Protein expression reagents

Abstract

Provided herein are methods of cell-free protein synthesis, optimised cell-free protein synthesis (CFPS) reagents for the expression of eukaryotic proteins and methods for optimising CFPS reagents to increase protein expression yields for mammalian proteins and subsequent expression in cells. 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 and then identify the optimum conditions for producing the stable folded protein in a cell-based system.

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 when transferred into correlated cell-based systems. The reagents can enable the expression of eukaryotic proteins that require codons rarely used in E. coli. The methods are applicable to protein expression on a microfluidic device having hydrophobic surfaces and help to correlate expression in such devices with expression in cell-based systems.

[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).

[0008] 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).

[0009] 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.

[0010] 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 / ).

[0011] Most amino acids are encoded by more than one codon, and each organism carries its own bias in the usage of the 61 available amino acid codons. In each cell, the tRNA population closely reflects the codon bias of the mRNA population. When the mRNA of heterologous target genes is overexpressed in E. coli, differences in codon usage can impede translation due to the demand for one or more tRNAs that may be rare or lacking in the population. Insufficient tRNA pools can lead to translational stalling, premature translation, termination, translation frameshifting and amino acid misincorporation. Heterologous protein expression can be very low when a gene encodes clusters of and / or numerous rare E. coli codons. Protein expression in E. coli derived expression systems can be improved by overcoming codon bias. Codon bias occurs when forced high-level expression of a gene containing codons rarely expressed in E. coli depletes internal tRNA pools.

[0012] Escherichia coli BL21 and BL21(DE3) cell extracts are common laboratory strains for recombinant protein production. The lack of Ion and ompT proteases, often regarded as common characteristics among B lineage, has driven the development of those strains for protein expression hosts. E. coli BL21(DE3), a derivative of BL21 , is probably the most widely used in high-level expression of recombinant proteins, and it harbors a prophage DE3 derived from a bacteriophage A, which carries the T7 RNA polymerase gene under the control of the / acllV5 promoter.

[0013] Rosetta host strains are BL21 derivatives designed to enhance the expression of eukaryotic proteins that contain codons rarely used in E. coli. These strains supply tRNAs for AGG, AGA, AUA, CUA, CCC, GGA codons on a compatible chloramphenicol-resistant plasmid. Thus the Rosetta strains provide for “universal” translation which is otherwise limited by the codon usage of E. coli. The tRNA genes are driven by their native promoters. In Rosetta(DE3)pLysS, the rare tRNA genes are present on the same plasmids that carries the T7 lysozyme gene.

[0014] Rosetta host strains are BL21 derivatives designed to enhance the expression of eukaryotic proteins that contain codons rarely used in E. coli. The systems have been extensively used as lysed compositions without purification and reconstitution of the purified components.

[0015] Reconstituted reagents for cell-free protein synthesis are expensive to use when large amount of proteins are required. A scale up reaction using for example 1 mL of reagent will likely give at most a few mg of the desired protein. For a significant amount of protein, expression in living cells is required.

[0016] WO2017 / 031399 discloses coupled transcription / translation (TX-TL) systems and methods for rapid prototyping of natural products, their analogs, and secondary metabolites. These systems can be used for the combinatorial biosynthesis of these compounds and for rapid prototyping of complex biosynthetic pathways. The patent also provides products of manufacture comprising mixtures of cytoplasmic and nuclear extracts from different cells, which can be used for in vitro transcription, translation, and / or coupled transcription and translation. The patent further provides processes for in vitro transcription / translation using these products of manufacture and substantially isolated or synthetic nucleic acids. The patent also provides methods for making, synthesizing, or altering the structure of compounds using these mixtures and processes. LIS2021163969 discloses a cell-free transcription and translation system derived from plants containing plastids, double-membrane organelles which are found in the cells of plants and algae. The patent discloses components, compositions, systems, kits, and methods for performing cell-free protein synthesis (CFPS) using isolated plant plastids and extracts thereof, specifically chloroplasts. This allows for high throughput genetic analysis and protein production. The systems and methods are suitable for automation and can be used for in vitro protein synthesis and prototyping of genetic expression in plants. The patent also relates to plants and plant materials, such as extracts prepared from isolated plastid of plants, including tobacco and maize. The patent further discloses components, compositions, systems, kits, and methods for performing in vitro transcription of mRNA and / or in vitro translation of a polypeptide. The methods may involve reacting a plant plastid cell-free protein synthesis mixture or system with a linear or plasmid template for transcription of the mRNA and / or a template for translation of the polypeptide. The patent also provides for the testing of the function of genetic components, codon-optimized constructs, constructs expressing several genes, and constructs of a biosynthetic pathway in the plant plastid cell-free protein synthesis system.

[0017] W02023 / 002187 discloses methods of loading aqueous fluids into hydrophobic electrowetting devices. This patent introduces a method for controlling the filling of a reservoir on a microfluidic device, specifically an electrowetting on dielectric (EWoD) device with hydrophobic surfaces. The method improves accuracy and efficiency in loading multiple aqueous reagents, particularly those with high surface tension, into devices with low surface energy. It prevents back-flow of fluids and allows for accurate control of the liquid volume in each reservoir. The method also includes the creation of virtual calibration structures to assist in the accurate loading of the reservoir with a defined area of liquid. Furthermore, the technology enables droplet-based nucleic acid synthesis, assembly, and cell-free expression of peptides or proteins.

