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

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