Protein expression reagents

Abstract

Provided herein are methods of cell-free protein synthesis, optimised cell-free protein synthesis (CFPS) reagents, and methods for optimising CFPS reagents to increase protein expression yields using additional NTPs and / or an additional energy source added with a nucleic acid template. The methods are applicable to protein expression on a microfluidic device having hydrophobic surfaces by merging droplets on the device in order to screen a selection of expression compositions in parallel.

Description

[0001] PROTEIN EXPRESSION REAGENTS

[0002] FIELD OF THE INVENTION

[0003] Provided herein are methods of cell-free protein synthesis, optimised cell-free protein synthesis (CFPS) reagents, and methods for optimising CFPS reagents to increase protein expression yields. The methods are applicable to protein expression on a microfluidic device having hydrophobic surfaces.

[0004] BACKGROUND TO THE INVENTION

[0005] Cell-free protein synthesis (CFPS) has become an important tool for molecular biologists by playing a central role in a wide variety of applications. Cell-free systems can be categorized into two main classes: cell extracts and recombinant systems. Cell extracts are highly functional but complex and undefined systems. In 2001 , Shimizu et al. demonstrated that a defined cell-free system called the “PURE” system (protein synthesis using recombinant elements) could be reconstituted from purified recombinant components.

[0006] The biggest advantage of CFPS is that it is the quickest way to obtain an expressed phenotype (protein) from a genotype (gene). Starting with a PCR or plasmid template, in-vitro protein synthesis and functional assays can be carried out in a few hours. Moreover, it is independent of host cells. However, extract-based systems are known to often contain nonspecific nucleases and proteases that adversely affect protein synthesis. CFPS systems are open systems that are suitable for modification by addition of external components.

[0007] Cell-free protein synthesis, also known as in-vitro protein synthesis or CFPS, is the production of peptides or proteins using biological machinery in a cell-free system, that is, without the use of living cells. The in-vitro protein synthesis environment is not constrained within a cell wall or limited by conditions necessary to maintain cell viability, and enables the rapid production of any desired protein from a nucleic acid template, usually plasmid DNA or RNA from an in- vitro transcription. CFPS has been known for decades, and many commercial systems are available. Cell-free protein synthesis encompasses systems based on crude lysate (Cold Spring Harb Perspect Biol. 2016 Dec; 8(12): a023853) and systems based on reconstituted, purified molecular reagents, such as the PURE system for protein production (Methods Mol Biol. 2014; 1118: 275-284). CFPS requires significant concentrations of biomacromolecules, including DNA, RNA, proteins, polysaccharides, molecular crowding agents, and more (Febs Letters 2013, 2, 58, 261-268). To date, digital microfluidics, electrowetting-on-dielectric (EWoD), and electrokinesis in general have only found limited uses in cell-free biological-based applications, mostly due to biofouling, where biological components such as proteins, nucleic acids, crude cell extracts and other bioproducts adsorb and / or denature to hydrophobic surfaces. Biofouling is well known in the art to limit the ability of EWoD devices to manipulate droplets containing biomacromolecules. Wheeler and colleagues report that the maximum actuation time for droplets on EWoD devices containing biological media is 30 min before biofouling inhibits EWoD-based droplet actuation (Langmuir 2011 , 27, 13, 8586-8594).

[0008] On an EWoD device, cell lysate expression leads to variable outcomes. The level of protein expression in different droplets on the same device is not consistent. The use of purified component mixtures gives reliable expression, but at a lower level than the best cell lysate mixtures. The inventors herein have improved the reliability of expression levels of proteins. Additionally, purified component mixtures are typically not capable of performing downstream maturation of the produced protein into the desired form. For example, purified component mixtures are not capable of effecting post-translational modification of proteins, such as glycosylation. The inventors herein have improved the utility of purified component mixtures for CFPS.

[0009] Reconstituted reagents for cell-free protein synthesis are commercially available. For example PUREfrex® kit is a reconstituted in-vitro Coupled Transcription / Translation Systems, completely different from an E.coli extract S30 system. By adding DNA or mRNA that encodes the target protein to the reaction solution, proteins can be synthesized easily and quickly without using living cells(https: / / purefrex.genefrontier.com / ).

[0010] SUMMARY

[0011] Disclosed herein is an improved reconstituted composition for the cell-free expression of proteins. The inventors herein have appreciated that during storage of the cell-free expression system, the enzymes required for protein expression are sufficiently active to metabolise the energy systems and nucleoside triphosphates (NTP’s) needed for protein expression despite the absence of any nucleic acid template. Thus when the template is added, the performance of the expression system is sub-optimal due to the lack of energy sources and / or NTP’s. The addition / replenishment of energy sources and / or NTP’s along with the nucleic acid templates improves the efficiency of expression of the protein of interest.

[0012] Disclosed is a method for the cell-free synthesis of proteins, the method comprising mixing a first composition containing reagents for cell-free protein expression, and a second composition containing a nucleic acid template and NTP’s and / or an energy source for the cell-free protein expression reagents. The nucleoside triphosphates may be selected from one or more of ATP, CTP, GTP or UTP. The NTP may be ATP. The NTP may be CTP. The NTP may be GTP. The NTP may be UTP. Two or more NTP’s may be added, for example ATP and GTP. Four NTP’s may be added.

[0013] Disclosed is a method for the cell-free synthesis of proteins, the method comprising mixing a first composition containing reagents for cell-free protein expression, and a second composition containing an energy source for the cell-free protein expression reagents. Either the first or second composition may contain a nucleic acid template. Disclosed is a method for the cell-free synthesis of proteins, the method comprising mixing a first composition containing reagents for cell-free protein expression, and a second composition containing a nucleic acid template and an energy source for the cell-free protein expression reagents. The second composition may comprise both an energy source and one or more NTP’s, for example ATP and / or GTP.

[0014] Disclosed is a method for the cell-free synthesis of proteins, the method comprising mixing a first composition containing reagents for cell-free protein expression, a second composition containing a nucleic acid template and a third composition containing NTP’s and / or an energy source for the cell-free protein expression reagents. The nucleoside triphosphates may be selected from one or more of ATP, CTP, GTP or UTP. The NTP may be ATP. The NTP may be CTP. The NTP may be GTP. The NTP may be UTP. Two or more NTP’s may be added, for example ATP and GTP. Four NTP’s may be added.

[0015] Disclosed is a method for the cell-free synthesis of proteins, the method comprising mixing a first composition containing reagents for cell-free protein expression, a second composition containing a nucleic acid template and a third composition containing an energy source for the cell-free protein expression reagents. The third composition may comprise both an energy source and one or more NTP’s, for example ATP and / or GTP.

[0016] The addition of the nucleic acid template may initiate the process of transcribing the template into RNA. The reduced / depleted energy source slows the process of translation into proteins. The energy source and optionally further NTP’s can be added after the template has been added to the CFPS reaction and the mRNA produced.

[0017] A reconstituted cell-free expression system which contains creatine phosphate initiates protein expression upon addition of a DNA template. Thus the only additional requirement is the template. As an alternative, where the reconstituted cell-free expression system is lacking creatine phosphate, addition of the DNA template initiates a certain level of transcription of RNA, but little translation of the RNA into protein. Thus the TX is uncoupled from the TL. Addition of CP after the DNA template thus actives the translation of the RNA into proteins. When CP is absent is used, significant translation does not start until CP is added to promote ATP / GTP recycling, but transcription occurs.

[0018] This pre-TX reaction displays additional advantages of creatine supplementation after transcription; the use of additives which are beneficial to translation but inhibitory to transcription. Additives may be included with the energy mix, for example chaperone proteins. Described herein are methods and compositions using chaperones such as GroE / L as an translation additive which are typically inhibitory to TX. The use of creatine deficient blends and subsequent addition of CP enables a convenient approach for splitting TX and TL reactions and compositions without requiring a separate in vitro transcription reaction and cleanup. Described herein are compositions including creatine phosphate and chaperone proteins.

[0019] The energy source may be one or more of phosphoenolpyruvate, acetyl phosphate, and creatine phosphate. The energy source may be creatine phosphate.

[0020] The method may be performed in tubes or plates. The method may be performed on an electrowetting-on-dielectric (EWoD) device. The method includes using a series of droplets having varying cell-free reagent compositions, including enzymes for protein synthesis and the template nucleic acid and energy source and monitoring the synthesis of the protein of interest in the various compositions, thereby identifying a composition suitable for expression of the protein of interest.

[0021] The cells lysates may be derived from mammalian cells, prokaryotic cells, yeast cells, plant cells or protozoa. The cell lysates may be derived from human embryonic kidney cells (HEK293), Chinese hamster ovary cells (CHO), HeLa, BHK21 , NSO, or Sp2 / 0 cells. The cell lysates may be derived from Escherichia coli cells, Saccharomyces cerevisiae or Pichia pastoris cells, tobacco or wheat cells, or Leishmania tarentolae.

[0022] The expression of proteins may be performed for at least 3 hours and the level of expressed protein is determined. Where the method is performed on an electrowetting device, the droplets may be periodically or continually moved for the period of expression. Disclosed is a composition comprising a nucleic acid template and an energy source for cell- free protein expression reagents selected from phosphoenolpyruvate, acetyl phosphate, and creatine phosphate, wherein the composition does not contain any enzymes that metabolise the energy sources.

[0023] The composition may contain a nucleic acid template and at least 50 mM creatine phosphate. The composition may contain a nucleic acid template and at least 100 mM creatine phosphate. The composition may contain a nucleic acid template and 120 mM to 360 mM creatine phosphate.

[0024] The composition may contain a nucleic acid template and at least 3 mM ATP.

[0025] The composition may contain a nucleic acid template and at least 3 mM GTP.

[0026] The composition may contain a nucleic acid template and at least 3 mM ATP and at least 3 mM GTP.

[0027] The composition may contain a nucleic acid template and at least 50 mM creatine phosphate and at least 3 mM ATP.

[0028] The composition may contain a nucleic acid template and at least 50 mM creatine phosphate and at least 3 mM GTP.

[0029] The composition may contain a nucleic acid template and at least 50 mM creatine phosphate and at least 3 mM ATP and at least 3mM GTP.

[0030] Disclosed is a composition comprising one or more chaperone proteins and an energy source for cell-free protein expression reagents selected from phosphoenolpyruvate, acetyl phosphate, and creatine phosphate, wherein the composition does not contain any enzymes that metabolise the energy sources. The chaperone protein may include GroE / L.

[0031] The composition may contain a chaperone protein and at least 50 mM creatine phosphate. The composition may contain a chaperone protein and at least 100 mM creatine phosphate. The composition may contain a chaperone protein and 120 mM to 360 mM creatine phosphate.