[0018] US2023 / 092310 discloses methods of cell-free protein synthesis on electrowetting devices. The patent describes improved methods for cell-free expression of peptides or proteins in a digital microfluidic device. This involves using droplets containing a nucleic acid template and a cell-free system with components for protein expression in an oil-filled environment. The droplets are moved using electrokinesis, which can be achieved using electrowetting-on- dielectric (EWoD). The oil used in the device can be any water immiscible liquid, such as mineral oil, silicone oil, an alkyl-based solvent, or a fluorinated oil. The oil can be oxygenated prior to or during the expression process. The droplets can be formed before entering the microfluidic device or merged on the device. The droplets can also be split on the device either before or after expression. The patent also discloses a kit for performing the method, which includes a digital microfluidic device, a reagent source to generate a plurality of droplets containing a cell-free system, and an oil. Such devices do not give substantial yields of the expressed protein.

[0019] US2006 / 084136 discloses the production of fusion proteins using cell-free protein synthesis. The patent describes systems and methods for in vitro transcription and translation, specifically focusing on in vitro protein synthesis (IVPS) compositions. The IVPS composition includes an extract of a cell or organism supplemented with one or more exogenously added transfer RNAs (tRNAs). The added tRNAs can be suppressor tRNAs, which promote readthrough of stop codons in a nucleic acid template used for protein synthesis, allowing for the incorporation of modified or nonnaturally-occurring amino acids into a protein. The invention also provides methods for increasing protein production in cases where the gene of interest includes one or more codons that are infrequently used in the cell or organism from which the translation extract was obtained. The invention further includes kits and instruments comprising the IVPS compositions, as well as methods of synthesizing proteins in vitro and methods of making fusion proteins using in vitro synthesis systems supplemented with suppressor tRNAs.

[0020] SUMMARY OF THE INVENTION

[0021] Disclosed herein is a method to correlate a composition for the cell-free expression of proteins with a cell-based expression system to produce the desired amino acid sequences in a stable and correctly folded form. The expression allows screening of additives which are applicable to cell-based scale-up. The process may allow use of additives which can be present in the cells or growing media, or may be proteins which can be co-expressed in the cells.

[0022] The composition may be prepared by adding certain tRNA’s that are otherwise limited in E. coli in order to overcome codon bias seen when making non E. coli proteins. Overcoming codon bias caused by the limited amounts of tRNA’s can enhance the expression of eukaryotic proteins that contain codons rarely used in E. coli, including for example tRNAs for AGG, AGA, AUA, CUA, CCC, GGA codons.

[0023] Once the optimal cell-free conditions are identified, the appropriate cell-based expression system can be selected for a scale-up of the desired protein, the protein being obtained in a stable form. Disclosed is a method of producing proteins comprising the steps of: a. taking nucleic sequences expressing the desired amino acid sequence; b. expressing the nucleic acid sequences in cell-free expression systems having varying compositions; c. identifying the nucleic sequence and conditions in which the desired protein is best expressed; d. based on the identified conditions, selecting a host-cell type, media composition and growing conditions in which to produce the protein; and e. producing the protein in said host-cell using the identified host cell type, media composition and growing conditions.

[0024] Disclosed is a method of producing proteins comprising the steps of: a. taking nucleic sequences expressing the desired amino acid sequence; b. expressing the nucleic acid sequences in cell-free expression systems having varying compositions optimised for different species; c. identifying the nucleic sequence and conditions in which the desired protein is best expressed; d. based on the identified conditions selecting a host-cell type in which to produce the protein; and e. producing the protein in said host-cell.

[0025] Disclosed is a method of producing proteins comprising the steps of: a. taking nucleic sequences expressing the desired amino acid sequence; b. expressing the nucleic acid sequences in cell-free expression systems having varying compositions optimised for different species; c. identifying the nucleic sequence and conditions in which the desired protein is best expressed; d. based on the identified conditions and media selecting a host-cell type and growing conditions in which to produce the protein; and e. producing the protein in said host-cell using the identified conditions and media.

[0026] Disclosed is a reconstituted cell-free protein expression system derived from E. coli comprising isolated tRNAs added to the expression system, wherein the ratio of the tRNA’s is optimised for use in expression of eukaryotic proteins. The ratio of the tRNA in the reconstituted system can be balanced with the regards to the codon usage needed by the template being expressed. Disclosed is a reconstituted cell-free protein expression system comprising tRNAs isolated from a mammalian codon tolerant or human codon tolerant E. coli strain. Disclosed is a reconstituted cell-free protein expression system comprising tRNAs purified after in-vitro transcription. Disclosed is a reconstituted cell-free protein expression system comprising tRNAs purified from a Rosetta BL21 E. coli strain. Rosetta host strains are BL21 derivatives designed to enhance the expression of eukaryotic proteins that contain codons rarely used in E. coli. The expression system may comprise tRNAs for AGG, AGA, AUA, CUA, CCC, GGA codons. Certain strains can supply tRNAs on a chloramphenicol-resistant plasmid.

[0027] 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.

[0028] Disclosed herein is the use of a reconstituted cell-free protein expression system comprising tRNAs purified from a codon enhanced strain such as a Rosetta BL21 E. coli strain for the expression of eukaryotic proteins.

[0029] Disclosed herein is the use of a reconstituted cell-free protein expression system comprising tRNAs after in-vitro transcription for the expression of eukaryotic proteins. The endogenous expression of tRNAs has been reported and used to produce tRNA population during CFPS. However the production of tRNA’s, and the addition of the isolated tRNA’s to a reconstituted cell-free protein expression system is described herein.

[0030] Disclosed is a method for the synthesis of proteins comprising adding a template nucleic acid encoding a protein of interest to a cell-free protein expression system comprising tRNAs which are codon balanced with the template sequence. Having a known template sequence with known codons means that the tRNA’s for those codons can be supplied in the correct ratio. The tRNA pool can be prepared in a way that is specific for each nucleic acid template. Starting from a generic pool of expression reagent and each tRNA required a desired composition can be prepared that is specific for the chosen template.

