An HLA-A2/beta 2-microglobulin/peptide complex assembled from subunits expressed separately in Escherichia coli.

The human class I histocompatibility antigen HLA-A2 has been assembled from subunits expressed separately in E. coli. A peptide that is known to be recognized by human cytotoxic T lymphocytes (CTLs) in association with HLA-A2 is a necessary component of the reconstitution mixture. The N-terminal extracellular fragment of the HLA-A2 heavy chain is initially synthesised as an insoluble aggregate. The aggregate is solubilized in denaturant, mixed with the influenza nucleoprotein 85-94 decapeptide (NP peptide), and diluted into a solution containing human beta 2-microglobulin (beta 2 m) isolated from the E. coli periplasm. The HLA-A2 heavy chain becomes soluble in physiological solutions if both beta 2m and the NP peptide are present. The reconstituted HLA-A2 complex is recognised by a monoclonal antibody that is specific for the native HLA-A2/beta 2m heterodimer, and is also recognised by a monoclonal antibody that recognises beta 2m. When other peptides known from CTL studies to associate with HLA-A2 are used, a significantly lower yield of reconstituted complex is obtained. The isoelectric point of the reconstituted complex depends on which peptide is used, confirming that the peptide is a component of the reconstituted complex.