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Reinventing Affinity Tags Bioprocessing Innovative Technology Designed for Routine Purification of C-terminal EPEA-Tagged Recombinant Proteins Pim Hermans, Remko Clasen, and Frank Detmers, Ph.D. Epitope tagging is a technique that employs genetic engineering to fuse a known epit- ope, called an affinity tag, to either the C or N terminus of a recombinant protein to facilitate affinity purification and detec- tion. This approach enables high selective Figure 1. Protein gel analysis: ST=starting material (400 μL E. coli-derived periplasmic fraction); FT=flow through (unbound fraction); lanes 1–4 show the elution fraction following addition of 2M MgCl2 . The lane marked pH 2 shows the strip of the column removing any bound protein from the column. capture and circumvents the multistep pu- rification 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 Figure 2: Protein gel analysis: ST=starting material (E. coli cytoplasmic fraction); FT=flow through (unbound fraction coming off of the column); W =fraction after washing the column; lanes 1–3 show the elution fraction following addition of 2 M MgCl2 . The lane marked pH2 shows the eluate from the column after removal of any bound protein TECH NOTE Figure 3. Chromatogram: Purification of a camelid domain antibody with a C-terminal GAA-EPEA tag on a 400 μL Tricorn column (2 cm bed height volume) using an Äkta Explorer system (GE Healthcare Lifesciences). protein or proteins that might be present in culture media. BIOPROCESSING STARTS HERE A number of commonly used and com- mercially available affinity tags require pairing with different purification resins. However each of these tag systems has limitations. ij 8S WR / ZRUNLQJ YROXPH ZLWK &2 DQG KXPLGLW\ FRQWURO ij 5HIULJHUDWLRQ OLJKWLQJ DQG KLJK WHPSHUDWXUH RSWLRQV ij &XVWRPL;]DEOH IRU VKDNLQJ PLFURSODWH FXOWXUHV ZLWK D PP RUELW DQG VSHHGV XS WR USP ij 'HOLYHUHG VWDFNHG DQG UHDG\ WR XVH ij $75ĬV XQSDUDOOHOHG VXSSRUW DQG FXVWRPHU VHUYLFH ij 7KH 0XOWLWURQ 3UR IHDWXUHV ŏ DWSDQHO GRXEOH SDQH GRRU IRU HDV\ FOHDQLQJ DQG UHGXFHG FRQGHQVDWLRQ ZZZ DWUELRWHFK FRP 2IŎ FH )D[ For example large tags can alter protein function and typically cannot be used for further functionality studies after purifica- tion. In contrast, smaller affinity tags do not enable purification of high purity recombi- nant protein from complex mixtures. Affinity tags that use monoclonal anti- bodies 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 chro- matographic setup. Furthermore, monoclo- nal antibody-based resins do not efficiently capture target proteins under denaturing conditions. CaptureSelect C-tag To overcome many of these limitations, researchers at BAC (www.bacbv.com), in Pim Hermans is director of ligand discov- ery, Remko Clasen is business development manager, and Frank Detmers, Ph.D. (frank. detmers@bac.nl), is director of ligand appli- cation at BAC (www.bacbv.com). 48 | October 1, 2012 | genengnews.com | Genetic Engineering & Biotechnology News