Protein expression systems

Abstract

The invention provided herein relates to methods for cell-free protein synthesis and characterisation. Provided is an automated system for analysing expression and purification of proteins, each protein having detection tags for detection and binding tags for purification. The measurements of expression level and purification efficiency are determined in parallel by adding a detector reagent binding to the detection tag to different aliquots of the purification process (eluted material and residual liquid volumes).

Description

[0001] PROTEIN EXPRESSION SYSTEMS

[0002] FIELD OF THE INVENTION

[0003] Provided herein are methods for improved expression of proteins in cell-free systems and purification thereof. Provided is an automated system for analysing expression and purification of proteins.

[0004] BACKGROUND

[0005] Proteins are biological macromolecules that maintain the structural and functional integrity of the cell, and many diseases are associated with protein malfunction. Protein purification is a fundamental step for analysing individual proteins and protein complexes and identifying interactions with other proteins, DNA or RNA. A variety of protein purification strategies exist to address desired scale, throughput and downstream applications. However, protein production can be challenging for many reasons. One major challenge is finding a suitable expression system, for example sourced from mammalian, bacterial, fungal, or plant cells. This can take months of work.

[0006] Cell-free protein synthesis (CFPS), also known as coupled or uncoupled in-vitro transcription and translation, is the production of peptides or proteins using biological machinery in a cell-free system, that is, without the use of living cells. The CFPS 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;

[0007] 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] Split detectors such as split green fluorescent protein (GFP) systems are known for use in protein expression. A protein of interest having a GFP subcomponent can be detected by complementing with a detector species having the remainder of the GFP. Cabantous and Waldo describe such a system (In-vivo and in-vitro protein solubility assays using split GFP (NATURE METHODS | VOL.3 NO.10 | OCTOBER 2006 | 845). The system described relies on expression in cells, from which the proteins of interest are lysed and then exposed to the detector in order to measure the level of expression.

[0009] WO2022 / 038353 describes cell-free expression of a protein having a GFP tag in the presence of a GFP detector species to measure signal as expression progresses. The application does not describe protein purification or screening.

[0010] WO2023 / 021295 describes reagents for droplet manipulation and uses in protein expression, but does not describe protein purification or screening.

[0011] W02021 / 161048 describes cell-free protein synthesis in droplets on electrowetting devices. The purification of the expressed proteins is not described.

[0012] WO2022 / 111661 describes the use of magnetic microspheres for binding.

[0013] Sullivan; Biotechnol J. vol 11 2015 pp238-248 describes a cell-free expression and purification process for rapid production of protein biologies.

[0014] US2015 / 099271 describes split fluorescent proteins and uses thereof.

[0015] Listwan; J Struct. Funct. Genomics vol 102009 pp47-55 describes a high-throughput platform for protein solubility screening using a split-GFP system.

[0016] Nguyen; Scientific Rep vol 11 2021 pp1-15 describes the preparation of the fluorescent proteins from Corynactis Californica (ccGFP).

[0017] Many proteins are expressed containing endogenous solubility factors. However factors such as for example maltose binding protein (MBP), Small Ubiquitin-like Modifier (SUMO), Glutathione S-transferase (GST) or thioredoxin (TRX) have a substantial size and may have undesirable effects on the proteins of interest to which they are attached. Additionally expression of large solubility elements uses the reagent resources during expression. Such sequences also take time to fold before acting at solubility enhancers, hence synthesising proteins of interest with solubility enhancing sequences attached thereto is far from ideal. To date protein purification and analysis typically requires complex analysis techniques involving electrophoresis or requiring purified proteins. The inventors herein have developed simplified protein analysis and purification methods allowing multiple ways of characterising expressed proteins in their crude form.

[0018] One of the current challenges for cell-free protein synthesis is to increase the soluble yield of the expressed and purified proteins and to avoid aggregation or insolubility.

[0019] WO2024208868 describes a workflow where only a subset of the expression conditions are selected for purification. The applicants herein have appreciated that it is desirable to measure both the level of expression and purification for all expression conditions rather than choosing a subset of the expressed proteins to purify.

[0020] SUMMARY OF THE INVENTION

[0021] Disclosed is a method that relies on parallel handling of volumes of fluid. Disclosed is a method for synthesising, purifying and characterising multiple expressed proteins, each protein having detection tags and binding tags. The invention relates to the ability to measure both protein expression level and purification efficiency in a single process. The measurements of expression level and purification efficiency are determined in parallel by adding a detector reagent to different aliquots of the reaction process. Rather than selecting a subset of proteins based on expression levels, all expressed conditions can be purified in parallel and the expression and purification levels measured by comparing the results as shown below:

[0022] Total expressed protein = protein immobilised and eluted after purification + protein not immobilised and remaining in solution

[0023] Purification efficiency = protein immobilised and eluted after purification I (protein immobilised and eluted after purification + protein not immobilised and remaining in solution)

[0024] In some instances proteins may remain immobilised or become stuck to the solid support, in which case:

[0025] Total expressed protein = protein immobilised and eluted after purification + protein not immobilised and remaining in solution + protein remaining immobilised Purification efficiency = protein immobilised and eluted after purification I (protein immobilised and eluted after purification + protein not immobilised and remaining in solution + protein remaining immobilised)

[0026] Alternatively, the measurements of expression level and purification efficiency can be determined by quantifying purification levels first and adding further aliquots of the reaction process subsequently. While slower than the method described above, this has the benefit of reducing the amount of required detection reagent. Two or more quantitation steps can be performed by adding additional aliquots to the detector species after a first measurement has been taken and measuring the changes in the same population of detector (normalised for an increase in volume).

[0027] The process described herein can be used to measure the expression level and purification efficiency by measuring the two or three distinct liquid volumes in parallel or sequentially and accounting for the level of protein appearing at each stage of the process.

[0028] Described herein is a method for synthesising, purifying and characterising a plurality of proteins having detection tags and binding tags, the method comprising the steps of:

[0029] i. mixing a protein expression system with one or more nucleic acid templates to form a plurality of merged liquid volumes capable of expressing different proteins; ii. allowing protein expression in the liquid volumes;

[0030] iii. separating the expressed proteins from the expression reagents in each of the plurality of proteins using the binding tags and a solid support which binds to the binding tags to form discreet liquid volumes having the solid supports and residual liquid volumes without the solid supports;

[0031] iv. optionally washing the solid supports;

[0032] v. eluting any expressed and optionally washed protein from the solid supports by disrupting the binding to the binding tags to form eluted material;

[0033] vi. adding a detector reagent which binds to the detection tags to either:

[0034] a. both the aliquots of eluted material and the residual liquid volumes without the solid supports; or

[0035] b. the eluted material, wherein the residual liquid volumes are added to the mix of detector reagent and eluted material after the level of eluted protein has been measured; in order to determine the level of protein expression by measuring the total amount of protein across each of the two aliquots, and the efficiency of immobilization and elution by determining the ratio of the two aliquots, thereby determining the conditions where protein is both expressed and purified.

