ENGINEERING PROTEIN FOR X-RAY CRYSTALLOGRAPHY - THE MURINE MAJOR HISTOCOMPATIBILITY COMPLEX CLASS-II MOLECULE I-A(D)

Class II Major Histocompatibility (MHC) molecules are cell surface het erodimeric glycoproteins that play a central role in the immune respon se by presenting peptide antigens for surveillance by T cells. Due to the inherent instability of the class II MHC heterodimer, and its depe ndence on bound peptide for proper assembly, the production of electro phoretically pure samples of class II MHC proteins in complex with spe cific peptides has been problematic. A soluble form of the murine clas s Il MHC molecule, I-A(d), with a leucine zipper tail added to each ch ain to enhance dimer assembly and secretion, has been produced in Dros ophila melanogaster SC2 cells. To facilitate peptide loading, a high a ffinity ovalbumin peptide was covalently engineered to be attached by a six-residue linker to the amino terminus of the I-A(d) beta chain. T his modified I-A(d) molecule was purified using preparative IEF and on e fraction, after removal of the leucine zipper tails, produced crysta ls suitable for X-ray crystallographic analysis. The protein engineeri ng and purification methods described here should be of general value for the expression of I-A and other class Il MHC-peptide complexes.