A novel method of affinity-purifying proteins using a bis-arsenical fluorescein

Genetically-encoded affinity tags constitute an important strategy for puri fying proteins. Here, we have designed a novel affinity matrix based on the bis-arsenical fluorescein dye FlAsH, which specifically recognizes short c t-helical peptides containing the sequence CCXXCC (Griffin BA, Adams SR, Ts ien RY, 1998, Science 281:269-272). We find that kinesin tagged with this c ysteine-containing helix binds specifically to FlAsH resin and can be elute d in a fully active form. This affinity tag has several advantages over pol yhistidine, the only small affinity tag in common use. The protein obtained with this single chromatographic step from crude Escherichia coli lysates is purer than that obtained with nickel affinity chromatography of 6xHis ta gged kinesin. Moreover, unlike nickel affinity chromatography, which requir es high concentrations of imidazole or pH changes for elution, protein boun d to the FlAsH column can be completely eluted by dithiothreitol. Because o f these mild elution conditions, FlAsH affinity chromatography is ideal for recovering fully active protein and for the purification of intact protein complexes.