[0036] Described herein is a method for synthesising, purifying and characterising a plurality of proteins having detection tags and binding tags, the method comprising the steps of:

[0037] i. mixing a protein expression system with one or more nucleic acid templates to form a plurality of merged liquid volumes capable of expressing different proteins; ii. allowing protein expression in the liquid volumes;

[0038] iii. separating the expressed proteins from the expression reagents in each of the plurality of proteins using the binding tags and a solid support which binds to the binding tags to form discreet liquid volumes having the solid supports and residual liquid volumes without the solid supports;

[0039] iv. optionally washing the solid supports;

[0040] v. eluting any expressed and optionally washed protein from the solid supports by disrupting the binding to the binding tags to form eluted material;

[0041] vi. adding a detector reagent which binds to the detection tags to both the aliquots of eluted material and the residual liquid volumes without the solid supports in order to determine the level of protein expression by measuring the total amount of protein across each of the two aliquots, and the efficiency of immobilization and elution by determining the ratio of the two aliquots, thereby determining the conditions where protein is both expressed and purified.

[0042] Described herein is a method for synthesising, purifying and characterising a plurality of proteins having detection tags and binding tags, the method comprising the steps of:

[0043] i. mixing a protein expression system with one or more nucleic acid templates to form a plurality of merged liquid volumes capable of expressing different proteins; ii. allowing protein expression in the liquid volumes;

[0044] iii. separating the expressed proteins from the expression reagents in each of the plurality of proteins using the binding tags and a solid support which binds to the binding tags to form discreet liquid volumes having the solid supports and residual liquid volumes without the solid supports;

[0045] iv. optionally washing the solid supports; v. eluting any expressed and optionally washed protein from the solid supports by disrupting the binding to the binding tags to form eluted material;

[0046] vi. adding a detector reagent which binds to the detection tags to the eluted material, to measure the amount of purified and eluted protein and subsequently adding the residual liquid volume to the mix of detector reagent and eluted material after the level of eluted protein has been measured in order to determine the level of protein expression by measuring the total amount of protein across each of the two aliquots, and the efficiency of immobilization and elution by determining the ratio of the two aliquots, thereby determining the conditions where protein is both expressed and purified.

[0047] In order to measure the level of residual immobilised material, the detector reagent may also be added to the solid supports after elution to measure the level of residual uneluted protein remaining on the solid supports.

[0048] In order to measure the level of residual immobilised / uneluted material, the remaining solid supports may be merged with the detector reagent after measurement of the eluted material and residual liquid volume to measure the level of residual uneluted protein remaining on the solid supports.

[0049] The solid supports may be in the form of beads. For example magnetic or paramagnetic beads. Alternatively the solid supports may be immobilised hydrogel patches. Alternatively the solid supports may be discreet locations on the surface of the device over which the liquids are moved.

[0050] Where the solid supports are beads, these may be present during the expression step, and therefore capture proteins as the binding tags are expressed. Alternatively the beads may be added after the protein expression has occurred.

[0051] The expression may be performed in cells. The cells may be transfected on the device prior to expression. The expression may be performed using cell-free protein synthesis. The cell-free protein synthesis can use cell-free lysates or may be performed using assembled components for transcription and translation in a system of purified recombinant elements (PURE). The expression may be performed for a sufficiently long period of time to allow levels of expression to occur before separating the expressed proteins from the expression reagents. The expression may be performed for at least 3 hours before separating the expressed proteins from the expression reagents.

[0052] Any method described herein can be performed in for example microtitre plates or microcentrifuge tubes. Any method may be performed on a microfluidic or digital microfluidic device. The digital microfluidic device may comprise an oil-filled or humidified gaseous environment, wherein the humidified gaseous environment is achieved by enclosing or sealing the digital microfluidic device and providing on-board reagent reservoirs.

[0053] The method may be performed in droplets on a digital microfluidic device, which may comprise active-matrix thin-film transistors. The digital microfluidic device may comprise an oil-filled or humidified gaseous environment, wherein the humidified gaseous environment is achieved by enclosing or sealing the digital microfluidic device and providing on-board reagent reservoirs.

[0054] In order to immobilise the expressed material, a binding tag is expressed in the protein sequence. 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:

[0055] Alfa-tag (SRLEEELRRRLTE)

[0056] Avi-tag (GLNDIFEAQKIEWHE)

[0057] C-tag (EPEA)

[0058] Calmodulin-tag (KRRWKKNFIAVSAANRFKKISSSGAL)

[0059] Dogtag (DIPATYEFTDGKHYITNEPIPPK)

[0060] E-tag (GAPVPYPDPLEPR)

[0061] FLAG (DYKDDDDK)

[0062] G4T (EELLSKNYHLENEVARLKK)

[0063] HA (YPYDVPDYA)

[0064] His (HHHHHH)

[0065] Isopeptag (TDKDMTITFTNKKDAE)

[0066] lanthanide binding tag (LBT) (FIDTNNDGWIEGDELLLEEG)

[0067] Myc (EQKLISEEDL)

[0068] NE-Tag (TKENPRSNQEESYDDNES) Poly Glutamate-tag (EEEEEEE)

[0069] Poly Arginine-tag (RRRRRRR)

[0070] Rho1D4-tag (TETSQVAPA)

[0071] SBP-tag (MDEKTTGWRGGHVVEGLAGELEQLRARLEHHPQGQREP)

[0072] Sdytag (DPIVMIDNDKPIT)

[0073] SH3 (STVPVAPPRRRRG)

[0074] SNAG (GSHHW)

[0075] Snooptag (KLGDIEFIKVNK)

[0076] Softag 1 (SLAELLNAGLGGS)

[0077] Softag 3 (TQDPSRVG)

[0078] Spot-tag (PDRVRAVSHWSS)

[0079] Spytag (AHIVMVDAYKPTK)

[0080] S-tag (KETAAAKFERQHMDS)

[0081] Strep-tag (AWAHPQPGG) (AWRHPQFGG)

[0082] Strep-tag II (WSHPQFEK)

[0083] T7tag (MASMTGGQQMG)

[0084] TC-tag (EVHTNQDPLD)

[0085] Ty-tag (CCPGCC)

[0086] VSV-tag (YTDIEMNRLGK)

[0087] Xpress-tag (DLYDDDDK).

[0088] The binding tag may be Strep-tag II (WSHPQFEK) or poly His. Suitable solid supports can be used with bind to the binding tag.

[0089] The detector may take the form of a split fluorescent protein. The detection tag may be one component of a fluorescent protein and the detector reagent 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 tag may be GFPn and the detector GFP1-10. The tag may be one component of sfCherry. The tag may be sfCherryn and the detector sfCherry-i-io. The tag may be CFASTn or CFAST10 and the detector NFAST in the presence of a hydroxybenzylidene rhodanine analog.

[0090] The tag may be ccGFPn and the detector ccGFPi.10. The complementary GFP peptide amino acid sequence could be the following:

[0091] 1. KRDHMVLLEFVTAAGITGT

[0092] 2. KRDHMVLHEFVTAAGITGT

[0093] 3. KRDHMVLHESVNAAGIT

[0094] 4. RDHMVLHEYVNAAGIT

[0095] 5. GDAVQIQEHAVAKYFTV

[0096] 6. GDTVQLQEHAVAKYFTV

[0097] 7. GETIQLQEHAVAKYFTE

[0098] or a truncated version thereof. Truncations may involve a shortening of up to 5 amino acids from the N terminus, the C terminus or a combination thereof.

[0099] GFPn or GFP-i-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.

