Genetically-encoded affinity tags constitute an
important strategy for purifying proteins. Here, we have
designed a novel affinity matrix based on the bis-arsenical
fluorescein dye FlAsH, which specifically recognizes short
α-helical peptides containing the sequence CCXXCC (Griffin
BA, Adams SR, Tsien RY, 1998, Science 281:269–272).
We find that kinesin tagged with this cysteine-containing
helix binds specifically to FlAsH resin and can be eluted
in a fully active form. This affinity tag has several advantages
over polyhistidine, 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 tagged kinesin. Moreover, unlike nickel affinity
chromatography, which requires high concentrations of imidazole
or pH changes for elution, protein bound to the FlAsH column
can be completely eluted by dithiothreitol. Because of
these mild elution conditions, FlAsH affinity chromatography
is ideal for recovering fully active protein and for the
purification of intact protein complexes.