Highly efficient tandem affinity purification of trypanosome protein complexes based on a novel epitope combination.
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.
Schimanski
Bernd
B
Department of Genetic and Developmental Biology, University of Connecticut Health Center, 263 Farmington Ave., Farmington, CT 06030-3301, USA.
Nguyen
Tu N
TN
Günzl
Arthur
A
D000818:Animals; D002846:Chromatography, Affinity / Q000379:methods*; D000939:Epitopes; D006801:Humans; D046912:Multiprotein Complexes; D015800:Protozoan Proteins / Q000235:genetics / Q000302:isolation & purification*; D012318:RNA Polymerase I / Q000302:isolation & purification; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D035561:Transcription Factor TFIIA / Q000302:isolation & purification; D014158:Transcription, Genetic; D014346:Trypanosoma brucei brucei / Q000235:genetics / Q000378:metabolism*
D000939:Epitopes; D046912:Multiprotein Complexes; D015800:Protozoan Proteins; D011993:Recombinant Fusion Proteins; D035561:Transcription Factor TFIIA; D012318:RNA Polymerase I