[0031] Disclosed is a method for the synthesis of proteins comprising adding a template nucleic acid encoding a protein of interest to a cell-free protein expression system comprising tRNAs purified from a Rosetta BL21 E. coli strain. Disclosed is a method for the synthesis of proteins comprising adding a template nucleic acid encoding a protein of interest to the composition described above. The synthesis may be performed 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 merging with a composition described herein. The method may use a series of droplets having varying cell-free reagent compositions, including enzymes for protein synthesis and the template nucleic acid 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.

[0032] The screening in a such a highly parallel system allows a variety of templates and tRNA populations to be screen in order to identify the best conditions for a desired protein sequence. The variety of templates may include the same nucleic acid sequence coding for the desired amino acid sequence flanked by a variety of different for example solubility tags or purification tags. Alternatively the codon sequences may be chosen such that varying codons express the desired amino acid sequence.

[0033] The method may identify conditions for protein synthesis, activity, folding and / or isolation which are aided by the presence of one or more protein sequences in addition to the desired amino acid sequence. In such instances the presence of the protein additives may be required for the cell-based expression. Where additional protein additives are beneficial, these may optionally be co-expressed in the cell. The co-expression may use the same plasmid as the desired amino acid sequence. Alternatively the cells used for expression may contain genes for the expression of the additional proteins which aid expression, activity, folding and / or isolation of the desired amino acid sequence.

[0034] The additional protein additives may be for example one or more of chaperones, glycosylating enzymes, proteases, redox active enzymes, phosphorylases and kinases.

[0035] The proteases may be for example 3C or TEV proteases. The proteases may be present during the expression screen. The proteases may be expressed in the cells for scale-up, for example either using a single template vector with the protein of interest or where the cells contain genes for the expression of the protease.

[0036] The cells used for expression may be of different origin from the cells from the expression system is derived. The cells may be for example mammalian cells, insect cells, prokaryotic cells, yeast cells, plant cells or protozoa. The cells may be derived from human embryonic kidney cells (HEK293), Chinese hamster ovary cells (CHO), HeLa cells, BHK21 cells, NSO cells, Sp2 / 0 cells, Escherichia coli cells, Saccharomyces cerevisiae cells, Pichia pastoris cells, tobacco cells, wheat cells, or Leishmania tarentolae cells.

[0037] The screening method may identify the optimum conditions for the growth of cells, for example the best temperature or growth media in order to obtain the desired amino acid sequence in a correctly folded state. The growth media may include for example metal ions or co-factors for expression.

[0038] Disclosed is a kit comprising: i. a nucleic acid template coding sequence for a protein of interest; ii. a reconstituted cell-free protein expression system; and iii. a cell-line for expressing the nucleic acid template coding sequence for a protein of interest.

[0039] Disclosed is a kit comprising: i. a nucleic acid template coding sequence for a protein of interest; ii. a reconstituted cell-free protein expression system comprising a protease; iii. a cell-line for expressing the nucleic acid template coding sequence for a protein of interest and the protease.

[0040] Disclosed is a kit comprising: i. a nucleic acid template coding sequence for a protein of interest; ii. a reconstituted cell-free protein expression system comprising a chaperone; iii. a cell-line for expressing the nucleic acid template coding sequence for a protein of interest and the chaperone.

[0041] The kit may comprise a variety of nucleic acid template sequences coding for the same amino acid sequence, for example having varying flanking sequences and / or varying coding sequences; a variety of a reconstituted cell-free protein expression systems comprising tRNAs which are codon balanced with each of the different nucleic acid templates and a variety of cell-lines for expressing the nucleic acid template coding sequence for a protein of interest.

[0042] Disclosed is a kit comprising: i. a reconstituted cell-free protein expression system comprising tRNAs purified from a BL21 E. coli strain; ii. a nucleic template coding sequence for a protein of interest; and iii. competent BL21 E. coli cells.

[0043] Disclosed is a kit comprising: i. a nucleic acid template coding sequence for a protein of interest; ii. a reconstituted cell-free protein expression system comprising tRNAs which are codon balanced with the nucleic acid template; and iii. a cell line expressing tRNAs which are codon balanced with the nucleic acid template.

[0044] In each of the kits the template may be a circular template that is suitable for both cell-free protein expression and cell based expression.

[0045] Disclosed is a kit comprising a variety of nucleic acid template sequences coding for the same amino acid sequence and a variety of a reconstituted cell-free protein expression systems comprising distinct tRNAs pools which are codon balanced with each of the different nucleic acid templates.

[0046] Where the synthesis is performed on a microfluidic device, the mixture can be optimised by combining reagents on the device. For example concentrations of reagents that give optimal expression can be identified by blending components at different ratios in a large number of droplets and the expression monitored in parallel to identify optimal compositions. For example a variety of tRNA compositions can be used to express each of the one or more sequences.

[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] 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. FIGURES

[0049] Figure 1 : In-vitro / vivo comparison: pmoles of purified protein normalised by expression volume. The pmoles of protein purified per iL of input CFPS / culture. Note that yields from E. coli cells are plotted on the secondary y-axis. The x-axis indicates the protein of interest and the construct used.

[0050] Figure 2: Comparison of pmoles of Protein per Volume - Device vs Cells. Comparison of the pmoles of purified protein produced per iL from CFPS (on device) or E. coli culture (cells).

[0051] Figure 3: IFN-g(23-166) - On device and in vivo correlation.

[0052] Figure 4: in situ expression and detagging (cells). Co-expression with 3C Protease results in solubility tag cleavage. (A) Total and soluble gel of GATA3 (254-341), BTK (214-377), and IFN-g (23-166) expressed alone or co-expressed with 3C Protease compared to the no plasmid and uninduced controls. (B) Unbound and elution gel of GATA3 (254-341), BTK (214- 377), and IFN-g (23-166) expressed alone or co-expressed with 3C Protease compared to the no plasmid control.