[0100] Also disclosed are nucleic acid sequences forexpressing particular tags. Nucleic acid sequences include

[0101] 5’GGTGATACCGTTCAGCTGCAAGAACATGCAGTTGCAAAATACTTTACCGTG

[0102] 5’GGTGAAACCATCCAGTTACAAGAACACGCCGTGGCCAAATATTTCACCGAA

[0103] or a truncated version thereof.

[0104] These sequences may be repeated one or more times to produce a protein having multiple GFPn domains.

[0105] For example, the sfCherry-1.10 polypeptide amino acid sequence could be:

[0106] MEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGHPYEGTQTAKLKVTKGGPLPFAWDILS PQFMYGSKAYVKHPADIPDYLKLSFPEGFTWERVMNFEDGGWTVTQDSSLQDGEFIYKVKLL GTNFPSDGPVMQKKTMGWEASTERMYPEDGALKGEINQRLKLKDGGHYDAEVKTTYKAKKP VQLPGAYNVDIKLDITSHNED The complementary sfCherryl 1 peptide amino acid sequence could be:

[0107] YTIVEQYERAEGRHSTGG

[0108] sfCherryl 1 or sfCherry-1.10 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.

[0109] For example, the NFAST polypeptide amino acid sequence could be:

[0110] MEHVAFGSEDIENTLAKMDDGQLDGLAFGAIQLDGDGNILQYNAAEGDITGRDPKQVIGKNFFK DVAPGTDSPEFYGKFKEGVASGNLNTMFEWMIPTSRGPTKVKVHMKKALS

[0111] The complementary CFASTn peptide amino acid sequence could be:

[0112] GDSYWVFVKRV

[0113] Or the complementary CFAST10 peptide amino acid sequence could be:

[0114] GDSYWVFVKR

[0115] NFAST, CFASTn, and / or CFAST10 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.

[0116] In order to improve solubility, the protein may be expressed as a fusion with a solubility enhancer. The solubility enhancer may be selected from:

[0117]

[0118]

[0119]

[0120] If desired the tag may be cleaved from the POI in order to remove the tag / detector. The cleavage may be performed for example using a protease.

[0121] The method may comprise expressing one or more proteins in one or more droplets on a digital microfluidic device having a two-dimensional array of planar microelectrodes, wherein the proteins have a ccGFPn peptide amino sequence tag and a Strep-tag, adding beads to the droplets to bind the Strep-tagged proteins, separating the beads from the non immobilised material and retaining the non-immobilised liquid, washing the beads, eluting the protein from the beads and adding a ccGFPi.-io detector reagent which binds to the ccGFPn detection tags to both the aliquots of eluted material and the residual liquid volumes from the non immobilised material in order to determine the level of protein expression by measuring the total amount of protein across each of the two aliquots, and the efficiency of immobilization and elution by determining the ratio of the two aliquots, thereby determining the conditions where protein is both expressed and purified.

[0122] Multiple nucleic acid templates and expression and purification conditions can be screened in parallel. For example at least 8 different nucleic acid templates can be screened against at least 4 different expression reagents on the same device. The device can separately manipulate a large number or droplets, for example at least 96 droplets or at least 192 droplets.

[0123] The device may be capable of immobilising magnetic beads such that the aqueous liquid volume can be moved away from where the beads are immobilised. For example the device may be able to manipulate magnetic beads in at least 32 locations. The device may be able to use the magnetic locations multiple times in order purify a larger number or droplets. For example the device can manipulate magnetic beads in at least 32 locations and each location is used at least twice in order to analyse at least 64 droplets. Each location may be used at least 3 times to purify at least 96 droplets.

[0124] The expression and purification screen can identify the optimal conditions for expression and purification of the desired protein in its most soluble and stable form.

[0125] FIGURES

[0126] Figure 1 shows a protein of interest with a peptide tag (detection tag) in the presence of a protein that binds to the peptide tag (detector protein). In the case that the detection tag and detector protein generate a detectable signal upon binding, then the quantity of the protein of interest can be measured.

[0127] Figure 2 shows a schematic workflow for protein expression. DNA expression constructs (ECon) can be mixed with expression reagents and merged. Protein expression progresses and beads added to the droplet to bind the expressed protein. The beads and liquid are separated. The residual unbound protein liquid is retained after removal of the beads. The beads may be washed, removing any residual undesired carry over from the solid supports. The bound material is eluted to give free protein. Detector reagent is added to both the residual protein liquids after bead removal and the ratio of bound and eluted protein to residual unbound protein signal is determined. Figure 3 shows a further schematic workflow for protein expression. DNA expression constructs (ECon) can be mixed with expression reagents and merged. Protein expression progresses and beads added to the droplet to bind the expressed protein. The beads and liquid are separated. The residual unbound protein liquid is retained after removal of the beads. The beads may be washed, removing any residual undesired carry over from the solid supports. The bound material is eluted to give free protein. Detector reagent is added to both the residual protein liquids after bead removal and added to the beads after elution to confirm if material remains on the beads. The ratio of bound and eluted protein to residual unbound protein signal and bound protein is determined.

[0128] Figure 4 shows a further schematic workflow for protein expression. DNA expression constructs (ECon) can be mixed with expression reagents and merged. Protein expression progresses and beads added to the droplet to bind the expressed protein. The beads and liquid are separated. The residual unbound protein liquid is retained after removal of the beads. The beads may be washed, removing any residual undesired carry over from the solid supports. The bound material is eluted to give free protein. Detector reagent is added to quantify the level of soluble purified protein. To measure the level of residual unbound protein signal the droplet can be subsequently merged after the first measurement in order to bind to the remaining detector. A second reading of the detector signal can be taken thus giving a total for soluble purified protein plus residual unbound protein.

[0129] Figure 5 shows a series of images using a purification zone multiple times. Each of the 32 magnetic zones remove the liquid from three droplets. The liquid is retained. The beads are washed and the bound material eluted. Thus 96 protein expression conditions can be analysed using 32 magnetic zones. The images show:

[0130] 1: Mixing 96 (8*12) DNA econstructs with 96 expression reagents.

[0131] 2: 96 sets of reagents blends next to 96 bead droplets

[0132] 3: 96 droplets having beads bound to proteins plus 96 droplets of wash buffer

[0133] 4: Each magnet position is used to strip sequentially 3 bead droplets. Each residual protein (dotted rectangle) is separated and retained. The beads are pinned, liquid removed, and resuspended with wash buffer (shaded rectangles) and moved away from the magnet zone. A fresh bead droplet is moved into the magnetic zone, stripped and resuspended with wash buffer. Thus by 4g, three droplets having resuspended beads in wash buffer are obtained, along with three residual protein droplets containing the residual expression reagents.

[0134] 5: 96 droplets in wash buffer and 96 droplets of elution buffer.

[0135] 6: Each magnet position is used to trap and elute sequentially 3 bead droplets. Each bound protein (diagonally shaded rectangle) is sequentially pinned, wash buffer removed and resuspended in elution buffer (horizontally shaded rectangle). After elution, the beads and elution buffer are separated and the eluate is retained for quantification. The beads are resuspended in the wash buffer and may also be retained (6a-d). The same process is then repeated for the second (6e) and third droplet. Thus by the end of the process, three droplets having eluted protein in elution buffer are obtained, along with eluted beads.

[0136] 7: 96 eluted proteins plus 96 droplets of detector moiety

[0137] 8: 96 droplets for protein quantitation plus 96 droplets of residual protein.

