A novel method of affinity-purifying proteins using a bis-arsenical fluorescein.
Genetically-encoded affinity tags constitute an important strategy for purifying proteins. Here, we have designed a novel affinity matrix based on the his-arsenical fluorescein dye FlAsH, which specifically recognizes short alpha-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.
Thorn
K S
KS
Department of Cellular and Molecular Pharmacology, University of California, San Francisco 94143, USA.
Naber
N
N
Matuska
M
M
Vale
R D
RD
Cooke
R
R
D000345:Affinity Labels; D000595:Amino Acid Sequence; D001151:Arsenic; D002846:Chromatography, Affinity / Q000379:methods*; D004926:Escherichia coli / Q000235:genetics; D005452:Fluoresceins / Q000138:chemical synthesis; D005456:Fluorescent Dyes / Q000138:chemical synthesis; D016547:Kinesins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D008969:Molecular Sequence Data; D009942:Organometallic Compounds / Q000138:chemical synthesis; D011506:Proteins / Q000737:chemistry / Q000302:isolation & purification*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification
C113276:4',5'-bis(1,3,2-dithioarsolan-2-yl)fluorescein; D000345:Affinity Labels; D005452:Fluoresceins; D005456:Fluorescent Dyes; D009942:Organometallic Compounds; D011506:Proteins; D011994:Recombinant Proteins; D016547:Kinesins; D001151:Arsenic