[0053] Figure 5: Table summarising screening data through 264 constructs in 2112 cell-free expression conditions and selected 33 distinct high and low expressors scaled up in cells.

[0054] Figure 6: Chart of correlation data from 33 distinct high and low expressors scaled up in cells (plotted data from Figure 5) (note that in order to plot on a log scale values of zero in the table are shown as T on the axis on the plot).

[0055] DETAILED DESCRIPTION OF THE INVENTION

[0056] Disclosed herein is an improved lysate or reconstituted composition for the cell-free expression of proteins and correlation thereof with a cell-based expression system. The proteins may be eukaryotic proteins The eukaryotic proteins may be mammalian or human. Disclosed is a reconstituted cell-free protein expression system comprising tRNAs isolated from a mammalian codon tolerant or human codon tolerant E coli strain. The reconstituted systems requires assembly of purified components. The assembly requires purification of the enzymes and tRNA’s needed for protein expression, and their subsequent combination into a reagents suitable for protein expression. This is in contrast to standard protocol where the cells are simply lysed and the contents resuspended without isolation of the discreet components. Disclosed is a mixture comprising purified components from E. coli such as enzymes and ribosomes, supplemented with a tRNA population which is codon balanced with the template being expressed. Certain codons may be designed out of the template, meaning that not all of the 61 possible codons are needed to express the desired sequence of 20 amino acids. Where certain codons are reduced or omitted, the tRNAs are not required or can be reduced.

[0057] The tRNA molecules can be purified from sources that have the desired balance of tRNA, such as engineered expression strains, or can be produced synthetically using in-vitro transcription (IVT). The IVT process allows each tRNA to be obtained and a specific pool of tRNAs to be prepared in concordance with the codons being used in the nucleic acid expression template.

[0058] The term ‘reconstituted’ refers to a system where the components are purified and the system re-assembled from purified discreet components. In contrast, lysate based systems are prepared from lysed cells and used in the ratio of components produced by the cells. The lysed cells may be treated, for example with centrifugation in order to pellet cellular debris, but the soluble components are not otherwise purified or separated from each other.

[0059] The existing prior-art reconstituted reagents such as PUREFREX are prepared from E. coli, and are thus limited by the tRNA mixtures purified from standard E. coli expression. The tRNA codons are not appropriate for the expression of eukaryotic proteins, many of which contain codons rarely used in E. coli. Certain strains of E. coli such as the Rosetta strain of BL21 supplies tRNAs for AGG, AGA, AUA, CUA, CCC, GGA codons, which are all under- represented in standard E. coli expression. Thus the Rosetta strains provide for “universal” translation which is otherwise limited by the codon usage of E. coli.

[0060] In contrast to a crude lysate derived from Rosetta BL21 E. coli or a reconstituted system derived from E. coli, disclosed herein is a reconstituted cell-free protein expression system comprising tRNAs purified from a Rosetta BL21 E. coli strain. Rosetta host strains are BL21 derivatives designed to enhance the expression of eukaryotic proteins that contain codons rarely used in E. coli.

[0061] 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. 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, blending droplets to form a series of droplets having varying cell-free reagent compositions, including enzymes for protein synthesis and the template nucleic acid 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. 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.

[0065] 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.

[0066] 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.

[0067] 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.

[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 two 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. 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.

[0069] 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).

[0070] 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 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.

[0071] 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.

[0072] 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. Such components may be added to the growing media for subsequent cell-based growth and expression. The additional protein components may be involved in any post-translational modification process, for example methylation, ubiquitinylation, sumoylation, isoprenylation or glycosylation.

[0073] 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).

[0074] 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.

[0075] 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.

[0076] 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 signaling, autophagy and apoptosis.

[0077] 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.

[0078] 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.

[0079] 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.

[0080] 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.

[0081] 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.

[0082] 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.

[0083] 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.

[0084] 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.

[0085] The expressed protein may contain a variety of flanking tags to enhance solubility or purification. The expressed protein may contain a sequence acting as a solubility enhancer, for example selected from:

[0086] The expressed protein may contain a sequence acting as binding moiety for purification. The binding moiety for purification may contain four or more amino acids. The binding sequences may contain 4-30 amino acids. The binding moiety may be selected from:

[0087] Alfa-tag (SRLEEELRRRLTE)

[0088] Avi-tag (GLNDIFEAQKIEWHE)

[0089] C-tag (EPEA)

[0090] Calmodulin-tag (KRRWKKNFIAVSAANRFKKISSSGAL)

[0091] Dogtag (DIPATYEFTDGKHYITNEPIPPK)

[0092] E-tag (GAPVPYPDPLEPR)

[0093] FLAG (DYKDDDDK)

[0094] G4T (EELLSKNYHLENEVARLKK)

[0095] HA (YPYDVPDYA)

[0096] His (HHHHHH)

[0097] Isopeptag (TDKDMTITFTNKKDAE) lanthanide binding tag (LBT) (FIDTNNDGWIEGDELLLEEG)

[0098] Myc (EQKLISEEDL)

[0099] NE-Tag (TKENPRSNQEESYDDNES)

[0100] Poly Glutamate-tag (EEEEEEE)

[0101] Poly Arginine-tag (RRRRRRR)

[0102] Rho1 D4-tag (TETSQVAPA)

[0103] SBP-tag (MDEKTTGWRGGHVVEGLAGELEQLRARLEHHPQGQREP)

[0104] Sdytag (DPIVMIDNDKPIT)

[0105] SH3 (STVPVAPPRRRRG)

[0106] SNAC (GSHHW)

[0107] Snooptag (KLGDIEFIKVNK)

[0108] Softag 1 (SLAELLNAGLGGS)

[0109] Softag 3 (TQDPSRVG)

[0110] Spot-tag (PDRVRAVSHWSS)

[0111] Spytag (AHIVMVDAYKPTK)

[0112] S-tag (KETAAAKFERQHMDS)

[0113] Strep-tag (AWAHPQPGG) (AWRHPQFGG)

[0114] Strep-tag II (WSHPQFEK)

[0115] T7tag (MASMTGGQQMG)

[0116] TC-tag (EVHTNQDPLD)

[0117] Ty-tag (CCPGCC)

[0118] VSV-tag (YTDIEMNRLGK) Xpress-tag (DLYDDDDK)

[0119] 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.