[0138] The total level of purification is measured after the components of step 7 have been mixed together and incubated. The total level of expression is measured after the components of step 8 have been mixed together and incubated.

[0139] Figure 6 shows total expressed, strep unbound, and strep elution yields for an expressed protein.

[0140] DETAILED DESCRIPTION OF THE INVENTION

[0141] The applicants have appreciated that the purification of protein sequences is not predictable and requires screening. Just because a protein is expressed, does not mean it can be obtained in a soluble purified form. The level of aggregation following protein expression is an important metric in determining whether a protein can be subsequently purified. A high level of protein aggregation indicates a lack of soluble protein for purification. Expressed protein may form a folded or aggregated structure such that the purification tags are within the structure, meaning purification yields are low. Proteins may bind to a solid support but then not elute and remains immobilised. The inventors have developed systems and methods that can monitor both expression levels and purification efficiency without having to artificially select a subset of the expression systems to undergo purification.

[0142] Disclosed is a method that relies on parallel handling of volumes of fluid. Described herein is a method for synthesising, purifying and characterising a plurality of proteins having detection tags and binding tags, the method comprising the steps of:

[0143] i. mixing a protein expression system with one or more nucleic acid templates to form a plurality of merged liquid volumes capable of expressing different proteins; ii. allowing protein expression in the liquid volumes;

[0144] iii. separating the expressed proteins from the expression reagents in each of the plurality of proteins using the binding tags and a solid support which binds to the binding tags to form discreet liquid volumes having the solid supports and residual liquid volumes without the solid supports;

[0145] iv. optionally washing the solid supports;

[0146] v. eluting any expressed and optionally washed protein from the solid supports by disrupting the binding to the binding tags to form eluted material;

[0147] vi. adding a detector reagent which binds to the detection tags to both the aliquots of eluted material and the residual liquid volumes without the solid supports in order to determine the level of protein expression by measuring the total amount of protein across each of the two aliquots, and the efficiency of immobilization and elution by determining the ratio of the two aliquots, thereby determining the conditions where protein is both expressed and purified.

[0148] In order to measure the level of residual immobilised material, the detector reagent may also be added to the solid supports after elution to measure the level of residual uneluted protein remaining on the solid supports.

[0149] When expressing proteins, various parameters are important for determining successful expression, both in terms of yield and function. One of the factors is the soluble yield of expressed proteins, as many proteins express in forms that are insoluble or become inactive through a lack of stability.

[0150] The assays for soluble yield described herein involve expression and detection of the expressed proteins to measure the degree of aggregation. The protein of interest (POI) may have a detection tag which binds to a detector to generate a signal. The detector are / or POI may have a further solubility factor. The method may be used as part of a process to determine whether an expressed protein is suitable for subsequent scale up reactions. For example the method may involve expressing a protein of interest (POI) in a first reagent volume and adding a solid support such as a bead. The reagent volume can be split in order to separate the solid support and produce liquid volumes with and without the solid supports. The solid supports may be washed, and the material eluted. The residual beads may be retained. The two liquid portions after bead removal (or three where beads are studied) may be merged with a detector moiety. The protein of interest may have a detector tag which complements a detector species and becomes fluorescent. The signal recorded and analysed as shown below:

[0151] Total expressed protein = protein immobilised and eluted after purification + protein not immobilised and remaining in solution

[0152] Purification efficiency = protein immobilised and eluted after purification I (protein immobilised and eluted after purification + protein not immobilised and remaining in solution)

[0153] In some instances proteins may remain immobilised or become stuck to the solid support, in which case:

[0154] Total expressed protein = protein immobilised and eluted after purification + protein not immobilised and remaining in solution + protein remaining immobilised

[0155] Purification efficiency = protein immobilised and eluted after purification I (protein immobilised and eluted after purification + protein not immobilised and remaining in solution + protein remaining immobilised)

[0156] The process described herein can be used to measure the expression level and purification efficiency by measuring the two or three distinct liquid volumes in parallel or sequentially and accounting for the level of protein appearing at each stage of the process.

[0157] In addition to determining overall signal, the uniformity of the signals within the droplets can be determined when the detector species is added to one or more of the aliquots and the signal recorded. The uniformity of the signal in the droplet determines the degree of aggregation. Where the signal is homogenous across the droplet, the POI is soluble, with little or no aggregation. Where the signal is non-homogenous across the droplet, seen in the form of clumps, the protein is aggregated and insoluble. Thus the level of overall signal indicates the level of expression and the signal uniformity determines aggregation. Thus the level of expression, and the level of soluble expression can be determined from looking at the amount of, and uniformity of the signal within the droplet. The degree of aggregation may be measured by using a measurement of uniformity, counting the number of aggregates, the area of the aggregates, the intensity of the aggregates or by using a measurement of dispersion.

[0158] Protein expression may give rise to aggregation if the proteins are unstable or poorly soluble. Protein aggregates give rise to a lack of homogeneous signal from the detector. Thus POIs having a high soluble yield give a uniform level of signal, proteins having a low soluble yield may have either a low or high level of signal, but the signal aggregates and is not homogenous throughout the reaction volume as the proteins are aggregated.

[0159] Assays for protein stability and potential for further purification may be performed on the device. Further assays for protein stability may include

[0160] • Solubility

[0161] • Aggregation (over time)

[0162] • Thermal Stability

[0163] Protein thermal stability does not necessarily predict aggregation rate at low temperature (and vice versa). For example, the melting temperature (Tm) of a monoclonal antibody does not necessarily correlate to the aggregation rate of said monoclonal antibody at room temperature (Mol. Pharmaceutics 2016, 13, 307-319). At high temperatures, the aggregation rate is dependent on the unfolded state whereas at low temperature, the aggregation rate is dependent on the native state. Thus denaturing a protein may or may not give an accurate measure of stability for the protein in its native state.

[0164] Providing a protein aggregation metric over time would be highly useful and present additional information in addition to simply a measurement of the expressed yield. Producing stable protein formulations is key to the development and manufacturing of proteins. The robustness of the formulations against external stress factors is crucial as proteins may be exposed to various types of stress such as temperature, pH, salts, mechanical stress, surface interaction or oxidation. Protein aggregation is a common issue faced during expression. Therefore, the prediction or control of protein aggregation during the production of a protein of interest is much needed. Described herein is a high throughput assay for protein aggregation / stability which measures the protein stability using the directly expressed and / or purified and eluted material.

[0165] The method for measuring stability avoids the need for complex purification steps or gel based separations prior to performing the assay. A fluorescence based assay can be used to measure the amount of expressed protein (soluble yield) in conjunction measuring protein stability. Thus both yield and stability can be determined after expression without needing to run gels or purify material.

[0166] This screening workflow enables users to rapidly screen different expression systems in the form cell-free lysates, or reconstituted systems. Having identified an optimal expression system, soluble yield, stability and purification conditions can be measured directly on the same device.

