Affinity purification of a difficult-sequence protein. Implications for the inclusion of capping in synthetic protocols.
A biotinylated derivative of a designed, difficult-sequence protein (the Minibody, Bianchi, E., Tramontano, A., Sollazzo, M. & Pessi, A. (1993) Int. J. Peptide Protein Res. 41, 385-393) which represented only 3.7% of the crude, cleaved material was quantitatively recovered with about 70% purity, in a single step, by affinity chromatography on immobilised avidin. Purification to homogeneity was then easily achieved by preparative HPLC. This highly effective purification scheme must be contrasted with the previously shown multidimensional, low-yield chromatographic protocol. Since no facilitation of the purification had been obtained by capping alone, this result suggests that capping is useful only in conjunction with affinity chromatography.
Bianchi
E
E
Department of Biochemistry, Instituto di Ricerche di Biologia Molecolare P. Angeletti (IRBM), Pomezia, Rome, Italy.
Sollazzo
M
M
Tramontano
A
A
Pessi
A
A
D000595:Amino Acid Sequence; D001360:Avidin; D001665:Binding Sites; D001710:Biotin; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D015195:Drug Design; D008670:Metals; D008969:Molecular Sequence Data; D011506:Proteins / Q000138:chemical synthesis / Q000737:chemistry / Q000302:isolation & purification*
D008670:Metals; D011506:Proteins; D001360:Avidin; D001710:Biotin