[0032] The composition may contain a chaperone protein and at least 3 mM ATP.

[0033] The composition may contain a chaperone protein and at least 3 mM GTP.

[0034] The composition may contain a chaperone protein and at least 3 mM ATP and at least 3 mM GTP.

[0035] The composition may contain a chaperone protein and at least 50 mM creatine phosphate and at least 3 mM ATP.

[0036] The composition may contain a chaperone protein and at least 50 mM creatine phosphate and at least 3 mM GTP. The composition may contain a chaperone protein and at least 50 mM creatine phosphate and at least 3 mM ATP and at least 3mM GTP.

[0037] Disclosed is a kit comprising: i. a first composition for the cell-free synthesis of proteins, the composition containing reagents for cell-free protein expression; and ii. a second composition comprising a nucleic template coding sequence for a protein of interest and NTP’s and / or an energy source selected from phosphoenolpyruvate, acetyl phosphate, and creatine phosphate.

[0038] Disclosed is a kit comprising: i. a first composition for the cell-free synthesis of proteins, the composition containing reagents for cell-free protein expression; ii. a second composition comprising a nucleic template coding sequence for a protein of interest; and iii. a third composition containing NTP’s and / or an energy source selected from phosphoenolpyruvate, acetyl phosphate, and creatine phosphate.

[0039] The third composition may contain one or more additives which are inhibitory to translation. The third composition may contain one or more chaperone proteins. The third composition may contain GroE / L.

[0040] The energy source in the second composition may be creatine phosphate. The energy source may be at least 50 mM creatine phosphate. The energy source may be 120 mM to 360 mM creatine phosphate. The energy source may be 240 mM creatine phosphate. The NTP’s may be for example ATP and / or GTP.

[0041] The first composition may contain no creatine phosphate or may contain a starting levels of creatine phosphate, which degrades over time. Hence the final concentration of creatine phosphate during expression is determined by the initial remaining amount of creatine phosphate from the first composition and the volume and concentration added by the second composition. The final working concentration of creatine phosphate should be in the range of 90 mM to 150 mM. Between 30 mM to 90 mM creatine phosphate is added at the time of nucleic template addition. Around 60 mM creatine phosphate can be added at the time of nucleic template addition.

[0042] The first composition contains NTP’s which degrade over time. The four NTP’s can degrade at different rates depending on which NTP is used for which process. Hence the final concentration of NTP’s during expression is determined by the initial remaining amount of NTP from the first composition and the volume and concentration added by the second composition. The final working concentration of ATP and GTP should be greater than 3 mM.

[0043] 3 mM ATP and / or GTP can be added at the time of nucleic template addition.

[0044] Disclosed herein is a stable composition comprising RNA polymerase, a ribosome, tRNA’s and amino acids, which is lacking one energy sources. Disclosed is a composition comprising RNA polymerase, a ribosome, tRNA’s, amino acids, ATP, GTP, UTP and CTP, which is missing creatine phosphate. The stable composition allows initiation of transcription and translation by the addition of creatine phosphate. The creatine phosphate can be added along with the nucleic acid template or can be added after the nucleic acid template. The transcription and translation process can be started by adding the missing creatine phosphate plus a nucleic acid template, either separately or as a single composition. The term missing implies insufficient concentration of the material is present to undergo enzymatic processes. A certain low concentration of material may be present in the assembled components as a result of carry-over from purification and re-constitution of components.

[0045] Disclosed herein is a stable composition comprising RNA polymerase, a ribosome, tRNA’s and amino acids, which is lacking one or more ribonucleoside triphosphates. Disclosed is a composition comprising RNA polymerase, a ribosome, tRNA’s, amino acids, UTP and CTP, which is missing ATP and GTP. The stable composition allows initiation of transcription and translation by the addition of one or more NTPs. Disclosed is a composition comprising RNA polymerase, a ribosome, tRNA’s, amino acids and three NTP’s, missing a fourth. Disclosed is a composition comprising RNA polymerase, a ribosome, tRNA’s, amino acids and UTP, CTP and GTP, having no ATP. Disclosed is a composition comprising RNA polymerase, a ribosome, tRNA’s, amino acids and UTP, CTP and ATP, having no GTP. The transcription and translation process can be started by adding the missing NTP plus a nucleic acid template, either separately or as a single composition. The term missing implies insufficient concentration of the material is present to undergo enzymatic processes. A certain low concentration of material may be present in the assembled components as a result of carryover from purification and re-constitution of components.

[0046] The transcription and translation system may be coupled or uncoupled. The composition may be a reconstituted coupled system for transcription and translation. The composition may contain synthesized or isolated ribosomes, initiation factors, elongation factors, aminoacyl- tRNA synthetases, methionyl tRNA transformylases, tRNAs, amino acids and ribonucleoside triphosphates. The composition may comprise one or more nucleic acid templates for protein expression.

[0047] The expressed protein may be fused to a peptide tag. The peptide tag may be one component of a fluorescent protein and the further polypeptide a complementary portion of the fluorescent protein. The fluorescent protein could include sfGFP, ccGFP, GFP, eGFP, deGFP, frGFP, eYFP, eBFP, eCFP, Citrine, Venus, Cerulean, Dronpa, DsRED, mKate, mCherry, mRFP, FAST, SmllRFP, miRFP670nano. For example the peptide tag may be GFPn and the further polypeptide GFP1.10. The peptide tag may be one component of sfCherry. The peptide tag may be sfCherryn and the further polypeptide sfCherryi- . The peptide tag may be CFASTn or CFAST10 and the further polypeptide NFAST in the presence of a hydroxybenzylidene rhodanine analog.

[0048] Alternatively, the GFP1.10 polypeptide amino acid sequence could be further mutated to become brighter more quickly upon complementation. The sequence may have a greater than 90 % homology to any sequence mentioned herein. The sequence may have a greater than 95 % homology to any sequence mentioned herein.

[0049] GFP11 or GFP1.10 can be fused to the protein of interest through an amino acid linker. In one embodiment, the oligopeptide, peptide, or polypeptide linker can be 0 - 50 amino acids.

[0050] DESCRIPTION OF FIGURES

[0051] Figure 1 shows the ccGFP protein yields from CFPS reactions formulated with a variety of reconstituted expression systems. Different batches of expression systems show different initial levels of expression (BO, B1 , B2, B4 and B6). Addition of increasing amounts of creatine phosphate to the expression template were measured over 17 hours. The protein expressed (ccGFP) is fluorescent and concentration can be determined directly against a standard solution. The yields of expressed protein peak when supplementing the CFPS reaction with an additional 60 mM of creatine phosphate. Additional creatine phosphate then starts to inhibit the expression.

[0052] Figure 2 shows expression yields ( .M) of each reformulated core reagent in the absence and presence of the missing component (R1 = + / -creatine phosphate, R2 = + / -ATP, R3 = + / -GTP, R4 = + / -ATP / GTP, R5 = + / -ATP / GTP / CP). Each composition with missing component(s) showed zero activity. The missing component(s) when added restored full activity of the expression system Figure 3 shows stability of the mixes at 20°C and measures the activity of each reaction normalised to the TO. Data are the average of three separately assembled reactions. Error bars show the standard deviation of the mean. The activity of the full mixture has reached zero after 3 days. The omission of reagents improves stability. Components missing creatine phosphate are the most stable.

[0053] Figure 4 shows activity of expression systems prepared with and without creatine phosphate (CP). Core_V2 activity is dependent on the presence of CP added with the nucleic acid template. Core_V1 is active, and adding extra CP has no impact on activity.

[0054] Figure 5a-d shows the storage stability of the cell-free expression blend with and without the CP present. 5a: Core_V1 loses activity in <1 day (stored at 20 °C prior to mixing DNA plus CP with Core). Core_V2 retains activity for >7 days (stored at 20oC prior to mixing DNA plus CP with Core). 5b 4 °C shelf life of Core V2 is greater than 3 days. 5c -20 °C shelf life of Core V2 is greater than 30 days. 5d 4 °C shelf life of Core V2 is greater than 12 months.

[0055] Figure 6 shows On Device Expression in Core V1 and Core V2 (+ / -CP), showing equivalent performance for expression on an electrowetting device.

[0056] Figure 7 shows SDS PAGE analysis to identify protein TL products.

[0057] Figure 8 shows Agarose gel analysis of RNA TX products. The gel was first imaged to identify mRNA products containing fluorescein-UTP (left), before staining to detect the ladder and other oligonucleotides present (right).

[0058] DETAILED DESCRIPTION OF THE INVENTION

[0059] Disclosed herein is an improved reconstituted composition for the cell-free expression of proteins. The inventors herein have appreciated that during storage of the cell-free expression system, the enzymes required for protein expression are sufficiently active to metabolise the NTP’s and energy systems needed for protein expression despite the absence of any nucleic acid template. Storage even at low temperature (-80 °C) is insufficient to prevent enzyme turnover. Thus when the template is added, the performance of the expression system is sub- optimal due to the lack of available NTPs and / or energy sources. The addition / replenishment of NTP’s and / or energy sources along with the nucleic acid templates improves the efficiency of expression of the protein of interest. Reagents for cell-free protein synthesis are well-known. However studies into the long-term stability of such reagents by the applicants have indicated that the efficiency of the reagents decreases over time. The inventors herein have identified and addressed the causes of the loss of reagent efficiency. Such improved reagents show greater storage stability and more efficient protein synthesis. Disclosed herein is a reagent for cell-free protein synthesis which is missing creatine phosphate.

[0060] Disclosed is a method of synthesising a protein in a digital microfluidic device. The droplets having the components required for cell-free protein synthesis (CFPS), otherwise known as in-vitro protein synthesis, can be manipulated by electrokinesis in order to effect and improve protein expression.

[0061] Protein expression is dependent on the conditions and reagents used for expression. The best expression system for a given protein of interest is not predictable, and may require screening of a large number of similar conditions in order to identify the optimal expression system. The use of EWoD devices can screen large numbers of closely related conditions in parallel in droplets on the device. The droplets can be blended on the device in order to prepare the reagents. For example a cell lysate or reconstituted protein system or mix thereof can be supplemented with a variety of additional components at a selection of concentrations. For example a salt screen, buffer screen or pH screen can be performed across a range of conditions and at variable concentrations. Described herein is the preparation of a variety of different conditions on a single device for the purposes of simultaneously screening a variety of expression conditions.