[0167] The protein can be expressed with a binding tag. The binding moiety can be a region of amino acid / peptide sequence. The affinity binding site can be a region of amino acid / peptide sequences specific to a particular antibody. The binding tag can be the Fc region of an antibody. The tag can be attached to the N or C terminus. For example the binding moiety can be selected from the list of exemplary peptide affinity binding sites below:

[0168] Alfa-tag (SRLEEELRRRLTE)

[0169] Avi-tag (GLNDIFEAQKIEWHE)

[0170] C-tag (EPEA)

[0171] Calmodulin-tag (KRRWKKNFIAVSAANRFKKISSSGAL)

[0172] Dogtag (DIPATYEFTDGKHYITNEPIPPK)

[0173] E-tag (GAPVPYPDPLEPR)

[0174] FLAG (DYKDDDDK)

[0175] G4T (EELLSKNYHLENEVARLKK)

[0176] HA (YPYDVPDYA)

[0177] His (HHHHHH)

[0178] Isopeptag (TDKDMTITFTNKKDAE)

[0179] lanthanide binding tag (LBT) (FIDTNNDGWIEGDELLLEEG)

[0180] Myc (EQKLISEEDL)

[0181] NE-Tag (TKENPRSNQEESYDDNES)

[0182] Poly Glutamate-tag (EEEEEEE) Poly Arginine-tag (RRRRRRR)

[0183] Rho1D4-tag (TETSQVAPA)

[0184] SBP-tag (MDEKTTGWRGGHVVEGLAGELEQLRARLEHHPQGQREP)

[0185] Sdytag (DPIVMIDNDKPIT)

[0186] SH3 (STVPVAPPRRRRG)

[0187] Snooptag (KLGDIEFIKVNK)

[0188] Softag 1 (SLAELLNAGLGGS)

[0189] Softag 3 (TQDPSRVG)

[0190] Spot-tag (PDRVRAVSHWSS)

[0191] Spytag (AHIVMVDAYKPTK)

[0192] S-tag (KETAAAKFERQHMDS)

[0193] Strep-tag (AWAHPQPGG) (AWRHPQFGG)

[0194] Strep-tag II (WSHPQFEK)

[0195] T7tag (MASMTGGQQMG)

[0196] TC-tag (EVHTNQDPLD)

[0197] Ty-tag (CCPGCC)

[0198] VSV-tag (YTDIEMNRLGK)

[0199] Xpress-tag (DLYDDDDK)

[0200] The binding moiety may include a small molecule affinity tag such as biotin. The binding moiety may include a particular sequence of nucleic acids.

[0201] The solid support which binds to the binding tags may be in the form of beads, for example magnetic beads. The solid support which binds to the binding tags may take the form of discreet locations on the surface of the device over which the droplets are moved.

[0202] The solid support which binds to the binding tags may be present during the expression process such that the expressed protein is bound, either as it is it expressed or immediately after expression. Alternatively the solid support may be added, or the droplet moved, after completion of the expression process. For example 192 expression conditions may be studied. Where the droplets contain beads, the beads may be immobilised and the liquid moved to separate the beads from the liquid. After the beads are separated and optionally washed, the immobilised material is eluted and the detector added to the eluate to enable the amount of eluted material to be measured. Washing removes any remaining CFPS reagents, and addition of the detector enables the amount of eluted material to be measured.

[0203] The expressed amino acid sequences can be bound to beads having an affinity to the tag. Suitable beads may be magnetic or paramagnetic beads. The beads may have for example metal ions to chelate to poly His or a streptavidin or modified streptavidin to bind to the strep tag. The beads may be of a size that can be handled within a droplet, for example 1-50 pM in average diameter. The beads may have a magnetic core and a polymer coating. The beads may be for example silica or agarose. Suitable beads are commercially available.

[0204] The solid supports / beads may contain for example protein A or protein G. Protein A and Protein G are bacterial proteins that primarily bind to the Fc (Fragment crystallizable) region of antibodies, particularly the Immunoglobulin G (IgG) class. Thus the expressed binding tag (Fc) of the antibody can be bound to the suitable solid support / beads. Disclosed herein is a method comprising expressing one or more antibodies having a binding Fc region. The expressed protein may be purified using affinity purification by binding to the FC region binding tag, for example using protein A or protein G.

[0205] The beads can be merged into droplets on the device. When exposed to a magnetic field, the beads can be immobilised whilst the droplets can be moved or split. Thus the beads can be removed from the unbound reagents. Washing steps can be performed by adding further reagents, agitating the beads in the droplets, immobilsing the beads and removing the droplet.

[0206] The binding can be reversed using suitable buffers, for example containing biotin to release a strep tag or imidazole to release poly-his.

[0207] The process can be performed in droplets, which can be manipulated by electrokinesis in order to effect and improve protein expression and analysis. 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.

[0208] Expression can be performed in cells or cell-free systems. 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.

[0209] After the nucleic acid template and cells are merged, the cells may be transfected to allow the nucleic acid to enter the cells. Transfection is the process of introducing foreign DNA into eukaryotic cells. Various methods can be used, each with its own advantages and applications. The methods can be broadly categorized into physical, chemical, and biological techniques, and may include for example electroporation, chemical methods such as calcium phosphate, lipofection, polymer based transfection using polymers such as polyethylenimine (PEI) or physical methods such as using magnets where DNA is attached to magnetic nanoparticles and guided into cells using a magnetic field or optoperforation, which uses a laser to create temporary pores in the cell membrane, allowing DNA to enter.

[0210] Transfection can add one or more plasmids. A single plasmid may be used to express multiple proteins. The plasmid may express both the protein of interest attached to a detector tag and a further protein which binds to the detector tag.

[0211] After transfection the cell is maintained in a viable state in order to express proteins.

[0212] Where the expression is performed in cells, the cells may be lysed in order to release the contents. The contents of the lysate may be mixed with the beads and later the detector species in order to measure the level of expression and residual unbound protein. Alternatively the protein may be secreted from the cells and purified and detected outside the cells.

[0213] Lysis conditions may involve physical or chemical treatments or combinations thereof. Chemical treatments may include:

[0214] Detergents: Detergents like Triton X-100, SDS, and NP-40 solubilize cell membranes, making them permeable. These are often used for eukaryotic cells and can be mild or harsh depending on the detergent.

[0215] Osmotic Lysis: Cells are placed in a hypotonic (low ionic strength) solution, causing them to swell and burst due to osmotic pressure.

[0216] Chaotropic Agents: Chemicals like urea and guanidine hydrochloride disrupt hydrogen bonds, denaturing proteins and lysing cells. Combinations of chemicals may be used as needed depending on the cells. Commercial buffers for cell lysis are available, for example RIPA Lysis and Extraction Buffer and M-PER™ Mammalian Protein Extraction Reagent, both from ThermoFisher. The device allows screening of different lysis conditions, for example to measure the level of expressed proteins after lysis under varying conditions.

[0217] Physical conditions may include heat or forcing the cells through gaps smaller than the size of the cells or related method of shearing. Physical method may include sonication. Physical method may include the high speed motion or heating of particles to which the cells are attached.

[0218] The cell-free expression of peptides or proteins can use a cell lysate having the reagents to enable protein expression ora reconstituted system. Common components of a cell-free reaction include an energy source, a supply of amino acids, cofactors such as magnesium, and the relevant enzymes. A cell extract is obtained by lysing the cells of interest and removing the cell walls, DNA genome, and other debris by centrifugation. The remains are the cell machinery including ribosomes, aminoacyl-tRNA synthetases, translation initiation and elongation factors, nucleases, etc. Once a suitable nucleic acid template is added, the nucleic acid template can be expressed as a peptide or protein using the cell derived expression machinery.

