Highly selective split intein method for efficient separation and purification of recombinant therapeutic proteins from mammalian cell culture fluid.

Biologics and vaccines have been successfully developed over the last few decades to treat many diseases. Each of these drugs must be highly purified for clinical use. Monoclonal antibodies (mAbs), the dominant therapeutic modality on the market, can be easily purified using the standard Protein A affinity platform. However, no generally applicable affinity platforms are available for the manufacture of other therapeutic proteins for clinical use. Thus, multicolumn chromatography processes for widely being used for product purification. These processes demand significant optimization to meet desired product quality attributes, where each step also decreases final yields. In this work, we demonstrate the novel self-removing iCapTag™ affinity tag, which provides a new platform for capturing, concentrating, and purifying recombinant proteins. Importantly, this system provides a tagless target protein, which is suitable for research and clinical use, where the only requirement for tag removal is a small change in buffer pH. No additional proteins, reagents or cofactors are required. We also present case studies demonstrating the use of iCapTag™ for highly efficient purification of untagged interferon alpha 2b, the ML39 single chain variable fragment (scFv), and the receptor binding domain (RBD) of SARS-CoV-2 spike protein. These proteins were expressed and secreted by Expi293 cells with the self-removing tag fused to their N-terminus. We were able to obtain highly pure (> 99 %) tagless protein in a single purification step with high clearance of host cell DNA, tagged precursor, higher and lower molecular weight impurities. Based on these preliminary results, we propose the iCapTag™ as a universal capture platform for diverse classes of recombinant therapeutic proteins.

Streamlining the Detection of Human Thyroid Receptor Ligand Interactions with XL1-Blue Cell-Free Protein Synthesis and Beta-Galactosidase Fusion Protein Biosensors.

Thyroid receptor signaling controls major physiological processes and disrupted signaling can cause severe disorders that negatively impact human life. Consequently, methods to detect thyroid receptor ligands are of great toxicologic and pharmacologic importance. Previously, we reported thyroid receptor ligand detection with cell-free protein synthesis of a chimeric fusion protein composed of the human thyroid receptor beta (hTRß) receptor activator and a ß-lactamase reporter. Here, we report a 60% reduction in sensing cost by reengineering the chimeric fusion protein biosensor to include a reporter system composed of either the full-length beta galactosidase (ß-gal), the alpha fragment of ß-gal (ß-gal-α), or a split alpha fragment of the ß-gal (split ß-gal-α). These biosensor constructs are deployed using E. coli XL1-Blue cell extract to (1) avoid the ß-gal background activity abundant in BL21 cell extract and (2) facilitate ß-gal complementation reporter activity to detect human thyroid receptor ligands. These results constitute a promising platform for high throughput screening and potentially the portable detection of human thyroid receptor ligands.

Mechanistic discoveries and simulation-guided assay optimization of portable hormone biosensors with cell-free protein synthesis.

Nuclear receptors (NRs) influence nearly every system of the body and our lives depend on correct NR signaling. Thus, a key environmental and pharmaceutical quest is to identify and detect chemicals which interact with nuclear hormone receptors, including endocrine disrupting chemicals (EDCs), therapeutic receptor modulators, and natural hormones. Previously reported biosensors of nuclear hormone receptor ligands facilitated rapid detection of NR ligands using cell-free protein synthesis (CFPS). In this work, the advantages of CFPS are further leveraged and combined with kinetic analysis, autoradiography, and western blot to elucidate the molecular mechanism of this biosensor. Additionally, mathematical simulations of enzyme kinetics are used to optimize the biosensor assay, ultimately lengthening its readable window by five-fold and improving sensor signal strength by two-fold. This approach enabled the creation of an on-demand thyroid hormone biosensor with an observable color-change readout. This mathematical and experimental approach provides insight for engineering rapid and field-deployable CFPS biosensors and promises to improve methods for detecting natural hormones, therapeutic receptor modulators, and EDCs.