Empty and peptide-loaded class II major histocompatibility complex proteins produced by expression in Escherichia coli and folding in vitro

The human class II major histocompatibility complex protein HLA-DR1 has bee n expressed in Escherichia coil as denatured alpha and beta subunits and fo lded in vitro to form the native structure. DR1 folding yields are 30-50% i n the presence or absence of tight-binding antigenic peptides. The protein produced in this manner is soluble and monomeric with the expected apparent molecular weight. It reacts with conformation-sensitive anti-DR antibodies and exhibits peptide-dependent resistance to SDS-induced chain dissociatio n and to proteolysis as does the native protein. The observed peptide speci ficity and dissociation kinetics are similar to those of native DR produced in B-cells and finally the protein exhibits circular dichroism spectra and cooperative thermal denaturation as expected for a folded protein. We conc lude that the recombinant DR1 has adopted the native fold. We have folded D R1 in the absence of peptide and isolated a soluble, peptide-free alpha bet a-heterodimer. The empty DR1 can bind antigenic peptide but exhibits altere d far UV-circular dichroism and thermal denaturation relative to the peptid e-bound form. (C) 1999 Academic Press.