Protein engineering: signaling and transport

To engineer artificial sensory and transport functions, our group specifically aims to (i) develop foundational technologies for protein engineering which includes new DNA assembly procedures and genetic screening systems, (ii) dissect the sequence-structure-function relationships underlying artificially engineered protein functions, (iii) map the evolutionary trajectories that connect distinct protein functions, and finally (iv) develop molecular tools for different applications in biotechnology, biomedicine and basic research.

Our primary goal concerns the construction of synthetic protein switches, sensors and thereof derived signalling circuits with tailored response functions. A specific area of interest concerns the development and application of tailored fluorescent protein sensors and protease switches. In particular, our group focusses on the role of linkers that underlie conformational transitions and other key functional properties of artificially engineered protein switches and sensors such as expression and folding. To facilitate linker engineering, we recently developed a linker toolbox along with a new DNA assembly process – termed iterative functional linker cloning (iFLinkC) – tailored to the needs of proteins. Combinatorial library screens have since demonstrated a large plasticity underlying functional linker space and highlighted the potential of unconventional Pro-rich linkers that frequently yield the most potent switches and sensors. Additional efforts now focus on implementing tailored sensors, switches and circuits in live cells for different biotechnological application. This is complemented by foundational projects that aim to examine how the operation of synthetic protein switches, sensors and circuits in live cells impacts host cell physiology.

Our second goal concerns the construction of artificial transport functions across biological membranes. Here, progress has been hampered by a lack of (i) molecular toolboxes of well characterised molecular components to construct tailored transport function, and (ii) genetic screening systems that enable us to assay nanopores, ion channels and transporters in high-throughput. Addressing these limitations, we recently developed a Functional Nanopore (FuN) screen to assay the functional properties of nanopores, ion channels and transporters in high-throughput. The assay features an optical read-out and can be applied across a range of experimental formats including conventional microtitre plates, on-plate colony, FACS and microfluidics. Current efforts aim to dissect the molecular and genetic factors that underlie the assembly, stoichiometry, permeability, specificity and size of pore-forming membrane peptides. Building on these foundational protein engineering efforts, our mid-term goal is to develop molecular toolboxes for nanopore engineering and develop them for different biotechnological applications.

Our third goal aims to develop systematic approaches to functionalise track-etched nanopores with tailored sensors, switches and receptors. Compared to biological membranes, nanoporous membranes are substantially more robust, but capable of generating equally sensitive and specific electrical read-outs with many potential applications as medical biosensors, in particular point-of-care diagnostics. Towards this goal, we recently developed a general approach to functionalise track-etched, nanoporous polymer foils with antibody fragments achieving quantitative and ultrasensitive electrical responses for a variety of analytes. Building on this capacity, we now aim to functionalise nanoporous materials with recombinant proteins in a systematic fashion. This includes both foundational studies and the developing biomedical applications looking to exploit the exquisite diversity of natural protein functions and examine how functionalisation impacts the responsiveness of track-etched nanopores in the context of biomedically-relevant analytes.