October 1, 2012 (Vol. 32, No. 17)
Frank Detmers, Ph.D.
Innovative Technology Designed for Routine Purification of C-terminal EPEA-Tagged Recombinant Proteins
Epitope tagging is a technique that employs genetic engineering to fuse a known epitope, called an affinity tag, to either the C or N terminus of a recombinant protein to facilitate affinity purification and detection. This approach enables high selective capture and circumvents the multistep purification processes that limit throughput during R&D.
The ideal affinity tag should be small in size and as inert as possible to limit any potential interaction with the recombinant protein or proteins that might be present in culture media.
A number of commonly used and commercially available affinity tags require pairing with different purification resins. However each of these tag systems has limitations.
For example large tags can alter protein function and typically cannot be used for further functionality studies after purification. In contrast, smaller affinity tags do not enable purification of high purity recombinant protein from complex mixtures.
Affinity tags that use monoclonal antibodies as binding agents provide a higher selectivity than Ni+ resin columns. However, the reusability of these resins is poor as the antibodies can become unstable in a chromatographic setup. Furthermore, monoclonal antibody-based resins do not efficiently capture target proteins under denaturing conditions.
To overcome many of these limitations, researchers at BAC, in close collaboration with the VIB Research Institute, have developed CaptureSelect C-Tag, a new affinity resin purification product. It is composed of a single domain camelid antibody that is produced in yeast and then immobilized onto a solid matrix support. CaptureSelect C-Tag has binding specificity due to a short four-amino-acid peptide tag: E-P-E-A (glutamic acid-proline-glutamic acid-alanine) that is fused in frame at the C-terminus of any target recombinant protein.
The affinity resin also recognizes the E-P-E-A tag sequence when fused to linkers such as GAA or GYQDY between the C-terminus and the E-P-E-A tag. The need for a linker is determined by the target protein. Other linkers can be applied providing the EPEA sequence is fused at the C-terminal end of the protein of interest.
The CaptureSelect C-tag affinity resin is designed to provide high selectivity for a single step purification of EPEA-tagged recombinant proteins from complex mixtures of periplasmic or cytoplasmic fractions from E. coli-derived expression systems, as well as mammalian or any other complex mixture.
Besides packed bed columns, the affinity resin also shows good compatibility with high-throughput purification setups such as pipette tips or miniature columns used in combination with robotics.
A schematic outlining the one-step purification procedure and data obtained from purifying a 12 kD camelid single domain antibody with either the C-terminal tag -GYQDY-EPEA (A) or -GAA-EPEA (B) from E. coli-derived periplasmatic fractions using a CaptureSelect C-tag affinity resin in a spin column format (MoBiTec) is shown in Figure 1.
These results illustrate that this one- step process yields good target protein purity and recovery from complex mixtures using a neutral pH-based elution buffer (20 mM Tris, 2 M MgCl2, pH 7).
Figure 2 shows a green fluorescent protein (GFP) containing a GYQDY-EPEA C-terminal tag purified using CaptureSelect C-tag affinity resin in a spin column format from an E. coli cytoplasmatic fraction. Protein gel analysis of the elution fraction following addition of 2M MgCl2 and analysis using UV light clearly illustrated the recovery of the GFP, which produced a green fluorescent signal in the tube containing the pooled elution fractions. Subsequent elution of the column at pH 2 confirmed that the bound protein had been successfully eluted in the previous steps.
For sensitive proteins that are not compatible with mild elution buffers the captured protein can be released by competition using the synthetic peptide S-E-P-E-A at a concentration of 2 mM in 20 mM Tris, pH 7. Typically, greater than 60% of the protein is released using this approach, which can be further optimized by the addition of NaCl and/or increasing the S-E-P-E-A peptide concentration.
The CaptureSelect C-tag system also allows binding of the protein-of-interest under denaturing conditions. Once the protein is bound to the resin in the presence of a denaturant (e.g., 8 M urea), the affinity resin can then be incubated with a suitable buffer to allow on-column re-folding prior to elution.
The CaptureSelect C-tag affinity resin can be used in a packed-bed format as well. The chromatogram in Figure 3 demonstrates the purification of a camelid domain antibody equipped with a C-terminal GAA-EPEA tag (MW 14.9 kDa, DBC ≈ 1.7 mg/mL).
In addition to the CaptureSelect C-tag affinity resin, the anti-C-tag affinity ligand is also provided as conjugated ligand (e.g., conjugated to Biotin). This facilitates easy detection and or quantitation of EPEA C-tagged proteins using techniques such as ELISA, Western blot, and label-free platforms (Biacore and Octet).
In summary, the EPEA tag, in combination with the CaptureSelect C-tag affinity matrix, is providing an alternative to existing technologies to facilitate the routine purification and analysis of proteins during R&D.