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

Abstract

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 CBPcalmodulin 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.Tandem affinity purification (TAP) has been developed as an efficient tool for protein complex purification under nondenaturing conditions (23,24). For this method, the protein of interest must be fused to the TAP tag and expressed in the organism or cell line under investigation. The TAP tag contains two immunoglobulin G (IgG)-binding domains of the Staphylococcus aureus protein A (ProtA) and the calmodulinbinding peptide (CBP). Both epitope types are separated by spacer regions and a cleavage site for tobacco etch virus (TEV) protease. In consecutive steps, TAP is achieved by binding of the tagged protein to an IgG column, release of the protein by TEV protease cleavage, binding of the CBP-tagged protein to a calmodulin column, and final elution of the bound protein by a buffer containing a chelating agent of divalent cations such as EGTA. The final elution step is facilitated by calcium cation dependence of CBP binding to calmodulin. The TAP method has been developed in the budding yeast Saccharomyces cerevisiae, and it has been outstandingly successful in this organism, enabling a thorough proteome characterization by systematic analysis of protein complexes (8). TAP has two great advantages over c...

Dynamic Localization of Trypanosoma brucei Mitochondrial DNA Polymerase ID

“…TbPOLID C-terminal coding sequence (1,635 bp) was PCR amplified from T. brucei 927 genomic DNA using forward (5=-ATA ATA GGG CCC TGC TCG TCA AGA GGT GCG-3=) and reverse (5=-ATA ATA CGG CCG CAG TGT CTC CTC AAT GAC AAC G-3=) primers containing ApaI and EagI sites, respectively. The PCR-amplified fragment was ligated into ApaI and NotI restriction sites of pC-PTP-NEO (44) to create the pPOLID-PTP-NEO vector.…”

The polyadenylation complex ofTrypanosoma brucei:Characterization of the functional poly(A) polymerase

“…40 The T. brucei open reading frames for Tb927.3.3160 (nts 672-1461), Tb927.7.3780 (nts 1172-2061), and CPSF160 (nts 3601-4356) were PCR-amplified and inserted in-frame into the pC-PTP-NEO vector upstream of the PTP tag sequence, using the ApaI and NotI restriction sites. For genomic integration, 10 mg of linearized constructs were transfected into procyclic T. brucei 427 cells and cloned by limiting dilution in the presence of G418 (40 mg/ml Geneticin; Gibco-BRL).…”

“…Tandem affinity purification of PTP-tagged proteins was done as described by Schimanski et al, 40 with minor modifications: Briefly, T. brucei cells were collected from 3-liter cultures (corresponding to »4 ml packed cell volume) and lysed in 7 ml extraction buffer (150 mM KCl, 20 mM Tris-Cl pH 7.7, 3 mM MgCl 2 , 0.5 mM DTT, containing a Complete Mini, EDTA-free protease inhibitor cocktail tablet [Roche]). For IgG affinity chromatography, 300 ml of IgG Sepharose 6 Fast Flow beads (packed bead volume; GE Healthcare) were incubated with extract for two hours at 4 C. Beads were washed extensively with PA-150 buffer (150 mM KCl, 20 mM Tris-Cl pH 7.7, 3 mM MgCl 2 , 0.5 mM DTT, 0.1% Tween20), followed by TEV protease buffer (150 mM KCl, 20 mM Tris-Cl pH 7.7, 3 mM MgCl 2 , 0.5 mM EDTA; 0.5 mM DTT, 0.1% Tween20).…”

A Novel Basal Body Protein That Is a Polo-like Kinase Substrate Is Required for Basal Body Segregation and Flagellum Adhesion in Trypanosoma brucei

“…To investigate whether SPBB1 and TbPLK interact in vivo in trypanosomes, we carried out co-immunoprecipitation. To this end, SPBB1 was endogenously tagged with a PTP (protein A-TEV-protein C) epitope (26) at its N terminus. Immunoprecipitation of PTP-SPBB1 was capable of pulling down TbPLK from the trypanosome cell lysate as detected by antiTbPLK antibody (Fig.…”