The production, purification and crystallization of a soluble form of the nonclassical MHC HLA-G: the essential role of cobalt.
HLA-G is a nonclassical class I major histocompatibility complex (MHC) molecule that is primarily expressed at the foetal-maternal interface. Although the role of HLA-G has not been fully elucidated, current evidence suggests it protects the foetus from the maternal immune response. In this report, HLA-G (44 kDa) is characterized by expression in Escherichia coli. The inclusion bodies were refolded in complex with a peptide derived from histone H2A (RIIPRHLQL), purified and subsequently crystallized. Correct refolding was determined using two conformation-dependent antibodies. Cobalt ions were shown to be an essential ingredient for obtaining diffraction-quality crystals. The crystals, which diffracted to 1.9 A resolution, belonged to space group P3(2)2(1), with unit-cell parameters a = b = 77.15, c = 151.72 A.
Clements
Craig S
CS
Department of Biochemistry and Molecular Biology, School of Biomedical Sciences, Monash University, Clayton, Victoria 3800, Australia.
Kjer-Nielsen
Lars
L
Kostenko
Lyudmila
L
McCluskey
James
J
Rossjohn
Jamie
J
D019943:Amino Acid Substitution / Q000235:genetics; D002413:Cations, Divalent / Q000737:chemistry; D045744:Cell Line, Tumor; D003035:Cobalt / Q000737:chemistry*; D003545:Cysteine / Q000235:genetics; D006680:HLA Antigens / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification*; D059951:HLA-G Antigens; D015395:Histocompatibility Antigens Class I / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification*; D006801:Humans; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D012694:Serine / Q000235:genetics; D012995:Solubility
D002413:Cations, Divalent; D006680:HLA Antigens; D059951:HLA-G Antigens; D015395:Histocompatibility Antigens Class I; D011994:Recombinant Proteins; D003035:Cobalt; D012694:Serine; D003545:Cysteine