[0219] 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.

[0220] 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.

[0221] By way of example, the screening reagents may include,

[0222] ■ Chaperone mix (e.g., PUREfrex GroE mix)

[0223] ■ Kinase 1 (e.g., NEB CK2)

[0224] ■ Kinase 2 (e.g., NEB PKA)

[0225] ■ Protease 1 (e.g., NEB TEV)

[0226] ■ Protease 2 (e.g., Merck HRV 3C)

[0227] ■ Common metal ions cocktail ■ Common co-factors cocktail

[0228] 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.

[0229] Any particular nucleic acid template (expression construct) can be expressed using the system described herein. Three types of nucleic acid templates used in cell-free protein synthesis (CFPS) include plasmids, linear expression constructs (LECs), and mRNA. Plasmids are circular templates, which can be produced either in cells or synthetically. LECs can be made via PCR. mRNA can be produced through in-vitro transcription systems. The methods can use a single nucleic acid template per droplet. The methods can use multiple nucleic acid templates per drop. The methods can use multiple droplets having a different nucleic acid template per droplet.

[0230] 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.

[0231] The cell-free extract having the components for protein expression includes everything required for protein expression apart from the nucleic acid template. Thus the term includes all the relevant ribosomes, enzymes, initiation factors, nucleotide monomers, amino acid monomers, metal ions and energy sources. Once the nucleic acid template is added, protein expression is initiated without further reagents being required.

[0232] Thus the cell-lysate can be supplemented with additional reagents prior to the template being added. The cell-free extract having the components for protein expression would typically be produced as a bulk reagent or ‘master mix’ which can be formulated into many identical droplets prior to the distinct template being separately added to separate droplets. Common cell extracts in use today are made from E. coli (ECE), rabbit reticulocytes (RRL), wheat germ (WGE), insect cells (ICE) and Yeast Kluyveromyces (the D2P system). All of these extracts are commercially available. Rather than originating from a cell extract, the cell-free system can be assembled from the required reagents. Systems based on reconstituted, purified molecular reagents are commercially available, for example the PURE system for protein production, and can be used as supplied. 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. Again the system can be supplemented with additives as desired.

[0233] The use of a population of droplets having different components allows the rapid screening of a variety of variable factors to identify optimal conditions for expression of a desired proteins. The protein can be contained with a sequence having other amino acid domains, for example solubility factors or binding tags.

[0234] The flank sequences attached to the target sequence may include elements required for transcription and translation, such as for example a promoter region such as a T7 promoter binding site, a ribosome binding site, start codons and stop codons. The flank sequences may optionally also include elements such as solubility tags, purification tags or detection tags.

[0235] Protein solubility sequences may be attached to the detector and / or the POI sequences. For example the expressed protein and the GFP-MO may be attached to further elements to improve solubility. The solubility enhancing sequence may be a peptide sequence ora naturally occurring sequence. The solubility enhancing sequence may be selected from for example maltose binding protein (MBP), Small Ubiquitin-like Modifier (SUMO), Glutathione S-transferase (GST) or thioredoxin (TRX). The tags may be attached to either the C or N terminus. Any example of a solubility enhancer may be used. A list of possible proteins is shown below. Any sequence selected from the list below may be chosen:

[0236]

[0237]

[0238]

[0239] Any fluorescent protein may be used. The term GFP is used herein to describe a group of green fluorescent proteins from different organisms including the proteins sfGFP, GFP, eGFP, ccGFP, deGFP, frGFP. The fluorescent protein may be sfGFP, GFP, eGFP, ccGFP, deGFP, frGFP, eYFP, eBFP, eGFP, 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 sfCherry-i-io. The peptide tag may be CFASTn or CFAST10 and the further polypeptide CFAST in the presence of a hydroxybenzylidene rhodanine analog. The peptide tag may be ccGFPn and the further polypeptide ccGFPi.10. The fluorescent protein may be GFP. The fluorescent protein may be sfGFP. The fluorescent protein may be ccGFP. The solubility enhancement moiety may comprise ccGFPi.-io and MBP.

[0240] The protein may be assembled and thereby become fluorescent as a result of the expressed protein binding with the binding partner. The affinity interaction results in the two sub-components of the fluorescent protein being near enough to each other to bind and induce fluorescence.

[0241] The complementary GFPn peptide amino acid sequence tag could be the following:

[0242] 1. KRDHMVLLEFVTAAGITGT

[0243] 2. KRDHMVLHEFVTAAGITGT

[0244] 3. KRDHMVLHESVNAAGIT

[0245] 4. RDHMVLHEYVNAAGIT

[0246] 5. GDAVQIQEHAVAKYFTV

[0247] 6. GDTVQLQEHAVAKYFTV

[0248] 7. GETIQLQEHAVAKYFTE

[0249] Or a truncated sequence thereof. Truncations may involve a shortening of up to 5 amino acids from the N terminus, the C terminus or a combination thereof.

[0250] Properties of the expressed protein may be characterised on the device. The screen may be based on the level of soluble and purified expression by measuring fluorescence formed on complementation of a detector with the expressed sequence before and after purification.

[0251] Once the protein is expressed the expression can be stopped by addition of chemical reagents to prevent further expression. The assay for purification should be independent of further expression, as increases in the amount of protein by further protein expression may bias the measurements of purification. For example the purification bead solution can be mixed with an agent that arrests protein synthesis, such as a Mg++ chelator (EDTA) in order to arrest and immobilise all in one. Alternatively the chelator / EDTA can be introduced first to all droplets and the beads introduced at different time points. The screening and analysis can be performed in liquid reagent volumes, for example in microtitre plates or strip-tubes of microfluidic channel systems. Reagent volumes can be split such that portions are purified and portions retained for further use.

[0252] Such screening, characterising and purification can all be performed on a single device, which may be a digital microfluidic device. The term digital microfluidic device refers to a device having a two-dimensional array of planar microelectrodes. The term digital microfluidic device 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 droplets 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.

[0253] 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.

[0254] 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.

[0255] 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.

[0256] 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 (J. Adhes. Sci. Technol., 2012, 26, 1747-1771).

[0257] 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. Alternatively the oil can be oxygenated.

[0258] The droplet 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 based or cell-free system having the components for protein expression to form the droplet.

[0259] 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 cells, expression 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 aqueous 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).

[0260] 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 expression system and to the DNA. This results in a dilution of the expression system, and it has been seen, in experiments, that diluting the expression system 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 of the expression system 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 GFP-i.

[0261] 10 detector polypeptide is hindered in the presence of surfactant.

[0262] Rather than adding high levels of surfactants to the aqueous sample, it is instead possible to add surfactant, such as Span85 (sorbitan trioleate), 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 / wSpan85 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. 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 system, so removing surfactants from the reagent mix and instead adding them to the oil can be beneficial.

[0263] 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 (DMPS). The filler liquid may contain a surfactant, for example a sorbitan ester such as Span 85. Disclosed is a method of taking multiple droplets having a protein with a GFPn tag and monitoring changes in the level of the GFPn tag in order to determine protein purification efficiency and stability. Disclosed is a method comprising expressing proteins in droplets on a digital microfluidic device having a two-dimensional array of planar microelectrodes wherein the proteins have a GFP peptide amino sequence tag, purifying the proteins, holding the droplets at a fixed temperature and monitoring the droplets for changes in the level of the amount of the GFPn peptide amino sequence tag in the droplets.