[0120] By way of example, the screening reagents may include,

[0121] ■ Chaperone mix (e.g.,PUREfrex GrpE mix)

[0122] ■ Kinase 1 (e.g., NEB CK2)

[0123] ■ Kinase 2 (e.g., NEB PKA)

[0124] ■ Protease 1 (e.g., NEB TEV)

[0125] ■ Protease 2 (e.g., Merck HRV 3C)

[0126] ■ N-acetyl transferase

[0127] ■ Common metal ions cocktail

[0128] ■ Common co-factors cocktail

[0129] 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.

[0130] Metal ions may include one or more of the following: MgCh, CuCh, ZnCh, CaCh, MnCh, NiCh, CoCI2.

[0131] 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.

[0132] 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).

[0133] The additive may be for example one or more reducing agents. The additive may be selected from DTT, glutathione (GSH) or glutathione disulfide (GSSG).

[0134] The added protease may cleave the protein of interest from flanking regions. The flanking regions may 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. 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.

[0135] 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.

[0136] 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.

[0137] 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.

[0138] 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.

[0139] 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. 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.

[0140] 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.

[0141] 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. The same circular nucleic acid template can be used in both the cell-free expression and cell expression stages.

[0142] 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.

[0143] The cell-free system is assembled from the required reagents. E Coli derived systems based on reconstituted, purified molecular reagents are commercially available, for example the PURE system for protein production (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.

[0144] 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.

[0145] 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.

[0146] 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, the expression conditions optimally including screening compositions having varying tRNA populations. The compositions may include varying template nucleic acid sequences which express a desired amino acid sequence.

[0147] 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.

[0148] 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.

[0149] 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. 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.

[0150] 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 two 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.

[0151] 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.

[0152] 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.

[0153] 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).

[0154] The oil in the device can be any water immiscible or hydrophobic liquid. The oil can be mineral oil, silicone oil, an alkyl-based solvent such as decane or dodecane, or a fluorinated oil. The air in the device can be any humidified gas. 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.

[0155] Alternatively the oil can be oxygenated. Alternatively the droplets can be presented in a humidified air filled device.

[0156] 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.

[0157] 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).

[0158] 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.

[0159] 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.

[0160] 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.

[0161] 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 GFP11 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- .

[0162] 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.

[0163] Devices

[0164] 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.

[0165] 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.V2 where 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.

[0166] 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.

[0167] 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.

[0168] 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.

[0169] 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.

[0170] 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. 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.

[0171] 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.

[0172] 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.

[0173] 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 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.

[0174] 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.

[0175] 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.

[0176] 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.

[0177] 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.

[0178] The method is particularly suitable for aqueous droplets with a volume of 1 pL or smaller.

[0179] 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.

[0180] 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;

[0181] 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;

[0182] 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.

[0183] Cell-Based Expression

[0184] Following the screening assay, the best conditions for production of soluble proteins are identified. The selected conditions can then be used to produce the protein in a host cell to which the nucleic acid sequence to be expressed is added, for example as an expression vector or plasmid. The screening assay may identify conditions for protein synthesis, activity, folding and / or isolation which are aided by the presence of one or more protein sequences in addition to the desired amino acid sequence. In such instances the presence of the protein additives may be required for the cell-based expression. Where additional protein additives are beneficial, these may optionally be co-expressed in the cell. The co-expression may use the same plasmid as the desired amino acid sequence. Alternatively the cells used for expression may contain genes for the expression of the additional proteins which aid expression, activity, folding and / or isolation of the desired amino acid sequence. The additional protein additives may be for example one or more of chaperones, glycosylating enzymes, proteases, redox active enzymes, phosphorylases and kinases.

[0185] The cells used for expression may be of different origin from the cells from the reconstituted expression system is derived. The cells may be for example mammalian cells, insect cells, prokaryotic cells, yeast cells, plant cells or protozoa. The cells may be derived from human embryonic kidney cells (HEK293), Chinese hamster ovary cells (CHO), HeLa cells, BHK21 cells, NSO cells, Sp2 / 0 cells, Escherichia coli cells, Saccharomyces cerevisiae cells, Pichia pastoris cells, tobacco cells, wheat cells, or Leishmania tarentolae cells.

[0186] The screening method may identify the optimum conditions for the growth of cells, for example the best temperature or growth media in order to obtain the desired amino acid sequence in a correctly folded state.

[0187] Disclosed is a kit comprising: i. a circular nucleic acid template coding sequence for a protein of interest; ii. a reconstituted cell-free protein expression system; and iii. a cell-line for expressing the nucleic acid template coding sequence for a protein of interest.

[0188] Disclosed is a kit comprising: i. a circular nucleic acid template coding sequence for a protein of interest; ii. a reconstituted cell-free protein expression system comprising a protease; iii. a cell-line for expressing the nucleic acid template coding sequence for a protein of interest and the protease.

[0189] The kit may comprise a variety of nucleic acid template sequences coding for the same amino acid sequence, for example having varying flanking sequences and / or varying coding sequences; a variety of a reconstituted cell-free protein expression systems comprising tRNAs which are codon balanced with each of the different nucleic acid templates and a variety of cell-lines for expressing the nucleic acid template coding sequence for a protein of interest.

