Replacement of the membrane proximal region of I-A(d) MHC class II molecule with I-E-derived sequences promotes production of an active and stable soluble heterodimer without altering peptide-binding specificity.
The MHC class II molecule I-A is the murine homologue of HLA-DQ in humans. The I-A and DQ heterodimers display considerable heterodimer instability compared with their I-E and HLA-DR counterparts. This isotype-specific behavior makes the production of soluble I-A and DQ molecules very difficult. We have developed a strategy for production of soluble I-A(d) molecules involving expression of I-A(d) as a glycosil phosphatidyl inositol (PI) anchored chimera in Chinese Hamster Ovary (CHO) cells. The regions comprising the membrane proximal segments of I-A(d) alpha and beta chains were substituted for the corresponding regions of I-E, and the derived constructs were expressed in CHO cells. Procedures for purification of the soluble class II molecules were optimized and the WT and chimeric molecule were compared for structure, biochemical stability and functionality. Our analysis revealed that the substitutions in the membrane proximal domains improved cell surface expression and thermal stability of I-A(d) without altering the peptide binding specificity of the class II molecule. The results suggest that similar strategies could be used to increase the stability of other unstable class II molecules for in vitro studies.
Chaves
Francisco A
FA
David H. Smith Center for Vaccine Biology and Immunology, Department of Microbiology and Immunology, University of Rochester, Rochester, NY 14642, USA.
Hou
Ping
P
Wu
Shenhong
S
Sant
Andrea J
AJ
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D016466:CHO Cells; D002846:Chromatography, Affinity; D006224:Cricetinae; D019281:Dimerization; D004355:Drug Stability; D005802:Genes, MHC Class II; D000949:Histocompatibility Antigens Class II / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D011485:Protein Binding; D015202:Protein Engineering; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012995:Solubility
D000911:Antibodies, Monoclonal; D000949:Histocompatibility Antigens Class II; C111389:I-Ad antigen; C024138:I-E-antigen; D011993:Recombinant Fusion Proteins