Our laboratory has a long-standing interest in immunochemical methods for cell separation, and in developing engineered macromolecular reagents for use in studying relatively low affinity interactions (such as those between MHC molecules and T cell receptors). Because balanced production of naturally multimeric proteins (such as antibodies) can be facilitated by engineering them as single chain molecules, we have engineered soluble recombinant single chain Class I MHC (MHC I) molecules. These recombinant molecules are useful for studying protein folding and assembly, and may serve as precursors for toxin-conjugates and T cell vaccines. During this year, our laboratory has prepared several recombinant soluble single chain MHC I molecules, which have their beta2-microglobulin (beta2m) domain covalently linked to the heavy chain. We have characterized two of them extensively. One molecule has wild type amino acid sequences. The other is a mutant molecule which lacks three salt bridges in the peptide- binding domains. The mutant molecule is not thermally stable and is secreted at 27 but not at 37. Both molecules contain endogenous peptides. This indicates that they have folded correctly, and suggests that a beta2m- free heavy chain/peptide complex is not an obligate intermediate in assembly. When pulsed with an antigenic peptide from the HIV gp120 envelope protein, both molecules can present it to an H-2Dd restricted antigen-specific T hybridoma. This indicated that the recombinant molecules are biologically active and have bound the peptide in the same configuration as in the wild-type two chain MHC I molecules. Because the single chain molecule, despite a tethered beta2m, retains sufficient flexibility to be loaded in vitro with antigenic peptides, it may be useful for structural studies, and as an intermediate in the formation of toxin- conjugates and T cell vaccines.
