In this study, we created a new epitope combination for tandem affinity purification, termed PTP. It was generated by fusing two ProtA domains and the TEV protease cleavage site to ProtC, thereby replacing the CBP of the original TAP tag. Introduction of ProtC resulted in several advantages. First, purification of N- or C- terminal PTP-tagged SNAP50 from crude extracts was considerably more efficient than purification of this protein harboring the original TAP tag. The higher efficiency may be crucial if the protein to be purified is expressed at low levels or if it is difficult or expensive to grow a large culture volume of the organism to be investigated. Second, the monoclonal antibody HPC4 was a sensitive and highly specific tool for the detection of ProtC-tagged proteins in our experiments. This is especially important if there is no antibody available for the endogenous protein and the ProtA domains have been removed from the tagged protein by TEV protease cleavage. Third, ProtC offers two options for protein elution. As with CBP, ProtC-tagged proteins can be efficiently eluted from HPC4 beads by EGTA. However, if depletion of divalent cations irreversibly interferes with protein function, elution in the presence of the ProtC peptide is likely to yield functional protein. Since peptide elution can be carried out in any native buffer required for subsequent applications, a dialysis step becomes unnecessary and the eluate can be used directly in a functional assay.
Although we demonstrated successful PTP purification of different protein complexes, the protein yield may still be improved. While in both affinity chromatography steps almost all of the tagged protein bound to the column, only about half of this material was recovered in each case. Elution of tagged protein from IgG Sepharose is mediated by TEV protease. In our standard procedure, TEV protease cleavage was conducted overnight at 4°C to conserve protein function. The recovery rate may be increased if the cleavage reaction is conducted at a higher temperature, e.g., 16°C (23
). Furthermore, as a modification to the original TAP protocol we replaced NP-40 by Tween 20 in the TEV protease cleavage buffer, due to our finding that NP-40 interferes with transcriptional activity in our extracts much more strongly than Tween 20 (data not shown). However, if the use of NP-40 is of no concern, this detergent may improve the yield of protein recovery by TEV protease. Protein loss in the second chromatography step cannot be attributed to inefficient elution because only minor amounts of tagged protein remained on the anti-ProtC matrix (data not shown). Therefore, protein loss most likely occurred during concentration of the final eluate. To minimize this loss, we evaluated different concentration procedures and found that a combination of reducing the eluate volume by evaporation and binding of proteins to a hydrophobic resin (32
) was most effective (data not shown).
An open question is why the calmodulin affinity step in the original TAP method is efficient in some cases but not in others even when studies are conducted in the same organism. Since endogenous calmodulin can interact with CBP and prevent binding of the epitope to the calmodulin column, it is possible that the efficiency of the calmodulin affinity step is directly dependent on the relative amounts of free endogenous calmodulin and TAP-tagged protein in an extract, e.g., the endogenous calmodulin amount may not suffice to block purification of a more abundant protein, whereas it may efficiently block purification of a protein of low abundance, such as gene expression factors. Successful TAP applications in T. brucei
are in accordance with such an explanation because the tagged exosome subunit was overexpressed for purification (6
) and RNA editing complexes were purified from mitochondrial extracts, which probably lacked calmodulin (1
). At any rate, the PTP method has eliminated this possibility.
Our experiments have been conducted with trypanosomal extracts but except for the extract preparation procedure, the method described here does not contain trypanosome-specific features. Thus, we anticipate that PTP tagging and purification can be applied equally well to other organisms. Protein C is expressed in mammals as a plasma component involved in the regulation of the blood coagulation cascade. The protein is expressed only in hepatocytes, due to transcriptional regulation by three liver-specific elements in its gene promoter (20
). Furthermore, the HPC4-binding site of protein C is not well conserved among mammals; accordingly, the antibody does not recognize bovine protein C (29
). Thus, specific detection and purification of ProtC-tagged proteins by the HPC4 antibody should be feasible in other organisms, including most mammalian cell lines.
In conclusion, tandem affinity purification of PTP-tagged proteins is a promising alternative to the well-established TAP method, especially in cases where calmodulin affinity chromatography is problematic or where purification efficiency is crucial.