EP4689663A1 - Protein expression systems - Google Patents

Protein expression systems

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Publication number
EP4689663A1
EP4689663A1 EP24714954.5A EP24714954A EP4689663A1 EP 4689663 A1 EP4689663 A1 EP 4689663A1 EP 24714954 A EP24714954 A EP 24714954A EP 4689663 A1 EP4689663 A1 EP 4689663A1
Authority
EP
European Patent Office
Prior art keywords
protein
tag
expression
droplets
binding
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP24714954.5A
Other languages
German (de)
French (fr)
Inventor
Eleonora BASSU
Marco Manni
Gordon Ross MCINROY
Tobias William Barr Ost
Sunidhi C Shetty
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Nuclera Ltd
Original Assignee
Nuclera Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Nuclera Ltd filed Critical Nuclera Ltd
Publication of EP4689663A1 publication Critical patent/EP4689663A1/en
Pending legal-status Critical Current

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
    • G01N33/6845—Methods of identifying protein-protein interactions in protein mixtures
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/71—Receptors; Cell surface antigens; Cell surface determinants for growth factors; for growth regulators
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1034—Isolating an individual clone by screening libraries
    • C12N15/1068—Template (nucleic acid) mediated chemical library synthesis, e.g. chemical and enzymatical DNA-templated organic molecule synthesis, libraries prepared by non ribosomal polypeptide synthesis [NRPS], DNA/RNA-polymerase mediated polypeptide synthesis
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
    • C07K14/43595—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from coelenteratae, e.g. medusae
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/60—Fusion polypeptide containing spectroscopic/fluorescent detection, e.g. green fluorescent protein [GFP]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/70—Fusion polypeptide containing domain for protein-protein interaction
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • G01N33/54326—Magnetic particles

Definitions

  • 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.
  • Cell-free protein synthesis 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.
  • CFPS requires significant concentrations of biomacromolecules, including DNA, RNA, proteins, polysaccharides, molecular crowding agents, and more (Febs Letters 2013, 2, 58, 261-268).
  • 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
  • split GFP NATURE METHODS
  • SUBSTITUTE SHEET (RULE 26) VOL.3 NO.10
  • 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.
  • 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.
  • WO2023/021295 describes reagents for droplet manipulation and uses in protein expression, but does not describe protein purification or screening.
  • W02021/161048 describes cell-free protein synthesis in droplets on electrowetting devices. The purification of the expressed proteins is not described.
  • WO2022/111661 describes the use of magnetic microspheres for binding.
  • MBP maltose binding protein
  • SUMO Small Ubiquitin-like Modifier
  • GST Glutathione S- transferase
  • TRX thioredoxin
  • 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.
  • the invention relates to determining the degree of aggregation of an expressed protein in a cell- free system comprising expressing a protein of interest (POI) in droplets on a digital microfluidic device, where the expressed POI is measured using a detectable signal and measuring the levels of soluble and insoluble POI.
  • the level of aggregation can be used to determine whether a protein has expressed as soluble or insoluble.
  • Soluble protein can be taken forward for purification, for example using magnetic beads. Soluble protein may be further purified, whereas proteins with a high degree of aggregation are unlikely to be purified or to be active.
  • a method for synthesising, characterising and purifying one or more proteins having detection tags and binding tags comprising the steps of: i mixing a cell-free protein expression system with one or more nucleic acid templates to form a plurality of merged liquid volumes; either iia splitting the merged volumes into at least two aliquots and allowing protein expression in at least two aliquots; or iib allowing protein expression then splitting into at least two aliquots; iii adding a detector reagent which binds to the detection tags to at least one of the aliquots in order to determine the level of protein expression and determining the conditions where protein is expressed; iv selecting one or more of the volumes where protein has been expressed for purification using a solid support which binds to the binding tags; v separating the expressed proteins from the expression reagents using the binding tags; vi eluting the expressed protein from the solid support by disrupting the binding to
  • SUBSTITUTE SHEET (RULE 26) vii adding a detector reagent which binds to the detection tags to at least one of the eluted aliquots in order to determine the level of protein purification and determining the conditions where purified protein is obtained.
  • a method for synthesising, characterising and purifying one or more proteins having detection tags and binding tags comprising the steps of: i mixing a cell-free protein expression system with one or more nucleic acid templates to form a plurality of merged liquid volumes; either iia splitting the merged volumes into at least two aliquots and allowing protein expression in at least two aliquots; or iib allowing protein expression then splitting into at least two aliquots; iii adding a detector reagent which binds to the detection tags to at least one of the aliquots in order to determine the level of protein expression and determining the conditions where protein is expressed in soluble form; iv selecting the volumes where protein has been solubly expressed and adding magnetic beads to a sub-population of the aliquots, wherein the beads bind to the binding tags; v separating the magnetic beads from the expression reagents; vi optionally adding wash reagents to the magnetic beads to remove unbound material; vii eluting
  • the degree of aggregation can be measured directly, for example using light scattering, or may be seen using visible labels.
  • the detection may be seen as optically detectable aggregates which cause light scatter, or may give rise to aggregates which can be detected by labelling, for example using fluorescence.
  • the light scattering may be measured at an angle different from the incident light.
  • the POI may have a binding sequence which binds to a detector moiety. The detector moiety can be added after the expression by combining additional droplets.
  • the degree of aggregation can be measured by counting the number of aggregates, the area of the aggregates, the intensity of the aggregates or by using a measurement of dispersion.
  • the POI may be expressed with a binding sequence which binds to a detector moiety. In which case a further droplet containing a detector moiety may be added to the droplet containing the POI to create an detectable signal in the combined droplet.
  • the binding sequences may contain four or more amino acids.
  • the binding sequences may contain 4-30 amino acids.
  • the detector moiety may be a protein.
  • the detector moiety may comprise a component of a fluorescent protein such as for example sfGFP, or ccGFP.
  • the expressed protein may contain a sequence acting as a solubility enhancer, for example selected from:
  • 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.
  • 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 sfCherryi-w.
  • the tag may be CFASTn or CFAST and the detector NFAST in the presence of a hydroxybenzylidene rhodanine analog.
  • the tag may be ccGFPn and the detector ccGFPi-i 0 .
  • GFP1.10 polypeptide amino acid sequence could be derived from sfGFP:
  • the GFP1.10 polypeptide amino acid sequence could be further mutated from the sequence above to become brighter more quickly upon complementation.
  • the sequence may have a greater than 90 % homology to any sequence mentioned herein.
  • the sequence may have a greater than 95 % homology to any sequence mentioned herein.
  • the GFP1.10 polypeptide amino acid sequence could also be derived from ccGFP, having a greater than 90 or 95% homology to:
  • the complementary GFPn peptide amino acid sequence could be the following:
  • KRDHMVLLEFVTAAGITGT (SEQ ID NO: 9)
  • Truncations may involve a shortening of up to 5 amino acids from the N terminus, the C terminus or a combination thereof.
  • GFPn or GFP1.10 can be fused to the protein of interest through an amino acid linker.
  • the oligopeptide, peptide, or polypeptide linker can be 0 - 50 amino acids.
  • nucleic acid sequences for expressing particular tags.
  • Nucleic acid sequences include
  • sequences may be repeated one or more times to produce a protein having multiple GFPn domains.
  • sfCherryi-io polypeptide amino acid sequence could be:
  • the complementary sfCherryl 1 peptide amino acid sequence could be:
  • YTIVEQYERAEGRHSTGG sfCherryl 1 or sfCherryi-io can be fused to the protein of interest through an amino acid linker.
  • the oligopeptide, peptide, or polypeptide linker can be 0 - 50 amino acids.
  • NFAST polypeptide amino acid sequence could be:
  • the complementary CFASTn peptide amino acid sequence could be:
  • NFAST, CFASTn, and/or CFASTw can be fused to the protein of interest through an amino acid linker.
  • the oligopeptide, peptide, or polypeptide linker can be 0 - 50 amino acids.
  • 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 of a metal cation.
  • the expression may be performed using cell-free protein synthesis reagents derived from whole cell extracts.
  • the expression may be performed using cell-free protein synthesis reagents derived from reconstituted systems comprising assembled components for transcription and translation in a system of purified recombinant elements (PURE).
  • 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.
  • 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:
  • Isopeptag (TDKDMTITFTNKKDAE) (SEQ ID NO: 33) lanthanide binding tag (LBT) (FIDTNNDGWIEGDELLLEEG) (SEQ ID NO: 34)
  • Rho1 D4-tag (TETSQVAPA) (SEQ ID NO: 39)
  • T7tag (MASMTGGQQMG) (SEQ ID NO: 52)
  • VSV-tag (YTDIEMNRLGK) (SEQ ID NO: 55)
  • 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 ccGFPn peptide amino sequence tag and a Strep-tag, splitting the droplets into at least two volumes, merging droplets containing ccGFPi-io into one of the droplets following the
  • SUBSTITUTE SHEET (RULE 26) expression process and measuring the uniformity of the fluorescent signal to determine the degree of aggregation of the expressed protein, taking corresponding droplets without ccGFPi-io which contain protein having been solubly expressed and adding magnetic beads which bind to the strep-tag, immobilizing and washing the beads to remove unbound material; eluting the POI by disrupting the strep binding, adding droplets containing ccGFPi-io following the elution process and measuring the fluorescent signal in order to determine the level of protein purification and determining the conditions where purified soluble protein is obtained.
  • the method may be performed on different sequences in parallel.
  • the method may use at least 8 different nucleic acid templates, which may be screened against at least 4 different expression reagents on the same device.
  • the device may be capable of handling many droplets in parallel, for example the device may separately manipulate at least 192 droplets.
  • the device may manipulate magnetic beads in a plurality of locations.
  • the device may manipulate magnetic beads in at least 30 locations.
  • the expression comparison and purification screen can identify the optimal conditions for expression and purification of the desired protein in its most soluble and stable form.
  • 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).
  • detection tag a protein that binds to the peptide tag
  • detector protein the detection tag and detector protein generate a detectable signal upon binding
  • Figure 2 shows a cartoon representation of protein expression of different proteins having varying soluble yields and levels of aggregation. Some conditions give no expression (dark). Some conditions give a high level of soluble protein (uniform intensity white squares). Some conditions give protein aggregates, meaning the expressed proteins are insoluble and clump together (white spots in dark background). Some give a mix of soluble and aggregated proteins (white spots in a grey background). Thus the level of soluble expression and level of aggregation can be determined by adding a detector species to the expressed proteins.
  • FIG. 3 shows a schematic workflow for protein expression.
  • DNA linear expression constructs LEG
  • the mixed reaction can be split into
  • SUBSTITUTE SHEET (RULE 26) multiple aliquots and left to undergo protein expression.
  • a soluble detector species (DET) is added to one drop after expression has occurred. Where the expressed protein is soluble and the degree of aggregation is low, the signal is uniform, showing the protein is solubly expressed.
  • Figure 4 shows a schematic workflow for protein expression.
  • DNA linear expression constructs LEG
  • EHT soluble detector species
  • FIG. 5 shows the experimental result from 24 different proteins expressed in a reconstituted cell-free protein synthesis system in droplets on an electrowetting on dielectric (EWoD) device.
  • Each construct contains a GFPn tag.
  • the GFP1.10 detector species is present from the start of expression.
  • the rows marked Endpoint shows the fluorescence signal from 10 hours expression in the absence of GFP1.10 detector species followed by 5 hours complementation with the GFP1.10 detector species.
  • This experiment showed significant differences between expression/complementation in Screen BioInk compared to Endpoint detection.
  • the detected protein clusters formed after expression only with endpoint detection mean that the protein aggregated after expression, thereby lowering the soluble yield.
  • Figure 6 shows a schematic workflow for protein expression and purification.
  • DNA linear expression constructs LEG
  • EHT soluble detector species
  • the detector gives a non-uniform signal, indicating the expressed protein is to some degree insoluble or aggregated.
  • the insoluble or aggregated POI is not likely to be purified from the droplets, and such droplets are not taken for purification.
  • the expressed protein is soluble and the degree of aggregation is low, the signal is uniform, showing the protein is solubly expressed.
  • Droplets, or a subset thereof are taken for purification.
  • SUBSTITUTE SHEET (RULE 26) containing beads can be added in order to capture the expressed POI having the binding tags.
  • the beads can be immobilised and washed by adding additional droplets and immobilizing the beads whilst the droplets are removed.