[0264] Disclosed is a method comprising expressing one or more proteins in one or more droplets on a digital microfluidic device having a two-dimensional array of planar microelectrodes, wherein the proteins have a ccGFP peptide amino sequence tag and a Strep-tag, adding beads to the droplets to bind the Strep-tagged proteins, separating the beads from the non immobilised material and retaining the non-immobilised liquid, washing the beads, eluting the protein from the beads and adding a ccGFPi.-io detector reagent which binds to the ccGFPn detection tags to both the aliquots of eluted material and the residual liquid volumes from the non immobilised material in order to determine the level of protein expression by measuring the total amount of protein across each of the two aliquots, and the efficiency of immobilization and elution by determining the ratio of the two aliquots, thereby determining the conditions where protein is both expressed and purified.

[0265] Devices

[0266] 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. Electrowetting occurs as result of a non-uniform electric field that influences the hydrostatic equilibrium of a dielectric liquid (dielectrophoresis or DEP) ora 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 semi-conductor film whose electrical properties can be modulated by an optical signal.

[0267] 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:

[0268] cosO - cos0o= (1 / 2yLG) c.V2

[0269] where 0Ois 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 er. £o / t, where Eris dielectric constant of the insulator / dielectric, E0is 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.

[0270] 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 / Er)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.

[0271] 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 forTFTs 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. 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. SU-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 SU-8; thus, the thickness of these materials is typically kept at 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.

[0272] 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.

[0273] 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.

[0274] 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.

[0275] 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.

[0276] 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.

[0277] The conformal layer may comprise a parylene, a siloxane, or an epoxy. It may be a thin protective parylene coating in between the insulating dielectric and the hydrophobic coating. Typically, parylene is used as a dielectric layer on simple devices. In this invention, the rationale for deposition of parylene is not to improve insulation / dielectric properties such as reduction in pinholes, but rather to act as a conformal layer between the dielectric and hydrophobic layers. The inventors find that parylene, as opposed to other similar insulating coatings of the same thickness such as PDMS (polydimethylsiloxane), prevent contact angle hysteresis caused by high conductivity solutions or solutions deviating from neutral pH for extended hours. The conformal layer may be between 10 nm and 100 pm thick. The conformal layer may be between 100 nm and 200 nm thick.

[0278] 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.

[0279] 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.

[0280] 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.

[0281] 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. 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.

[0282] The method is particularly suitable for aqueous droplets with a volume of 1 pL or smaller.

[0283] 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.

[0284] Described herein are electrokinetic devices, including:

[0285] 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:

[0286] a dielectric layer in contact with the matrix electrodes,

[0287] a conformal layer in contact with the dielectric layer, and

[0288] a hydrophobic layer in contact with the conformal layer;

[0289] a second substrate comprising a top electrode;

[0290] a spacer disposed between the first substrate and the second substrate and defining an electrokinetic workspace; and

[0291] a voltage source operatively coupled to the matrix electrodes;

[0292] Described herein is an electrokinetic device, including:

[0293] 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:

[0294] one or more dielectric layer(s) comprising silicon nitride, hafnium oxide or aluminum oxide in contact with the matrix electrodes,

[0295] a conformal layer comprising parylene in contact with the dielectric layer, and

[0296] a hydrophobic layer in contact with the conformal layer;

[0297] a second substrate comprising a top electrode; a spacer disposed between the first substrate and the second substrate and defining an electrokinetic workspace; and

[0298] a voltage source operatively coupled to the matrix electrodes;

[0299] 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.

[0300] "Droplet” refers to a volume of liquid that electrowets a hydrophobic surface and is at least partially bounded by carrier fluid and / or, in some instances, a gas or gaseous mixture such as ambient air. For example, a droplet may be completely surrounded by carrier fluid or may be bounded by carrier fluid and one or more surfaces of an EWoD device. Droplets may take a wide variety of shapes; non-limiting examples include generally disc shaped, slug shaped, truncated sphere, ellipsoid, spherical, partially compressed sphere, hemispherical, ovoid, cylindrical, and various shapes formed during droplet operations, such as merging or splitting or formed as a result of contact of such shapes with one or more working surface of an EWoD device. Droplets may include typical polar fluids such as water, as is the case for aqueous or non-aqueous compositions, or may be mixtures or emulsions including aqueous and non-aqueous components. Droplets may also include dispersions and suspensions, for example magnetic beads in an aqueous solvent. Droplets may also include one or more surfactants. In various embodiments, a droplet may include a biological sample, such as whole blood, lymphatic fluid, serum, plasma, sweat, tear, saliva, sputum, cerebrospinal fluid, amniotic fluid, seminal fluid, vaginal excretion, serous fluid, synovial fluid, pericardial fluid, peritoneal fluid, pleural fluid, transudates, exudates, cystic fluid, bile, urine, gastric fluid, intestinal fluid, fecal samples, liquids containing single or multiple cells, liquids containing organelles, fluidized tissues, fluidized organisms, liquids containing multi-celled organisms, biological swabs and biological washes.

[0301] “Droplet operation” refers to any manipulation of one or more droplets on a microfluidic device. A droplet operation may, for example, include: loading a droplet into the DMF device; dispensing one or more droplets from a source reservoir; splitting, separating or dividing a droplet into two or more droplets; moving a droplet from one location to another in any direction; merging or combining two or more droplets into a single droplet; diluting a droplet; mixing a droplet; agitating a droplet; deforming a droplet; holding a droplet in position; incubating a droplet; heating a droplet; vaporizing a droplet; cooling a droplet; disposing of a droplet; transporting a droplet out of a microfluidic device; other droplet operations described herein; and / or any combination of the foregoing. The terms “merge,” “merging,” “combine,” “combining” and the like are used to describe the creation of one droplet from two or more droplets. It should be understood that when such a term is used in reference to two or more droplets, any combination of droplet operations that are sufficient to result in the combination of the two or more droplets into one droplet may be used. For example, “merging droplet A with droplet B,” can be achieved by transporting droplet A into contact with a stationary droplet B, transporting droplet B into contact with a stationary droplet A, or transporting droplets A and B into contact with each other. The terms “splitting,” “separating” and “dividing” are not intended to imply any particular outcome with respect to volume of the resulting droplets (i.e. , the volume of the resulting droplets can be the same or different) or number of resulting droplets (the number of resulting droplets may be 2, 3, 4, 5 or more). The term “mixing” refers to droplet operations which result in more homogenous distribution of one or more components within a droplet. Examples of “loading” droplet operations includes but is not limited to microdialysis loading, pressure assisted loading, robotic loading, passive loading, and pipette loading. Droplet operations may be electrode-mediated. In some cases, droplet operations are further facilitated by the use of hydrophilic and / or hydrophobic regions on surfaces and / or by physical obstacles.

