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Protein Science, Vol 7, Issue 2 413-418, Copyright © 1998 by Cold Spring Harbor Laboratory Press

ARTICLE

Engineering protein for X-ray crystallography: The murine Major Histocompatibility Complex class II molecule I-A(d)

C. A. SCOTT, K. C. GARCIA, E. A. STURA, P. A. PETERSON, I. A. WILSON and L. TEYTON
Department of Molecular Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037

Class II Major Histocompatibility (MHC) molecules are cell surface heterodimeric glycoproteins that play a central role in the immune response by presenting peptide antigens for surveillance by T cells. Due to the inherent instability of the class II MHC heterodimer, and its dependence on bound peptide for proper assembly, the production of electrophoretically pure samples of class II MHC proteins in complex with specific peptides has been problematic. A soluble form of the murine class II MHC molecule, I-A(d), with a leucine zipper tail added to each chain to enhance dimer assembly and secretion, has been produced in Drosophila melanogaster SC2 cells. To facilitate peptide loading, a high affinity ovalbumin peptide was covalently engineered to be attached by a six-residue linker to the amino terminus of the I-A(d){beta} chain. This modified I-A(d) molecule was purified using preparative IEF and one fraction, after removal of the leucine zipper tails, produced crystals suitable for X-ray crystallographic analysis. The protein engineering and purification methods described here should be of general value for the expression of I-A and other class II MHC-peptide complexes.
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