  • the bound material can be eluted from the beads by disrupting the binding.
  • the process of addition of detector can be repeated. Where a high signal is seen from soluble material, the protein has been purified and eluted. Where the signal is low, absent or aggregated, limited soluble purified POI has been obtained.
  • the results from detector screen 1 and 2 can be compared in order to establish conditions for expression and purification.
  • Figure 7 shows a cartoon schematic of the workflow in Figure 6.
  • the protein can be produced using a scale up to obtain a larger volume of material.
  • 24 DNA constructs can be screened in 8 different conditions to determine the best expression.
  • the 24 constructs may be 24 independent sequences for different POIs or may be a single POI with 24 varying flanks (solubility tags etc).
  • the 8 conditions may be lysates or reconstituted systems have different components.
  • reagents can be selected for a purification screen.
  • the screen may select for example 30 liquid volumes (droplets) to which beads are added.
  • the conditions giving the best purification can be scaled up in tubes off device to allow soluble proteins to be obtained.
  • Figure 8 shows an expression screen for protein VEGF with varying flank sequences.
  • An expression and solubility screen was first performed to determine the optimal expression of human VEGF using three different cell-free reagents in combination with constructs containing 8 different nucleic acid template variations corresponding to VEGF with various solubility tags in the N- and C- terminus.
  • These three cell-free blends consist of different cell-free protein synthesis reagents.
  • Blend-3 is designed to promote disulfide bond formation while Blend-4 contains chaperones that promote protein folding.
  • Blend-2 contains the core component of the cell-free synthesis reagents without any additional components.
  • Each construct was run in duplicate. 192 droplets were run in total on a single cartridge.
  • the dotted lines denote the cell-free blends run with the corresponding constructs.
  • the position of the detector (DET) is also noted. Bright white fluorescent spots indicate expression of soluble proteins.
  • Figure 9 shows a graphical representation of spot intensity in Figure 8. Concentration of proteins expressed is calculated based on fluorescent intensity measured. Construct A5 (SUMO_VEGF_DET) produced the highest expression in Blend-3. Constructs with the highest yield (mg/mL) were selected for on-cartridge purification (highlighted in green dashed box).
  • FIG. 10 shows an image of thirty soluble candidates that presented the highest expression yield which were automatically selected and taken forward for an on-cartridge purification step. Among these candidates were SUMO-VEGF expressed in Blend- 2, Blend-3 and Blend-4. These expression candidates were purified on the cartridge using magnetic beads.
  • Figure 5 shows the location of SUMO_VEGF_STREP_DET purified on the cartridge. Fluorescent intensity measurement was used to determine yield of the purified proteins.
  • Figure 11 shows a graphical representation of Figure 10 and shows a side by side comparison of the expression yield and purified yield for SUMO-VEGF-STREP_DET. The results show that this construct, expressed in all three cell-free blends, is soluble and can be purified. Blend-3 yields the highest protein expression and purified yield and therefore was chosen for off-cartridge scale up expression.
  • Figure 12 shows a gel showing off cartridge, scaled-up expression (500 pL CFPS reaction) and purification of SUMO_VEGF_STREP_DET.
  • SDS-PAGE illustrates the VEGF protein with a molecular weight slightly higher than the predictive weight of 36.32 kDa as the SUMO tag normally shows higher than its calculated molecular weight on an SDS-PAGE gel.
  • Figure 13 shows results of an expression screen for protein CD19. The dots highlighted were selected for purification.
  • Figure 14 shows predicted yields for CD19 for a 200 pl scale-up.
  • Figure 15 shows SDS-PAGE gels for a 200 pl scale-up of CD19 constructs.
  • Figure 16 shows the purity (16a) and yield (16b) for the scale-up purifications of CD19.
  • Figure 17 shows the screening images for expression of PARK7
  • Figure 18 shows results of an expression screen for protein PARK7. The dots highlighted were selected for purification.
  • Figure 20 shows SDS-PAGE gels for a 200 pl scale-up of PARK7 constructs.
  • Figure 21 shows the scale-up purifications of PARK7.
  • Figure 22 shows the expression reaction occurring in 12 columns of droplets in 16 rows.
  • Each of the central 192 droplets is a different combination of nucleic acid template and expression blend (the edge droplets are controls and reagents for later steps in the process.
  • the lower panel shows the 192 droplets split in half, and one portion of each droplet being mixed with the detector reagent.
  • Figure 23 shows the expression screening result.
  • the top panel is white light, and shows all the droplets.
  • the 192 droplets having the detector are fluorescent (shown in the fluorescent blue light image in the lower panel). The droplets without the detector are available for subsequent steps.
  • Figure 24 shows the processing step of droplet selection. 32 of the droplets are chosen for purification. The screened droplets, and the unselected droplets are all removed to leave only 32 droplets behind. These chosen droplets are then evenly spaced out on the device for a purification screen.
  • Figure 25 shows a purification screen.
  • the droplets are bound to magnetic beads, which are repeatedly immobilised and washed with fresh wash buffer.
  • the top panel shows the 32 bead locations and wash droplets.
  • the bound material is eluted with an elution buffer to remove the protein from the beads.
  • Detector droplets are added and the fluorescence measured.
  • the blue light image is shown in the lower panel of Figure 25.
  • SUBSTITUTE SHEET indicates a lack of soluble protein for purification.
  • a method for determining the degree of aggregation of an expressed protein in a cell-free system comprising expressing a protein of interest (POI) in droplets on a digital microfluidic device, where the expressed POI is measured by an optically detectable signal and measuring the levels of insoluble POI.
  • POI protein of interest
  • Disclosed is a method that relies on parallel handling of volumes of fluid.
  • a method for synthesising, characterising and purifying one or more proteins having detection tags and binding tags comprising the steps of: i mixing a cell-free protein expression system with one or more nucleic acid templates to form a plurality of merged liquid volumes; either iia splitting the merged volumes into at least two aliquots and allowing protein expression in at least two aliquots; or iib allowing protein expression then splitting into at least two aliquots; iii adding a detector reagent which binds to the detection tags to at least one of the aliquots in order to determine the level of protein expression and determining the conditions where protein is expressed; iv selecting one or more of the volumes where protein has been expressed for purification using a solid support which binds to the binding tags; v separating the expressed proteins from the expression reagents using the binding tags; vi eluting the expressed protein from the solid support by disrupting the binding to the binding tags; and vii adding a detector reagent which binds
  • a method for synthesising, characterising and purifying one or more proteins having detection tags and binding tags comprising the steps of: i mixing a cell-free protein expression system with one or more nucleic acid templates to form a plurality of merged liquid volumes; either iia splitting the merged volumes into at least two aliquots and allowing protein expression in at least two aliquots; or iib allowing protein expression then splitting into at least two aliquots;
  • SUBSTITUTE SHEET (RULE 26) iii adding a detector reagent which binds to the detection tags to at least one of the aliquots in order to determine the level of protein expression and determining the conditions where protein is expressed in soluble form; iv selecting the volumes where protein has been solubly expressed and adding magnetic beads to a sub-population of the aliquots, wherein the beads bind to the binding tags; v separating the magnetic beads from the expression reagents; vi optionally adding wash reagents to the magnetic beads to remove unbound material; vii eluting the bound protein from the magnetic beads by disrupting the binding to the binding tag; and viii adding a detector reagent which binds to the detection tags to at least one of the eluted aliquots in order to determine the level of protein purification and determining the conditions where purified soluble protein is obtained.
  • 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 purification testing.
  • the method may involve expressing a protein of interest (POI) in a first reagent volume and splitting the reagent volume into multiple aliquots. The splitting may be performed before or after the expression process.
  • the protein of interest may have a detector tag which complements a detector species and becomes fluorescent. 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.
  • the protein is aggregated and insoluble.
  • the level of overall signal indicates the level of expression and the signal uniformity determines aggregation.
  • SUBSTITUTE SHEET (RULE 26) 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.
  • 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.
  • Assays for protein stability and potential for further purification may be performed on the device. Further assays for protein stability may include
  • Protein thermal stability does not necessarily predict aggregation rate at low temperature (and vice versa).
  • the melting temperature (T m ) 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).
  • T m melting temperature
  • the aggregation rate is dependent on the unfolded state whereas at low temperature, the aggregation rate is dependent on the native state.
  • denaturing a protein may or may not give an accurate measure of stability for the protein in its native state.
  • 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 material.
  • Disclosed is a method for protein synthesis comprising expressing a protein, wherein the expressed protein contains a sub-component of a fluorescent protein, the method comprising monitoring stability of the protein over time by creating the fluorescent protein in different batches of material at different times such as before and after purification.
  • 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.
  • the fluorescence measurement can act as a real time measure of soluble protein expression.
  • the presence of the remaining fluorescent protein as a detector species then allows real-time measurement of the tag sequence and the amount of soluble protein.
  • the fluorescence can be measured by adding the detector species after expression to measure insoluble protein. The fluorescence can be retained and monitored during the stability assay.
  • further detector protein can be added after the stability step to determine the remaining protein level. Both yield of synthesis and stability of expressed protein can thus be determined.
  • GFPn can be attached to the expressed protein and GFPi. 10 used as the detector species.
  • 192 expression conditions may be split into 384 droplets to which one of each pair has a detector moiety added.
  • the 192 ‘detected’ droplets are separated and not purified.
  • a subset of the 192 screening droplets to which the detector was not added are chosen for purification. For example 30 of the droplets may be used
  • SUBSTITUTE SHEET (RULE 26) 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.
  • 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 tag can be attached to the N or C terminus.
  • the binding moiety can be selected from the list of exemplary peptide affinity binding sites below:
  • Isopeptag (TDKDMTITFTNKKDAE) (SEQ ID NO: 33) lanthanide binding tag (LBT) (FIDTNNDGWIEGDELLLEEG) (SEQ ID NO: 34)
  • Rho1 D4-tag (TETSQVAPA) (SEQ ID NO: 39)
  • T7tag (MASMTGGQQMG) (SEQ ID NO: 52)
  • VSV-tag (YTDIEMNRLGK) (SEQ ID NO: 55)
  • the binding moiety may include a small molecule affinity tag such as biotin.
  • the binding moiety may include a particular sequence of nucleic acids.
  • 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.
  • 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.
  • 192 expression conditions may be split into 384 droplets to which one of each pair has a detector moiety added. A subset of the 192 screening droplets to which the detector was not added are chosen for purification. For example 30 of the droplets may be used.
  • the chosen droplets may be washed to separate the beads from the liquid.
  • the immobilised material is eluted and the detector added to the eluate to enable the amount of eluted material to be measured.
  • the beads may be added to the chosen droplets to enable capture of the expressed protein from the expression solution. Again washing removes the CFPS reagents, and elution and addition of the detector enables the amount of eluted material to be measured.
  • 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
  • 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.
  • 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.
  • the binding can be reversed using suitable buffers, for example containing biotin to release a strep tag or imidazole to release poly-his.
  • 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).
  • EWoD electrowetting on dielectric
  • the electrical signal on the EWoD or optical EWoD device can be delivered through segmented electrodes, active-matrix thin-film transistors, or digital micromirrors.
  • the cell-free expression of peptides or proteins can use a cell lysate having the reagents to enable protein expression or a 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.
  • the nucleic acid template can be expressed as a peptide or protein using the cell derived expression machinery.
  • 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.
  • 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.
  • the screening reagents may include,
  • Chaperone mix e.g., PUREfrex GroE mix
  • ⁇ Kinase 1 e.g., NEB CK2
  • ⁇ Kinase 2 e.g., NEB PKA
  • ⁇ Protease 1 e.g., NEB TEW
  • compositions can be blended by the user and the level of expression of the protein of interest monitored in each of the blended conditions.
  • nucleic acid template 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.
  • 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.
  • cell-lysate can be supplemented with additional reagents prior to the template being added.
  • cell-free extract having the components for protein expression would typically be
  • SUBSTITUTE SHEET (RULE 26) 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.
  • EAE E. coli
  • RRL rabbit reticulocytes
  • WGE wheat germ
  • ICE insect cells
  • Yeast Kluyveromyces the D2P system
  • 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.
  • 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.
  • 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.
  • Protein solubility sequences may be attached to the detector and the POI sequences.
  • the GFP1.10 may be attached to further elements to improve solubility.
  • the solubility enhancing sequence may be a peptide sequence or a 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).
