title: Purification and characterization of the carboxyl-terminal transactivation domain of Vmw65 from herpes simplex virus type 1.
abstract: A glutathione S-transferase fusion to the COOH-terminal acidic transactivation domain of Vmw65 from herpes simplex virus type 1 was overexpressed in Escherichia coli and isolated by affinity chromatography on glutathione-Sepharose. Following cleavage of the fusion protein with thrombin, the transactivation domain was purified to homogeneity by ion exchange chromatography yielding approximately 0.6 mg of protein/liter of bacterial culture. Equilibrium sedimentation analysis showed the purified polypeptide to be monomeric; however, it displayed aberrant electrophoretic and chromatographic properties. Contrary to secondary structure predictions, circular dichroism spectroscopy demonstrated that this transactivation domain was devoid of significant alpha-helical structure at physiological conditions. The polypeptide, however, became notably more structured under hydrophobic conditions or at low pH, suggesting that it was sensitive to its environment. Near-UV circular dichroism suggested that phenylalanyl and tyrosyl residues were under influence from tertiary structure.
authors.affiliation: Department of Biochemistry, McMaster University, Hamilton, Ontario, Canada.
authors.forename: L
authors.initials: L
authors.lastname: Donaldson
authors.forename: J P
authors.initials: JP
authors.lastname: Capone
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D002942:Circular Dichroism; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D005982:Glutathione Transferase / Q000235:genetics; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D010957:Plasmids; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D018139:Simplexvirus / Q000235:genetics / Q000378:metabolism*; D015534:Trans-Activators / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
chemical_list: D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D015534:Trans-Activators; D005982:Glutathione Transferase