Design of a methotrexate-controlled chemical dimerization system and its use in bio-electronic devices

Abstract

Natural evolution produced polypeptides that selectively recognize chemical entities and their polymers, ranging from ions to proteins and nucleic acids. Such selective interactions serve as entry points to biological signaling and metabolic pathways. The ability to engineer artificial versions of such entry points is a key goal of synthetic biology, bioengineering and bioelectronics. We set out to map the optimal strategy for developing artificial small molecule:protein complexes that function as chemically induced dimerization (CID) systems. Using several starting points, we evolved CID systems controlled by a therapeutic drug methotrexate. Biophysical and structural analysis of methotrexate-controlled CID system reveals the critical role played by drug-induced conformational change in ligand-controlled protein complex assembly. We demonstrate utility of the developed CID by constructing electrochemical biosensors of methotrexate that enable quantification of methotrexate in human serum. Furthermore, using the methotrexate and functionally related biosensor of rapamycin we developed a multiplexed bioelectronic system that can perform repeated measurements of multiple analytes. The presented results open the door for construction of genetically encoded signaling systems for use in bioelectronics and diagnostics, as well as metabolic and signaling network engineering.

Protein engineering of a nanoCLAMP antibody mimetic scaffold as a platform for producing bioprocess-compatible affinity capture ligands

“…Their general compatibility with high temperature, organic solvent, and pH extremes may enable industrial applications where extreme conditions are required. For example, nC-A class nanoCLAMPs have recently been utilized in electrochemical biosensors ( 23 ). The robustness of the improved nC-B class has the potential to enable the development of sensors and smart materials which encounter extreme conditions during manufacture or use.…”

A simeprevir-inducible molecular switch for the control of cell and gene therapies

“…In another study, two sequential phage display selections were used to identify both protein components of a CID system 8 . An alternative approach is to start from a known protein:small molecule complex and identify specific protein binding partners by selection from phage display libraries of antibody (or antibody-like scaffold) proteins 9 – 11 or computational design 12 . Protein engineering and directed evolution have also been used to alter the ligand-binding specificity of the natural ABA-based CID, thus creating a variety of CID modules 13 – 15 .…”

Highly Porous Gold Electrodes – Preparation and Characterization

“…[61] Among many other examples of roughened electrode surfaces, [62][63][64][65] microporous Au electrodes developed recently using the DHBT method [13,66] have found various bioelectrochemical applications, especially in biosensors. [67][68][69][70] These electrodes are particularly attractive for their use in bioelectrochemical systems due to the increased load of biomolecules (e. g., biocatalytic enzymes) at the enlarged surface area and easy diffusional access of reactive species (e. g., enzyme cofactors, substrates or analytes) through broadly open microcavities. The use of the Au conductive support is also convenient for biomolecule immobilization using a thiol selfassembling method for creating a linker to bind biomolecules.…”