While protein purification has long been dominated by standard chromatography, the relatively high cost and complex scale-up have promoted the development of alternative non-chromatographic separation methods. Here, we reported a new non-chromatographic affinity method for the purification of proteins by fusing the target protein with a dockerin domain from Clostridium thermocellum. Direct purification and recovery can be achieved using thermo-responsive elastin-like protein (ELP) scaffold containing cohesin domain from the same species. By exploiting the highly specific interaction between the dockerin and cohesin domain and the reversible aggregation property of ELP, highly purified and active dockerin-tagged proteins, such as endoglucanase CelA from C. thermocellum, chloramphenicol acetyl transferase (CAT) and EGFP, were recovered directly from crude cell extracts in a single purification step with yields achieving over 90%. Incorporation of a self-cleaving intein domain enabled rapid removal of the affinity tag from the target proteins by an addition cycle of thermal precipitation.The purification cost can be further reduced by regenerating and recycling of the ELP-cohesin capturing scaffold. However, due to the high binding affinity between cohesin and dockerin domains, the bound dockerin-intein tag cannot be completely disassociated from ELP-cohesin scaffold after binding. Therefore, a truncated version of dockerin with the calcium-coordinating function impaired was used in replace of the original full length dockerin domain. The truncated dockerin domain functioned effectively as an affinity tag, and the efficient EDTA-mediated dissociation of the bound dockerin-intein tag was also realized. The regenerated ELP-cohesin scaffold was reused for additional purification cycles without any decrease in the efficiency.

Engineering a high-affinity and recyclable scaffold for protein purification

245th National Meeting (2013)

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