  • MBP maltose binding protein
  • SUMO Small Ubiquitin-like Modifier
  • GST Glutathione S-transferase
  • TRX thioredoxin
  • 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:
  • 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,
  • SUBSTITUTE SHEET (RULE 26) SmURFP, miRFP670nano.
  • 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-w.
  • the peptide tag may be CFASTn or CFAST and the further polypeptide CFAST in the presence of a hydroxybenzylidene rhodanine analog.
  • the peptide tag may be ccGFPn and the further polypeptide ccGFPi-i 0 .
  • 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.
  • 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.
  • the complementary GFPn peptide amino acid sequence tag could be the following:
  • KRDHMVLLEFVTAAGITGT (SEQ ID NO: 9)
  • Properties of the expressed protein may be characterised on the device.
  • An initial screen may be based on the level of soluble expression by measuring fluorescence formed on complementation of a detector with the expressed sequence.
  • the protein may remain fluorescent during immobilisation, at which point the level of affinity purification can be determined.
  • a non-fluorescent portion of the protein can be purified, and the detector added to measure the soluble yield of the purified material.
  • SUBSTITUTE SHEET (RULE 26) 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 expression yield.
  • the methods can measure aggregation by measuring solubility before and after purification. For example,
  • a mother droplet containing nucleic acid templates and protein expression reagents expresses the POI.
  • the mother droplet is split into multiple daughter droplets (either before, during or after expression).
  • a droplet containing detector is added to daughter droplet 1 .
  • a subset of the daughter 2 droplets is chosen to have beads added, binding the expressed protein.
  • the beads are washed to remove unbound material.
  • the fluorescence is measured from both droplets and compared. Protein soluble yield is measured from the pre and post purification in order to determine purification yield.
  • 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.
  • the detector solution can be mixed with an agent that arrests protein synthesis, such as a Mg++ chelator (EDTA) in order to arrest and detect all in one.
  • EDTA Mg++ chelator
  • the chelator/EDTA can be introduced first to all daughter droplets and the detector introduced at different time points.
  • the screening and analysis can be performed in liquid reagent volumes, for example in microtitre plates or strip-tubes. Reagent volumes can be split such that portions are tested and portions retained for further use.
  • Such screening, characterising and purification can all be performed on a single device, which may be a digital microfluidic device.
  • digital microfluidic device refers to a device having a two-dimensional array of planar microelectrodes.
  • digital microfluidic device excludes any devices simply having droplets in a flow of oil in a channel. The droplets are moved over the
  • SUBSTITUTE SHEET (RULE 26) 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.
  • 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.
  • Digital microfluidics refers to a two-dimensional planar surface platform for lab-on-a-chip systems that is based upon the manipulation of microdroplets. Droplets can be dispensed, moved, stored, mixed, reacted, or analyzed on a platform with a set of insulated electrodes. Digital microfluidics can be used together with analytical analysis procedures such as mass spectrometry, colorimetry, electrochemical, and electrochemiluminescense.
  • the droplet can be moved using any means of electrokinesis.
  • the aqueous droplet can be moved using electrowetting-on-dielectric (EWoD).
  • Electrowetting on a dielectric is a variant of the electrowetting phenomenon that is based on dielectric materials.
  • EWoD Electrowetting on a dielectric
  • 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.
  • 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).
  • a source of supplemental oxygen can be supplied to the droplets.
  • droplets or gas bubbles containing gaseous or dissolved oxygen can be merged with the aqueous droplets during the protein expression.
  • the source of oxygen can be a molecular source which releases oxygen.
  • the droplets can be moved to an air/liquid boundary to enable increased diffusion of oxygen from a gaseous environment.
  • the oil can be oxygenated.
  • 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-free system having the components for protein expression to form the droplet.
  • 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).
  • PTFE polytetrafluoroethylene
  • Teflon AF DuPont Inc
  • CYTOP APC Chemicals Inc
  • 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).
  • 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).
  • SLIPS slippery liquid infused porous surface
  • 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 detector polypeptide is hindered in the presence of surfactant.
  • SUBSTITUTE SHEET (RULE 26) 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.
  • surfactant such as Span85 (sorbitan trioleate)
  • 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.
  • 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.
  • the filler liquid may be a hydrophobic or non-ionic liquid.
  • the filler liquid may be decane or dodecane.
  • the filler fluid may be a silicone oil such as dodecamethylpentasiloxane (DMPS).
  • DMPS dodecamethylpentasiloxane
  • the filler liquid may contain a surfactant, for example a sorbitan ester such as Span 85.
  • 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 GFPn peptide amino sequence tag, 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.
  • 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 ccGFPn peptide amino sequence tag and a Strep-tag, splitting the droplets into at least two volumes, merging droplets containing ccGFPi-io into one of the droplets following the expression process and measuring the uniformity of the fluorescent signal to determine the degree of aggregation of the expressed protein, taking corresponding droplets without ccGFPi-io
  • SUBSTITUTE SHEET (RULE 26) which contain protein having been solubly expressed and adding magnetic beads which bind to the strep-tag, immobilizing and washing the beads to remove unbound material; eluting the POI by disrupting the strep binding, adding droplets containing ccGFPi-io following the elution process and measuring the fluorescent signal in order to determine the level of protein purification and determining the conditions where purified soluble protein is obtained.
  • Electrowetting 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 semi-conductor film whose electrical properties can be modulated by an optical signal.
  • EWoD phenomena occur when droplets are actuated between two parallel electrodes covered with a hydrophobic insulator or dielectric.
  • the electric field at the electrode-electrolyte interface induces a change in the surface tension, which results in droplet motion as a result of a change in droplet contact angle.
  • Eo/t where E r is dielectric constant of the insulator/dielectric, E 0 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.
  • the minimum voltage applied to balance the electrowetting force with the sum of all drag forces is variably determined by the thickness-to-dielectric contact ratio of the insulator/dielectric, (t/s r ) 1/2 .
  • (t/E r ) 1/2 i.e. , increase dielectric constant or decrease insulator/dielectric thickness.
  • thin insulator/dielectric layers must be used.
  • 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.
  • High voltage EWoD-based devices with thick dielectric films 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.
  • 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.
  • 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
  • SUBSTITUTE SHEET (RULE 26) 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).
  • 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).
  • 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).
  • SUBSTITUTE SHEET (RULE 26) 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.
  • 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.
  • the dielectric layer may comprise silicon dioxide, silicon oxynitride, silicon nitride, hafnium oxide, yttrium oxide, lanthanum oxide, titanium dioxide, aluminum oxide, tantalum oxide, hafnium silicate, zirconium oxide, zirconium silicate, barium titanate, lead zirconate titanate, strontium titanate, or barium strontium titanate.
  • the dielectric layer may be between 10 nm and 100 pm thick. Combinations of more than one material may be used, and the dielectric layer may comprise more than one sublayer that may be of different materials.
  • the conformal layer may comprise a parylene, a siloxane, or an epoxy. It may be a thin protective parylene coating in between the insulating dielectric and the hydrophobic coating. Typically, parylene is used as a dielectric layer on simple devices. In this invention, the rationale for deposition of parylene is not to improve insulation/dielectric properties such as reduction in pinholes, but rather to act as a conformal layer between the dielectric and hydrophobic layers. The inventors find that parylene, as opposed to other similar insulating coatings of the same thickness such as PDMS (polydimethylsiloxane), prevent contact angle hysteresis caused by high conductivity solutions or solutions deviating from neutral pH for extended hours.
  • the conformal layer may be between 10 nm and 100 pm thick.
  • the hydrophobic layer may comprise a fluoropolymer coating, fluorinated silane coating, manganese oxide polystyrene nanocomposite, zinc oxide polystyrene nanocomposite,
  • SUBSTITUTE SHEET (RULE 26) precipitated calcium carbonate, carbon nanotube structure, silica nanocoating, or slippery liquid- infused porous coating.
  • 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.
  • 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.
  • 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.
  • the method is particularly suitable for aqueous droplets with a volume of 1 pL or smaller.
  • the EWoD-based devices shown and described below are active matrix thin film transistor devices containing a thin film dielectric coating with a Teflon hydrophobic top coat. These devices are based on devices described in the E Ink Corp patent filing on “Digital microfluidic devices including dual substrate with thin-film transistors and capacitive sensing”, US patent application no 2019/0111433, incorporated herein by reference.
  • electrokinetic devices including:
  • SUBSTITUTE SHEET (RULE 26) 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;
  • 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;
  • 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.
  • 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.
  • 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
  • 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.
  • 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.
  • 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, ex
  • 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
  • SUBSTITUTE SHEET (RULE 26) further facilitated by the use of hydrophilic and/or hydrophobic regions on surfaces and/or by physical obstacles.
  • the 24 constructs can be screened in 8 different conditions to determine the best construct and conditions for expression.
  • the 24 constructs may be 24 independent sequences for different POIs or may be a single POI with 24 varying flanks (solubility tags etc) or a combination thereof (e.g 2 POI’s with 12 flanks, 3 POI’s with 8 flanks, 4 POI’s with 6 flanks etc).
  • the 8 conditions may be lysates or reconstituted expression systems having different components or additives. 192 conditions may be screened in parallel. After identification of the best expression systems for a chosen POI, reagents/droplets can be selected for a purification screen. The screen may select for example 30 liquid volumes (droplets) to which beads are then added.
  • the beads may be washed to remove unbound material.
  • the POI’s may be eluted from the beads. Once detector reagent is added to the eluted POI’s, soluble material can be measured. A lack of signal indicates either the POI did not bind to the bead or was not eluted from the bead. If the detectable signal is seen in clumps or patches, the eluted POI has aggregated during purification/elution and is not a good candidate for scale-up. The conditions where a high level of uniform signal is seen can be chosen for a scale-up using the same identified conditions for expression and purification.
  • the conditions giving the best purification can be scaled up in tubes off device using the desired volumes of reagents to allow sufficient amounts of soluble proteins to be obtained.
  • the level of signal measured by the purification screen allows the correct scale up volume to be identified to obtain the desired mas of protein.
  • Such a scale up may allow for example greater than 20 pg of protein in a 100 pL volume.
  • mg of protein may be obtained depending on the level of expression of the construct and the volume of solution used.
  • the construct screen with different flank sequences allows placement of for example solubility tags at the C and/or N terminus along with varying configurations of the binding and detection tags, thereby increasing the likelihood that a particular POI can be expressed and purified in soluble form.
  • FIG. 5 shows the experimental result from 24 different proteins expressed in a reconstituted cell-free protein synthesis system in droplets on an electrowetting on dielectric (EWoD) device.
  • Each construct contains a GFPn tag.
  • the GFP1.10 detector species is present from the start of expression.
  • the rows marked Endpoint shows the fluorescence signal from 10 hours expression in the absence of GFP1.10 detector species followed by 5 hours complementation with the GFP1.10 detector species. This experiment showed significant
  • SUBSTITUTE SHEET (RULE 26) differences between expression/complementation in Screen BioInk compared to Endpoint detection.
  • the detected protein clusters formed after expression only with endpoint detection mean that the protein aggregated after expression, thereby lowering the soluble yield. It is evident from the image the presence of speckles for several constructs, indicating the likely presence of protein aggregates.
  • the level of aggregation enables identification of conditions which are worthy of further testing, and identification of conditions having high level of aggregated protein from which further purification is unlikely to give material.
  • VEGF Human vascular endothelial growth factor
  • VEGF protein has previously been recombinantly produced in vivo using yeast, insect and mammalian cell expression systems. Expressing this protein in E.coli has proven to be challenging due to the inherent cysteine-knot motif in human VEGF proteins and can result in protein misfolding and the formation of inclusion bodies. Expressing soluble VEGF in E.coli is only possible in the presence of solubility fusion tags such as maltose-binding protein (MBP). Moreover, MBP-VEGF proteins can only express successfully in E.coli Origami 2 (CE3) strains which carry specific gene mutations that facilitate proper disulfide bond formation. Optimizing protein expression in E.coli cells can be laborious and time consuming, taking days to weeks to discover the ideal condition to express and purify soluble proteins.
  • MBP maltose-binding protein
  • E.coli protein expression involves cloning the protein-of-interest in various expression plasmids encoding different solubility tags, trying expression in different E.coli cell lines and optimizing different expression conditions.
  • the inventors have produced VEGF in a microfluidic system using cell-free reagents derived from E.coli.
