title: Engineering protein for X-ray crystallography: the murine Major Histocompatibility Complex class II molecule I-Ad.
abstract: 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-Ad, 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-Adbeta chain. This modified I-Ad 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.
authors.affiliation: Department of Molecular Biology, The Scripps Research Institute, La Jolla, California 92037, USA.
authors.forename: C A
authors.initials: CA
authors.lastname: Scott
authors.forename: K C
authors.initials: KC
authors.lastname: Garcia
authors.forename: E A
authors.initials: EA
authors.lastname: Stura
authors.forename: P A
authors.initials: PA
authors.lastname: Peterson
authors.forename: I A
authors.initials: IA
authors.lastname: Wilson
authors.forename: L
authors.initials: L
authors.lastname: Teyton
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D003001:Cloning, Molecular; D018360:Crystallography, X-Ray; D004331:Drosophila melanogaster / Q000235:genetics; D000949:Histocompatibility Antigens Class II / Q000737:chemistry* / Q000235:genetics; D051379:Mice; D008969:Molecular Sequence Data; D010047:Ovalbumin / Q000737:chemistry; D010446:Peptide Fragments / Q000737:chemistry; D015202:Protein Engineering
chemical_list: D000949:Histocompatibility Antigens Class II; C111390:OVA 323-339; D010446:Peptide Fragments; D010047:Ovalbumin