Expression and crystallization of the complex of HLA-DR2 (DRA, DRB1*1501) and an immunodominant peptide of human myelin basic protein.
HLA-DR2 is associated with susceptibility to multiple sclerosis (MS). A peptide from human myelin basic protein (MBP, residues 85-99) was previously found to bind to purified HLA-DR2 (DRA, DRB1*1501) and to be recognized by human MBP-specific T cell clones. Soluble HLA-DR2 was expressed in the baculovirus system by replacing the hydrophobic transmembrane regions and cytoplasmic segments of DRalpha and DRbeta with leucine zipper dimerization domains from the transcription factors Fos and Jun. In the expression construct, the MBP(85-99) sequence was covalently linked to the N terminus of the mature DRbeta chain. The recombinant protein was secreted by Sf9 cells infected with the recombinant baculovirus and purified by affinity chromatography. The leucine zipper dimerization domains were then cleaved from the assembled HLA-DR2/MBP peptide complex with V8 protease, and the protein was further purified by anion-exchange HPLC. Analysis by HPLC gel filtration indicated that the HLA-DR2/MBP peptide complex did not have a tendency to aggregate. The purified HLA-DR2/MBP peptide complex readily crystallized by the hanging drop method in 15-18% polyethylene glycol 6000/100 mM glycine, pH 3.5. At a synchrotron radiation source, a crystal with a tetragonal space group diffracted to a resolution of 2.6 A. The expression of such homogenous HLA-DR/peptide complexes may facilitate cocrystallization with T cell receptors as well as other molecules involved in T cell receptor recognition and signaling.
Gauthier
L
L
Department of Cancer Immunology & AIDS, Dana-Farber Cancer Institute, Boston, MA 02115, USA.
Smith
K J
KJ
Pyrdol
J
J
Kalandadze
A
A
Strominger
J L
JL
Wiley
D C
DC
Wucherpfennig
K W
KW
D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D001483:Base Sequence; D002460:Cell Line; D003460:Crystallization; D017931:DNA Primers / Q000235:genetics; D019281:Dimerization; D015870:Gene Expression; D015802:HLA-DR2 Antigen / Q000235:genetics* / Q000302:isolation & purification*; D006801:Humans; D016056:Immunodominant Epitopes / Q000235:genetics / Q000302:isolation & purification; D016350:Leucine Zippers; D008969:Molecular Sequence Data; D009103:Multiple Sclerosis / Q000235:genetics / Q000276:immunology; D004676:Myelin Basic Protein / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification*; D010446:Peptide Fragments / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification; D012995:Solubility; D018411:Spodoptera; D013601:T-Lymphocytes / Q000276:immunology
D017931:DNA Primers; D015802:HLA-DR2 Antigen; D016056:Immunodominant Epitopes; D004676:Myelin Basic Protein; D010446:Peptide Fragments; D011994:Recombinant Proteins