(477d) Engineering a Reversible and High-Affinity Scaffold for Protein Purification

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 developed a new non-chromatographic affinity method for the purification of secreted proteins.  The approach is to engineer E. coli cells to secrete target proteins that are fused with an affinity tag. Direct purification and recovery can be achieved using a thermo-responsive elastin-like protein (ELP) scaffold containing the capturing ligands. Naturally occurring cohesin-dockerin pairs, which are high-affinity protein complex responsible for the formation of cellulosome in anaerobic bacteria, were used as the 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 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. This method offers great flexibility as a wide range of affinity tags and ligands can be used.