Seth Thompson

Membrane proteins achieve a remarkable range of cellular functions, yet their characterization at high throughputs remains difficult with standard reconstitution methods. Here, we develop on-bead reconstitution into bilayers via cell-free transcription and translation (ORB-TXTL), a platform that uses compositionally tunable lipid bilayer-coated silica beads as scaffolds for cell-free synthesized interacting and integral membrane proteins. ORB-TXTL is fast as it takes only a few hours to integrate membrane proteins onto the beads, which can be extensively washed and seamlessly transferred between reaction buffers to perform assays that are read out by standard laboratory equipment without tagging and sophisticated protocols. We first characterized the lipid interactions of the mechanosensitive channel MscL, then screened 169 Escherichia coli proteins, and identified a systematic dependence of membrane integration efficiency on the number of transmembrane domains. Finally, we functionally reconstituted the E. coli phospholipid synthesis pathway, demonstrating that ORB-TXTL is a tractable and cheap chassis for multienzyme membrane biochemistry.

Membrane proteins achieve a remarkable range of cellular functions, yet their characterization at high throughputs remains difficult with standard reconstitution methods. Here, we develop On-bead Reconstitution into Bilayers via Cell-free Transcription and Translation (ORB-TXTL), a platform that uses compositionally tunable lipid bilayer-coated silica beads as scaffolds for cell-free synthesized interacting and integral membrane proteins. ORB-TXTL is fast as it just takes a few hours to integrate membrane proteins onto the beads, which can be extensively washed and seamlessly transferred between reaction buffers, to perform assays that are read out by standard laboratory equipment without tagging and sophisticated equipment. We first characterized the lipid interactions of the mechanosensitive channel MscL, then screened 169 E. coli proteins and identified a systematic dependence of membrane integration efficiency on the number of transmembrane domains. Finally, we functionally reconstituted the E. coli phospholipid synthesis pathway, demonstrating that ORB-TXTL is a tractable and cheap chassis for multi-enzyme membrane biochemistry.

Cell-free synthetic biology approaches offer biosafe, low-cost and versatile genetic tools to advance therapeutic research and development. Measuring the antibody response to a range of target and off-target proteins is essential for deep immuno-profiling of therapeutic antibodies and individual patient immune responses. Here we extend a previously developed microfluidic-free biochip platform to quantitatively reconstitute interactions of cell-free synthesized antigens with antibodies in miniaturized, photolithographically patterned compartments from localized gene brushes. This creates a continuous density gradient of antigens displayed on the surface, generating multiple antibody binding curves, one in each single nanolitre-volume compartment for affinity determination. We used SARS-CoV-2 antigens to profile the specificity and affinity of monoclonal antibodies to more than 30 viral epitopes, which were synthesized simultaneously on a single chip. We also profiled polyclonal antibodies in a total of 1 μl of human serum, revealing patient-specific epitope profiles that are difficult to detect by conventional approaches. By spatially separating gene brushes in the compartment, we extended the gradient approach to reconstitute the interaction of on-chip cell-free expressed human ACE2 receptor with the viral receptor-binding domain in a specific manner. This on-chip genetically programmed approach enables rapid and quantitative interrogation of complex protein–protein interactions, without protein purification steps, for human immuno-profiling and preparedness for emerging pathogens.