Purification, Crystallization and X-ray Diffraction Study of the C-terminal Domain of Human Herpesvirus 6A Immediate Early Protein 2

摘要

Human herpesvirus 6A (HHV-6A) starts its replication cycle following the action of immediate earlyproteins that transactivate viral promoters. Immediate early protein 2 (IE2) of HHV-6A is a 1500 aminoacid polypeptide with a C-terminal region that is conserved among beta-herpesvirus subfamily members.In this study, a structural domain in the homologous C-terminal region was subjected to secondarystructure prediction, and residues 1324?1500 were subsequently designated as the C-terminal domain ofIE2 (IE2-CTD). The gene fragment encoding IE2-CTD was inserted into an E. coli expression vector andexpressed as a fusion protein with maltose binding protein (MBP) at the N-terminus. IE2-CTD has atheoretical isoelectric point (pI) of 9.29, and strong cation exchange column chromatography was effectivefor purification. Needle-shaped crystals of IE2-CTD were obtained using the sitting-drop vapour diffusionmethod, and larger selenomethionine-labelled crystals of space group P21 diffracted X-rays to 2.5 ?resolution using synchrotron radiation. Data were collected at the selenium absorption peak wavelengthfor experimental phasing by the single anomalous dispersion method. The resulting electron density mapclearly shows the protein backbone, and full structural determination and refinement are in progress.