[0062] Disclosed herein is a method for the synthesis of a protein on an electrowetting-on-dielectric (EWoD) device, the method comprising taking a reaction system having at least one template nucleic acid encoding a protein of interest and an energy source such as phosphoenolpyruvate, acetyl phosphate, or creatine phosphate, blending droplets to form a series of droplets having varying cell-free reagent compositions, a first droplet including enzymes for protein synthesis and a second droplet including the template nucleic acid and energy source and monitoring the synthesis of the protein of interest in the various compositions, thereby identifying a composition suitable for expression of the protein of interest. The blend may be a mixture of cell lysate and reconstituted expression systems.

[0063] Electrowetting is the modification of the wetting properties of a surface (which is typically hydrophobic) with an applied electric field. Microfluidic devices for manipulating droplets or magnetic beads based on electrowetting have been extensively described. In the case of droplets in channels this can be achieved by causing the droplets, for example in the presence of an immiscible carrier fluid, to travel through a microfluidic channel defined by the walls of a cartridge or microfluidic tubing. Embedded in the walls of the cartridge or tubing are electrodes covered with a dielectric layer each of which are connected to an A / C biasing circuit capable of being switched on and off rapidly at intervals to modify the electrowetting field characteristics of the layer. This gives rise to the ability to steer the droplet along a given path.

[0064] As an alternative to microfluidic channel systems, droplets can also be generated and manipulated on planar surfaces using digital microfluidics (DMF). In contrast to channel based microfluidics, DMF utilizes alternating currents on an electrode array for moving fluid on the surface of the array. Liquids can thus be moved on an open-plan device by electrowetting. Digital microfluidics allows precise control over the droplet movements including droplet fusion and separation.

[0065] Digital microfluidics can be carried out in an air-filled system where the liquid drops are manipulated on the surface in air. However, at elevated temperatures or over prolonged periods, the volatile aqueous droplets simply dry onto the surface by evaporation. This issue is compounded by the high surface area to volume ratio of nanoliter and microliter sized drops. Hence air-filled systems are generally not suitable for protein expression where the temperature of the system needs to be maintained at a temperature suitable for enzyme activity and the duration of the synthesis needs to be prolonged for synthesized protein levels to be detectable.

[0066] Protein expression typically requires an ample supply of oxygen. The most convenient and high yielding way to power CFPS is via oxidative phosphorylation where O2 serves as the final electron acceptor; however, there are other ways that involve replenishing with energy molecules not involved in oxidative phosphorylation. In a confined microfluidic or digital microfluidic system of droplets, insufficient oxygen is available to enable efficient protein synthesis.

[0067] The requirement for oxygen is highest when a cellular lysate system is used for expression. Reconstituted systems do not require as much oxygen and do not consume oxygen via metabolic pathways. For expression in sealed droplets where hypoxia may be a problem, blending of the two expression systems is advantageous as the protein expression levels are consistent and metabolic oxidation is reduced. Described herein are improved methods allowing for the cell-free expression of peptides or proteins in a digital microfluidic device. Included is a method for the cell-free expression of peptides or proteins in a microfluidic device wherein the method comprises one or more droplets containing a nucleic acid template (i.e., DNA or RNA) 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 components for the cell- free protein synthesis droplet can comprise both reconstituted systems (PURE reagents) and cell lysates.

[0068] The droplet can be repeatedly moved for at least a period of 30 minutes whilst the protein is expressed. The droplet can be repeatedly moved for at least a period of three hours whilst the protein is expressed. The droplet can be repeatedly moved for at least a period of twelve hours whilst the protein is expressed. The act of moving the droplet allows oxygen to be supplied to the droplet and dispersed throughout the droplet. The act of moving improves the level of protein expression over a droplet which remains static.

[0069] The droplet can be moved using any means of electrokinesis. The droplet can be moved using electrowetting-on-dielectric (EWoD). The electrical signal on the EWoD or optical EWoD device can be delivered through segmented electrodes, active-matrix thin-film transistors, or digital micromirrors.

[0070] The filler liquid may be a hydrophobic or non-ionic liquid. For example the filler liquid may be decane or dodecane. The filler fluid may be a silicone oil such as dodecamethylpentasiloxane (DM PS). The filler liquid may contain a surfactant, for example a sorbitan ester such as Span 85. The oil can be oxygenated prior to or during the expression process. Alternatively, the device can be an air-filled device where droplets containing cell-free protein synthesis reagents are rapidly moved into position and fixed into an array under a humidified gas to prevent evaporation. Humidification can be achieved by enclosing or sealing the digital microfluidic device and providing on-board reagent reservoirs. Additionally, humidification can be achieved by connecting an aqueous reservoir to an enclosed or sealed digital microfluidic device. The aqueous reservoir can have a defined temperature or solute concentration in order to provide specific relative humidities (e.g., a saturated potassium sulfate solution at 30 °C).

[0071] The droplets can be formed before entering the microfluidic device and flowed into the device. Alternatively the droplets can be merged on the device. Included is a method comprising merging a first droplet containing a nucleic acid template such as a plasmid and an additional energy source with a second droplet containing a cell-free extract having the components for protein expression to form a combined droplet capable of cell-free protein synthesis.

[0072] The droplets can be split on the device either before or after expression. Included herein is a method further comprising splitting the aqueous droplet into multiple droplets. If desired the split droplets can be screened with further additives. Included is a method wherein one or more of the split droplets are merged with additive droplets for screening.

[0073] The cell-free expression of peptides or proteins can use a cell lysate having the reagents to enable protein expression. Common components of a cell-free reaction include an energy source, a supply of amino acids, cofactors such as magnesium, and the relevant enzymes. A cell extract is obtained by lysing the cell of interest and removing the cell walls, DNA genome, and other debris by centrifugation. The remains are the cell machinery including ribosomes, aminoacyl-tRNA synthetases, translation initiation and elongation factors, nucleases, etc. Once a suitable nucleic acid template is added, the nucleic acid template can be expressed as a peptide or protein using the cell derived expression machinery.

[0074] Storage of the cell-free expression mix allows reduction of the energy sources over time. The energy sources can be replenished by adding a mixture containing both the nucleic acid template and one or more additional energy sources.

[0075] In order to optimise expression, the expression system can be supplemented with additional components, including purified enzymes. The additional components may include salts, cofactors, buffers, surfactants, chaperones or additional protein components. The additional protein components may be selected from for example chaperones, glycosylating enzymes, proteases, redox active enzymes, phosphorylases and kinases.

[0076] The additional protein components may be involved in any post-translational modification process, for example methylation, ubiquitinylation, sumoylation, isoprenylation or glycosylation.

[0077] Common post-translation modifications include phosphorylation, methylation, sulfation, acetylation, ubiquitylation, prenylation, myristoylation, SUMOylation, palmitoylation, different types of glycosylation (N-glycosylation, O-glycosylation, C-glycosylation and S-glycosylation), phosphoglycosylation and glycosylphosphatidylinositol (GPI anchored). Protein phosphorylation is an important reversible regulatory mechanism that plays a key role in the activities of many enzymes, membrane channels and many other proteins in prokaryotic and eukaryotic organisms. Phosphorylation target sites are Ser, Thr, Tyr, His, Pro, Arg, Asp and Cys amino acid residues, but mainly happens on Ser, Thr, Tyr and His residues. Phosphorylation involves transferring a phosphate group from adenosine triphosphate to the receptor residues by kinase enzymes. Conversely, dephosphorylating or removal of a phosphate group is an enzymatic reaction catalyzed by phosphatases. Phosphorylation can change the function of proteins via one of the two principal ways: by allostery or by binding to interaction domains.

[0078] Acetylation is typically catalyzed via lysine acetyltransferase (KAT) and histone acetyltransferase (HAT) enzymes. Acetyltransferases use acetyl CoA as a cofactor for adding an acetyl group (COCH3) to the e-amino group of lysine side chains, whereas deacetylases (HDACs) remove an acetyl group on lysine side chains. Forms of acetylation include Na- acetylation, Ne-acetylation and O-acetylation, and may occur on Lys, Ala, Arg, Asp, Cys, Gly, Glu, Met, Pro, Ser, Thr and Vai residues with different frequencies, although the acetylation is more reported on Lysine residue. Ne-acetylation is more biologically significant compared to the other types of acetylation. Acetylation has an essential role in biological processes such as chromatin stability, protein-protein interaction, cell cycle control, cell metabolism, nuclear transport and actin nucleation.

[0079] Ubiquitylation is an important reversible PTM and can occur on all 20 amino acids, however, it occurs on lysine more frequently. This PTM has a major role in the degradation of intracellular proteins via the ubiquitin (Ub)-proteasome pathway. Ubiquitylation is catalyzed by an enzyme complex that contains ubiquitin-activating (E1), ubiquitin-conjugating (E2) and ubiquitin ligase (E3) enzymes. Ubiquitinated proteins may be acetylated on Lys, or phosphorylated on Ser, Thr or Tyr residues. Ubiquitylation modification in substrate proteins can be removed by several specialized families of proteases called deubiquitinases. Ubiquitination plays important roles in stem cell preservation and differentiation by regulation of the pluripotency, and plays a role in many various cell activities such as proliferation, regulation of transcription, DNA repair, replication, intracellular trafficking and virus budding, the control of signal transduction, degradation of the protein, innate immune signalling, autophagy and apoptosis.

[0080] Methylation is a reversible PTM, which often occurs in the cell nucleus and on the nuclear proteins such as histone proteins. Methylation occurs on the Lys, Arg, Ala, Asn, Asp, Cys, Gly, Glu, Gin, His, Leu, Met, Phe and Pro residues in target proteins, with lysine and arginine the two main target residues, at least in eukaryotic cells. One important role of methylation is in histone modification. Histone proteins, after synthesis of their polypeptide chains, are methylated at Lys, Arg, His, Ala or Asn residues. NMysine methylation is one of the most abundant histone modifications in eukaryotic chromatin, involves transferring the methyl groups from S-adenosylmethionine to histone proteins via methyltransferase enzyme. In eukaryotes, methylated arginine has been observed in histone and non-histone proteins. Recent studies have shown that methylation is associated with fine tuning of various biological processes ranging from transcriptional regulation to epigenetic silencing via heterochromatin assembly.

[0081] Glycosylation occurs in multiple subcellular locations, such as endoplasmic reticulum, the Golgi apparatus, cytosol and the sarcolemma membrane. Glycosylation occurs in eukaryotic and prokaryotic membranes and secreted proteins, and nearly 50% of the plasma proteins are glycosylated. In this modification, oligosaccharide chains are linked to specific residues by a covalent bond. This enzymatic process, which is catalyzed by a glycosyltransferase enzyme, usually occurs in the side chain of residues such as Trp, Ala, Arg, Asn, Asp, lie, Lys, Ser, Thr, Vai, Glu, Pro, Tyr, Cys and Gly; however, it occurs more frequently on Ser, Thr, Asn and Trp residues in proteins and lipoproteins. According to the target residues, glycosylation can be classified into six groups: N-glycosylation, O-glycosylation, C-glycosylation, S-glycosylation, phosphoglycosylation and glypiation (GPI-anchored). N-glycosylation and O-glycosylation are two major types of glycosylation and have important roles in the maintenance of protein conformation and activity. Glycosylation has a great role in many important biological processes such as cell adhesion, cell-cell and cell-matrix interactions, molecular trafficking, receptor activation, protein solubility effects, protein folding and signal transduction, protein degradation, and protein intracellular trafficking and secretion.

