[29] Purification and kinetic characterization of human cytomegalovirus assemblin

Publisher Summary This chapter describes expression and preparation of pure human cytomegalovirus (HCMV) assemblin with the help of a chelating peptide (CP) purification handle. Chelating peptide-immobilized metal ion affinity chromatography (CP-IMAC) uses an engineered metal-binding site or CP at either the N terminus or C terminus of a recombinant protein for a one-step affinity purification using IMAC. The CP sequences are easily incorporated into the protein with recombinant DNA cloning techniques. Cells are lysed with lysozyme and sonicated. Inclusion bodies are collected by centrifugation, lyophilized, and 10 mg/ml of solids dissolved in 0.5 M Tris, 7 M urea, pH 8.2. All purification steps are carried out in buffers containing 7 M urea, which maintains the enzyme in an unfolded inactive conformation. Blocking the cysteine residues as S-sulfonates facilitates the purification by solubilizing more protein from the inclusion bodies and preventing the formation of intermolecular disulfide bonds and aggregates. The activity of HCMV CP-assemblin is measured by following the hydrolysis of FITC, fluorescein isothiocyanate, a labeled peptide mimic of the maturational site of the assembly protein precursor.