[0190] Disclosed is a kit comprising: i. a reconstituted cell-free protein expression system comprising tRNAs purified from a BL21 E. coli strain; ii. a nucleic template coding sequence for a protein of interest; and iii. competent BL21 E. coli cells. Disclosed is a kit comprising: i. a nucleic acid template coding sequence for a protein of interest; ii. a reconstituted cell-free protein expression system comprising tRNAs which are codon balanced with the nucleic acid template; and iii. a cell line expressing tRNAs which are codon balanced with the nucleic acid template.

[0191] Disclosed is a kit comprising a variety of nucleic acid template sequences coding for the same amino acid sequence and a variety of a reconstituted cell-free protein expression systems comprising distinct tRNAs pools which are codon balanced with each of the different nucleic acid templates.

[0192] Examples

[0193] Example 1 : Comparison of expression using cell-free conditions and cell based scale up.

[0194] Six proteins of interest (shown in Table 1) were expressed using the Nuclera eProtein Discovery system using 7 solubility tags and a no solubility tag construct (8 gene sequences per POI) and non supplemented cell-free expression blend. Each POI contains a detector tag (ccGFPn) and a purification tag (STREP). The predicted yield from the eProtein run for each POI was ranked and the top, bottom and intermediate recipe was selected as "exemplars". MLKL (190-471) Kinase domain

[0195] Table 1

[0196] Full length plasmids were synthesised for the three constructs (High, Int, Low) for each of the 6x POI (so 18 in total) and subcloned in pET41 (a+). These were sequence verified as perfect. The vector used was immediately compatible with BL21 cells. The plasmids were transformed into BL21 and 2 mL cultures were grown, induced, harvested and purified using strep magbeads on a robotic liquid handling system. The eluted yield was calculated by complementation with a ccGFPi-io detector protein. Comparison of the device CFPS and tube cells are shown in Figure 1 and 2. The pmoles of protein purified per iL of input CFPS / culture. Note that yields from E. coli cells are plotted on the secondary y-axis. The x-axis indicates the protein of interest and the construct used. As would be expected, the CFPS reaction generates a higher per iL protein yield than the E. coli culture (Figure 1). This is expected because CFPS is a more concentrated expression reaction than E. coli cells in culture medium. Between proteins there is a positive correlation between the two expression methods - i.e. the proteins with the highest purification yield on device (GATA and BTK) also have the highest purification yield from cells.

[0197] SDS PAGE gels of the Soluble, Flowthrough (Unbound) and Eluted protein are shown in Figure 3 for 2 replicates and a high and low expressor.

[0198] Example 2: Co-expression of 3C-protease and POI’s (in-situ cleavage of solubility tags).

[0199] BL21 (DE3) cells were transformed with a plasmids containing the target proteins alone or cotransformed with a plasmid containing NT*3C Protease. For the co-expression, cells were grown in TB media and the plasmids with the target proteins were induced with 0.2 mM IPTG and expressed overnight at 18 °C. The plasmids containing the NT*3C Protease were not induced as the basal expression was sufficient to cleave the target protein. After overnight expression the cells were pelleted and lysed with a chemical lysis buffer. Total and soluble fractions of the lysed cells were run on a gel. The proteins expressed + / - protease were purified using STREP magnetic beads from the soluble lysate. Unbound and elution samples were run on the gel (Figure 4). Figure 4 shows co-expression with 3C Protease results in solubility tag cleavage. (A) Total and soluble gel of GATA3 (254-341), BTK (214-377), and IFN-g (23-166) expressed alone or co-expressed with 3C Protease compared to the no plasmid and uninduced controls. (B) Unbound and elution gel of GATA3 (254-341), BTK (214-377), and IFN-g (23-166) expressed alone or co-expressed with 3C Protease compared to the no plasmid control. The in vivo co-expression of GATA3 (254-341), BTK (214-377), and IFN-g (23-166) with NT*3CP protease resulted in cleaved protein which is not present when the NT*3CP protease is not co-expressed.

[0200] Example 3: Correlating cell-free expression in an electrowetting cartridge with protein scale- up in E.coli.

[0201] 11 protein sequences were selected for evaluation:

[0202] BRCA1

[0203] CALM1

[0204] CD135 / FLT3

[0205] CD19

[0206] DHX36

[0207] HIF1A

[0208] IDH1

[0209] IKZF1

[0210] MAPK1

[0211] MGAT2

[0212] PARK7

[0213] Each protein was studied using 3 length variants. 33 variants amino acid sequences were therefore studied. Each of the 33 sequences was studied using one of 7 solubility tags or as a no tag variant. Thus 33 x 8 (264) different nucleic acid sequences were prepared for screening using Nuclera’s eProtein Discovery system. 8 different cell-free expression conditions were studied for each of the 264 constructs, giving a total of 2112 expression conditions. Based on the predicted cell-free expression yield, a selection of high and low expressors for cell-based scale up were selected. 33 constructs were evaluated in BL21 cells and SHuffle cells using the chosen additives where identified. The linear expression templates were ligated into suitable circular plasmids for cell-based expression and transformed into the cells. The expression data and yield from cell expression is shown in Figure 5 in tabular form and plotted in Figure 6 (note that in order to plot on a log scale values of zero in the table are shown as T on the axis on the plot). For Figure 6, the thresholds were selected as shown:

[0214] BL-21 : 10 mg / L purified yield in cells Cartridge: 85 mg / L purified yield on cartridge (equivalent to 85 pg of purified protein in 1 mL cell free expression)

[0215] Data shows that there is a high correlation (82%) between eProtein Discovery and E. coli cells, which can be further enhanced by providing the correct conditions for growing the cells. In some instances the proteins can be produced in cell-free mixtures, but fail in cells (2 instances). In some instances proteins can be produced in cells in higher than anticipated yields. However in many instances where the expression is low in cell-free blends, the results in cells are also low, hence enabling a rapid elimination of cell-culture conditions that would fail.