  • Figure 8 shows an expression screen for protein VEGF with varying flank sequences. These 8 protein constructs were generated with different solubility tags attachment in either the N- or C- terminus. Strep II tag was included for purification purposes (Table 1):
  • An expression and solubility screen was first performed to determine the optimal expression of human VEGF using three different cell-free reagents in combination with constructs containing 8 different nucleic acid template variations corresponding to VEGF with various solubility tags in the N- and C- terminus.
  • These three cell-free blends consist of different cell-free protein synthesis reagents.
  • Blend-3 is designed to promote disulfide bond formation while Blend-4 contains chaperones that promote protein folding.
  • Blend-2 contains the core component of the cell-free synthesis reagents without any additional components.
  • Each construct was run in duplicate. 192 droplets were run in total on a single cartridge. The dotted lines denote the cell-free blends run with the corresponding constructs.
  • the position of the detector (DET) is also noted. Bright white fluorescent spots indicate expression of soluble proteins.
  • Figure 9 shows a graphical representation of spot intensity in Figure 8. Concentration of proteins expressed is calculated based on fluorescent intensity measured. Construct A5 (SUMO_VEGF_DET) produced the highest expression in Blend-3. Constructs with the highest yield (mg/mL) were selected for on-cartridge purification (highlighted in green dashed box).
  • Figure 10 shows an image of thirty soluble candidates that presented the highest expression yield which were automatically selected and taken forward for an on-cartridge purification step. Among these candidates were SUMO-VEGF expressed in Blend-2, Blend-3 and Blend-4. These
  • SUBSTITUTE SHEET (RULE 26) expression candidates were purified on the cartridge using magnetic beads.
  • Figure 5 shows the location of SUMO_VEGF_STREP_DET purified on the cartridge. Fluorescent intensity measurement was used to determine yield of the purified proteins.
  • Figure 11 shows a graphical representation of Figure 10 and shows a side by side comparison of the expression yield and purified yield for SUMO-VEGF-STREP_DET. The results show that this construct, expressed in all three cell-free blends, is soluble and can be purified. Blend-3 yields the highest protein expression and purified yield and therefore was chosen for off-cartridge scale up expression.
  • Linear DNA construct corresponding to SUMO-VEGF_Strep_DET was added to 500 pL of Blend- 3 expression reagent in tubes and incubated overnight. Expressed proteins were purified using magnetic beads. Sumo-tagged VEGF proteins were eluted in 100 pL elution buffer and the eluted proteins were >95% pure as seen in figure 12 SDS-PAGE gel analysis. The final purified yield of SUMO_VEGF is 32.1 pg of proteins obtained.
  • Figure 12 shows a gel showing off cartridge, scaled-up expression (500 pL CFPS reaction) and purification of SUMO_VEGF_STREP_DET.
  • SDS-PAGE illustrates the VEGF protein with a molecular weight slightly higher than the predictive weight of 36.32 kDa as the SUMO tag normally shows higher than its calculated molecular weight on an SDS-PAGE gel.
  • VEGF is a difficult candidate to express in the E.coli expression system. Expression of tag-less VEGF tends to end up with inclusion body formation. To obtain soluble proteins, expressing VEGF with multiple combinations of solubility tags is required to determine the right construct for successful expression and purification. The conventional cell-based expression and purification approaches can be time consuming and laborious, considering the variety of constructs that need to be tested. We have demonstrated that a droplet microfluidic platform enables rapid construct expression screening and selection of soluble, purifiable candidates. VEGF with N-terminus SUMO tag gave the highest expression yield in Blend-3 expression reagent. Results generated on the platform were validated through an off-cartridge cell free expression. The expressed
  • SUBSTITUTE SHEET (RULE 26) proteins were purified to high purity and yield; comparable to VEGF proteins produced in cellbased expression platforms.
  • CD19 B-lymphocyte antigen CD19
  • CD19 is a transmembrane glycoprotein.
  • the transmembrane domain of CD19 crosses the membrane with a single transmembrane helix.
  • CD19 is involved in B cell signalling and is required for an optimal immune response.
  • the N-terminal domain is extracellular and the C-terminal domain is cytoplasmic.
  • the C-terminal domain of CD19 is required for the correct humoral immune response.
  • CD19 has a role in autoimmune disorders making CD19 a target for treatment of rheumatoid arthritis and multiple sclerosis.
  • CD19 antibodies have a potential use as therapies for lymphoma, leukaemia, and autoimmune disorders. Therefore, production of CD19 would be beneficial to a pharmaceutical organisation.
  • CD19 is 556 amino acids in length.
  • the first 20 amino acids of CD19 comprise a signal sequence.
  • the N-terminal domain is the most likely candidate for obtainability.
  • the X-ray structures including CD19 are of this domain suggesting it can be purified well and is stable.
  • the N-terminal domain was not extended to the start of the transmembrane helix at residue 292. This was because the Alphafold structure suggested a small helix prior to the transmembrane helix. It was noted that this small helix contained an abundance of hydrophobic residues on the side towards the transmembrane helix. For this reason the truncation was stopped at residue 277, which was the end of the N-terminal domain.
  • Templates for the 3 amino acid sequences for CD19 and each of seven solubility tags and a no solubility tag were prepared using the commercially available eGene Prep Kit NC3001 and associated protocol. Electrophoresis agarose gels indicated each of the 24 templates was obtained as a single band after purification to remove amplification primers.
  • CD19 contains disulphide bonds in the N-terminal domain
  • the following additives were selected: o PDI+/GSSG - Chaperone and to support disulfide bond formation o GSSG - Redox modification to oxidizing environment o TRXB1 - acts as a reductase in redox regulation o 3C protease - cleavage of solubility tag o DnaK - Chaperone mix to support folding and prevent aggregation o Cofactor Mix - assist in folding, stability and activity o Calcium - C-terminal domain might have some interaction with Calcium.
  • the expression system screens 192 sets of conditions to identify the highest expressing protein.
  • a graph was created of the 192 expression screening results ( Figure 13).
  • SUBSTITUTE SHEET (RULE 26) 30 conditions showing the highest level of expression (10 per protein variant) were selected for the purification screen.
  • the predicted scale up results for purification are shown in Figure 14. Based on the purification screen, the following construct and expression pairs were selected for scale-up (Table 2):
  • Table 2 Predicted yields for a 200 l scale-up for CD19.
  • Scale-up purifications were performed using commercially available scale-up and additive kits from Nuclera (NC3004 & NC3005) and associated protocols for expression and bead based purification. After overnight incubation at 29°C, samples were purified using magnetic STREP beads. Samples of the total expressed material, bead flow through (FT) and bead elution were studied by SDS-PAGE ( Figure 15).
  • the purity is shown in Figure 16a and the yield in Figure 16b.
  • the 21-227 template had the best production and purification of the nucleic acid templates tested. However, the full length template was also purified despite there being a transmembrane domain present in the centre of the protein.
  • PARK7 (also called DJ-1 or Parkinsonism associated deglycase) is a pleiotropic protein belonging to the peptidase C56 family. It acts as a positive regulator of androgen receptor-dependent transcription, redox-sensitive chaperone, sensor for oxidative stress, and apparently protects neurons against oxidative stress and cell death. Dysfunctions in PARK7 are related to autosomal recessive early-onset Parkinson disease 7 and cancer forms.
  • Templates for the 3 amino acid sequences for CD19 and each of seven solubility tags and a no solubility tag were prepared using the commercially available eGene Prep Kit NC3001 and associated protocol.
  • the PGR products for each sample were purified using NUCPure beads, then the purified eGene products were run on an agarose gel. Gel showed clean DNA constructs after PGR.
  • the DNA was quantified and the concentrations of each construct in pg/mL were converted to nM.
  • Each sample was diluted to 5 nM using UMAMI elution buffer (HEPES 10 mM pH8, 0.05% F127) to make it compatible with the eProtein Discovery workflow.
  • the constructs were stored at -20°C until use.
  • Figure 22 shows the expression reaction occurring in 12 columns of droplets in 16 rows.
  • Each of the central 192 droplets is a different combination of nucleic acid template and expression blend (the edge droplets are controls and reagents for later steps in the process.
  • the lower panel shows the 192 droplets split in half, and one portion of each droplet being mixed with the detector reagent.
  • Figure 23 shows the expression screening result.
  • the top panel is white light, and shows all the droplets.
  • the 192 droplets having the detector are fluorescent (shown in the fluorescent blue light image in the lower panel). The droplets without the detector are available for subsequent steps.
  • Figure 24 shows the processing step of droplet selection. 32 of the droplets are chosen for purification. The screened droplets, and the unselected droplets are all removed to leave only 32 droplets behind. These chosen droplets are then evenly spaced out on the device for a purification screen.
  • Figure 25 shows a purification screen.
  • the droplets are bound to magnetic beads, which are repeatedly immobilised and washed with fresh wash buffer.
  • the top panel shows the 32 bead locations and wash droplets. Two of the locations are purification and elution controls, thus enabling screening of 30 of the expression reactions.
  • the bound material is eluted with an elution buffer to remove the protein from the beads.
  • Detector droplets are added and the fluorescence measured.
  • the blue light image is shown in the lower panel of Figure 25.
  • SUBSTITUTE SHEET (RULE 26) beads. Samples of the total expressed material, bead flow through (FT) and bead elution were studied by SDS-PAGE ( Figure 20). Arrows indicate the target proteins based on the absence of the band from the no DNA control and the expected molecular mass of the protein. The expected molecular mass for the purified samples were 30, 27, and 34 kDa respectively. As seen from the SDS-PAGE, the purity was good for the scaled-up proteins and was >80% by the Gelanalyzer.
  • Figure 21 shows the scale-up purifications of PARK7 as pg of obtained proteins.
  • the tested constructs were successfully obtained using the eProtein Discovery protocol, which could successfully predict the best conditions to obtain the protein in the chosen cell-free blends.

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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.

Description

PROTEIN EXPRESSION SYSTEMS
FIELD OF THE INVENTION
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.
BACKGROUND
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.
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; 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).
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 |
1
SUBSTITUTE SHEET (RULE 26) 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.
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.
WO2023/021295 describes reagents for droplet manipulation and uses in protein expression, but does not describe protein purification or screening.
W02021/161048 describes cell-free protein synthesis in droplets on electrowetting devices. The purification of the expressed proteins is not described.
WO2022/111661 describes the use of magnetic microspheres for binding.
Sullivan; Biotechnol J. vol 11 2015 pp238-248 describes a cell-free expression and purification process for rapid production of protein biologies.
US2015/099271 describes split fluorescent proteins and uses thereof.
Listwan; J Struct. Funct. Genomics vol 10 2009 pp47-55 describes a high-throughput platform for protein solubility screening using a split-GFP system.
Nguyen; Scientific Rep vol 11 2021 pp1-15 describes the preparation of the fluorescent proteins from Corynactis Californica (ccGFP).
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.
2
SUBSTITUTE SHEET (RULE 26) 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.
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.
SUMMARY OF THE INVENTION
The invention relates to determining the degree of aggregation of an expressed protein in a cell- free system comprising expressing a protein of interest (POI) in droplets on a digital microfluidic device, where the expressed POI is measured using a detectable signal and measuring the levels of soluble and insoluble POI. The level of aggregation can be used to determine whether a protein has expressed as soluble or insoluble. Soluble protein can be taken forward for purification, for example using magnetic beads. Soluble protein may be further purified, whereas proteins with a high degree of aggregation are unlikely to be purified or to be active.
Disclosed is a method that relies on parallel handling of volumes of fluid. Disclosed is a method for synthesising, characterising and purifying one or more proteins having detection tags and binding tags, the method comprising the steps of: i mixing a cell-free protein expression system with one or more nucleic acid templates to form a plurality of merged liquid volumes; either iia splitting the merged volumes into at least two aliquots and allowing protein expression in at least two aliquots; or iib allowing protein expression then splitting into at least two aliquots; iii adding a detector reagent which binds to the detection tags to at least one of the aliquots in order to determine the level of protein expression and determining the conditions where protein is expressed; iv selecting one or more of the volumes where protein has been expressed for purification using a solid support which binds to the binding tags; v separating the expressed proteins from the expression reagents using the binding tags; vi eluting the expressed protein from the solid support by disrupting the binding to the binding tags; and
3
SUBSTITUTE SHEET (RULE 26) vii adding a detector reagent which binds to the detection tags to at least one of the eluted aliquots in order to determine the level of protein purification and determining the conditions where purified protein is obtained.
