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.
Therapeutic protein drugs from biotechnology processes have exerienced explosive growth over the past decades and are estimated to reach a market size of over $165 billion by 2018. Many of these protein products are glycoproteins and their human use require exhaustive purifications characterized by multiple chromatography steps. In this NSF-sponsored research, we developed a new glycoprotein purification scheme that could change how affinity purification is conducted and potentially lower the cost of purification and meanwhile reduces the complexity in downstreme processes. We developed themo-responsive bacterial lectin-based affinity ligands that recognize and bind sugars on a glycoprotein. Upon binding, a mild temperature shift allows the protien complex to precipitate, which could be collected by centrifugation or filtration and subsequntly the target protein is separated from the ligand by a midl temperature change. We showed that the target glycoprotein is separated from the ligand by a mild temperature change. We showed that the target glycoprotein could be recovered in high yield and high purity owing to the high spedificity of the affinity ligand, Besides glycoprotein purification, the ligand and the method developed is expected to be widely useful in the rapidly growing field of glycomics. A US provisional patent was filed for the non-chromatographic glycoprotein purification technology. Further studies are ongoing to test therapeutic protein drugs from major biotechnology firms. Besides lectin-based ligands, we also developed other recombinant thermal-responsive ligands that incorporate a naturally existing cohesin-dockerine affinity interaction as a new mechanism of protein purification. In this approach, the target protein is fused with a tag (spedifically docekrine), which is recognized by the engineered ligand containing cognate cohesin. Although we have usedd the new ligands for purification of only a handful of target proteins, the new ligands and the method developed are generally applicable. Furthermore, using an intein-based tag removal, we demonstrated that the tag introduced in the process of purification could be self-cleaved to generate an authentic protein.
