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Biological Sciences Division
Research Highlights

May 2005

A fluorescent probe finds a second use in a resin for protein pull-downs

With the ready availability of genomic information, the focus of biology is shifting toward the study of protein complexes and protein-protein interactions. Researchers at Pacific Northwest National Laboratory (PNNL) have shown that FlAsH, a Fluorescein Arsenical Helix binder, can be used to find a protein's binding partners (1). This probe was previously used to label genetically tagged proteins in vivo, allowing monitoring of the protein's location within the cell (2,3), and it permits structural analysis and validation of isolated complexes (4,5). Advantages are that 1) the same protein has to then be genetically modified only once for several very different purposes, and 2) researchers do not have to perform the laborious purification process to raise antibodies for protein pull-downs or imaging experiments.

Requirements for the perfect protein tag/affinity reagent system are 1) a small genetic tag size, 2) an uncharged tag, 3) tight tag/probe binding with minimal background interactions, and 4) a mild elution methodology orthogonal to the wash methods. The FlAsH approach fulfills all of these requirements. The protein of interest is tagged genetically with a tetracysteine motif, which allows it to bind to the FlAsH resin. The protein complex can then be released in a mild, non-denaturing, one-step procedure with a competing dithiol. The small tag size enables ease of cloning, as the genetic tag can be added directly on the primers and thus be incorporated easily into either terminus of any protein expressed in an organism of choice. The PNNL team chose the bacterial RNA polymerase (Rpo) from Shewanella oneidensis as the first model system, with the alpha subunit as bait. The core enzyme is a stable complex of two alpha (A) subunits, the beta (B) subunit, and the beta prime (C) subunit.

PNNL researchers synthesized a new FlAsH resin with a longer linker on a glass support. For the pull-down, the tetracysteine-tagged RNA polymerase A subunit was overexpressed in S. oneidensis, and the lysed cells were exposed to the affinity resin. After washing, the complex was eluted, and the eluted protein was run on sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE). As shown in Figure 1A, the main protein bands above background are the correct sizes for RpoA (36 kDa), RpoB (150 kDa), and RpoC (155 kDa). Protein identity was further verified by capillary liquid chromatograph-tandem mass spectrometry with methods established at PNNL. Analysis using conservative filters gave good sequence coverage for the core enzyme in the bait experiments (Figure 1B) with no background for the target proteins RpoB and RpoC and a low background for RpoA.

This work is published in the inaugural issue of Molecular Biosystems, a new journal directed at research on the interface of chemistry and systems biology. It is sponsored by the U.S. Department of Energy's Genomics: Genomes to Life program through DOE's Office of Science. PNNL team members are M. Uljana Mayer, Liang Shi, and Thomas C. Squier. The researchers, joined by Baowei Chen, Haishi Cao, Ping Yan, Seema Verma and Yijia Xiong, are currently refining the tags and probes and extending the range of applications.


Figure 1. A) SDS-PAGE gel of combined elutions: 1) lysate only and 2) 4Cys-RpoA bait; B) Schematic showing sequence coverage for the holoenzyme subunits (3 runs): top bar 4Cys-RpoA bait, bottom bar control. Red: one or two peptides; yellow: 3 to 5 peptides; green: 6 to 9 peptides; blue: 10+ peptides. Full Image

References

  1. Mayer MU, L Shi, and TC Squier. 2005. "One-Step, Non-Denaturing Isolation of an RNA Polymerase Enzyme Complex using an Improved Multi-Use Affinity Probe Resin." Molecular BioSystems 1(1):53-56. DOI:10.1039/b500950b.
  2. Griffin BA, SR Adams, and RY Tsien. 1998. "Specific Covalent Labeling of Recombinant Protein Molecules Inside Live Cells." Science 281(5374):269-272. DOI:10.1126/science.281.5374.269.
  3. Gaietta G, TJ Deerinck, SR Adams, J Bouwer, O Tour, DW Laird, GE Sosinsky, RY Tsien, and MH Ellisman. 2002. "Multicolor and Electron Microscopic Imaging of Connexin Trafficking." Science 296(5567):503-507. DOI:10.1126/science.1068793. 
  4. Chen B, MU Mayer, and TC Squier. 2005. "Structural Uncoupling Between Opposing Domains of Oxidized Calmodulin Underlies Enhanced Binding Affinity and Inhibition of the Plasma Membrane Ca-ATPase." Biochemistry 44(12):4737-4747. DOI:10.1021/bi0474113.
  5. Chen B, MU Mayer, LM Markillie, DL Stenoien, and TC Squier. 2005. "Dynamic Motion of Helix A in the Amino-Terminal Domain of Calmodulin is Stabilized upon Calcium Activation." Biochemistry 44(3):905-914. DOI:10.1021/bi048332u.

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