Expression, refolding and preliminary X-ray crystallographic analysis of equine MHC class I molecule complexed with an EIAV-Env CTL epitope

Abstract

In order to clarify the structure and the peptide-presentation characteristics of the equine major histocompatibility complex (MHC) class I molecule, a complex of equine MHC class I molecule (ELA-A1 haplotype, 7-6 allele) with mouse 2 -microglobulin and the cytotoxic T lymphocyte (CTL) epitope Env-RW12 (RVEDVTNTAEYW) derived from equine infectious anaemia virus (EIAV) envelope protein (residues 195-206) was refolded and crystallized. The crystal, which belonged to space group P2 1 , diffracted to 2.3 Å resolution and had unitcell parameters a = 82.5, b = 71.4, c = 99.8 Å , = 102.9 . The crystal structure contained two molecules in the asymmetric unit. These results should help to determine the first equine MHC class I molecule structure presenting an EIAV CTL epitope.

Crystallization and preliminary crystallographic studies of the complement 1qA globular domain from zebrafish,Dare-C1qAgD

“…The Dare-C1qAgD protein was expressed as inclusion bodies. The inclusion bodies were extracted and washed, and then dissolved in guanidine-HCl buffer [6 M guanidine-HCl, 50 mM Tris-HCl pH 8.0, 10 mM EDTA, 100 mM NaCl, 10%(v/v) glycerol, 10 mM DTT] to a concentration of 30 mg ml À1 (Yao et al, 2012).…”

Complex assembly, crystallization and preliminary X-ray crystallographic analysis of the bovine CD8αα–BoLA-2*02201 complex

“…These proteins were expressed as inclusion bodies. The bacteria were harvested and lysed by sonication (Yao et al, 2012). The samples were centrifuged for 10 min at 11 000 rev min À1 and the pellets were washed three times with a solution consisting of 20 mM Tris-HCl pH 8.0, 1 mM DTT, 1 mM EDTA, 100 mM NaCl, 0.5% Triton X-100.…”

“…Two components of the complex were prepared. First of all, the BoLA-2*02201-FMDV-VP1 YY9 complex was prepared essentially as described previously by Garboczi et al (1992) with some modifications (Yao et al, 2012). Briefly, the peptide FMDV-VP1 YY9 (YRMKRAETY) derived from FMDV structural protein VP1 (FMDV-VP1) residues 902-910 was synthesized and purified by HPLC (Scilight-Peptide).…”

“…The proteins were mixed in a 1:1 molar ratio and incubated for 12 h at 277 K. Finally, the complex was diluted to 5 and 10 mg ml À1 for crystallization. Crystallization trials were carried out by the sitting-drop method using Crystal Screen 2 (Hampton Research, Riverside, California, USA) at 291 K (Yao et al, 2012). Drops were prepared by mixing 1 ml protein solution with 1 ml reservoir solution and were equilibrated against 150 ml of the same reservoir solution in 48-well VDX plates (Hampton Research).…”