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TECH NOTE Bioprocessing close collaboration with the VIB Research In- stitute, have developed CaptureSelect C-Tag, a new affinity resin purification product. It is composed of a single domain camelid anti- body that is produced in yeast and then im- mobilized onto a solid matrix support. Cap- tureSelect C-Tag has binding specificity due to a short four-amino-acid peptide tag: E-P- E-A (glutamic acid-proline-glutamic acid-ala- nine) 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. Applications The CaptureSelect C-tag affinity resin is designed to provide high selectivity for a single step purification of EPEA-tagged re- combinant 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 affin- ity 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 puri- fication 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 pro- tein (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. Pro- tein gel analysis of the elution fraction fol- lowing addition of 2M MgCl2 and analysis using UV light clearly illustrated the recovery of the GFP, which produced a green fluores- cent 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 com- patible with mild elution buffers the cap- tured 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 con- centration. Genetic Engineering & Biotechnology News | genengnews.com | October 1, 2012 | 49 For aseptic connection…think Saint-Gobain. Think Saint-Gobain…for sterile transfer. www.biopharm.saint-gobain.com/media/G10C The CaptureSelect C-tag system also al- lows binding of the protein-of-interest under denaturing conditions. Once the protein is bound to the resin in the presence of a dena- turant (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 5 1.7 mg/mL). In addition to the CaptureSelect C-tag af- finity resin, the anti-C-tag affinity ligand is also provided as conjugated ligand (e.g., con- jugated to Biotin). This facilitates easy detec- tion and or quantitation of EPEA C-tagged ® proteins using techniques such as ELISA, Western blot, and label-free platforms (Bi- acore and Octet). In summary, the EPEA tag, in combina- tion with the CaptureSelect C-tag affinity matrix, is providing an alternative to ex- isting technologies to facilitate the routine purification and analysis of proteins during R&D.; 5x increase in seal reliability Think Saint-Gobain when you need reliable and secure aseptic connection and sterile fluid transfer. NEW 1" SIZE FEATURES/BENEFITS: > Ultra-secure O-ring seal > Easy to assemble > Widest range of sizes in the industry > Patented Pure-Fit® SIB® technology > Fully validated for use in BioProduction > Animal derivative-free and BPA-free materials > Triple-redundant locking mechanism - simplifies validation > Can be sterilized via gamma irradiation or autoclave > Available as part of a presterilized Single Use Bio-Simplex™ custom manifold