title: [Construction and characterization of soluble HLA-A*0201-PR1 complex].
abstract: This study was aimed to construct the soluble HLA-A*0201-PR1 complex for preparation of HLA-A*0201-PR1 tetramer. The recombinant HLA-A*0201-BSP (BirA substrate peptide) fusion protein as heavy chain and beta(2)-microglobulin (beta(2) m) as light chain were expressed highly as insoluble aggregates in Escherichia coli and then purified with gel filtration, and the final purity reached above 90%. The two subunits were refolded to form an HLA-A*0201-peptide complex by dilution method in the presence of an antigenic peptide PR1, a HLA-A2-restricted peptide from proteinase 3 (aa 169 - 177, VLQELNVTV). Refolded HLA-A*0201-PR1 complex was biotinylated using a BirA enzyme and purified by anion exchange chromatography on a Q-Sepharose (fast flow) column. The extent of reconstitution of the HLA-A*0201-PR1 complex was analyzed by HPLC gel filtration. The refolded and biotinylated products were detected by Western blot and ELISA with monoclonal antibody BB7.2 that recognized the natural conformations of HLA-A2 and streptavidin. The results showed that the refolded complex was composed of HLA-A*0201-BSP aggregate, HLA-A*0201-PR1 complex and beta(2) m, and reconstitution yields of 18% with PR1 was obtained. Refolded HLA-A*0201-PR1 complex could be confirmed by practical immunological method and biotinylated efficiently. It is concluded that the refolding and biotinylation of HLA-A*0201-PR1 complex is successfully obtained. This work provides the basis for the preparation of HLA-A*0201-PR1 tetramer.
authors.affiliation: Department of Hematology, Affiliated Hospital, Academy of Military Medical Sciences, Beijing 100071, China.
authors.forename: Wan-Jun
authors.initials: WJ
authors.lastname: Sun
authors.forename: Dong-Gang
authors.initials: DG
authors.lastname: Xu
authors.forename: Hai-Lan
authors.initials: HL
authors.lastname: Hu
authors.forename: Min-Ji
authors.initials: MJ
authors.lastname: Zou
authors.forename: Jian-Fang
authors.initials: JF
authors.lastname: DU
authors.forename: Jin-Feng
authors.initials: JF
authors.lastname: Wang
authors.forename: Xin
authors.initials: X
authors.lastname: Cai
authors.forename: Jia-Xi
authors.initials: JX
authors.lastname: Wang
authors.forename: Hui-Sheng
authors.initials: HS
authors.lastname: Ai
mesh_terms: D017931:DNA Primers / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D015234:HLA-A Antigens / Q000032:analysis / Q000096:biosynthesis / Q000235:genetics; D015789:HLA-A2 Antigen; D006801:Humans; D009842:Oligopeptides / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D017510:Protein Folding; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000096:biosynthesis; D001613:beta 2-Microglobulin / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics
chemical_list: D017931:DNA Primers; D015234:HLA-A Antigens; C435939:HLA-A*02:01 antigen; D015789:HLA-A2 Antigen; D009842:Oligopeptides; D011993:Recombinant Fusion Proteins; D001613:beta 2-Microglobulin