title: [Improvement of refolding efficiency of HLA-A2-peptide complex in vitro by preformed disulfide bonds in heavy chain].
abstract: OBJECTIVE
  To improve the refolding efficiency of soluble HLA-A2-peptide complex in vitro.
  
  
  METHODS
  The heavy chain (HC) of MHC class I was extracted from bacteria under denaturing and non-reducing conditions. Anion-exchange and (NH4)2SO4 precipitation were applied to purify the HC. Then the purified HC, beta2m and an antigenic peptide (N-YMDGTMSQV-COOH of Try(369-377)) were refolded to form an HLA-A2-peptide complex by dilution method in the buffer of pH 6.6. The refolded products were detected by Western blot and ELISA with W6/32 and anti-human beta2m antibody.
  
  
  RESULTS
  The refolded products consisted of HLA-A2-peptide complex, beta2m, and a little amount of HC polymer. The refolding efficiency was 2.5 fold higher than that of the conventional method.
  
  
  CONCLUSIONS
  This study confirmed that the refolding efficiency of the method reported in this paper is higher as compared with the conventional method, which is of importance to the preparation of HLA-peptide tetramers and artificial antigen presenting cells.
authors.affiliation: Department of Immunology, Tongji Medical College, Huazhong University of Sciences and Technology, Wuhan 430030, China.
authors.forename: Shi-jiang
authors.initials: SJ
authors.lastname: Fei
authors.forename: Xiong-wen
authors.initials: XW
authors.lastname: Wu
authors.forename: Xiu-fang
authors.initials: XF
authors.lastname: Wong
authors.forename: Lei
authors.initials: L
authors.lastname: Cai
authors.forename: Zhi-hui
authors.initials: ZH
authors.lastname: Liang
authors.forename: Jun-yan
authors.initials: JY
authors.lastname: Han
authors.forename: Fei-li
authors.initials: FL
authors.lastname: Gong
mesh_terms: D000595:Amino Acid Sequence; D000645:Ammonium Sulfate / Q000494:pharmacology; D000818:Animals; D000938:Antigen-Presenting Cells / Q000166:cytology; D004220:Disulfides / Q000737:chemistry*; D004591:Electrophoresis, Polyacrylamide Gel; D015789:HLA-A2 Antigen / Q000737:chemistry* / Q000378:metabolism*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D002479:Inclusion Bodies / Q000378:metabolism; D010455:Peptides / Q000737:chemistry* / Q000378:metabolism*; D017510:Protein Folding; D020673:Protein Renaturation / Q000187:drug effects*; D012995:Solubility
chemical_list: D004220:Disulfides; D015789:HLA-A2 Antigen; D010455:Peptides; D000645:Ammonium Sulfate