[0302] Example

[0303] Protein TAts[31AR-GFP containing a strep-tag was expressed using a MSP1E3D1 DOPG nanodisc composition in a variety of different expression conditions according to the table below:

[0304]

[0305] 100 pL expression reactions were assembled and incubated at 29°C overnight. 10 pL of each reaction was retained as the “total” fraction. The remaining 90 pL were purified using strep beads using wash and elution buffers as described below:

[0306]

[0307]

[0308] Proteins were eluted in 90 pL elution buffer. For each of 5 separate additive conditions, the total expressed, unbound and bound / eluted fractions of GFP were measured against a GFP control. The amount of expressed protein was calculated as shown:

[0309]

[0310] Figure 6 shows that for each of the 5 sets of conditions, the sum of the unbound and bound / eluted matches the total expression. Thus the amount of expressed protein can be measured by adding together the elution and residual unbound fractions as shown below:

[0311]

Claims

CLAIMS1. A method for synthesising, purifying and characterising a plurality of proteins having detection tags and binding tags, the method comprising the steps of:i. mixing a protein expression system with one or more nucleic acid templates to form a plurality of merged liquid volumes capable of expressing different proteins; ii. allowing protein expression in the liquid volumes;iii. separating the expressed proteins from the expression reagents in each of the plurality of proteins using the binding tags and a solid support which binds to the binding tags to form discreet liquid volumes having the solid supports and residual liquid volumes without the solid supports;iv. optionally washing the solid supports;v. eluting any expressed and optionally washed protein from the solid supports by disrupting the binding to the binding tags to form eluted material;vi. adding a detector reagent which binds to the detection tags to either:a. both the aliquots of eluted material and the residual liquid volumes without the solid supports; orb. the eluted material, wherein the residual liquid volumes are added to the mix of detector reagent and eluted material after the level of eluted protein has been measured;in order to determine the level of protein expression by measuring the total amount of protein across each of the two aliquots, and the efficiency of immobilization and elution by determining the ratio of the two aliquots, thereby determining the conditions where protein is both expressed and purified.

2. The method according to claim 1, wherein the detector reagent is also added to the solid supports after elution to measure the level of residual uneluted protein remaining on the solid supports.

3. The method according to claim 1, wherein the detector reagent is added to both the aliquots of eluted material and the residual liquid volumes without the solid supports.

424. The method according to claim 1, wherein the residual liquid volumes are added to the mix of detector reagent and eluted material after the level of eluted protein has been measured to determine the total amount of protein expressed.

5. The method according to claim 4, wherein the solid supports after elution are added to the aliquots containing detection reagent after the level of protein within the aliquot has been measured to determine the level of residual uneluted protein remaining on the solid supports.

6. The method according to any one of claims 1 to 5, wherein the solid support is in the form of beads.

7. The method according to any one of claims 1 to 6, wherein the solid support takes the form of discreet locations on the surface of the device over which the liquids are moved.

8. The method according to claim 6, wherein the beads are present during the expression step.

9. The method according to any one of claims 1 to 8, wherein the proteins are expressed in cells.

10. The method according to any one of claims 1 to 8, wherein the proteins are expressed using cell-free protein synthesis.

11. The method according to any one of claims 1 to 10, wherein the method is performed in droplets on a digital microfluidic device.

12. The method according to claim 11, wherein the digital microfluidic device comprises active-matrix thin-film transistors.

13. The method according to anyone of claims 1 to 12, wherein each of the binding sequences contains four or more amino acids.

14. The method according to any one of claims 1 to 13, wherein the detector reagent is a component of a fluorescent protein.4315. The method according to claim 14, wherein the protein of interest has a ccGFPn tag and the detector reagent comprises ccGFPi.-io.

16. The method according to claim 15, wherein the POI contains a ccGFPn peptide amino sequence tag selected from:KRDHMVLLEFVTAAGITGT KRDHMVLHEFVTAAGITGT KRDHMVLHESVNAAGIT RDHMVLHEYVNAAGIT GDAVQIQEHAVAKYFTV GDTVQLQEHAVAKYFTV GETIQLQEHAVAKYFTEor a truncated version thereof.

17. The method according to any one of claims 1 to 16, wherein the POI is expressed with a solubility enhancer selected from:

18. The method according to any one of claims 1 to 17, wherein the expression is performed using cell-free lysates or is performed using assembled components for transcription and translation in a system of purified recombinant elements (PURE).

19. The method according to any one preceding claim, wherein the expression is performed for at least 3 hours before separating the expressed proteins from the expression reagents.

20. The method according to any one of claims 10 to 19, wherein the digital microfluidic device comprises an oil-filled or humidified gaseous environment, wherein the humidified gaseous environment is achieved by enclosing or sealing the digital microfluidic device and providing on-board reagent reservoirs.

21. The method according to any one of claims 1 to 20, wherein the binding moiety is selected from:Alfa-tag (SRLEEELRRRLTE)Avi-tag (GLNDIFEAQKIEWHE)C-tag (EPEA)Calmodulin-tag (KRRWKKNFIAVSAANRFKKISSSGAL)Dogtag (DIPATYEFTDGKHYITNEPIPPK)E-tag (GAPVPYPDPLEPR)FLAG (DYKDDDDK)G4T (EELLSKNYHLENEVARLKK)HA (YPYDVPDYA)His (HHHHHH)Isopeptag (TDKDMTITFTNKKDAE)lanthanide binding tag (LBT) (FIDTNNDGWIEGDELLLEEG)Myc (EQKLISEEDL)NE-Tag (TKENPRSNQEESYDDNES)Poly Glutamate-tag (EEEEEEE)Poly Arginine-tag (RRRRRRR)Rho1D4-tag (TETSQVAPA)SBP-tag (MDEKTTGWRGGHVVEGLAGELEQLRARLEHHPQGQREP) Sdytag (DPIVMIDNDKPIT)SH3 (STVPVAPPRRRRG)SNAG (GSHHW)Snooptag (KLGDIEFIKVNK)Softag 1 (SLAELLNAGLGGS)Softag 3 (TQDPSRVG)Spot-tag (PDRVRAVSHWSS)Spytag (AHIVMVDAYKPTK)S-tag (KETAAAKFERQHMDS)Strep-tag (AWAHPQPGG) (AWRHPQFGG)Strep-tag II (WSHPQFEK)T7tag (MASMTGGQQMG)TC-tag (EVHTNQDPLD)Ty-tag (CCPGCC)VSV-tag (YTDIEMNRLGK)Xpress-tag (DLYDDDDK).4722. The method according to any one preceding claim comprising expressing one or more antibodies having a binding Fc region.

23. The method according to any one preceding claim comprising expressing one or more proteins in one or more droplets on a digital microfluidic device having a two-dimensional array of planar microelectrodes, wherein the proteins have a ccGFPn peptide amino sequence tag and a Strep-tag, adding beads to the droplets to bind the Strep-tagged proteins, separating the beads from the non immobilised material and retaining the nonimmobilised liquid, washing the beads, eluting the protein from the beads and adding a ccGFPi-io detector reagent which binds to the ccGFPn detection tags to both the aliquots of eluted material and the residual liquid volumes from the non immobilised material in order to determine the level of protein expression by measuring the total amount of protein across each of the two aliquots, and the efficiency of immobilization and elution by determining the ratio of the two aliquots, thereby determining the conditions where protein is both expressed and purified.

24. The method according to any one preceding claim, wherein at least 8 different nucleic acid templates are screened against at least 4 different expression reagents on the same device.

25. The method according to any one preceding claim, wherein the device can separately manipulate at least 96 droplets.

26. The method according to any one preceding claim, wherein the device can manipulate magnetic beads in at least 32 locations and the locations can optionally be used at least twice in order to analyse at least 64 droplets.48

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