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

Published online by Cambridge University Press:  01 February 2000

KURT S. THORN ,
NARIMAN NABER ,
MARIJA MATUSKA ,
RONALD D. VALE  and
ROGER COOKE
KURT S. THORN
Affiliation:
Department of Cellular and Molecular Pharmacology, University of California, San Francisco, California 94143
NARIMAN NABER
Affiliation:
Department of Biochemistry and Biophysics, University of California, San Francisco, California 94143
MARIJA MATUSKA
Affiliation:
Department of Biochemistry and Biophysics, University of California, San Francisco, California 94143
RONALD D. VALE
Affiliation:
Department of Cellular and Molecular Pharmacology, University of California, San Francisco, California 94143 Department of Biochemistry and Biophysics, University of California, San Francisco, California 94143 The Howard Hughes Medical Institute, University of California, San Francisco, California 94143
ROGER COOKE
Affiliation:
Department of Biochemistry and Biophysics, University of California, San Francisco, California 94143
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Abstract

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.

Keywords

affinity tagFlAsHprotein purification
Type
ACCELERATED COMMUNICATION
Information
Protein Science , Volume 9 , Issue 2 , February 2000 , pp. 213 - 217
Copyright
© 2000 The Protein Society

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