Expression, purification and preliminary X-ray crystallographic analysis of the chicken MHC class I molecule YF1*7.1

Abstract

YF1*7.1 is an allele of a polymorphic major histocompatibility complex (MHC) class I-like locus within the chicken Y gene complex. With the aim of understanding the possible role of the YF1*7.1 molecule in antigen presentation, the complex of YF1*7.1 heavy chain and beta(2)-microglobulin was reconstituted and purified without a peptide. Crystals diffracted synchrotron radiation to 1.32 A resolution and belonged to the monoclinic space group P2(1). The phase problem was solved by molecular replacement. A detailed examination of the structure may provide insight into the type of ligand that could be bound by the YF1*7.1 molecule.

Figure 1

Assessment of the quality of the purified YF1*7.1–β₂m complex using size-exclusion chromatography and SDS–PAGE under reducing conditions (inset). The blue curve represents the sample absorbance at 280 nm; numbered red fields indicate the eluted fractions. The single symmetrical peak indicates the purity and homogeneity of the sample. Inset: SDS–PAGE under reducing conditions with fractions collected from FPLC. The two bands observed in each of lanes a, b and c represent YF1*7.1 heavy chain (31.4 kDa) and β₂m (11.6 kDa), respectively. Molecular-weight markers (kDa) are shown in the left lane.

Figure 2

Photograph of the YF1*7.1–β₂m crystal used for the diffraction analyses. The scale bar indicates 100 µm.

Figure 3

Diffraction pattern of the YF1*7.1–β₂m crystal.