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

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: g.roder@immi.ku.dk OR http://www.tapasin.com

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

Maj 2006 IS Årsmøde Århus

The Human MHC Project

Cloning Expression Purification Active HCs

MHC-I Heavy Chain Manufacturing

Maj 2006 IS Årsmøde Århus

Production of recombinant HLA complexes

Maj 2006 IS Årsmøde Århus

Production of recombinant HLA complexes

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

Maj 2006 IS Årsmøde Århus

Production of recombinant HLA complexes

Purification by IMAC

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

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

Maj 2006 IS Årsmøde Århus

Crystallization and structure determination

Hanging Drops

MHC-I Complex

Maj 2006 IS Årsmøde Århus

Crystallization and structure determination

Model Building

Maj 2006 IS Årsmøde Århus

Crystallization and structure determination

Maj 2006 IS Årsmøde Århus

Crystallization and structure determination

Maj 2006 IS Årsmøde Århus

Crystallization and structure determination

ILGPPGSVY

LEKARGSTY

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

Human ubiquitinConjugating enz.(83-91)

Maj 2006 IS Årsmøde Århus

Modeling and structure based affinity prediction

Maj 2006 IS Årsmøde Århus

Acknowledgements

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

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