The production, purification and crystallization of a soluble form of the nonclassical MHC HLA-G: the essential role of cobalt

Abstract

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.

Figure 1

HLA-G refolding and functional testing. (a) SDS–PAGE analysis of refolded HLA-­G–RIIPRHLQL complex following anion-exchange and size-exclusion chromatography. (b) ELISA of refolded HLA-G. Two antibodies specific for human MHC molecules were used in a sandwich ELISA with HLA-G (filled squares) and HLA-B8 (filled triangles) as positive controls and LC13 (filled circles) as a negative control. Open symbols indicate the control experiment without primary antibody. Data represents the mean of three experiments.

Figure 2

Preliminary crystals of HLA-G grown in 20% PEG 3350, 0.1 M HEPES pH 7.0, 0.2 M potassium formate at 277 K.

Figure 3

Diffracting crystals of HLA-G grown in 18–21% PEG 3350, 0.1 M HEPES pH 6.8, 0.2 M potassium formate and 10 mM CoCl₂ at 277 K.