[0216] In certain instances proteins are expressed in SHuffle cells (SHuffle cells are competent E. coli cells suitable for T7 protein expression with enhanced capacity to correctly fold proteins with multiple disulfide bonds). SHuffle cells generally express at a lower level than BL 21 cells, however in several cases (seen in Figure 5 and shown below) expression is improved in SHuffle. The use of SHuffle may be indicated where cell-free expression is highest in the presence of redox agents such as GSSG or chaperones that fold proteins with disulfide bonds.

[0217] Using the cell-free additive composition which best expresses a particular protein allows selection of the cell type which best expresses the protein. Data from 4 proteins is shown in the table below:

[0218] Where the protein expresses best in compositions containing GSSG, this is suggestive that expression may benefit from a SHuffle type cell line, for example as seen in the first two rows. Where the protein doers not benefit from redox environments, and cell-free expression is highest for other combinations of reagents such as metal ions, there is no benefit in using SHuffle cells (lowest 2 rows). Thus the results obtained for examining the cell-free blends to best produce proteins can be used to scale up reactions in cells.

[0219] Example 4: Co-expression with chaperones to increase yield

[0220] Certain proteins benefit from the presence of chaperones for correct folding. Where the yield of cell-free expression is seen as being best in the presence of chaperones, this can only easily be translated into cells if the cells co-express the chaperone and the protein of interest. An example of co-expression of a protein of interest and the chaperone TRXB1 is shown in the table below:

[0221] The protein expresses optimally in the presence of TRXB1 in the cell-free system. Cell based expression in the absence of TRXbl gives no protein in either BL21 or SHuffle. When TRXB1 is co-expressed with the FH8 protein in BL21 , the FH8 protein can be obtained.

[0222] The disclosure includes the following embodiments:

[0223] 1 . A method of producing proteins comprising the steps of: a. taking nucleic sequences expressing the desired amino acid sequence; b. expressing the nucleic acid sequences in cell-free expression systems having varying compositions; c. identifying the nucleic sequence and conditions in which the desired protein is best expressed; d. based on the identified conditions, selecting a host-cell type, media composition and growing conditions in which to produce the protein; and e. producing the protein in said host-cell using the identified host cell type, media composition and growing conditions.

[0224] 2. The method according to embodiment 1 , wherein the cell-free expression systems are E. coli lysates or reconstituted E. coli cell-free protein expression systems and comprise isolated tRNAs added to the expression system, wherein the ratio of the tRNA’s is optimised for use in expression of eukaryotic proteins

[0225] 3. The method according to embodiment 2 which is optimised for use in the expression of human proteins.

[0226] 4. The method according to embodiment 2 or embodiment 3 which comprises tRNAs for AGG, AGA, AUA, CUA, CCC, GGA codons.

[0227] 5. The method according to any one of embodiments 2 to 4, wherein tRNA’s are derived from a codon supplemented BL21 strain.

[0228] 6. The method according to embodiment 5, wherein tRNA’s are derived from a Rosetta BL21 strain.

[0229] 7. The method according to any one of embodiments 2 to 4, wherein tRNA’s are synthesised using in-vitro transcription (IVT).

[0230] 8. The method according to any one of embodiments 1 to 7, wherein each reconstituted cell-free expression system is a reconstituted coupled system for transcription and translation.

[0231] 9. The method according to any one of embodiments 1 to 8, wherein each reconstituted cell-free expression system contains synthesized or isolated ribosomes, initiation factors, elongation factors, aminoacyl-tRNA synthetases, methionyl tRNA transformylases, tRNAs, amino acids and ribonucleoside triphosphates.

[0232] 10. The method according to any one of embodiments 1 to 9 comprising adding a template nucleic acid encoding a protein of interest to a reconstituted cell-free protein expression system comprising tRNAs which are codon balanced with the template sequence.

[0233] 11. Use of a method according to any one of embodiments 1 to 10 for the expression of eukaryotic or human proteins.

[0234] 12. The use according to embodiment 10, wherein the eukaryotic proteins require tRNAs rarely used in E. coli.

[0235] 13. The method or use according to any one of embodiments 1 to 12, wherein the cell-free protein expression is performed on an electrowetting-on-dielectric (EWoD) device.

[0236] 14. The method according to embodiment 13 comprising blending droplets to form a series of droplets having varying cell-free reagent compositions, including enzymes for protein synthesis and the template nucleic acid 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.

[0237] 15. The method or use according to any one of embodiments 1 to 14, wherein the identified conditions for protein synthesis, activity, folding and / or isolation are aided by the presence of one or more protein sequences in addition to the desired amino acid sequence.

[0238] 16. The method according to embodiment 15, wherein the additional proteins required are co-expressed in the cell.

[0239] 17. The method according to embodiment 15, wherein the additional proteins are expressed using the same plasmid as the desired amino acid sequence.

[0240] 18. The method according to any one of embodiments 15 to 17, wherein the additional proteins are one or more of chaperones, glycosylating enzymes, proteases, redox active enzymes, phosphorylases and kinases.

[0241] 19. The method according to any one of embodiments 1 to 18, wherein the cells used for expression are selected from mammalian cells, insect cells, prokaryotic cells, yeast cells, plant cells or protozoa.