Disclosed is a method for synthesising, characterising and purifying one or more proteins having detection tags and binding tags, the method comprising the steps of: i mixing a cell-free protein expression system with one or more nucleic acid templates to form a plurality of merged liquid volumes; either iia splitting the merged volumes into at least two aliquots and allowing protein expression in at least two aliquots; or iib allowing protein expression then splitting into at least two aliquots; iii adding a detector reagent which binds to the detection tags to at least one of the aliquots in order to determine the level of protein expression and determining the conditions where protein is expressed in soluble form; iv selecting the volumes where protein has been solubly expressed and adding magnetic beads to a sub-population of the aliquots, wherein the beads bind to the binding tags; v separating the magnetic beads from the expression reagents; vi optionally adding wash reagents to the magnetic beads to remove unbound material; vii eluting the bound protein from the magnetic beads by disrupting the binding to the binding tag; and viii adding a detector reagent which binds to the detection tags to at least one of the eluted aliquots in order to determine the level of protein purification and determining the conditions where purified soluble protein is obtained.
The degree of aggregation can be measured directly, for example using light scattering, or may be seen using visible labels. The detection may be seen as optically detectable aggregates which cause light scatter, or may give rise to aggregates which can be detected by labelling, for example using fluorescence. The light scattering may be measured at an angle different from the incident light. The POI may have a binding sequence which binds to a detector moiety. The detector moiety can be added after the expression by combining additional droplets.
The degree of aggregation can be measured by counting the number of aggregates, the area of the aggregates, the intensity of the aggregates or by using a measurement of dispersion.
4
SUBSTITUTE SHEET (RULE 26) The POI may be expressed with a binding sequence which binds to a detector moiety. In which case a further droplet containing a detector moiety may be added to the droplet containing the POI to create an detectable signal in the combined droplet. The binding sequences may contain four or more amino acids. The binding sequences may contain 4-30 amino acids.
The detector moiety may be a protein. The detector moiety may comprise a component of a fluorescent protein such as for example sfGFP, or ccGFP.
The expressed protein may contain a sequence acting as a solubility enhancer, for example selected from:
5
SUBSTITUTE SHEET (RULE 26)
SUBSTITUTE SHEET (RULE 26)
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 sfCherryi-w. The tag may be CFASTn or CFAST and the detector NFAST in the presence of a hydroxybenzylidene rhodanine analog.
The tag may be ccGFPn and the detector ccGFPi-i0.
For example, the GFP1.10 polypeptide amino acid sequence could be derived from sfGFP:
SEQ ID NO: 1
MSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLKFICTTGKLPVPWPTLVTTL TYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDGTYKTRAEVKFEGDTLVNRIELKG IDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKANFKIRHNVEDGSVQLADHYQQNTPIGDG PVLLPDNHYLSTQSVLSKDPNEK
Alternatively, the GFP1.10 polypeptide amino acid sequence could be further mutated from the sequence above to become brighter more quickly upon complementation. The sequence may have a greater than 90 % homology to any sequence mentioned herein. The sequence may have a greater than 95 % homology to any sequence mentioned herein.
SEQ ID NO: 2
MSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATIGKLTLKFICTTGKLPVPWPTLVTTLT YGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDGKYKTRAWKFEGDTLVNRIELKGT
7
SUBSTITUTE SHEET (RULE 26) DFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKANFTVRHNVEDGSVQLADHYQQNTPIGDG PVLLPDNHYLSTQTVLSKDPNEK
The GFP1.10 polypeptide amino acid sequence could also be derived from ccGFP, having a greater than 90 or 95% homology to:
SEQ ID NO: 3 (ccGFPi-n)
MSLSKQWKEDMKMTYHMDGCVNGHYFTIEGEGTGKPFKGQKTLKLRVTEGGPLPFAFDILSA TFTYGNRCFCDYPEDMPDYFKQSLPEGYSWERTMMYEDGACGTASAHISLDKNGFVHNSTFH GVNFPANGPVMKKKGVNWEPSSEKITACDGILKGDVTMFLVLEGGHRLKCLFQTTYKADKWK MPPNHIIEHRLVRSEDGDAVQI QEHAVAKYFTV
SEQ ID NO: 4 (ccGFPi-io)
MSLSKQWKEDMKMTYHMDGCVNGHYFTIEGEGTGKPFKGQKTLKLRVTEGGPLPFAFDILSA TFTYGNRCFCDYPEDMPDYFKQSLPEGYSWERTMMYEDGACGTASAHISLDKNGFVHNSTFH GVNFPANGPVMKKKGVNWEPSSEKITACDGILKGDVTMFLVLEGGHRLKCLFQTTYKADKWK MPPNHIIEHRLVRSED
SEQ ID NO 5 (ccGFPi-io)
MSMEKQVLKENMKTTYHMDGSVDGHYFEIEGEGTGNPFKGEQELKLRVTKGGPLPFAFDILSP TFTYGNRVFTDYPEDMPDYFKQSLPEGYSWERTMMYEDGATATASARISLDKNGFVHKSTFH GENFPANGPVMKKKGVDWEPSSETITPEDGILKGDVEMFLVLEGGQRLKALFQTTYKANKWK MPPRHKIEHRLVRS
Nucleic acid sequence to express seq ID No 5 ccGFPi-i0; SEQ ID NO: 6
5’atgagcatggaaaaacaggtgctgaaagaaaacatgaaaaccacctatcacatggatggtagcgttgatggtcactattttgaaat tgaaggtgaaggcaccggcaatccgtttaaaggtgaacaagaactgaaactgcgtgttaccaaaggtggtccgctgccgtttgcattt gatattctgagcccgacctttacctatggtaatcgtgtttttaccgactatccggaagatatgccggattatttcaaacagagcctgccgga aggttatagctgggaacgtaccatgatgtatgaagatggtgcaaccgcaaccgccagcgcacgtattagcctggataaaaatggtttt gtgcataagagcacctttcacggtgaaaactttccggcaaatggtccggttatgaaaaagaaaggtgttgattgggaaccgagcagc gaaaccattacaccggaagatggtattctgaaaggtgatgttgaaatgtttctggttctggaaggtggtcagcgtctgaaagccctgtttc agaccacctataaagccaataaagtggttaaaatgcctccgcgtcataaaattgaacatcgtctggttcgtagc
SEQ ID NO 7
8
SUBSTITUTE SHEET (RULE 26) MSMSKQVLKENMKTTYHMDGSVNGHYFTIEGEGTGNPFKGQQSLKLRVTKGGPLPFAFDILSP TFTYGNRVFTDYPEDMPDYFKQSLPEGYSWERTMMYEDGATATASARISLDKNGFVHKSTFH GENFPANGPVMKKKGVNWEPSSETITPSDGILKGDVTMFLVLEGGQRLKALFQTTYKANKWK
MPPRHKIEHRLVRS
Nucleic acid sequence to express seq ID No 7 ccGFPi-i0; SEQ ID NO: 8
5’atgagcatgagcaaacaggtgctgaaagaaaatatgaaaaccacctatcacatggatggtagcgttaatggtcactattttaccatt gaaggtgaaggcaccggtaatccgtttaaaggtcagcagagcctgaaactgcgtgttaccaaaggtggtccgctgccgtttgcatttg atattctgagcccgacctttacctatggtaatcgtgtttttaccgactatccggaagatatgccggattatttcaaacagagcctgccggaa ggttatagctgggaacgtaccatgatgtatgaagatggtgcaaccgcaaccgccagcgcacgtattagcctggataaaaatggttttgt gcataagagcacctttcacggtgaaaactttccggcaaatggtccggttatgaaaaagaaaggtgttaattgggaaccgagcagcga aaccattacaccgagtgatggtattctgaaaggtgatgttaccatgtttctggttctggaaggtggtcagcgtctgaaagccctgtttcaga ccacctataaagccaataaagtggttaaaatgcctccgcgtcataaaattgaacatcgtctggttcgtagc
The complementary GFPn peptide amino acid sequence could be the following:
1 . KRDHMVLLEFVTAAGITGT (SEQ ID NO: 9)
2. KRDHMVLHEFVTAAGITGT (SEQ ID NO: 10)
3. KRDHMVLHESVNAAGIT (SEQ ID NO: 11)
4. RDHMVLHEYVNAAGIT (SEQ ID NO: 12)
5. GDAVQIQEHAVAKYFTV (SEQ ID NO: 13)
6. GDTVQLQEHAVAKYFTV (SEQ ID NO: 14)
7. GETIQLQEHAVAKYFTE (SEQ ID NO: 15) 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.
GFPn 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.
Also disclosed are nucleic acid sequences for expressing particular tags. Nucleic acid sequences include
SEQ ID NO: 16
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SUBSTITUTE SHEET (RULE 26) 5’GGTGATACCGTTCAGCTGCAAGAACATGCAGTTGCAAAATACTTTACCGTG
SEQ ID NO: 17
5’GGTGAAACCATCCAGTTACAAGAACACGCCGTGGCCAAATATTTCACCGAA or a truncated version thereof.
These sequences may be repeated one or more times to produce a protein having multiple GFPn domains.
For example, the sfCherryi-io polypeptide amino acid sequence could be:
SEQ ID NO: 18
MEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGHPYEGTQTAKLKVTKGGPLPFAWDILS
PQFMYGSKAYVKHPADIPDYLKLSFPEGFTWERVMNFEDGGWTVTQDSSLQDGEFIYKVKLL
GTNFPSDGPVMQKKTMGWEASTERMYPEDGALKGEINQRLKLKDGGHYDAEVKTTYKAKKP
VQLPGAYNVDIKLDITSHNED
The complementary sfCherryl 1 peptide amino acid sequence could be:
SEQ ID NO: 19
YTIVEQYERAEGRHSTGG sfCherryl 1 or sfCherryi-io 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.
For example, the NFAST polypeptide amino acid sequence could be:
SEQ IS NO: 20
MEHVAFGSEDIENTLAKMDDGQLDGLAFGAIQLDGDGNILQYNAAEGDITGRDPKQVIGKNFFK DVAPGTDSPEFYGKFKEGVASGNLNTMFEWMIPTSRGPTKVKVHMKKALS
The complementary CFASTn peptide amino acid sequence could be:
SEQ ID NO: 21
GDSYWVFVKRV
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SUBSTITUTE SHEET (RULE 26) Or the complementary CFASTw peptide amino acid sequence could be:
SEQ ID NO: 22
GDSYWVFVKR
NFAST, CFASTn, and/or CFASTw 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.
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 of a metal cation.
The expression may be performed using cell-free protein synthesis reagents derived from whole cell extracts. The expression may be performed using cell-free protein synthesis reagents derived from reconstituted systems comprising assembled components for transcription and translation in a system of purified recombinant elements (PURE).
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.
Disclosed is a method for expressing proteins in droplets on a digital microfluidic device having a two-dimensional array of planar microelectrodes wherein the proteins have a ccGFPn peptide amino sequence tag, wherein a portion of the droplets contain ccGFPi-w during the expression process and a further portion have ccGFPi-w added after expression and comparing the level of ccGFP1.11 signal from the droplets.
