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.
Parker
K C
KC
Biological Resources Branch, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892.
Silver
M L
ML
Wiley
D C
DC
D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics*; D015789:HLA-A2 Antigen / Q000302:isolation & purification; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009842:Oligopeptides; D010957:Plasmids; D011994:Recombinant Proteins; D014162:Transfection; D014764:Viral Proteins; D001613:beta 2-Microglobulin
D015789:HLA-A2 Antigen; D009842:Oligopeptides; D011994:Recombinant Proteins; D014764:Viral Proteins; D001613:beta 2-Microglobulin