This Article
Right arrow Full Text
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Schimanski, B.
Right arrow Articles by Günzl, A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Schimanski, B.
Right arrow Articles by Günzl, A.

 Previous Article  |  Next Article 

Eukaryotic Cell, November 2005, p. 1942-1950, Vol. 4, No. 11
1535-9778/05/$08.00+0     doi:10.1128/EC.4.11.1942-1950.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Highly Efficient Tandem Affinity Purification of Trypanosome Protein Complexes Based on a Novel Epitope Combination

Bernd Schimanski, Tu N. Nguyen, and Arthur Günzl*

Department of Genetic and Developmental Biology and Department of Molecular, Microbial and Structural Biology, University of Connecticut Health Center, 263 Farmington Ave., Farmington, Connecticut 06030-3301

Received 13 July 2005/ Accepted 22 August 2005

Tandem affinity purification (TAP) allows for rapid and efficient purification of epitope-tagged protein complexes from crude extracts under native conditions. The method was established in yeast and has been successfully applied to other organisms, including mammals and trypanosomes. However, we found that the original method, which is based on the TAP tag, consisting of a duplicate protein A epitope, a tobacco etch virus protease cleavage site, and the calmodulin-binding peptide (CBP), did not yield enough recovery of transcription factor SNAPc (for small nuclear RNA-activating protein complex) from crude trypanosome extracts for protein identification. Specifically, the calmodulin affinity chromatography step proved to be inefficient. To overcome this problem, we replaced CBP by the protein C epitope (ProtC) and termed this new epitope combination PTP tag. ProtC binds with high affinity to the monoclonal antibody HPC4, which has the unique property of requiring calcium for antigen recognition. Thus, analogous to the calcium-dependent CBP-calmodulin interaction, ProtC-tagged proteins can be released from immobilized HPC4 by a chelator of divalent cations. While this property was retained, epitope substitution improved purification in our experiments by eliminating the inefficiency of calmodulin affinity chromatography and by providing an alternative way of elution using the ProtC peptide in cases where EGTA inactivated protein function. Furthermore, HPC4 allowed highly sensitive and specific detection of ProtC-tagged proteins after protease cleavage. Thus far, we have successfully purified and characterized the U1 small nuclear ribonucleoprotein particle, the transcription factor complex TATA-binding protein related factor 4 (TRF4)/SNAPc/transcription factor IIA (TFIIA), and RNA polymerase I of Trypanosoma brucei.

* Corresponding author. Mailing address: Department of Genetic and Developmental Biology, University of Connecticut Health Center, 263 Farmington Ave., Farmington, CT 06030-3301. Phone: (860) 679-8878. Fax: (860) 679-8345. E-mail: gunzl{at}uchc.edu.

