This NSF award by the Chemical and Biological Separations program supports work by Professors Wilfred Chen and Rachel Chen at University of California, Riverside and Georgia Institute of Technology, respectively, to develop an integrated platform for the extracellular secretion of affinity tagged proteins and their non-chromatographic purification. The integration of the development of genetically engineered capturing scaffolds with protein secretion represents a unique effort that expands the fundamental development of protein engineering with the implementation of purification technology.
The proposed research will provide a flexible platform for the direct purification of mature authentic proteins from the growth medium while retaining the high specificity of the affinity-based interaction. The integrated platform is expected to simplify and lower the cost of protein production and can be easily adaptable for any affinity tagged protein of interest. This research will combine the expertise of the two PIs in order to provide a proof of concept study for the implementation of this transformative technology for protein purification. Moreover, graduate students participating in this research will gain an integrated perspective of the important interfaces and synergies connecting biochemistry, modern genetics, and process engineering.
The ever increasing demand of novel protein drugs necessitates the use of more effective processes for their large-scale production. Development of a cost-effective and highly efficient purification processes has become one of the most pressing challenges. While protein purification has long been dominated by standard chromatography, the limitations of high cost and complex scale-up have prompted the development of alternative non-chromatographic separation methods. Our approach is to develop a non-chromatographic purification scheme based the use of affinity tagged proteins. The target proteins was fused with an affinity tag and self-cleaving intein sequence. Direct purification and recovery was achieved using a thermo-responsive ELP scaffold containing the capturing ligands for affinity-based protein purification 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 from Clostridium thermocellum and the reversible aggregation property of ELP, highly purified and active dockerin-tagged proteins, such as the endoglucanase CelA, chloramphenicol acetyl transferase (CAT) and enhanced green fluorescence protein (EGFP), were recovered directly from crude cell extracts in a single thermal precipitation step with yields achieving over 90%. Incorporation of a self-cleaving intein domain enabled rapid removal of the affinity tag from the target proteins, which was subsequently removed by another cycle of thermal precipitation. This method offers great flexibility as a wide range of affinity tags and ligands can be used. In order to further reduce the purification cost by recycling the ELP capturing scaffold, a truncated dockerin domain with the calcium-coordinating function partially impaired was employed. We demonstrated that the truncated dockerin domain was sufficient to function as an effective affinity tag, and the target protein was purified directly from cell extracts in a single binding step followed by intein cleavage. The efficient EDTA-mediated dissociation of the bound dockerin-intein tag from the ELP-cohesin capturing scaffold was realized, and the regenerated ELP capturing scaffold was reused in another purification cycle without any decrease in the purification efficiency. This recyclable non-chromatographic based affinity method provides an attractive approach for efficient and cost-effective protein purification.
