title: HLA-A2-peptide complexes: refolding and crystallization of molecules expressed in Escherichia coli and complexed with single antigenic peptides.
abstract: The two subunits of the human class I histocompatibility antigen (HLA)-A2 have been expressed at high levels (20-30 mg/liter) as insoluble aggregates in bacterial cells. The aggregates were dissolved in 8 M urea and then refolded to form an HLA-A2-peptide complex by removal of urea in the presence of an antigenic peptide. Two peptides from the matrix protein and nucleoprotein of influenza virus are known to bind to HLA-A2, and both support the refolding of the recombinant HLA-A2 molecule. An additional peptide, a nonamer from the gp120 envelope protein of human immunodeficiency virus type 1, also supported refolding. Yields of purified recombinant HLA-A2 are 10-15%. In the absence of an HLA-A2-restricted peptide, a stable HLA-A2 complex was not formed. Monoclonal antibodies known to bind to native HLA-A2 also bound to the recombinant HLA-A2-peptide complex. Three purified HLA-A2-peptide complexes refolded from bacterially produced protein aggregates crystallize under the identical conditions as HLA-A2 purified from human lymphoblastoid cells. Crystals of the recombinant HLA-A2 molecule in complex with the influenza matrix nonamer peptide, Mp(58-66), diffract to greater than 1.5-A resolution.
authors.affiliation: Department of Biochemistry and Molecular Biology, Howard Hughes Medical Institute, Harvard University, Cambridge, MA 02138-2092.
authors.forename: D N
authors.initials: DN
authors.lastname: Garboczi
authors.forename: D T
authors.initials: DT
authors.lastname: Hung
authors.forename: D C
authors.initials: DC
authors.lastname: Wiley
mesh_terms: D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000378:metabolism; D001483:Base Sequence; D003001:Cloning, Molecular; D003460:Crystallization; D004926:Escherichia coli / Q000235:genetics; D015699:HIV Envelope Protein gp120 / Q000737:chemistry; D015789:HLA-A2 Antigen / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D006801:Humans; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D009698:Nucleoproteins / Q000737:chemistry; D009838:Oligodeoxyribonucleotides; D010455:Peptides / Q000737:chemistry* / Q000276:immunology; D010957:Plasmids; D016133:Polymerase Chain Reaction; D011485:Protein Binding; D011487:Protein Conformation; D011489:Protein Denaturation; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry; D015183:Restriction Mapping; D014763:Viral Matrix Proteins / Q000737:chemistry; D014764:Viral Proteins / Q000737:chemistry* / Q000235:genetics / Q000276:immunology
chemical_list: D000911:Antibodies, Monoclonal; D015699:HIV Envelope Protein gp120; D015789:HLA-A2 Antigen; D046911:Macromolecular Substances; D009698:Nucleoproteins; D009838:Oligodeoxyribonucleotides; D010455:Peptides; D011994:Recombinant Proteins; D014763:Viral Matrix Proteins; D014764:Viral Proteins