[0082] Small Ubiquitin-Related Modifier (SUMO) ylation has been discovered in a wide range of eukaryotic organisms. SUMOylation can occur in both cytoplasm and nucleus on lysine residues. SUMOylation occurs as a modifier in e-amino group of lysine residues in target protein through a multi-enzymatic cascade. In this reaction, SUMO is connected to a lysine residue in substrate protein by covalent linkage via three enzymes, namely activating (E1), conjugating (E2) and ligase (E3). Often, SUMOylation modifications occur at a consensus motif WKxE (where W represents Lys, lie, Vai or Phe and X any amino acid). SUMOylation plays a major role in many basic cellular processes like transcription control, chromatin organization, accumulation of macromolecules in cells, regulation of gene expression and signal transduction and is necessary for the conservation of genome integrity. Lipidation involves the covalent attachment of lipids to proteins. These PTMs may use a variety of lipids, including octanoic acid, myristic acid, palmitic acid, palmitoleic acid, stearic acid, cholesterol, etc. Myristoylation, palmitoylation and prenylation can be considered as the three main types of these lipid modifications. Palmitoylation is the covalent attachment of fatty acids, like palmitic acid on the Cys, Gly, Ser, Thr and Lys. S-palmitoylation contains a reversible covalent addition of a 16-carbon fatty acid chains, palmitate, to a cysteine via a thioester linkage. Palmitoyl-CoA (as the lipid substrate) is attached to the target protein. Mostly, S-palmitoylation occurs in eukaryotic cells and plays critical roles in many different biological processes including protein function regulation, protein-protein interaction, membrane-protein associations, neuronal development, signal transduction, apoptosis and mitosis.

[0083] Myristoylation (N-myristoylation) is an irreversible PTM that occurs mainly on cytoplasmic eukaryotic proteins. Myristoylation happens approximately in 0.5-1.5% of eukaryotic proteins. In myristoylation, after removal of the initiating Met, a 14-carbon saturated fatty acid, called myristic acid, is attached to the N-terminal glycine residue via a covalent bond. This attachment is often observed in Met-Gly-X-X-X- Ser / Thr motif and is catalyzed by an N- myristoyl transferase (NMT) (there are at least two types of NMT enzymes, NMT 1 and NMT2, in humans). Proteins that undergo this PTM play critical roles in regulating the cellular structure and many biological processes such as stabilizing the protein structure maturation, signalling, extracellular communication, metabolism and regulation of the catalytic activity of the enzymes.

[0084] Prenylation is another important lipid-based PTM, which occurs after translation as an irreversible covalent linkage mainly in the cytosol. This reaction occurs on cysteine and near the carboxyl-terminal end of the substrate protein. Prenylation has two main forms: farnesylation and geranylation. These two forms contain the addition of two different types of isoprenoids to cysteine residues: farnesyl pyrophosphate (15-carbon) and geranylgeranyl pyrophosphates (20-carbon), respectively. In prenylated proteins, one can find a consensus motif at the C-terminal; the motif is CAAX where C is cysteine, A is an aliphatic amino acid and X is any amino acid. This process is catalyzed by three prenyltransferase enzymes: farnesyltransferase (FT) and two geranyl transferases (GT1 and GT2). Prenylation is known as a crucial physiological process for facilitating many cellular processes such as proteinprotein interactions, endocytosis regulation, cell growth, differentiation, proliferation and protein trafficking. N-sulfation or O-sulfation includes the addition of a negatively charged sulfate group by nitrogen or oxygen to an exposed residue on the target protein. Currently, PTS is observed mainly in secreted and transmembrane proteins in multicellular eukaryotes. This reaction is catalyzed by two transmembrane enzymes, tyrosyl protein sulfotransferases 1 and 2 (TPST1 and TPST2). TPSTs govern the transfer of an activated sulfate from 3-phospho adenosine 5- phosphosulfate to residues within acidic motifs of polypeptides.

[0085] The expressed protein may contain an AVI tag. The Avi-tag peptide (GLNDIFEAQKIEWHE) is recognized by BirA ligase which enzymatically attaches a biotin molecule to a single lysine residue within the Avi-tag sequence. The enzyme can be present during expression to attach the biotin to the expressed tag after expression of the POI.

[0086] The expression composition may be assembled on the device from mixing a variety of droplets in order to screen a variety of compositions in parallel.

[0087] By way of example, the screening reagents may include,

[0088] ■ Chaperone mix (e.q.,PUREfrex GrpE mjx)

[0089] ■ Kinase 1 (e.g., NEB CK2)

[0090] ■ Kinase 2 (e.g., NEB PKA)

[0091] ■ Protease 1 (e.g., NEB. TEV)

[0092] ■ Protease 2 (e.g., Merck HRV 3C)

[0093] ■ N-acetyl transferase

[0094] ■ Common metal ions cocktail

[0095] ■ Common co-factors cocktail

[0096] The compositions can be blended by the user and the level of expression of the protein of interest monitored in each of the blended conditions.

[0097] Metal ions may include one or more of the following: MgCh, CuCh, ZnCh, CaCh, MnCh, NiCh, CoCI2.

[0098] Co-factors may include one or more of Nicotinamide adenine dinucleotide (NAD), Flavin adenine dinucleotide (FAD), S-adenosyl methionine (SAM), pyridoxal phosphate (PLP) Coenzyme A (CoA), thiamine pyrophosphate (TPP) or haem. Chaperones may include one or more of DnaK, DnaJ, GroE, GrpE, heat shock proteins, protein disulfide isomerase (PDI), human protein disulfide isomerase (hPDI), disulfide bond C (DsbC), a thioredoxin, such as TRXB1 , Caseinolytic peptidase B protein homolog (CLPB) or FK506 binding proteins (FKBPs).

[0099] The additive may be for example one or more reducing agents. The additive may be selected from DTT, glutathione (GSH) or glutathione disulfide (GSSG).

[0100] The added protease may cleave the protein of interest from flanking regions. The flanking regions mat include tags used for detection or solubility or other buffer regions. The flanking regions may be cleaved by any protease. The protease may be a TEV or 3C protease.

[0101] The TEV protease may act upon the amino acid sequence ENLYFQS. The template for expression of the POI may include the nucleic acid sequence GAGAACCTGTACTTCCAGAGC.

[0102] The 3C protease may act upon the amino acid sequence LEVLFQGP. The template for expression of the POI may include the nucleic acid sequence CTCGAGGTTCTGTTCCAAGGACCT.

[0103] The method may be used to perform a surfactant screen to identify the best surfactant for expression of a particular protein. The surfactant may be ionic, nonionic, or zwitterionic.

[0104] Surfactant molecules are composed of a hydrophobic region and a hydrophilic region. This amphiphilic structure enables surfactants to obtain a discoidal conformation in the solution, known as micelles. Micelles solubilize membrane proteins by encompassing the transmembrane domains of integral membrane proteins, with the loops and hydrophilic regions exposed to solvent. The minimum concentration of a surfactant necessary to form micelles and extract membrane proteins is called critical micelle concentration or CMC.

[0105] Depending on the charge of hydrophilic group, surfactants are classified into three groups: ionic, nonionic, and zwitterionic surfactants. Ionic surfactants carry a charged group, either negative (anionic) or positive (cationic), and historically have been the most efficient group of detergents in extracting membrane proteins from lipid bilayers. However, ionic detergents can have deleterious effects on protein-protein interactions and often lead to protein denaturation. Sodium dodecyl sulfate (SDS) and sodium cholate are two common examples of ionic detergents. Nonionic surfactants are currently the most popular and successful group of surfactants in solubilizing membrane proteins for both functional and structure determination purposes. This is due to their nondisruptive nature, which enables them to preserve the native structure of the target protein by breaking protein-lipid interactions instead of protein-protein interactions. Alkyl glycoside surfactants such as n-dodecyl-B-D-maltoside (DDM), n-decyl-B-D-maltoside (DM), n-Octyl-B-D-Glucopyranoside (OG), and n-Nonyl-B-D-Glucopyranoside (NG) by contributing to the purification and crystallization of about 70% of membrane proteins are the most common nonionic surfactants for protein studies. Another advantage of nonionic surfactants is that they do not interfere with optical measurements, which enables fluorescence-based experiments on expressed proteins.

[0106] Zwitterionic surfactants typically have an intermediate level of harshness between ionic and nonionic detergents. They carry both positive and negative charged groups in their polar regions with an overall net charge of zero. An example of a Zwitterionic surfactant is lauryldimethylamine-N-oxide or LDAO.

[0107] Membrane mimetic systems, such as nanodiscs and styrene malic acid lipid particles (SMALPs), provide an alternative platform for stabilization of membrane proteins and hence eliminate the deleterious effects of detergents on these macromolecules. Nanodiscs are composed of phospholipid patches surrounded by two copies of membrane scaffold protein (MSP), a genetically engineered version of human serum apolipoprotein A-l.

[0108] Any particular nucleic acid template can be expressed using the system described herein. Three types of nucleic acid templates used in CFPS include plasmids, linear expression templates (LETs), and mRNA. Plasmids are circular templates, which can be produced either in cells or synthetically. LETs can be made via PCR. While LETs are easier and faster to make, plasmid yields are usually higher in CFPS. mRNA can be produced through in-vitro transcription systems. The methods use a single nucleic acid template per droplet. The methods can use multiple droplets having a different nucleic acid template per droplet.

[0109] An energy source is an important part of a cell-free reaction. Usually, a separate mixture containing the needed energy source, along with a supply of amino acids, is added to the extract for the reaction. Common sources are phosphoenolpyruvate, acetyl phosphate, and creatine phosphate. The energy source can be replenished during the expression process by adding further reagents to the droplet during the process.

[0110] Thus the cell-lysate can be supplemented with additional reagents along with the template being added. The cell-free extract having the components for protein expression would typically be produced as a bulk reagent or ‘master mix’ which can be formulated into many identical droplets prior to the distinct template being separately added to separate droplets. Common cell extracts in use today are made from E. coli (ECE), rabbit reticulocytes (RRL), wheat germ (WGE), insect cells (ICE) and Yeast Kluyveromyces (the D2P system). All of these extracts are commercially available.

