Methods for cell-free protein expression
Inventors
Chen, Michael Chun Hao • Chen, Sihong • Dulson, Christopher • Kalsi, Sumit • Mcinroy, Gordon Ross • Reikine, Stephanie
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Assignees
Member
NucleraNuclera develops automated benchtop platforms and integrated systems for rapid protein expression, optimization, and purification, utilizing cell-free synthesis, digital microfluidics, and software-driven workflows. Their technology enables miniaturized and scalable protein prototyping—including challenging targets such as membrane proteins—directly at the lab bench. Nuclera serves academic and industrial researchers, focusing on reducing turnaround time for functional protein access and streamlining screening and production. The company has secured significant funding to enable broad commercialization, expanded their leadership team to support scale-up, and continues to drive advancements in drug discovery, proteomics, and experimental automation.
Nuclera develops automated benchtop platforms and integrated systems for rapid protein expression, optimization, and purification, utilizing cell-free synthesis, digital microfluidics, and software-driven workflows. Their technology enables miniaturized and scalable protein prototyping—including challenging targets such as membrane proteins—directly at the lab bench. Nuclera serves academic and industrial researchers, focusing on reducing turnaround time for functional protein access and streamlining screening and production. The company has secured significant funding to enable broad commercialization, expanded their leadership team to support scale-up, and continues to drive advancements in drug discovery, proteomics, and experimental automation.
Publication Number
US-11821018-B2
Publication Date
2023-11-21
Expiration Date
Abstract
Disclosed is a method for the cell-free expression of peptides or proteins in a liquid filled digital microfluidic device. The droplets having the components required for cell-free protein expression can be manipulated by electrokinesis in order to enhance levels of protein expression in the droplets.
Core Innovation
Disclosed is a method for the cell-free expression of peptides or proteins in a digital microfluidic device. The method comprises one or more droplets containing a nucleic acid template and a cell-free system having components for protein expression in an oil-filled environment, and moving said droplets using electrokinesis. The components for the cell-free protein synthesis droplet can be pre-mixed prior to introduction to or mixed on the digital microfluidic device.
The background identifies limitations that have prevented practical use of electrowetting-on-dielectric (EWoD) devices for cell-free biological applications, including biofouling that irreversibly pins aqueous droplets, evaporation of droplets in air-filled systems, and insufficient oxygen in confined microfluidic droplets. Prior art has only suggested desirability of performing CFPS in microfluidic devices and practical working systems had not been demonstrated.
The invention addresses these problems by operating CFPS droplets in an oil-filled EWoD or a sealed humidified gaseous environment and by moving droplets using electrokinesis to supply and disperse oxygen and to mix reagents, which improves levels of protein expression compared with static droplets. The inventors demonstrate that CFPS reagents can be actuated on an oil-filled EWoD device for extended periods with minimal surface fouling, and that droplets can be merged, split, and screened on the device.
Claims Coverage
Independent claim identified: claim 1. The following 6 main inventive features are extracted from the independent claim.
Digital microfluidic device environment
A digital microfluidic device having a two-dimensional array of planar microelectrodes and having an oil-filled environment or a sealed humidified gaseous environment.
Plurality of nucleic acid droplets with different templates
Taking a plurality of nucleic acid droplets having a different nucleic acid template.
Plurality of cell-free expression system droplets
Taking a plurality of cell-free expression system droplets, each containing a cell-free system having components for protein expression.
Combination by electrowetting-on-dielectric
Combining the nucleic acid droplets and the cell-free expression system droplets using electrowetting-on-dielectric (EWoD) to produce a plurality of protein expression droplets capable of expressing proteins.
Mixing protein expression droplets during expression
Mixing the protein expression droplets for [procedural detail omitted for safety] to enable cell-free expression of synthesized proteins that include a detectable tag.
Merging with additive droplets to form a fluorescent protein for screening
Merging the protein expression droplets with additive droplets having an entity which binds to the detectable tag to form a fluorescent protein, in order to screen protein expression levels by measuring fluorescence from the fluorescent protein, thereby detecting the expression of proteins within individual droplets.
Claim 1 covers methods of preparing and manipulating distinct nucleic acid and cell-free reagent droplets on a digital microfluidic device with an oil-filled or sealed humidified environment using EWoD to form, mix, and screen protein expression droplets, including formation of a detectable fluorescent readout within individual droplets.
Stated Advantages
Enables the cell-free expression of peptides or proteins in a digital microfluidic device.
Allows long-term actuation of CFPS droplets on an oil-filled EWoD device with minimal surface fouling or biofouling.
Actuation (movement) during expression supplies and disperses oxygen and mixes reagents, improving levels of protein expression compared with static droplets.
Permits matrix screening with small volumes of expensive reagent and enables reagents to be added to droplets after expression.
Enables methods that mimic continuous flow (CF) and continuous exchange (CE) CFPS to scale up CFPS production.
Documented Applications
Cell-free expression of peptides or proteins in a digital microfluidic device using droplets containing nucleic acid templates and cell-free expression components.
Forming, splitting, merging and actuating droplets on a digital microfluidic device to initiate and control cell-free protein synthesis, including formation of droplet arrays and initiation by adding plasmid droplets.
Screening protein expression levels within individual droplets by merging expression droplets with additive droplets that bind a detectable tag to form a fluorescent protein and measuring fluorescence.
Immobilizing CFPS-expressed proteins via affinity tags to a solid-support affinity resin and delivering fresh batches of CFPS reagent over the resin to mimic continuous flow and continuous exchange CFPS for scale-up.
Providing kits comprising a digital microfluidic device, a reagent source to generate droplets containing a cell-free extract system, and an oil or oxygenated oil for preparing a plurality of peptides or proteins.
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