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:
Alfa-tag (SRLEEELRRRLTE) (SEQ ID NO: 23)
Avi-tag (GLNDIFEAQKIEWHE) (SEQ ID NO: 24)
C-tag (EPEA) (SEQ ID NO: 25)
Calmodulin-tag (KRRWKKNFIAVSAANRFKKISSSGAL) (SEQ ID NO: 26)
1 1
SUBSTITUTE SHEET (RULE 26) Dogtag (DIPATYEFTDGKHYITNEPIPPK) (SEQ ID NO: 27)
E-tag (GAPVPYPDPLEPR) (SEQ ID NO: 28)
FLAG (DYKDDDDK) (SEQ ID NO: 29)
G4T (EELLSKNYHLENEVARLKK) (SEQ ID NO: 30)
HA (YPYDVPDYA) (SEQ ID NO: 31)
His (HHHHHH) (SEQ ID NO: 32)
Isopeptag (TDKDMTITFTNKKDAE) (SEQ ID NO: 33) lanthanide binding tag (LBT) (FIDTNNDGWIEGDELLLEEG) (SEQ ID NO: 34)
Myc (EQKLISEEDL) (SEQ ID NO: 35)
NE-Tag (TKENPRSNQEESYDDNES) (SEQ ID NO: 36)
Poly Glutamate-tag (EEEEEEE) (SEQ ID NO: 37)
Poly Arginine-tag (RRRRRRR) (SEQ ID NO: 38)
Rho1 D4-tag (TETSQVAPA) (SEQ ID NO: 39)
SBP-tag (MDEKTTGWRGGHVVEGLAGELEQLRARLEHHPQGQREP) (SEQ ID NO: 40)
Sdytag (DPIVMIDNDKPIT) (SEQ ID NO: 41)
SH3 (STVPVAPPRRRRG) (SEQ ID NO: 42)
Snooptag (KLGDIEFIKVNK) (SEQ ID NO: 43)
Softag 1 (SLAELLNAGLGGS) (SEQ ID NO: 44)
Softag 3 (TQDPSRVG) (SEQ ID NO: 45)
Spot-tag (PDRVRAVSHWSS) (SEQ ID NO: 46)
Spytag (AHIVMVDAYKPTK) (SEQ ID NO: 47)
S-tag (KETAAAKFERQHMDS) (SEQ ID NO: 48)
Strep-tag (AWAHPQPGG) (SEQ ID NO: 49) (AWRHPQFGG) (SEQ ID NO: 50)
Strep-tag II (WSHPQFEK) (SEQ ID NO: 51)
T7tag (MASMTGGQQMG) (SEQ ID NO: 52)
TC-tag (EVHTNQDPLD) (SEQ ID NO: 53)
Ty-tag (CCPGCC) (SEQ ID NO: 54)
VSV-tag (YTDIEMNRLGK) (SEQ ID NO: 55)
Xpress-tag (DLYDDDDK) (SEQ ID NO: 56)
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 ccGFPn peptide amino sequence tag and a Strep-tag, splitting the droplets into at least two volumes, merging droplets containing ccGFPi-io into one of the droplets following the
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SUBSTITUTE SHEET (RULE 26) expression process and measuring the uniformity of the fluorescent signal to determine the degree of aggregation of the expressed protein, taking corresponding droplets without ccGFPi-io which contain protein having been solubly expressed and adding magnetic beads which bind to the strep-tag, immobilizing and washing the beads to remove unbound material; eluting the POI by disrupting the strep binding, adding droplets containing ccGFPi-io following the elution process and measuring the fluorescent signal in order to determine the level of protein purification and determining the conditions where purified soluble protein is obtained.
The method may be performed on different sequences in parallel. The method may use at least 8 different nucleic acid templates, which may be screened against at least 4 different expression reagents on the same device. The device may be capable of handling many droplets in parallel, for example the device may separately manipulate at least 192 droplets. The device may manipulate magnetic beads in a plurality of locations. The device may manipulate magnetic beads in at least 30 locations.
The expression comparison and purification screen can identify the optimal conditions for expression and purification of the desired protein in its most soluble and stable form.
FIGURES
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.
Figure 2 shows a cartoon representation of protein expression of different proteins having varying soluble yields and levels of aggregation. Some conditions give no expression (dark). Some conditions give a high level of soluble protein (uniform intensity white squares). Some conditions give protein aggregates, meaning the expressed proteins are insoluble and clump together (white spots in dark background). Some give a mix of soluble and aggregated proteins (white spots in a grey background). Thus the level of soluble expression and level of aggregation can be determined by adding a detector species to the expressed proteins.
Figure 3 shows a schematic workflow for protein expression. DNA linear expression constructs (LEG) can be mixed with expression reagents and merged. The mixed reaction can be split into
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SUBSTITUTE SHEET (RULE 26) multiple aliquots and left to undergo protein expression. A soluble detector species (DET) is added to one drop after expression has occurred. Where the expressed protein is soluble and the degree of aggregation is low, the signal is uniform, showing the protein is solubly expressed.
Figure 4 shows a schematic workflow for protein expression. DNA linear expression constructs (LEG) can be mixed with expression reagents and merged. The mixed reaction can be split into multiple aliquots and left to undergo protein expression. A soluble detector species (DET) is added to one drop after expression has occurred. Where the expressed protein is aggregated, the detector gives a non-uniform signal, indicating the expressed protein is to some degree insoluble or aggregated. The insoluble or aggregated POI is not likely to be purified from the droplets.
Figure 5 shows the experimental result from 24 different proteins expressed in a reconstituted cell-free protein synthesis system in droplets on an electrowetting on dielectric (EWoD) device. Each construct contains a GFPn tag. In the rows marked Screen, the GFP1.10 detector species is present from the start of expression. The rows marked Endpoint shows the fluorescence signal from 10 hours expression in the absence of GFP1.10 detector species followed by 5 hours complementation with the GFP1.10 detector species. This experiment showed significant differences between expression/complementation in Screen BioInk compared to Endpoint detection. The detected protein clusters formed after expression only with endpoint detection mean that the protein aggregated after expression, thereby lowering the soluble yield. It is evident from the image the presence of speckles for several constructs, indicating the likely presence of protein aggregates. The level of aggregation enables identification of conditions which are worthy of further testing, and identification of conditions having high level of aggregated protein from which further purification is unlikely to give material.
Figure 6 shows a schematic workflow for protein expression and purification. DNA linear expression constructs (LEG) can be mixed with expression reagents and merged. The mixed reaction can be split into multiple aliquots and left to undergo protein expression. A soluble detector species (DET) is added to one drop after expression has occurred. Where the expressed protein is aggregated, the detector gives a non-uniform signal, indicating the expressed protein is to some degree insoluble or aggregated. The insoluble or aggregated POI is not likely to be purified from the droplets, and such droplets are not taken for purification. Where the expressed protein is soluble and the degree of aggregation is low, the signal is uniform, showing the protein is solubly expressed. Such droplets, or a subset thereof are taken for purification. Droplets
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SUBSTITUTE SHEET (RULE 26) containing beads can be added in order to capture the expressed POI having the binding tags. The beads can be immobilised and washed by adding additional droplets and immobilizing the beads whilst the droplets are removed. The bound material can be eluted from the beads by disrupting the binding. The process of addition of detector can be repeated. Where a high signal is seen from soluble material, the protein has been purified and eluted. Where the signal is low, absent or aggregated, limited soluble purified POI has been obtained. The results from detector screen 1 and 2 can be compared in order to establish conditions for expression and purification.
Figure 7 shows a cartoon schematic of the workflow in Figure 6. After the optimal conditions have been identified, the protein can be produced using a scale up to obtain a larger volume of material. For example 24 DNA constructs can be screened in 8 different conditions to determine the best expression. The 24 constructs may be 24 independent sequences for different POIs or may be a single POI with 24 varying flanks (solubility tags etc). The 8 conditions may be lysates or reconstituted systems have different components. After identification of the best expression systems, reagents can be selected for a purification screen. The screen may select for example 30 liquid volumes (droplets) to which beads are added. The conditions giving the best purification can be scaled up in tubes off device to allow soluble proteins to be obtained.
Figure 8 shows an expression screen for protein VEGF with varying flank sequences. An expression and solubility screen was first performed to determine the optimal expression of human VEGF using three different cell-free reagents in combination with constructs containing 8 different nucleic acid template variations corresponding to VEGF with various solubility tags in the N- and C- terminus. These three cell-free blends consist of different cell-free protein synthesis reagents. Blend-3 is designed to promote disulfide bond formation while Blend-4 contains chaperones that promote protein folding. Blend-2 contains the core component of the cell-free synthesis reagents without any additional components. Each construct was run in duplicate. 192 droplets were run in total on a single cartridge. The dotted lines denote the cell-free blends run with the corresponding constructs. The position of the detector (DET) is also noted. Bright white fluorescent spots indicate expression of soluble proteins.
Figure 9 shows a graphical representation of spot intensity in Figure 8. Concentration of proteins expressed is calculated based on fluorescent intensity measured. Construct A5 (SUMO_VEGF_DET) produced the highest expression in Blend-3. Constructs with the highest yield (mg/mL) were selected for on-cartridge purification (highlighted in green dashed box).
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SUBSTITUTE SHEET (RULE 26) Figure 10 shows an image of thirty soluble candidates that presented the highest expression yield which were automatically selected and taken forward for an on-cartridge purification step. Among these candidates were SUMO-VEGF expressed in Blend- 2, Blend-3 and Blend-4. These expression candidates were purified on the cartridge using magnetic beads. Figure 5 shows the location of SUMO_VEGF_STREP_DET purified on the cartridge. Fluorescent intensity measurement was used to determine yield of the purified proteins.
Figure 11 shows a graphical representation of Figure 10 and shows a side by side comparison of the expression yield and purified yield for SUMO-VEGF-STREP_DET. The results show that this construct, expressed in all three cell-free blends, is soluble and can be purified. Blend-3 yields the highest protein expression and purified yield and therefore was chosen for off-cartridge scale up expression.
Figure 12 shows a gel showing off cartridge, scaled-up expression (500 pL CFPS reaction) and purification of SUMO_VEGF_STREP_DET. SDS-PAGE illustrates the VEGF protein with a molecular weight slightly higher than the predictive weight of 36.32 kDa as the SUMO tag normally shows higher than its calculated molecular weight on an SDS-PAGE gel. Lane 1 molecular weight ladder, lane 2 Blend-3 negative control, lane 3 CFPS total after expression, lane 4 CPFS (soluble) obtained following centrifugation and soluble fraction obtained, lane 5 Unbound; flow through sample not bound to beads, lane 6 Purified (elute); sample eluted off of the resin bead.
Figure 13 shows results of an expression screen for protein CD19. The dots highlighted were selected for purification.
Figure 14 shows predicted yields for CD19 for a 200 pl scale-up.
Figure 15 shows SDS-PAGE gels for a 200 pl scale-up of CD19 constructs.
Figure 16 shows the purity (16a) and yield (16b) for the scale-up purifications of CD19.
Figure 17 shows the screening images for expression of PARK7
Figure 18 shows results of an expression screen for protein PARK7. The dots highlighted were selected for purification.
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SUBSTITUTE SHEET (RULE 26) Figure 19 shows predicted yields for PARK7 for a 200 pl scale-up.
Figure 20 shows SDS-PAGE gels for a 200 pl scale-up of PARK7 constructs.
Figure 21 shows the scale-up purifications of PARK7.
Figure 22 (top panel) shows the expression reaction occurring in 12 columns of droplets in 16 rows. Each of the central 192 droplets is a different combination of nucleic acid template and expression blend (the edge droplets are controls and reagents for later steps in the process. The lower panel shows the 192 droplets split in half, and one portion of each droplet being mixed with the detector reagent.
Figure 23 shows the expression screening result. The top panel is white light, and shows all the droplets. The 192 droplets having the detector are fluorescent (shown in the fluorescent blue light image in the lower panel). The droplets without the detector are available for subsequent steps.
Figure 24 shows the processing step of droplet selection. 32 of the droplets are chosen for purification. The screened droplets, and the unselected droplets are all removed to leave only 32 droplets behind. These chosen droplets are then evenly spaced out on the device for a purification screen.
Figure 25 shows a purification screen. The droplets are bound to magnetic beads, which are repeatedly immobilised and washed with fresh wash buffer. The top panel shows the 32 bead locations and wash droplets. The bound material is eluted with an elution buffer to remove the protein from the beads. Detector droplets are added and the fluorescence measured. The blue light image is shown in the lower panel of Figure 25. Thus the expressed proteins which can best be purified and eluted are identified.
DETAILED DESCRIPTION OF THE INVENTION
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
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SUBSTITUTE SHEET (RULE 26) indicates a lack of soluble protein for purification. Disclosed herein is a method for determining the degree of aggregation of an expressed protein in a cell-free system comprising expressing a protein of interest (POI) in droplets on a digital microfluidic device, where the expressed POI is measured by an optically detectable signal and measuring the levels of insoluble POI.
Disclosed is a method that relies on parallel handling of volumes of fluid.