[0111] Rather than originating from a cell extract, the cell-free system can be assembled from the required reagents. Systems based on reconstituted, purified molecular reagents are commercially available, for example the PURE system for protein production, and can be used as supplied (e.g. PURE-FREX). The PURE system is composed of all the enzymes that are involved in transcription and translation, as well as highly purified 70S ribosomes. The protein synthesis reaction of the PURE system lacks proteases and ribonucleases, which are often present as undesired molecules in cell extracts.

[0112] The expression system may be assembled from a mixture of sources, such as for example a mixture of eukaryotic lysates, or a mix of eukaryotic and prokaryotic lysates. The system may be a mixture of a lysate system and a reconstituted system (such as PUREFrex).

[0113] The term digital microfluidic device refers to a device having a two-dimensional array of planar microelectrodes. The term excludes any devices simply having droplets in a flow of oil in a channel. The droplets are moved over the surface by electrokinetic forces by activation of particular electrodes. Upon activation of the electrodes the dielectric layer becomes less hydrophobic, thus causing the droplet to spread onto the surface. A digital microfluidic (DMF) device set-up is known in the art, and depends on the substrates used, the electrodes, the configuration of those electrodes, the use of a dielectric material, the thickness of that dielectric material, the hydrophobic layers, and the applied voltage.

[0114] Once the CFPS reagents have been enclosed in the droplets, additional reagents can be supplied by merging the original droplet with a second droplet. The second droplet can carry any desired additional reagents, including for example oxygen or ‘power’ sources, or test reagents to which it is desired to expose to the expressed protein.

[0115] The droplets can be aqueous droplets. The droplets can contain an oil immiscible organic solvent such as for example DMSO. The droplets can be a mixture of water and solvent, providing the droplets do not dissolve into the bulk oil. The droplets can be in a bulk oil layer. A dry gaseous environment simply dries the bubbles onto the surface during the expression process, leaving comet type smears of dried material by evaporation. Thus the device is filled with liquid for the expression process. Alternatively, the aqueous droplets can be in a humidified gaseous environment. A device filled with air can be sealed and humidified in order to provide an environment that reduces evaporation of CFPS droplets.

[0116] The droplets containing the cell-free extract having the components for protein expression will therefore typically be in the oil filled environment before the nucleic acid templates are added to the droplets. The templates can be added by merging droplets on the microfluidic device. Alternatively, the templates can be added to the droplets outside the device and then flowed into the device for the expression process. For example the expression process can be initiated on the device by increasing the temperature. The expression system typically operates optimally at temperatures above standard room temperatures, for example at or above 29 °C.

[0117] The expression process typically takes many hours. Thus the process should be left for at least 30 minutes or 1 hour, typically at least 2 hours. Expression can be left for at least 12 hours. During the process of expression the droplets should be moved within the device. The moving improves the process by mixing the reagents and ensuring sufficient oxygen is available within the droplet. The moving can be continuous, or can be repeated with intervening periods of non-movement.

[0118] Thus the aqueous droplet can be repeatedly moved for at least a period of 30 minutes or one hour whilst the protein is expressed. The aqueous droplet can be repeatedly moved for at least a period of three hours whilst the protein is expressed. The aqueous droplet can be repeatedly moved for at least a period of twelve hours whilst the protein is expressed. The act of moving the droplet allows mixing within the droplet, and allows oxygen or other reagents to be supplied to the droplet. The act of moving improves the level of protein expression over a droplet which remains static.

[0119] Digital microfluidics (DMF) refers to a two-dimensional planar surface platform for lab-on-a- chip systems that is based upon the manipulation of microdroplets. Droplets can be dispensed, moved, stored, mixed, reacted, or analyzed on a platform with a set of insulated electrodes. Digital microfluidics can be used together with analytical analysis procedures such as mass spectrometry, colorimetry, electrochemical, and electrochemiluminescense. The droplet can be moved using any means of electrokinesis. The aqueous droplet can be moved using electrowetting-on-dielectric (EWoD). Electrowetting on a dielectric (EWoD) is a variant of the electrowetting phenomenon that is based on dielectric materials. During EWoD, a droplet of a conducting liquid is placed on a dielectric layer with insulating and hydrophobic properties. Upon activation of the electrodes the dielectric layer becomes less hydrophobic, thus causing the droplet to spread onto the surface.

[0120] The electrical signal on the EWoD or optically-activated amorphous silicon (a-Si) EWoD device can be delivered through segmented electrodes, active-matrix thin-film transistors or digital micromirrors. Optically-activated s-Si EWoD devices are well known in the art for actuating droplets ( . Adhes. Sci. Technol., 2012, 26, 1747-1771).

[0121] A source of supplemental oxygen can be supplied to the droplets. For example droplets or gas bubbles containing gaseous or dissolved oxygen can be merged with the aqueous droplets during the protein expression. Alternatively the source of oxygen can be a molecular source which releases oxygen. Alternatively the droplets can be moved to an air / liquid boundary to enable increased diffusion of oxygen from a gaseous environment. Additionally, a source of supplemental oxygen can be found by oxygenating the oil that is used as the filler medium. It is well-known in the art that oils such as hexadecane, HFE-7500, and others can be oxygenated to support the oxygen requirements of cell growth, especially E. coli cell growth (RSC Adv., 2017, 7, 40990-40995). Oxygenation can be achieved by aerating the oil with pure oxygen or atmospheric air.

[0122] Alternatively the oil can be oxygenated. Alternatively the droplets can be presented in a humidified air filled device.

[0123] Through an affinity tag, such as a FLAG-tag, HIS-tag, GST-tag, MBP-tag, STREP-tag, or other form of affinity tag, CFPS-expressed proteins can be immobilized to a solid-support affinity resin and fresh batches of CFPS reagent can be delivered over the said resin. Thus, renewed reagents can be used to carry out protein synthesis, closely mimicking industrial methods of continuous flow (CF) and continuous exchange (CE) CFPS. By mimicking CF- and CE-CFPS, users can scale up their CFPS production methods.

[0124] The droplets can be actuated on a hydrophobic surface on the digital microfluidic device (ACS Nano 2018, 12, 6, 6050-6058). The hydrophobic surface can be a hydrophobic surface such as polytetrafluoroethylene (PTFE), Teflon AF (DuPont Inc), CYTOP (AGC Chemicals Inc), or FluoroPei (Cytonix LLC). The hydrophobic surface may be modified in such a way to reduce biofouling, especially biofouling resulting from exposure to CFPS reagents or nucleic acid reagents. The hydrophobic surface may also be superhydrophobic, such as NeverWet (NeverWet LLC) or Ultra-Ever Dry (Flotech Performance Systems Ltd). Superhydrophobic surfaces prevent biofouling compared with typical fluorocarbon-based hydrophobic surfaces. Superhydrophobic surfaces thus prolong the capability of digital microfluidic devices to move CFPS droplets and general solutions containing biopolymers (RSC Adv., 2017, 7, 49633- 49648). The hydrophobic surface can also be a slippery liquid infused porous surface (SLIPS), which can be formed by infusing Krtox-103 oil (DuPont) with porous PTFE film (Lab Chip, 2019, 19, 2275).

[0125] Droplets can also contain additives to reduce the effects of biofouling on digital microfluidic surfaces. Specifically, droplets containing CFPS components can also contain additives such as surfactants or detergents to reduce the effects of biofouling on the hydrophobic or superhydrophobic surface of a digital microfluidic device (Langmuir 2011 , 27, 13, 8586-8594). Such droplets may use antifouling additives such as TWEEN 20, Triton X-100, and / or Pluronic F127. Specifically, droplets containing CFPS components may contain TWEEN 20 at 0.1% v / v, Triton X-100 at 0.1 % v / v, and / or Pluronic F127 at 0.08% w / v.

[0126] For electrowetting on dielectrics (EWoD), the change in contact angle of reagent upon the application of electric potential is an inverse function of surface tension. Thus, for low voltage EWoD operations, reduction in surface tension is achieved by addition of surfactants to reagents, which for CFPS reactions means to the lysate and to the DNA. This results in a dilution of the lysate, and it has been seen, in experiments, that diluting or otherwise adulterating the lysate results in a decrease in expression level of the protein of interest. Thus performing CFPS on DMF where the surfactants are added to the solutions being moved will necessarily result in a dilution and adulteration of the lysate and thus a decrease in the level of protein expression. In addition to being a problem in its own right, this further complicates extrapolation of on-DMF results to in-tube predictions of protein yield. An additional detriment of having to add surfactants to the samples is that this increases the time required for sample preparation, as well as increasing the potential for inconsistent results due to ‘user error,’ as there is more handling of reagents. An additional detriment of having to add surfactants to the samples is that certain downstream operations are hindered. For example, if a protein of interest is expressed in a cell-free system with a GFPn (or similar) peptide tag, it’s downstream complementation with a GFP1.10 (or similar) detector polypeptide is hindered in the presence of surfactant. Removal of the surfactant from the aqueous phase is therefore advantageous. Rather than adding surfactants to the aqueous sample, it is instead possible to add surfactant, such as a sorbitan ester such as Span85 (e.g. Sorbitan trioleate, Sigma Aldrich, SKU 8401240025), to the oil. This has the advantages of enabling CFPS reactions to proceed on- DMF without dilution or adulteration. Additionally, it simplifies the sample preparation procedure for setting up the reactions, increasing the ease of use and the consistency of results. Using 1 % w / w Span85 in dodecane allows for dilution-free CFPS reactions on-DMF, as well as dilution-free detection of the expressed non-fluorescent proteins. Other surfactants besides Span85, and oils other than dodecane could be used. A range of concentrations of Span85 could be used. Surfactants could be nonionic, anionic, cationic, amphoteric or a mixture thereof. Oils could be mineral oils or synthetic oils, including silicone oils, petroleum oils, and perfluorinated oils. Surfactants can have a detrimental effect on (1) the CFPS reactions and (2) the efficiency of the detection system (if the detection system involves complementation of a tag and detector). For example, by performing the CFPS reaction on- DMF with oil-surfactant mix, the detection of the expressed protein can also proceed without dilution and without adding aqueous surfactant. It has been shown that surfactants reduce the efficiency of some detection systems, including but not limited to the Split GFP (e.g. GFP11 / GFP1.10) system, so removing surfactants from the reagent mix and instead adding them to the oil can be beneficial.

[0127] The peptide tag can be attached to the C or N terminus of the protein. The peptide tag may be one component of a green fluorescent protein (GFP). For example the peptide tag may be GFPn and the further polypeptide GFP1.10. The peptide tag may be one component of sfCherry. The peptide tag may be sfCherryn and the further polypeptide sfCherryi- .