Eukaryotic Cell, November 2005, p. 1942-1950, Vol. 4, No. 11
1535-9778/05/$08.00+0     doi:10.1128/EC.4.11.1942-1950.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Preusser, C., Palfi, Z., Bindereif, A. (2009). Special Sm Core Complex Functions in Assembly of the U2 Small Nuclear Ribonucleoprotein of Trypanosoma brucei. Eukaryot Cell 8: 1228-1234 [Abstract] [Full Text]  
  • Palfi, Z., Jae, N., Preusser, C., Kaminska, K. H., Bujnicki, J. M., Lee, J. H., Gunzl, A., Kambach, C., Urlaub, H., Bindereif, A. (2009). SMN-assisted assembly of snRNP-specific Sm cores in trypanosomes. Genes Dev. 23: 1650-1664 [Abstract] [Full Text]  
  • Luz Ambrosio, D., Lee, J. H., Panigrahi, A. K., Nguyen, T. N., Cicarelli, R. M. B., Gunzl, A. (2009). Spliceosomal Proteomics in Trypanosoma brucei Reveal New RNA Splicing Factors. Eukaryot Cell 8: 990-1000 [Abstract] [Full Text]  
  • Lee, J. H., Jung, H. S., Gunzl, A. (2009). Transcriptionally active TFIIH of the early-diverged eukaryote Trypanosoma brucei harbors two novel core subunits but not a cyclin-activating kinase complex. Nucleic Acids Res 37: 3811-3820 [Abstract] [Full Text]  
  • Fisk, J. C., Sayegh, J., Zurita-Lopez, C., Menon, S., Presnyak, V., Clarke, S. G., Read, L. K. (2009). A Type III Protein Arginine Methyltransferase from the Protozoan Parasite Trypanosoma brucei. J. Biol. Chem. 284: 11590-11600 [Abstract] [Full Text]  
  • Zamudio, J. R., Mittra, B., Chattopadhyay, A., Wohlschlegel, J. A., Sturm, N. R., Campbell, D. A. (2009). Trypanosoma brucei Spliced Leader RNA Maturation by the Cap 1 2'-O-Ribose Methyltransferase and SLA1 H/ACA snoRNA Pseudouridine Synthase Complex. Mol. Cell. Biol. 29: 1202-1211 [Abstract] [Full Text]  
  • Li, Z., Wang, C. C. (2008). KMP-11, a Basal Body and Flagellar Protein, Is Required for Cell Division in Trypanosoma brucei. Eukaryot Cell 7: 1941-1950 [Abstract] [Full Text]  
  • Kramer, S., Queiroz, R., Ellis, L., Webb, H., Hoheisel, J. D., Clayton, C., Carrington, M. (2008). Heat shock causes a decrease in polysomes and the appearance of stress granules in trypanosomes independently of eIF2{alpha} phosphorylation at Thr169. J. Cell Sci. 121: 3002-3014 [Abstract] [Full Text]  
  • Umeyama, T., Wang, C. C. (2008). Polo-Like Kinase Is Expressed in S/G2/M Phase and Associated with the Flagellum Attachment Zone in both Procyclic and Bloodstream Forms of Trypanosoma brucei. Eukaryot Cell 7: 1582-1590 [Abstract] [Full Text]  
  • Panigrahi, A. K., Zikova, A., Dalley, R. A., Acestor, N., Ogata, Y., Anupama, A., Myler, P. J., Stuart, K. D. (2008). Mitochondrial Complexes in Trypanosoma brucei: A Novel Complex and a Unique Oxidoreductase Complex. Mol. Cell. Proteomics 7: 534-545 [Abstract] [Full Text]  
  • Li, Z., Gourguechon, S., Wang, C. C. (2007). Tousled-like kinase in a microbial eukaryote regulates spindle assembly and S-phase progression by interacting with Aurora kinase and chromatin assembly factors. J. Cell Sci. 120: 3883-3894 [Abstract] [Full Text]  
  • Rogers, K., Gao, G., Simpson, L. (2007). Uridylate-specific 3' 5'-Exoribonucleases Involved in Uridylate-deletion RNA Editing in Trypanosomatid Mitochondria. J. Biol. Chem. 282: 29073-29080 [Abstract] [Full Text]  
  • Kamil, J. P., Coen, D. M. (2007). Human Cytomegalovirus Protein Kinase UL97 Forms a Complex with the Tegument Phosphoprotein pp65. J. Virol. 81: 10659-10668 [Abstract] [Full Text]  
  • Nguyen, T. N., Schimanski, B., Gunzl, A. (2007). Active RNA Polymerase I of Trypanosoma brucei Harbors a Novel Subunit Essential for Transcription. Mol. Cell. Biol. 27: 6254-6263 [Abstract] [Full Text]  
  • Lee, J. H., Nguyen, T. N., Schimanski, B., Gunzl, A. (2007). Spliced Leader RNA Gene Transcription in Trypanosoma brucei Requires Transcription Factor TFIIH. Eukaryot Cell 6: 641-649 [Abstract] [Full Text]  
  • Takebe, S., Witola, W. H., Schimanski, B., Gunzl, A., Ben Mamoun, C. (2007). Purification of Components of the Translation Elongation Factor Complex of Plasmodium falciparum by Tandem Affinity Purification. Eukaryot Cell 6: 584-591 [Abstract] [Full Text]  
  • Hernandez, G. Jr, Valafar, F., Stumph, W. E. (2007). Insect small nuclear RNA gene promoters evolve rapidly yet retain conserved features involved in determining promoter activity and RNA polymerase specificity. Nucleic Acids Res 35: 21-34 [Abstract] [Full Text]  
  • Schimanski, B., Brandenburg, J., Nguyen, T. N., Caimano, M. J., Gunzl, A. (2006). A TFIIB-like protein is indispensable for spliced leader RNA gene transcription in Trypanosoma brucei. Nucleic Acids Res 34: 1676-1684 [Abstract] [Full Text]  


Copyright © 2009 by the American Society for Microbiology.   For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»