Disclosed is a method for synthesising, characterising and purifying one or more proteins having detection tags and binding tags, the method comprising the steps of: i mixing a cell-free protein expression system with one or more nucleic acid templates to form a plurality of merged liquid volumes; either iia splitting the merged volumes into at least two aliquots and allowing protein expression in at least two aliquots; or iib allowing protein expression then splitting into at least two aliquots; iii adding a detector reagent which binds to the detection tags to at least one of the aliquots in order to determine the level of protein expression and determining the conditions where protein is expressed; iv selecting one or more of the volumes where protein has been expressed for purification using a solid support which binds to the binding tags; v separating the expressed proteins from the expression reagents using the binding tags; vi eluting the expressed protein from the solid support by disrupting the binding to the binding tags; and vii adding a detector reagent which binds to the detection tags to at least one of the eluted aliquots in order to determine the level of protein purification and determining the conditions where purified protein is obtained.
Disclosed is a method for synthesising, characterising and purifying one or more proteins having detection tags and binding tags, the method comprising the steps of: i mixing a cell-free protein expression system with one or more nucleic acid templates to form a plurality of merged liquid volumes; either iia splitting the merged volumes into at least two aliquots and allowing protein expression in at least two aliquots; or iib allowing protein expression then splitting into at least two aliquots;
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SUBSTITUTE SHEET (RULE 26) iii adding a detector reagent which binds to the detection tags to at least one of the aliquots in order to determine the level of protein expression and determining the conditions where protein is expressed in soluble form; iv selecting the volumes where protein has been solubly expressed and adding magnetic beads to a sub-population of the aliquots, wherein the beads bind to the binding tags; v separating the magnetic beads from the expression reagents; vi optionally adding wash reagents to the magnetic beads to remove unbound material; vii eluting the bound protein from the magnetic beads by disrupting the binding to the binding tag; and viii adding a detector reagent which binds to the detection tags to at least one of the eluted aliquots in order to determine the level of protein purification and determining the conditions where purified soluble protein is obtained.
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.
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 purification testing. For example the method may involve expressing a protein of interest (POI) in a first reagent volume and splitting the reagent volume into multiple aliquots. The splitting may be performed before or after the expression process. The protein of interest may have a detector tag which complements a detector species and becomes fluorescent. 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
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SUBSTITUTE SHEET (RULE 26) 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.
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.
Assays for protein stability and potential for further purification may be performed on the device. Further assays for protein stability may include
• Solubility
• Aggregation (over time)
• Thermal Stability
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.
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 material.
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SUBSTITUTE SHEET (RULE 26) Protein purification can be challenging and time consuming, often resulting in a significant loss of material or a failure to obtain material in purified form. Therefore it is desirable to measure protein stability directly after expression before having to purify the proteins from the proteins used in expression. Measurement in crude cell lysates or directly in cell-free expression systems are possible using optical based analysis techniques, as these can tolerate the presence of other proteins which are stable to the conditions being measured or are not otherwise detectable.
Disclosed is a method for protein synthesis comprising expressing a protein, wherein the expressed protein contains a sub-component of a fluorescent protein, the method comprising monitoring stability of the protein over time by creating the fluorescent protein in different batches of material at different times such as before and after purification.
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.
The fluorescence measurement can act as a real time measure of soluble protein expression. As the tag sequence is produced, the presence of the remaining fluorescent protein as a detector species then allows real-time measurement of the tag sequence and the amount of soluble protein. Alternatively the fluorescence can be measured by adding the detector species after expression to measure insoluble protein. The fluorescence can be retained and monitored during the stability assay. Alternatively further detector protein can be added after the stability step to determine the remaining protein level. Both yield of synthesis and stability of expressed protein can thus be determined. For example GFPn can be attached to the expressed protein and GFPi. 10 used as the detector species.
For example 192 expression conditions may be split into 384 droplets to which one of each pair has a detector moiety added. In such instances the 192 ‘detected’ droplets are separated and not purified. A subset of the 192 screening droplets to which the detector was not added are chosen for purification. For example 30 of the droplets may be used
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SUBSTITUTE SHEET (RULE 26) 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.
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 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:
Alfa-tag (SRLEEELRRRLTE) (SEQ ID NO: 23)
Avi-tag (GLNDIFEAQKIEWHE) (SEQ ID NO: 24)
C-tag (EPEA) (SEQ ID NO: 25)
Calmodulin-tag (KRRWKKNFIAVSAANRFKKISSSGAL) (SEQ ID NO: 26)
Dogtag (DIPATYEFTDGKHYITNEPIPPK) (SEQ ID NO: 27)
E-tag (GAPVPYPDPLEPR) (SEQ ID NO: 28)
FLAG (DYKDDDDK) (SEQ ID NO: 29)
G4T (EELLSKNYHLENEVARLKK) (SEQ ID NO: 30)
HA (YPYDVPDYA) (SEQ ID NO: 31)
His (HHHHHH) (SEQ ID NO: 32)
Isopeptag (TDKDMTITFTNKKDAE) (SEQ ID NO: 33) lanthanide binding tag (LBT) (FIDTNNDGWIEGDELLLEEG) (SEQ ID NO: 34)
Myc (EQKLISEEDL) (SEQ ID NO: 35)
NE-Tag (TKENPRSNQEESYDDNES) (SEQ ID NO: 36)
Poly Glutamate-tag (EEEEEEE) (SEQ ID NO: 37)
Poly Arginine-tag (RRRRRRR) (SEQ ID NO: 38)
Rho1 D4-tag (TETSQVAPA) (SEQ ID NO: 39)
SBP-tag (MDEKTTGWRGGHVVEGLAGELEQLRARLEHHPQGQREP) (SEQ ID NO: 40)
Sdytag (DPIVMIDNDKPIT) (SEQ ID NO: 41)
SH3 (STVPVAPPRRRRG) (SEQ ID NO: 42)
Snooptag (KLGDIEFIKVNK) (SEQ ID NO: 43)
Softag 1 (SLAELLNAGLGGS) (SEQ ID NO: 44)
Softag 3 (TQDPSRVG) (SEQ ID NO: 45)
Spot-tag (PDRVRAVSHWSS) (SEQ ID NO: 46)
Spytag (AHIVMVDAYKPTK) (SEQ ID NO: 47)
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SUBSTITUTE SHEET (RULE 26) S-tag (KETAAAKFERQHMDS) (SEQ ID NO: 48)
Strep-tag (AWAHPQPGG) (SEQ ID NO: 49) (AWRHPQFGG) (SEQ ID NO: 50)
Strep-tag II (WSHPQFEK) (SEQ ID NO: 51)
T7tag (MASMTGGQQMG) (SEQ ID NO: 52)
TC-tag (EVHTNQDPLD) (SEQ ID NO: 53)
Ty-tag (CCPGCC) (SEQ ID NO: 54)
VSV-tag (YTDIEMNRLGK) (SEQ ID NO: 55)
Xpress-tag (DLYDDDDK) (SEQ ID NO: 56)
The binding moiety may include a small molecule affinity tag such as biotin. The binding moiety may include a particular sequence of nucleic acids.
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.
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 split into 384 droplets to which one of each pair has a detector moiety added. A subset of the 192 screening droplets to which the detector was not added are chosen for purification. For example 30 of the droplets may be used. Where the droplets already contain beads, the chosen droplets may be washed to separate the beads from the liquid. The immobilised material is eluted and the detector added to the eluate to enable the amount of eluted material to be measured. Alternatively the beads may be added to the chosen droplets to enable capture of the expressed protein from the expression solution. Again washing removes the CFPS reagents, and elution and addition of the detector enables the amount of eluted material to be measured.
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
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SUBSTITUTE SHEET (RULE 26) 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.
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.
The binding can be reversed using suitable buffers, for example containing biotin to release a strep tag or imidazole to release poly-his.
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.
The cell-free expression of peptides or proteins can use a cell lysate having the reagents to enable protein expression or a 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.
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.
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.
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SUBSTITUTE SHEET (RULE 26) By way of example, the screening reagents may include,
■ Chaperone mix (e.g., PUREfrex GroE mix)
■ Kinase 1 (e.g., NEB CK2)
■ Kinase 2 (e.g., NEB PKA)
■ Protease 1 (e.g., NEB TEW)
■ Protease 2 (e.g., Merck HRV 30)
■ Common metal ions cocktail
■ Common co-factors cocktail
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.
Any particular nucleic acid template 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.
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.
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.
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
25
SUBSTITUTE SHEET (RULE 26) 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.
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.
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.
Protein solubility sequences may be attached to the detector and the POI sequences. For example the GFP1.10 may be attached to further elements to improve solubility. The solubility enhancing sequence may be a peptide sequence or a 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:
26
SUBSTITUTE SHEET (RULE 26)
27
SUBSTITUTE SHEET (RULE 26)
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,
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SUBSTITUTE SHEET (RULE 26) SmURFP, 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-w. The peptide tag may be CFASTn or CFAST and the further polypeptide CFAST in the presence of a hydroxybenzylidene rhodanine analog. The peptide tag may be ccGFPn and the further polypeptide ccGFPi-i0.
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.
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.
The complementary GFPn peptide amino acid sequence tag could be the following:
1 . KRDHMVLLEFVTAAGITGT (SEQ ID NO: 9)
2. KRDHMVLHEFVTAAGITGT (SEQ ID NO: 10)
3. KRDHMVLHESVNAAGIT (SEQ ID NO: 11)
4. RDHMVLHEYVNAAGIT (SEQ ID NO: 12)
5. GDAVQIQEHAVAKYFTV (SEQ ID NO: 13)
6. GDTVQLQEHAVAKYFTV (SEQ ID NO: 14)
7. GETIQLQEHAVAKYFTE (SEQ ID NO: 15)
Or a truncated sequence thereof, 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.
Properties of the expressed protein may be characterised on the device. An initial screen may be based on the level of soluble expression by measuring fluorescence formed on complementation of a detector with the expressed sequence. The protein may remain fluorescent during immobilisation, at which point the level of affinity purification can be determined. Alternatively a non-fluorescent portion of the protein can be purified, and the detector added to measure the soluble yield of the purified material.
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SUBSTITUTE SHEET (RULE 26) 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 expression yield.
The methods can measure aggregation by measuring solubility before and after purification. For example,
1 . A mother droplet containing nucleic acid templates and protein expression reagents expresses the POI.
2. The mother droplet is split into multiple daughter droplets (either before, during or after expression).
3. A droplet containing detector is added to daughter droplet 1 .
4. A subset of the daughter 2 droplets is chosen to have beads added, binding the expressed protein.
5. The beads are washed to remove unbound material.
6. The POI is removed from the beads.
7. Another droplet containing detector is added to the eluted POI.
8. The complementations from (7) and (3) are run for the same period of time each.
9. The fluorescence is measured from both droplets and compared. Protein soluble yield is measured from the pre and post purification in order to determine purification yield.
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 detector solution can be mixed with an agent that arrests protein synthesis, such as a Mg++ chelator (EDTA) in order to arrest and detect all in one. Alternatively the chelator/EDTA can be introduced first to all daughter droplets and the detector introduced at different time points.
The screening and analysis can be performed in liquid reagent volumes, for example in microtitre plates or strip-tubes. Reagent volumes can be split such that portions are tested and portions retained for further use.
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
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SUBSTITUTE SHEET (RULE 26) 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.
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.
Digital microfluidics (DMF) refers to a two-dimensional planar surface platform for lab-on-a-chip systems that is based upon the manipulation of microdroplets. Droplets can be dispensed, moved, stored, mixed, reacted, or analyzed on a platform with a set of insulated electrodes. Digital microfluidics can be used together with analytical analysis procedures such as mass spectrometry, colorimetry, electrochemical, and electrochemiluminescense.
The droplet can be moved using any means of electrokinesis. The aqueous droplet can be moved using electrowetting-on-dielectric (EWoD). Electrowetting on a dielectric (EWoD) is a variant of the electrowetting phenomenon that is based on dielectric materials. During EWoD, a droplet of a conducting liquid is placed on a dielectric layer with insulating and hydrophobic properties. Upon activation of the electrodes the dielectric layer becomes less hydrophobic, thus causing the droplet to spread onto the surface.
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).
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.
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SUBSTITUTE SHEET (RULE 26) 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-free system having the components for protein expression to form the droplet.
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).
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 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 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 detector polypeptide is hindered in the presence of surfactant.
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SUBSTITUTE SHEET (RULE 26) 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/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. 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.
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 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 GFPn peptide amino sequence tag, 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.