[0128] The protein may be fused to multiple tags. For example the protein may be fused to multiple GFP11 peptide tags and the synthesis occurs in the presence of multiple GFP1.10 polypeptides. For example the protein may be fused to multiple sfCherryn peptide tags and the synthesis occurs in the presence of multiple sfCherryi- polypeptides. The protein of interest may be fused to one or more sfCherryn peptide tags and one or more GFPn peptide tags and the synthesis occurs in the presence of one or more GFP1.10 polypeptides and one or more sfCherryi- polypeptides.

[0129] Disclosed is a kit comprising: i. a first composition for the cell-free synthesis of proteins, the composition containing reagents for cell-free protein expression; and ii. a second composition comprising a nucleic template coding sequence for a protein of interest and NTP’s and / or an energy source selected from phosphoenolpyruvate, acetyl phosphate, and creatine phosphate. Disclosed is a kit comprising: i. a first composition for the cell-free synthesis of proteins, the composition containing reagents for cell-free protein expression; ii. a second composition comprising a nucleic template coding sequence for a protein of interest; and iii. a third composition containing NTP’s and / or an energy source selected from phosphoenolpyruvate, acetyl phosphate, and creatine phosphate.

[0130] The energy source in the kit may be creatine phosphate. The concentration in the second composition may be in the range of 120 mM to 360 mM. The concentration is diluted upon mixing the first and second composition. The final concentration can be supplemented with an additional amount of creatine phosphate in the range 30 mM to 90 mM. The final concentration can be supplemented with an additional amount of creatine phosphate in the range 50 mM to 70 mM. The final concentration can be supplemented with an additional amount of 60 mM creatine phosphate.

[0131] The NTP’s in the kit may be ATP, CTP, GTP or UTP. The NTP’s in the kit may be ATP and GTP. The NTP’s may be present at a concentration of greater than 3 mM. The NTP’s may be present at a concentration of 3 mM to 5 mM. the NTP’s may be present at a concentration of 3 mM.

[0132] The kit may comprise other reagents to enable use on electrowetting devices such as basefluids containing suitable detergents, magnetic or paramagnetic beads to enable protein purification, washing buffers, elution buffers etc.

[0133] Devices

[0134] The manipulation of droplets by the application of electrical potential can be achieved on electrodes covered with an insulator or a dielectric or a series of insulators or dielectrics. Droplet manipulation as a result of an applied electrical potential is known as electrowetting. Electrokinesis occurs as result of a non-uniform electric field that influences the hydrostatic equilibrium of a dielectric liquid (dielectrophoresis or DEP) or a change in the contact angle of the liquid on solid surface (electrowetting-on-dielectric or EWoD). DEP can also be used to create forces on polarizable particles to induce their movement. The electrical signal can be transmitted to a discrete electrode, a transistor, an array of transistors, or a sheet of semiconductor film whose electrical properties can be modulated by an optical signal. EWoD phenomena occur when droplets are actuated between two parallel electrodes covered with a hydrophobic insulator or dielectric. The electric field at the electrode-electrolyte interface induces a change in the surface tension, which results in droplet motion as a result of a change in droplet contact angle. The electrowetting effect can be quantitatively treated using Young- Lippmann equation: cos0 - cos0o= (1 / 2 / LG) c.V2where 0o is the contact angle when the electric field across the interfacial layer is zero, yLG is the liquid-gas tension, c is the specific capacitance (given as sr. so / t, where sris dielectric constant of the insulator / dielectric, so is permittivity of vacuum, t is thickness) and V is the applied voltage or electrical potential. The change in contact angle (inducing droplet movement) is thus a function of surface tension, electrical potential, dielectric thickness, and dielectric constant.

[0135] When a droplet is actuated by EWoD, there are two opposing sets of forces that act upon it: an electrowetting force induced by electric field and resistant forces that include the drag forces resulting from the interaction of the droplet with filler medium and the contact line friction (ref). The minimum voltage applied to balance the electrowetting force with the sum of all drag forces (threshold voltage) is variably determined by the thickness-to-dielectric contact ratio of the insulator / dielectric, (t / Sr )1 / 2. Thus, to reduce actuation voltage, it is required to reduce (t / Sr )1 / 2(i.e. , increase dielectric constant or decrease insulator / dielectric thickness). To achieve low voltage actuation, thin insulator / dielectric layers must be used. However, the deposition of high quality thin insulator / dielectric layers is a technical challenge, and these thin layers are easily damaged before the desired electrowetting contact angle is large enough to drive the droplet is achieved. Most academic studies thus report the use of much higher voltages >100V on easily fabricated, thick dielectric films (>3 pm) to effect electrowetting.

[0136] High voltage EWoD-based devices with thick dielectric films, however, have limited industrial applicability largely due to their limited droplet multiplexing capability. The use of low voltage devices including thin-film transistors (TFT) and optically-activated amorphous silicon layers (a-Si) have paved the way for the industrial adoption of EWoD-based devices due to their greater flexibility in addressing electrical signals in a highly multiplex fashion. The driving voltage for TFTs or optically-activated a-Si are low (typically <15 V). The bottleneck for fabrication and thus adoption of low voltage devices has been the technical challenge of depositing high quality, thin film insulators / dielectrics. Hence there has been a particular need for improving the fabrication and composition of thin film insulator / dielectric devices.

[0137] Typically, the electrodes (or the array elements) used for EWoD are covered with (i) a hydrophilic insulator / dielectric and a hydrophobic coating or (ii) a hydrophobic insulator / dielectric. Commonly used hydrophobic coatings comprise of fluoropolymers such as Teflon AF 1600 or CYTOP. The thickness of this material as a hydrophobic coating on the dielectric is typically <100 nm and can have defects in the form of pinholes or a porous structure; hence, it is particularly important that the insulator / dielectric is pinhole free to avoid electrical shorting. Teflon has also been used as an insulator / dielectric, but it has higher voltage requirements due to its low dielectric constant and the thickness required to make it pinhole free. Other hydrophobic insulator / dielectric materials can include polymer-based dielectrics such as those based on siloxane, epoxy (e.g. Sll-8), or parylene (e.g., parylene N, parylene C, parylene D, or parylene HT). Due to minimal contact angle hysteresis and a higher contact angle with aqueous solutions, Teflon is still used as a hydrophobic topcoat on these insulator / dielectric polymers. However, there are difficulties in reliably producing <1 micron pinhole-free coatings of parylene or Sll-8; thus, the thickness of these materials is typically kept at a 2-5 microns at the cost of increased voltage requirements for electrowetting. It has also been reported that traditional EWoD devices with parylene C are easily broken and unstable for repeated droplet manipulation with cell culture medium. Multi-layer insulator devices deposited with metal-oxide and parylene C films have been used to produce a more robust insulator / dielectric and enable operations with lower applied voltages. Inorganic materials, such metal oxides and semiconductor oxides, commonly used in the CMOS industry as “gate dielectrics”, have been used as insulator / dielectric for EWoD devices. They offer the advantage of utilizing standard cleanroom processes for thin film depositions (<100 nm). These materials are inherently hydrophilic, requiring an additional hydrophobic coating, and can be prone to pinhole formation as a result of thin film layer deposition process. Together with the need for lower voltage operations of EWoD, recent developmental work has focused on (1) using materials with improved dielectric properties (e.g., using high-dielectric constant insulators / dielectrics), (2) optimizing the fabrication process to make the insulator / dielectric pinhole free to avoid dielectric breakdown.

[0138] Operation of EWoD devices suffers from contact angle saturation and hysteresis, which is believed to be brought about by either one or combination of these phenomena: (1) entrapment of charges in the hydrophobic film or insulator / dielectric interface, (2) adsorption of ions, (3) thermodynamic contact angle instabilities, (4) dielectric breakdown of dielectric layer, (5) the electrode-electrode-insulator interface capacitance (arising from the double layer effect), and (6) fouling of the surface (such as by biomacromolecules). One of the adverse effects of this hysteresis is reduced operational lifetime of the EWoD-based device.

[0139] Contact angle hysteresis is believed to be a result of charge accumulation at the interface or within the hydrophobic insulator after several operations. The required actuation voltage increases due to this charging phenomenon resulting in eventual catastrophic dielectric breakdown. The most probable explanation is that pinholes at the insulator / dielectric may allow the liquid to come into contact with the electrode causing electrolysis. Electrolysis is further facilitated by pinhole-prone or porous hydrophobic insulators.

[0140] Most of the studies to understand contact angle hysteresis on EWoD have been conducted on short time scales and with low conductivity solutions. Long duration actuations (e.g., >1 hour) and high conductivity solutions (e.g., 1 M NaCI) could produce several effects other than electrolysis. The ions in solution can permeate through the hydrophobic coat (under the applied electric field) and interact with the underlying insulator / dielectric. Ion permeation can result in (1) change in dielectric constant due to charge entrapment (which is different from interfacial charging) and (2) change in surface potential of a pH sensitive metal oxide. Both can result in reduction of electrowetting forces to manipulate aqueous droplets, leading to contact angle hysteresis. The inventors have previously found that the damage from high conductivity solutions reduces or disables electrowetting on electrodes by inhibiting the modulation of contact angle when an electric field is applied.

[0141] An electrokinetic device includes a first substrate having a matrix of electrodes, wherein each of the matrix electrodes is coupled to a thin film transistor, and wherein the matrix electrodes are overcoated with a functional coating comprising: a dielectric layer in contact with the matrix electrodes, a conformal layer in contact with the dielectric layer, and a hydrophobic layer in contact with the conformal layer; a second substrate comprising a top electrode; a spacer disposed between the first substrate and the second substrate and defining an electrokinetic workspace; and a voltage source operatively coupled to the matrix electrodes.

[0142] The dielectric layer may comprise silicon dioxide, silicon oxynitride, silicon nitride, hafnium oxide, yttrium oxide, lanthanum oxide, titanium dioxide, aluminum oxide, tantalum oxide, hafnium silicate, zirconium oxide, zirconium silicate, barium titanate, lead zirconate titanate, strontium titanate, or barium strontium titanate. The dielectric layer may be between 10 nm and 100 pm thick. Combinations of more than one material may be used, and the dielectric layer may comprise more than one sublayer that may be of different materials. The conformal layer may comprise a parylene, a siloxane, or an epoxy. It may be a thin protective parylene coating in between the insulating dielectric and the hydrophobic coating. Typically, parylene is used as a dielectric layer on simple devices. In this invention, the rationale for deposition of parylene is not to improve insulation / dielectric properties such as reduction in pinholes, but rather to act as a conformal layer between the dielectric and hydrophobic layers. The inventors find that parylene, as opposed to other similar insulating coatings of the same thickness such as PDMS (polydimethylsiloxane), prevent contact angle hysteresis caused by high conductivity solutions or solutions deviating from neutral pH for extended hours. The conformal layer may be between 10 nm and 100 pm thick. The conformal layer may be between 100 nm and 200 nm thick.

