Purification of polyglutamine proteins.
The misfolding and formation of fibrillar-like aggregates by polyglutamine proteins is believed to be a key factor in the development of the neurodegenerative polyglutamine diseases; however, relatively little is known about structural and conformational aspects of polyglutamine-induced misfolding and aggregation. This is largely attributable to the fact that polyglutamine proteins have proved difficult to purify in quantities suitable for biochemical and biophysical analyses, thus limiting the extent to which the proteins can be conformationally characterized. Recent advances, however, have seen the development of a number of protocols enabling the expression and purification of these proteins in more significant quantities. In this report, we describe a purification protocol for ataxin-3, which, in its polyglutamine-expanded form, causes Machado-Joseph disease. Purification of different length ataxin-3 variants, including one of pathological length, is facilitated by an N-terminal hexa-histidine tag, which enables binding to a nickel-chelated agarose resin. A key issue that arose during purification was the undesirable proteolysis of ataxin-3 by a trace contaminant protease. We solved this problem by the addition of a benzamidine-binding step during purification, which greatly reduced the level of proteases present. We found that the inclusion of this step had a significant positive impact on the quality of the purified protein product. We also inactivated trace amounts of proteases during experiments by the addition of specific protease inhibitors. Finally, we also describe initial structural and functional analyses that confirm the integrity of the purified protein.
Chow
Michelle K M
MK
Monash University, Department of Biochemistry and Molecular Biology, Clayton, Victoria, Australia.
Ellisdon
Andrew M
AM
Cabrita
Lisa D
LD
Bottomley
Stephen P
SP
D000067699:Ataxin-3; D001483:Base Sequence; D002850:Chromatography, Gel; D002942:Circular Dichroism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000302:isolation & purification*; D009687:Nuclear Proteins / Q000235:genetics / Q000302:isolation & purification*; D010455:Peptides / Q000302:isolation & purification*; D011480:Protease Inhibitors / Q000494:pharmacology; D011487:Protein Conformation; D020836:Protein Structure, Quaternary; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification; D012097:Repressor Proteins / Q000235:genetics / Q000302:isolation & purification*
D009419:Nerve Tissue Proteins; D009687:Nuclear Proteins; D010455:Peptides; D011480:Protease Inhibitors; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; C097188:polyglutamine; C092341:ATXN3 protein, human; D000067699:Ataxin-3