Reprint of: Expression and purification of ataxin-1 protein.

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.

In silico tandem affinity purification refines an Oct4 interaction list.

INTRODUCTION: Octamer-binding transcription factor 4 (Oct4) is a master regulator of early mammalian development. Its expression begins from the oocyte stage, becomes restricted to the inner cell mass of the blastocyst and eventually remains only in primordial germ cells. Unearthing the interactions of Oct4 would provide insight into how this transcription factor is central to cell fate and stem cell pluripotency. METHODS: In the present study, affinity-tagged endogenous Oct4 cell lines were established via homologous recombination gene targeting in embryonic stem (ES) cells to express tagged Oct4. This allows tagged Oct4 to be expressed without altering the total Oct4 levels from their physiological levels. RESULTS: Modified ES cells remained pluripotent. However, when modified ES cells were tested for their functionality, cells with a large tag failed to produce viable homozygous mice. Use of a smaller tag resulted in mice with normal development, viability and fertility. This indicated that the choice of tags can affect the performance of Oct4. Also, different tags produce a different repertoire of Oct4 interactors. CONCLUSIONS: Using a total of four different tags, we found 33 potential Oct4 interactors, of which 30 are novel. In addition to transcriptional regulation, the molecular function associated with these Oct4-associated proteins includes various other catalytic activities, suggesting that, aside from chromosome remodeling and transcriptional regulation, Oct4 function extends more widely to other essential cellular mechanisms. Our findings show that multiple purification approaches are needed to uncover a comprehensive Oct4 protein interaction network.

Expression and purification of ataxin-1 protein.

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.

The protein kinase CK2 phosphorylates SNAP190 to negatively regulate SNAPC DNA binding and human U6 transcription by RNA polymerase III.

Human U6 small nuclear RNA gene transcription by RNA polymerase III requires the general transcription factor SNAP(C), which binds to human small nuclear RNA core promoter elements and nucleates pre-initiation complex assembly with the Brf2-TFIIIB complex. Multiple components in this pathway are phosphorylated by the protein kinase CK2, including the Bdp1 subunit of the Brf2-TFIIIB complex, and RNA polymerase III, with negative and positive outcomes for U6 transcription, respectively. However, a role for CK2 phosphorylation of SNAP(C) in U6 transcription has not been defined. In this report, we investigated the role of CK2 in modulating the transcriptional properties of SNAP(C) and demonstrate that within SNAP(C), CK2 phosphorylates the N-terminal half of the SNAP190 subunit at two regions (amino acids 20-63 and 514-545) that each contain multiple CK2 consensus sites. SNAP190 phosphorylation by CK2 inhibits both SNAP(C) DNA binding and U6 transcription activity. Mutational analyses of SNAP190 support a model wherein CK2 phosphorylation triggers an allosteric inhibition of the SNAP190 Myb DNA binding domain.

Production of constitutively acetylated recombinant p53 from yeast and Escherichia coli by tethered catalysis.

Post-translational modification of proteins is a dynamic way of generating new protein-protein interaction interfaces that are critical for signaling networks in diverse cellular functions. Purified recombinant proteins frequently lack these signature modifications. Using the tumor suppressor p53 as the model protein, we present here a tethered catalysis approach for the production of acetylated p53 in vivo. P53 is a major tumor suppressor protein that protects the cell from various oncogenic stresses. Upon DNA damage, p53 is stabilized and activated by a plethora of post-translational modifications, including acetylation. Here, we show that constitutively acetylated p53 can be expressed and purified from both yeast and Escherichia coli. This method is highly suitable for studying protein-protein interactions in the conventional yeast two-hybrid screen that requires a constitutively acetylated state of p53. Furthermore, effective production and purification of acetylated p53 from E. coli supports future biochemical and structural characterization. The method described in this work can be applied to other proteins and modifications, and thus has widespread use in the fields of signal transduction and proteomic research.