[0143] The hydrophobic layer may comprise a fluoropolymer coating, fluorinated silane coating, manganese oxide polystyrene nanocomposite, zinc oxide polystyrene nanocomposite, precipitated calcium carbonate, carbon nanotube structure, silica nanocoating, or slippery liquid-infused porous coating.

[0144] The elements may comprise one or more of a plurality of array elements, each element containing an element circuit; discrete electrodes; a thin film semiconductor in which the electrical properties can be modulated by incident light; and a thin film photoconductor whose properties can be modulated by incident light.

[0145] The functional coating may include a dielectric layer comprising silicon nitride, a conformal layer comprising parylene, and a hydrophobic layer comprising an amorphous fluoropolymer. This has been found to be a particularly advantageous combination.

[0146] The electrokinetic device may include a controller to regulate a voltage provided to the individual matrix electrodes. The electrokinetic device may include a plurality of scan lines and a plurality of gate lines, wherein each of the thin film transistors is coupled to a scan line and a gate line, and the plurality of gate lines are operatively connected to the controller. This allows all the individual elements to be individually controlled.

[0147] The second substrate may also comprise a second hydrophobic layer disposed on the second electrode. The first and second substrates may be disposed so that the hydrophobic layer and the second hydrophobic layer face each other, thereby defining the electrokinetic workspace between the hydrophobic layers.

[0148] The method is particularly suitable for aqueous droplets with a volume of 1 pL or smaller. The EWoD-based devices shown and described below are active matrix thin film transistor devices containing a thin film dielectric coating with a Teflon hydrophobic top coat. These devices are based on devices described in the E Ink Corp patent filing on “Digital microfluidic devices including dual substrate with thin-film transistors and capacitive sensing”, US patent application no 2019 / 0111433, incorporated herein by reference.

[0149] Described herein are electrokinetic devices, including: a first substrate having a matrix of electrodes, wherein each of the matrix electrodes is coupled to a thin film transistor, and wherein the matrix electrodes are overcoated with a functional coating comprising: a dielectric layer in contact with the matrix electrodes, a conformal layer in contact with the dielectric layer, and a hydrophobic layer in contact with the conformal layer; a second substrate comprising a top electrode; a spacer disposed between the first substrate and the second substrate and defining an electrokinetic workspace; and a voltage source operatively coupled to the matrix electrodes;

[0150] Described herein is an electrokinetic device, including: a first substrate having a matrix of electrodes, wherein each of the matrix electrodes is coupled to a thin film transistor, and wherein the matrix electrodes are overcoated with a functional coating comprising: one or more dielectric layer(s) comprising silicon nitride, hafnium oxide or aluminum oxide in contact with the matrix electrodes, a conformal layer comprising parylene in contact with the dielectric layer, and a hydrophobic layer in contact with the conformal layer; a second substrate comprising a top electrode; a spacer disposed between the first substrate and the second substrate and defining an electrokinetic workspace; and a voltage source operatively coupled to the matrix electrodes;

[0151] The electrokinetic devices as described may be used with other elements, such as for example devices for heating and cooling the device or reagent cartridges for the introduction of reagents as needed.

[0152] Examples

[0153] Figure 1 shows the ccGFP protein expression yields from CFPS reactions formulated with a variety of reconstituted expression systems. Different batches of expression systems show different initial levels of expression (BO, B1 , B2, B4 and B6). Addition of increasing amounts of creatine phosphate to the expression template were measured in reaction volumes of 12 pL in 1.5 mL Eppendorf tubes for 17 hours. The solutions were transferred to a microtitre plate for fluorescence intensity measurements on a fluorescence plate reader (485 nm Ex 520 nm Em).

[0154] The protein expressed (ccGFP) is fluorescent and concentration can be determined directly against a standard solution. The yields of expressed protein peak when supplementing the CFPS reaction with an additional 60 mM of creatine phosphate (therefore theoretically doubling the concentration creatine phosphate in the reaction to 120 mM as the starting solution contained 60 mM [final]). Additional creatine phosphate then starts to inhibit the expression. The optimum range of added creatine phosphate is between 30 mM and 90 mM added to the initial 60 mM to give a final working concentration of 90 to 150 mM.

[0155] Figure 2 shows activity date where certain essential components are removed from the reconstituted CFPS mixture. The experiment shows the activity of the reformulated core reagents in the absence and presence of the missing component (ATP / GTP / CP). Experiments were performed in a 96 well plate using a nucleic acid template which expresses a fluorescent protein, incubated at 29°C for an overnight incubation. Formulations for cell-free protein expression were prepared with certain of the required reagents missing from the composition. The formulations were used to express a fluorescent protein, thus allowing comparison of the expression activity directly in microwell plates. R1 = -Creatine Phosphate (CP); R2 = -ATP, R3 = -GTP, R4 = -ATP and GTP, R5 = -CP, ATP and GTP.

[0156] The expression activity can be measured in iM by comparing with a titration curve of the same protein being expressed. Data shown plotted on Figure 2 is below:

[0157] The formulated core reagent expression mastermix (S1 Comp) was fully active. Adding in DNA without adding back the missing components results in inactive CFPS reactions (e.g. R1-, R2- etc.). Spiking the missing components back into the remixes with added DNA restored activity, visible by the expression of the fluorescent protein. All of the restored mixes worked and the activity were comparable. R1 = -CP; R2 = -ATP, R3 = -GTP, R4 = -ATP and GTP, R5 = -CP, ATP and GTP.

[0158] Figure 3 show the stability on storage at 20°C. Compositions lacking the various components were stored at 20°C then the additives needed and the nucleic acids were added and the activity was measured. As with the example above, expression of a fluorescent protein was performed overnight at 29°C in a microtitre plate, and compared with a standard curve of the same fluorescent protein. The date shown in Figure 3 is normalised to the activity of the full system at time 0. R1 = -CP; R2 = -ATP, R3 = -GTP, R4 = -ATP and GTP, R5 = -CP, ATP and GTP.

[0159] The complete core mastermix is completely dead after 3 days stored at 20°C. Solutions aged without ATP (R2) were almost as bad, retaining less than 20% of the starting activity. Those aged without GTP, ATP and GTP or all three components (CP and ATP and GTP) degraded to ca. 40-60% of a non-aged control. The most stable mix (R1 ) was the one that was aged in the absence of creatine phosphate. This only lost 10% of its starting activity when aged at 20°C for 3 days. Thus CFPS mixes having missing components show an improvement in storage stability, and that the full activity can be restored on re-addition of the omitted components. Figure 4 shows activity the activity of systems with missing creatine phosphate (CP) is eliminated and can be restored when CP is added:

[0160] Experimental design:

[0161] • Compare the activity of CoreV2 (expression mix without CP) to CoreVI (expression mix with CP) to express a nucleic acid sequence coding for a fluorescent protein (ccGFP).

[0162] • Reactions set up with either no DNA, ccGFP template (C_6816), or ccGFP template + CP (60 mM final in CoreV2).

[0163] • All reactions set up in triplicate (27 in total).

[0164] DNA mixes were prepared as described below:

[0165] Table 1. Preparation of DNA mixes used to set up expression reactions.

[0166] Reactions were set up directly in a low volume, clear bottom, 384-well, black plate.

[0167] 10 jiL reactions were assembled as described below:

[0168] Table 2. Reaction set up.

[0169] A control dilution of the ccGFP protein was added to the plate to prepare a concentration standard.

[0170] The plate was sealed, vortexed, and centrifuged at 500 xg for 30 seconds, before incubation at 29 °C overnight. The reaction plate was retrieved, vortexed briefly and centrifuged at 500xg for 30 seconds. The plate was scanned using a fluorescence plate reader. Data is shown in Figure 4, and shows that the formulated CoreV2 activity is dependent on supplementation with CP. Once supplemented, coreV2 displays comparable activity to the CoreVI having CP. Figure 5 shows long term stability studies on the expression mix with and without creatine phosphate (CP), and tests the stability of expression mixes without and without CP using an stability study at four temperatures. For each day of the 20 °C stability time course 1 aliquot of CoreVI , CoreV2, DNA (C_6816), and DNA (C_6816) + CP were transferred from -80oC to 20oC. After 7 days the contents were prepared for expression and fluorescence analysis as described above and compared to a control standard ccGFP dilution series. CoreVI was inactive after incubation at 20 °C for one day. CoreV2 still possessed between 80-100% of their original activity after one week at 20 °C (Figure 5a). In a similar way aliquots were stored for the respective times and temperatures then used for expression. Figure 5d shows no major changes in activity after storage at -80 °C. 5c shows that after storage at -20 °C for 56 days, the average activity of CoreVI and CoreV2 was 71.3% and 81.8%, respectively. This shows that both Core reagents have reduced activity when stored at -20 °C for a prolonged period, and CoreV2 retains slightly more activity. Figure 5b shows that CoreVI is inactive after storage at 4 °C for 7 days. CoreV2 activity has significantly dropped between day 28 (82.75%) and 56 (22.5%) of storage at 4 °C.

[0171] Figure 6 shows expression in droplets on a microfluidic device. As expected, in the absence of creatine phosphate (CP) little detectable expression is seen. When CP is added via the nucleic acid activity is restored. The omission of CP from the expression mix has no impact on droplet handling. The CP can be added using the nucleic acid droplet to form the complete expression mix. The mixing of droplets occurs at greater than 20 °C and make several hours of time to be completed on the microfluidic device. Addition of the CP via the nucleic acid means that the activity of the expression system is not compromised by the CP being consumed prior to the template and expression system droplets being merged together on device.

[0172] Figure 7 and 8 show the effect of creatine phosphate on transcription and translation using reconstituted cell-free blends. The experiment tests whether pre-incubation of a reconstituted cell-free expression system which lacks creatine phosphate (Core V2) with DNA promotes transcription (TX) in the absence of translation (TL).

[0173] A reconstituted cell-free expression system which contains creatine phosphate (+CP) (Core V1) initiates protein expression upon addition of a DNA template. Thus the only additional requirement is the template. As an alternative, where the reconstituted cell-free expression system is lacking creatine phosphate (-CP) (Core V2), addition of the DNA template initiates a certain level of transcription of RNA, but limited translation of the RNA into protein. Thus the TX is uncoupled from the TL. Addition of CP after the DNA template thus actives the translation of the RNA into proteins. When CoreV2 is used, significant translation does not start until CP is added to promote ATP / GTP recycling, but transcription occurs. This pre-TX reaction displays additional advantages of CoreV2 e.g. the possibility to use GroE / L as an additive which is typically inhibitory to TX. The use of creatine deficient blends and subsequent addition of CP enables a convenient approach for splitting TX and TL reactions without requiring a separate in vitro transcription reaction and cleanup.

