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 secreted proteins. The approach is to engineer cells to secrete target proteins that are fused with an affinity tag. Direct purification and recovery can be achieved using thermo-responsive elastin-like protein (ELP) scaffolds containing different capturing domains. Naturally occurring cohesin-dockerin pairs, which are high-affinity protein complex responsible for the formation of cellulosome in anaerobic bacteria, were used as the initial model. 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 additional cycle of thermal precipitation. Because of the modular nature of method, we will discuss the use of smaller interaction domains for the direct purification of secreted proteins from a wide range of recombinant hosts.

Engineering high-affinity scaffolds for protein purification

243rd National Meeting (2012)

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