Expression and Purification of ataxin-1 Protein

J Neurosci Methods. 2010 May 30;189(1):30-5. doi: 10.1016/j.jneumeth.2010.03.008. Epub 2010 Mar 19.

Abstract

Ataxin-1 is part of a larger family of polyglutamine-containing proteins that is linked to nine distinct neurodegenerative disorders. There are no known effective therapies for any of these expanded polyglutamine tract disorders. One possible reason for this is the lack of sufficient amounts of pure polyglutamine-containing proteins suitable for biochemical and conformational studies. Here, we show that we were able to successfully purify a non-pathological, wild-type human ataxin-1 protein containing a 30-glutamine repeat sequence. This ataxin-1 protein was expressed in Escherichia coli as a fusion protein with a GST tag at the N-terminus and a double (His)(6) tag at the C-terminus. The devised dual affinity tag strategy allowed successful purification of the full-length ataxin-1 fusion protein to 90% homogeneity as confirmed by Western blot analysis using the two monoclonal ataxin-1 antibodies developed in our laboratory. In addition, the GST tag was successfully removed from the purified ataxin-1 fusion protein by treatment with Tobacco etch virus (TEV) protease. Since polyglutamine-containing proteins tend to aggregate, solvents/buffers that minimize aggregation have been used in the purification process. This dual affinity purification protocol could serve as a useful basis for purifying aggregation-prone proteins that are involved in other neurodegenerative diseases.

MeSH terms

  • Affinity Labels / chemistry*
  • Amino Acid Sequence / physiology
  • Antibodies, Monoclonal / isolation & purification
  • Antibody Specificity / immunology
  • Ataxin-1
  • Ataxins
  • Biochemistry / methods
  • Biological Assay / methods*
  • Blotting, Western
  • Electrophoresis, Polyacrylamide Gel
  • Endopeptidases / chemistry
  • Escherichia coli / genetics
  • Escherichia coli / metabolism
  • Humans
  • Nerve Tissue Proteins / isolation & purification*
  • Nerve Tissue Proteins / metabolism
  • Nuclear Proteins / isolation & purification*
  • Nuclear Proteins / metabolism
  • Proteomics / methods*
  • Recombinant Fusion Proteins / chemical synthesis*

Substances

  • ATXN1 protein, human
  • Affinity Labels
  • Antibodies, Monoclonal
  • Ataxin-1
  • Ataxins
  • Nerve Tissue Proteins
  • Nuclear Proteins
  • Recombinant Fusion Proteins
  • Endopeptidases
  • TEV protease