[0174] Reactions were assembled using CoreVI or CoreV2 in the absence or presence of CP and DNA. To visualize TX, fluorescein-12-UTP (Jena, NU-821-FAMX) was added to reactions. Fluorescein-12-UTP is readily incorporated by T7 RNA polymerase during TX and allows visualisation of mRNA without interference from ribsosomal / tRNAs which would occur if gel staining was performed. TL products were analyzed by SDS-PAGE from reactions not containing Fluorescein-12-UTP.

[0175] A nucleic acid template for untagged DHODH was used for expression since the protein can be easily distinguished from background bands on SDS PAGE gels without purification. The construct was diluted to 5 nM in reconstituted cell-free blends V1 or V2.

[0176] 20 pL reactions were assembled according to Table 1 below:

[0177] Reaction 1 2 3 4 5 6

[0178] CoreVI ' 12 ' 12 'O 0 0 0

[0179] CoreV2 0 0 12 12 12 12

[0180] DNA V1 (5 nM stock) 0 5 0

[0181] DNA V2 (5 nM stock) 0 0 0

[0182] EBV1 5 0 5 0 0 0

[0183] EBV2 0 0 0 5 0 0

[0184] Fluor-UTP (5 mM stock) 1.5 1.5 1,5 1,5 1,5 1,5

[0185] Additive Buffer 1,5 1,5 1,5 1,5 1,5 1,5

[0186] Final volume 2020 20 20 20 20'

[0187] Reactions were overlaid with 20 pL of base fluid and incubated at 29°C overnight.

[0188] 3 pL were removed, diluted to 12 pL with 3.6x SDS loading dye and water, and heated to 95°C for 5 minutes. 3 pL of each diluted sample was analysed by SDS PAGE (Figure 7). 2 pL were removed and added to 8 pL of water + 2 pL of 6x purple DNA loading dye. 10 pL was run on an 1 % agarose gel (no intercalating dye added). After running, the bottom of the gel was cut off to remove signal from the free fluorescein-12 UTP and the gel was imaged using SYBRgold settings on the Gel Doc. The gel was then stained with SYBRsafe for 1 hour, washed with TAE and re-imaged using SYBRsafe settings (two images seen in Figure 8).

[0189] As predicted, incubation of CoreV2 with DNA template, in the absence of CP, promotes TX but not TL. The level of transcription appeared to be reduced in the absence of CP. This may be due to a combination of polymerase-mediated RNA hydrolysis / exonuclease activity / pyrophosphorolysis and nucleotide consumption from tRNA charging / limited TL.

[0190] The predominant TX products generated in the absence of CP migrated more slowly than those generated in its presence. This may be due to the remaining association of RNA polymerase / DNA with the mRNA product if stalling has occurred due to a depleted nucleotide pool. Similarly, the larger molecular weight smear in the +CP lanes may be mRNA transcripts associated with TL machinery.

[0191] Embodiments

[0192] 1. A method for the cell-free synthesis of proteins, the method comprising mixing a first composition containing reagents for cell-free protein expression, and a second composition containing a nucleic acid template and NTP’s and / or an energy source for the cell-free protein expression reagents. 2. The method according to embodiment 1 , wherein the energy source is one or more of phosphoenolpyruvate, acetyl phosphate, and creatine phosphate.

[0193] 3. The method according to embodiment 1 , wherein the energy source is creatine phosphate.

[0194] 4. The method according to any one of embodiments 1 to 3, wherein the second composition contains ATP.

[0195] 5. The method according to any one of embodiments 1 to 4, wherein the second composition contains GTP.

[0196] 6. The method according to embodiment 4 or embodiment 5, wherein the second composition contains creatine phosphate.

[0197] 7. The method according to any one of embodiments 1 to 6, wherein the synthesis of a protein is performed on an electrowetting-on-dielectric (EWoD) device.

[0198] 8. The method according to embodiment 7 comprising using a series of droplets having varying cell-free reagent compositions, including enzymes for protein synthesis and the template nucleic acid and energy source and monitoring the synthesis of the protein of interest in the various compositions, thereby identifying a composition suitable for expression of the protein of interest.

[0199] 9. The method according to any one of embodiments 1 to 8, wherein the first composition is reconstituted coupled system for transcription and translation.

[0200] 10. The method according to any one of embodiments 1 to 9, wherein the first composition contains synthesized or isolated ribosomes, initiation factors, elongation factors, aminoacyl-tRNA synthetases, methionyl tRNA transformylases, tRNAs, amino acids and ribonucleoside triphosphates.

[0201] 11. The method according to any one of embodiments 1 to 10, wherein the reagents for cell-free protein expression are derived from mammalian cells, insect cells, prokaryotic cells, yeast cells, plant cells or protozoa. 12. The method according to embodiment 11 , wherein the cells are HEK293, HeLa, BHK21, NSO, Sp2 / 0, or CHO.

[0202] 13. The method according to embodiment 11, wherein the cells are derived from Escherichia Coli, Leishmania Tarentolae, Saccharomyces Cerevisiae or Pichia Pastoris.

[0203] 14. The method according to any one of embodiments 1 to 13, wherein the first reagent composition is a mixture of cell lysate and reconstituted expression systems.

[0204] 15. The method according to any one of embodiments 1 to 14, wherein the expression of proteins is performed for at least 3 hours and the level of expressed protein is determined.

[0205] 16. A composition comprising a nucleic acid template and an energy source for the cell- free protein expression reagents selected from phosphoenolpyruvate, acetyl phosphate, and creatine phosphate, wherein the composition does not contain any enzymes that metabolise the energy sources.

[0206] 17. The composition according to embodiment 16 comprising a nucleic acid template and at least 120 mM creatine phosphate.

[0207] 18. The composition according to embodiment 16 comprising a nucleic acid template and 120 mM to 360 mM creatine phosphate.

[0208] 19. The composition according to any one of embodiments 16 to 18, further comprising ATP and / or GTP.

[0209] 20. The composition according to embodiment 19, further comprising ATP at a concentration of 3 mM to 5 mM and / or GTP at a concentration of 3 mM to 5 mM.

[0210] 21. A kit comprising: i. a first composition for the cell-free synthesis of proteins, the composition containing reagents for cell-free protein expression; and ii. a second composition comprising a nucleic template coding sequence for a protein of interest and NTP’s and / or an energy source selected from phosphoenolpyruvate, acetyl phosphate, and creatine phosphate. 22. The kit according to embodiments 21 , wherein the energy source is 120 mM to 360 mM creatine phosphate. 23. The kit according to embodiment 21 or embodiment 22, wherein the kit contains ATP and / or GTP.

[0211] 24. The kit according to embodiment 23, further comprising ATP at a concentration of 3 mM to 5 mM and / or GTP at a concentration of 3 mM to 5 mM.

Claims

Claims1. A composition comprising a nucleic acid template and at least 120 mM creatine phosphate.

2. The composition according to claim 1 comprising a nucleic acid template and 120 mM to 360 mM creatine phosphate.

3. The composition according to claim 1 or claim 2 further comprising ATP and / or GTP.

4. The composition according to claim 3, further comprising ATP at a concentration of 3 mM to 5 mM and / or GTP at a concentration of 3 mM to 5 mM.

5. A kit comprising: i. a first composition for the cell-free synthesis of proteins, the composition containing reagents for cell-free protein expression; and ii. a second composition comprising creatine phosphate, wherein either the first or second composition contains a nucleic acid template.

6. A kit comprising: i. a first composition for the cell-free synthesis of proteins, the composition containing reagents for cell-free protein expression; ii. a second composition comprising a nucleic template coding sequence for a protein of interest; and iii. a third composition containing NTP’s and / or an energy source selected from phosphoenolpyruvate, acetyl phosphate, and creatine phosphate.

7. The kit according to claim 6, wherein the third composition further contains a chaperone protein.

8. The kit according to any one of claims 5 to 7, wherein the concentration of creatine phosphate is 120 mM to 360 mM.

9. The kit according to any one of claims 5 to 8, wherein the second composition contains ATP and / or GTP.

10. The kit according to claim 9, wherein the second composition contains ATP at a concentration of 3 mM to 5 mM and / or GTP at a concentration of 3 mM to 5 mM.

11. A method for the cell-free synthesis of proteins, the method comprising mixing a first composition containing reagents for cell-free protein expression, and a second composition containing creatine phosphate, wherein either the first or second composition contains a nucleic acid template.

12. The method according to claim 11 comprising mixing a first composition containing reagents for cell-free protein expression, and a second composition containing a nucleic acid template and at least 120 mM creatine phosphate.

13. The method according to claim 11 comprising mixing a first composition containing reagents for cell-free protein expression, and a second composition containing a nucleic acid template and a third composition containing at least 120 mM creatine phosphate.

14. The method according to any one of claims 10 to 13, wherein the synthesis of a protein is performed on an electrowetting-on-dielectric (EWoD) device.

15. The method according to claim 14, comprising using a series of droplets having varying cell-free reagent compositions, including enzymes for protein synthesis and the template nucleic acid and energy source and monitoring the synthesis of the protein of interest in the various compositions, thereby identifying a composition suitable for expression of the protein of interest.

16. The method according to any one of claims 10 to 15, wherein the first composition is reconstituted coupled system for transcription and translation.

17. The method according to claim 16, wherein the first composition contains synthesized or isolated ribosomes, initiation factors, elongation factors, aminoacyl-tRNA synthetases, methionyl tRNA transformylases, tRNAs, amino acids and ribonucleoside triphosphates.

18. The method according to any one of claims 10 to 15, wherein the reagents for cell-free protein expression are derived from mammalian cells, insect cells, prokaryotic cells, yeast cells, plant cells or protozoa.

19. The method according to claim 18, wherein the cells are HEK293, HeLa, BHK21 , NSO, Sp2 / 0, or CHO, Escherichia Coli, Leishmania Tarentolae, Saccharomyces Cerevisiae or Pichia Pastoris.

20. The method according to any one of claims 10 to 19, wherein the expression of proteins is performed for at least 3 hours and the level of expressed protein is determined. 21 . The method according to according to any one of claims 10 to 20, wherein the second composition contains 120 mM to 360 mM creatine phosphate.

22. The method according to according to any one of claims 10 to 21 , wherein the second composition contains 240 mM creatine phosphate.

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