[0242] 20. The method according to embodiment 19, wherein the cells are derived from human embryonic kidney cells (HEK293), Chinese hamster ovary cells (CHO), HeLa cells, BHK21 cells, NSO cells, Sp2 / 0 cells, Escherichia coli cells, Saccharomyces cerevisiae cells, Pichia pastoris cells, tobacco cells, wheat cells, or Leishmania tarentolae cells.

[0243] 21. The method according to any one of embodiments 15 to 20, wherein the cells contain genes for the expression of the additional proteins which aid expression, activity, folding and / or isolation of the desired amino acid sequence.

[0244] 22. The method according to any one of embodiments 1-21 which identify the optimum expression parameters for growth of cells in order to obtain the desired amino acid sequence in a correctly folded state.

[0245] 23. A kit comprising: i. a nucleic acid template coding sequence for a protein of interest; ii. a reconstituted cell-free protein expression system comprising tRNAs which are codon balanced with the nucleic acid template iii. a cell-line for expressing the nucleic acid template coding sequence for a protein of interest.

[0246] 24. The kit according to embodiment 23 comprising a variety of nucleic acid template sequences coding for the same amino acid sequence; a variety of a reconstituted cell- free protein expression systems comprising tRNAs which are codon balanced with each of the different nucleic acid templates and a variety of cell-lines for expressing the nucleic acid template coding sequence for a protein of interest.

Claims

Claims1. A method of producing proteins comprising the steps of: a. taking nucleic sequences expressing the desired amino acid sequence; b. expressing the nucleic acid sequences in cell-free expression systems having varying compositions; c. identifying the nucleic sequence and conditions in which the desired protein is best expressed; d. based on the identified conditions, selecting a host-cell type, media composition and growing conditions in which to produce the protein; and e. producing the protein in said host-cell using the identified host cell type, media composition and growing conditions.

2. The method according to claim 1 , wherein the identified expression conditions for protein synthesis, activity, folding and / or isolation are aided by the presence of one or more protein sequences in addition to the desired amino acid sequence.

3. The method according to claim 2, wherein the additional proteins are one or more of chaperones, glycosylating enzymes, proteases, redox active enzymes, phosphorylases and kinases which are present in the expression compositions.

4. The method according to claim 2 or claim 3, wherein the additional proteins required are co-expressed in the cell.

5. The method according to claim 1 , wherein the desired protein is expressed in the presence of a protease and / or chaperone.

6. The method according to claim 1, wherein the desired protein and the protease and / or chaperone are co-expressed in the cell.

7. The method according to claim 5 or claim 6, wherein the protease is 3C protease.

8. The method according to any one of claims 3 to 7, wherein the additional proteins are expressed using the same plasmid as the desired amino acid sequence.

9. The method according to any one of claims 1 to 8, wherein the cell-free expression systems are E. coli lysates or reconstituted E. coli cell-free protein expression systems.

10. The method according to any one of claims 1 to 9, wherein isolated tRNAs are added to the expression system, wherein the ratio of the tRNA’s is optimised for use in expression of eukaryotic proteins11. The method according to claim 10 which is optimised for use in the expression of human proteins.

12. The method according to claim 10 or claim 11 which comprises tRNAs for AGG, AGA, AUA, CUA, CCC, GGA codons.

13. The method according to any one of claims 10 to 12, wherein tRNA’s are synthesised using in-vitro transcription (IVT).

14. The method according to any one of claims 1 to 13, wherein each reconstituted cell- free expression system contains synthesized or isolated ribosomes, initiation factors, elongation factors, aminoacyl-tRNA synthetases, methionyl tRNA transformylases, tRNAs, amino acids and ribonucleoside triphosphates.

15. The method according to any one of claims 1 to 14 comprising adding a template nucleic acid encoding a protein of interest to a reconstituted cell-free protein expression system comprising tRNAs which are codon balanced with the template sequence.

16. Use of a method according to any one of claims 1 to 15 for the expression of eukaryotic or human proteins.

17. The use according to claim 16, wherein the eukaryotic proteins require tRNAs rarely used in E. coli.

18. The method or use according to any one of claims 1 to 17, wherein the cell-free protein expression is performed on an electrowetting-on-dielectric (EWoD) device.

19. The method according to claim 18 comprising using a series of droplets having varying cell-free reagent compositions, including enzymes for protein synthesis and the template nucleic acid 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.

20. The method according to any one of claims 1 to 19, wherein the cells used for expression are selected from mammalian cells, insect cells, prokaryotic cells, yeast cells, plant cells or protozoa.

21. The method according to claim 20, wherein the cells are derived from human embryonic kidney cells (HEK293), Chinese hamster ovary cells (CHO), HeLa cells, BHK21 cells, NSO cells, Sp2 / 0 cells, Escherichia coli cells, Saccharomyces cerevisiae cells, Pichia pastoris cells, tobacco cells, wheat cells, or Leishmania tarentolae cells.

22. The method according to any one of claims 2 to 21 , wherein the cells contain genes for the expression of the additional proteins which aid expression, activity, folding and / or isolation of the desired amino acid sequence.

23. A kit comprising: i. a nucleic acid template coding sequence for a protein of interest; ii. a reconstituted cell-free protein expression system comprising a protease; iii. a cell-line for expressing the nucleic acid template coding sequence for a protein of interest and the protease.

24. A kit comprising:iv. a nucleic acid template coding sequence for a protein of interest; v. a reconstituted cell-free protein expression system comprising a chaperone; vi. a cell-line for expressing the nucleic acid template coding sequence for a protein of interest and the chaperone.

25. The kit according to claim 23 or claim 24 comprising a variety of nucleic acid template sequences coding for the same amino acid sequence; a variety of a reconstituted cell- free protein expression systems comprising tRNAs which are codon balanced with each of the different nucleic acid templates and a variety of cell-lines for expressing the nucleic acid template coding sequence for a protein of interest.

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