Blicher,Ole Kristensen,Jette Kastrup,Michael Gajhede

Presentation at Immunologisk Selskab, 2006

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