Purification, crystallographic analysis of rhesus MHC-I Mamu-A*02 complexed with simian immunodeficiency virus nonapeptide

Abstract:

Rhesus macaque (Macaca mulatta) is the best model to study of human immunodeficiency virus (HIV) infection and to develop acquired immunodeficiency syndrome (AIDS) vaccine. The crystal structure of its major histocompatibility antigen complex (MHC) is helpful to understand the mechanism of HIV immune evasion. In this study, we cloned the light chain (β2m) of MHC class I allele of rhesus macaques, Mamu-A*02, and inserted it into pET21a(+) vector. We transfected the recombinant plasmid pET21a(+)-Mamu-β2m and pET21a(+)-Mamu-α into BL21(DE3). Mamu-A*02 and β2m were expressed in the form of inclusion bodies in BL21 (DE3). We co-refolded the inclusion bodies of Mamu-α and Mamu-β2m with SIV nonapeptide YY9 and obtained the correct refolded protein complex. Then we purified the protein complex by the gel filtration and anion-exchange column. With hanging-drop method, we screened and optimized for the protein crystal. We managed to collect a X-ray diffraction with the resolution to 2.8 ? in the condition of 0.1 mol/L BIS-TRIS (pH5.5), 2.0 mol/L(NH4)2SO4. This crystal belong to perpendicular space group P212121, with unit-cell parameters a=128.99 ?, b=129.01 ?, c=129.03 ?. This data is available for the structure determination.