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 ccGFPn peptide amino sequence tag and a Strep-tag, splitting the droplets into at least two volumes, merging droplets containing ccGFPi-io into one of the droplets following the expression process and measuring the uniformity of the fluorescent signal to determine the degree of aggregation of the expressed protein, taking corresponding droplets without ccGFPi-io
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SUBSTITUTE SHEET (RULE 26) which contain protein having been solubly expressed and adding magnetic beads which bind to the strep-tag, immobilizing and washing the beads to remove unbound material; eluting the POI by disrupting the strep binding, adding droplets containing ccGFPi-io following the elution process and measuring the fluorescent signal in order to determine the level of protein purification and determining the conditions where purified soluble protein is obtained.
Devices
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) 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 semi-conductor film whose electrical properties can be modulated by an optical signal.
EWoD phenomena occur when droplets are actuated between two parallel electrodes covered with a hydrophobic insulator or dielectric. The electric field at the electrode-electrolyte interface induces a change in the surface tension, which results in droplet motion as a result of a change in droplet contact angle. The electrowetting effect can be quantitatively treated using Young- Lippmann equation: cos0 - cos0o= (1/2yLG) 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 Er. Eo/t, where Er is dielectric constant of the insulator/dielectric, E0 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.
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).
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SUBSTITUTE SHEET (RULE 26) 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.
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.
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
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SUBSTITUTE SHEET (RULE 26) 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.
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.
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
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SUBSTITUTE SHEET (RULE 26) 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.
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.
The dielectric layer may comprise silicon dioxide, silicon oxynitride, silicon nitride, hafnium oxide, yttrium oxide, lanthanum oxide, titanium dioxide, aluminum oxide, tantalum oxide, hafnium silicate, zirconium oxide, zirconium silicate, barium titanate, lead zirconate titanate, strontium titanate, or barium strontium titanate. The dielectric layer may be between 10 nm and 100 pm thick. Combinations of more than one material may be used, and the dielectric layer may comprise more than one sublayer that may be of different materials.
The conformal layer may comprise a parylene, a siloxane, or an epoxy. It may be a thin protective parylene coating in between the insulating dielectric and the hydrophobic coating. Typically, parylene is used as a dielectric layer on simple devices. In this invention, the rationale for deposition of parylene is not to improve insulation/dielectric properties such as reduction in pinholes, but rather to act as a conformal layer between the dielectric and hydrophobic layers. The inventors find that parylene, as opposed to other similar insulating coatings of the same thickness such as PDMS (polydimethylsiloxane), prevent contact angle hysteresis caused by high conductivity solutions or solutions deviating from neutral pH for extended hours. The conformal layer may be between 10 nm and 100 pm thick.
The hydrophobic layer may comprise a fluoropolymer coating, fluorinated silane coating, manganese oxide polystyrene nanocomposite, zinc oxide polystyrene nanocomposite,
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SUBSTITUTE SHEET (RULE 26) precipitated calcium carbonate, carbon nanotube structure, silica nanocoating, or slippery liquid- infused porous coating.
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.
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.
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.
The method is particularly suitable for aqueous droplets with a volume of 1 pL or smaller.
The EWoD-based devices shown and described below are active matrix thin film transistor devices containing a thin film dielectric coating with a Teflon hydrophobic top coat. These devices are based on devices described in the E Ink Corp patent filing on “Digital microfluidic devices including dual substrate with thin-film transistors and capacitive sensing”, US patent application no 2019/0111433, incorporated herein by reference.
Described herein are electrokinetic devices, including:
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SUBSTITUTE SHEET (RULE 26) 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;
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;
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.
"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
39
SUBSTITUTE SHEET (RULE 26) 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.
“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
40
SUBSTITUTE SHEET (RULE 26) further facilitated by the use of hydrophilic and/or hydrophobic regions on surfaces and/or by physical obstacles.
For example 24 DNA constructs can be screened in 8 different conditions to determine the best construct and conditions for expression. The 24 constructs may be 24 independent sequences for different POIs or may be a single POI with 24 varying flanks (solubility tags etc) or a combination thereof (e.g 2 POI’s with 12 flanks, 3 POI’s with 8 flanks, 4 POI’s with 6 flanks etc). The 8 conditions may be lysates or reconstituted expression systems having different components or additives. 192 conditions may be screened in parallel. After identification of the best expression systems for a chosen POI, reagents/droplets can be selected for a purification screen. The screen may select for example 30 liquid volumes (droplets) to which beads are then added. The beads may be washed to remove unbound material. The POI’s may be eluted from the beads. Once detector reagent is added to the eluted POI’s, soluble material can be measured. A lack of signal indicates either the POI did not bind to the bead or was not eluted from the bead. If the detectable signal is seen in clumps or patches, the eluted POI has aggregated during purification/elution and is not a good candidate for scale-up. The conditions where a high level of uniform signal is seen can be chosen for a scale-up using the same identified conditions for expression and purification. The conditions giving the best purification can be scaled up in tubes off device using the desired volumes of reagents to allow sufficient amounts of soluble proteins to be obtained. The level of signal measured by the purification screen allows the correct scale up volume to be identified to obtain the desired mas of protein. Such a scale up may allow for example greater than 20 pg of protein in a 100 pL volume. At a suitable scale mg of protein may be obtained depending on the level of expression of the construct and the volume of solution used. The construct screen with different flank sequences allows placement of for example solubility tags at the C and/or N terminus along with varying configurations of the binding and detection tags, thereby increasing the likelihood that a particular POI can be expressed and purified in soluble form.
Experimental & Results
Figure 5 shows the experimental result from 24 different proteins expressed in a reconstituted cell-free protein synthesis system in droplets on an electrowetting on dielectric (EWoD) device. Each construct contains a GFPn tag. In the rows marked Screen, the GFP1.10 detector species is present from the start of expression. The rows marked Endpoint shows the fluorescence signal from 10 hours expression in the absence of GFP1.10 detector species followed by 5 hours complementation with the GFP1.10 detector species. This experiment showed significant
41
SUBSTITUTE SHEET (RULE 26) differences between expression/complementation in Screen BioInk compared to Endpoint detection. The detected protein clusters formed after expression only with endpoint detection mean that the protein aggregated after expression, thereby lowering the soluble yield. It is evident from the image the presence of speckles for several constructs, indicating the likely presence of protein aggregates. The level of aggregation enables identification of conditions which are worthy of further testing, and identification of conditions having high level of aggregated protein from which further purification is unlikely to give material.
VEGF Expression
Human vascular endothelial growth factor (VEGF) is a key regulator of angiogenesis and plays a central role in the process of tumor growth and metastatic dissemination. VEGF also has involvement with neovascular age-related macular degeneration and rheumatoid arthritis.
VEGF protein has previously been recombinantly produced in vivo using yeast, insect and mammalian cell expression systems. Expressing this protein in E.coli has proven to be challenging due to the inherent cysteine-knot motif in human VEGF proteins and can result in protein misfolding and the formation of inclusion bodies. Expressing soluble VEGF in E.coli is only possible in the presence of solubility fusion tags such as maltose-binding protein (MBP). Moreover, MBP-VEGF proteins can only express successfully in E.coli Origami 2 (CE3) strains which carry specific gene mutations that facilitate proper disulfide bond formation. Optimizing protein expression in E.coli cells can be laborious and time consuming, taking days to weeks to discover the ideal condition to express and purify soluble proteins. E.coli protein expression involves cloning the protein-of-interest in various expression plasmids encoding different solubility tags, trying expression in different E.coli cell lines and optimizing different expression conditions. The inventors have produced VEGF in a microfluidic system using cell-free reagents derived from E.coli.
Figure 8 shows an expression screen for protein VEGF with varying flank sequences. These 8 protein constructs were generated with different solubility tags attachment in either the N- or C- terminus. Strep II tag was included for purification purposes (Table 1):
42
SUBSTITUTE SHEET (RULE 26)
Table 1. DNA constructs corresponding to VEGF with various solubility tags in the N- and C- terminus.
An expression and solubility screen was first performed to determine the optimal expression of human VEGF using three different cell-free reagents in combination with constructs containing 8 different nucleic acid template variations corresponding to VEGF with various solubility tags in the N- and C- terminus. These three cell-free blends consist of different cell-free protein synthesis reagents. Blend-3 is designed to promote disulfide bond formation while Blend-4 contains chaperones that promote protein folding. Blend-2 contains the core component of the cell-free synthesis reagents without any additional components. Each construct was run in duplicate. 192 droplets were run in total on a single cartridge. The dotted lines denote the cell-free blends run with the corresponding constructs. The position of the detector (DET) is also noted. Bright white fluorescent spots indicate expression of soluble proteins.
Figure 9 shows a graphical representation of spot intensity in Figure 8. Concentration of proteins expressed is calculated based on fluorescent intensity measured. Construct A5 (SUMO_VEGF_DET) produced the highest expression in Blend-3. Constructs with the highest yield (mg/mL) were selected for on-cartridge purification (highlighted in green dashed box).
Figure 10 shows an image of thirty soluble candidates that presented the highest expression yield which were automatically selected and taken forward for an on-cartridge purification step. Among these candidates were SUMO-VEGF expressed in Blend-2, Blend-3 and Blend-4. These
43
SUBSTITUTE SHEET (RULE 26) expression candidates were purified on the cartridge using magnetic beads. Figure 5 shows the location of SUMO_VEGF_STREP_DET purified on the cartridge. Fluorescent intensity measurement was used to determine yield of the purified proteins.
Figure 11 shows a graphical representation of Figure 10 and shows a side by side comparison of the expression yield and purified yield for SUMO-VEGF-STREP_DET. The results show that this construct, expressed in all three cell-free blends, is soluble and can be purified. Blend-3 yields the highest protein expression and purified yield and therefore was chosen for off-cartridge scale up expression.
Linear DNA construct corresponding to SUMO-VEGF_Strep_DET was added to 500 pL of Blend- 3 expression reagent in tubes and incubated overnight. Expressed proteins were purified using magnetic beads. Sumo-tagged VEGF proteins were eluted in 100 pL elution buffer and the eluted proteins were >95% pure as seen in figure 12 SDS-PAGE gel analysis. The final purified yield of SUMO_VEGF is 32.1 pg of proteins obtained. Figure 12 shows a gel showing off cartridge, scaled-up expression (500 pL CFPS reaction) and purification of SUMO_VEGF_STREP_DET. SDS-PAGE illustrates the VEGF protein with a molecular weight slightly higher than the predictive weight of 36.32 kDa as the SUMO tag normally shows higher than its calculated molecular weight on an SDS-PAGE gel. Lane 1 molecular weight ladder, lane 2 Blend-3 negative control, lane 3 CFPS total after expression, lane 4 CPFS (soluble) obtained following centrifugation and soluble fraction obtained, lane 5 Unbound; flow through sample not bound to beads, lane 6 Purified (elute); sample eluted off of the resin bead.
VEGF protein validation data summary and conclusions
VEGF is a difficult candidate to express in the E.coli expression system. Expression of tag-less VEGF tends to end up with inclusion body formation. To obtain soluble proteins, expressing VEGF with multiple combinations of solubility tags is required to determine the right construct for successful expression and purification. The conventional cell-based expression and purification approaches can be time consuming and laborious, considering the variety of constructs that need to be tested. We have demonstrated that a droplet microfluidic platform enables rapid construct expression screening and selection of soluble, purifiable candidates. VEGF with N-terminus SUMO tag gave the highest expression yield in Blend-3 expression reagent. Results generated on the platform were validated through an off-cartridge cell free expression. The expressed
44
SUBSTITUTE SHEET (RULE 26) proteins were purified to high purity and yield; comparable to VEGF proteins produced in cellbased expression platforms.
CD19 expression screening and scale-up
B-lymphocyte antigen CD19 (CD19) is a transmembrane glycoprotein. The transmembrane domain of CD19 crosses the membrane with a single transmembrane helix. CD19 is involved in B cell signalling and is required for an optimal immune response. The N-terminal domain is extracellular and the C-terminal domain is cytoplasmic. The C-terminal domain of CD19 is required for the correct humoral immune response. CD19 has a role in autoimmune disorders making CD19 a target for treatment of rheumatoid arthritis and multiple sclerosis. Additionally, CD19 antibodies have a potential use as therapies for lymphoma, leukaemia, and autoimmune disorders. Therefore, production of CD19 would be beneficial to a pharmaceutical organisation.
Protein structure & design of nucleic acid templates
• CD19 is 556 amino acids in length.
• The first 20 amino acids of CD19 comprise a signal sequence.
• Uniprot reports a single transmembrane helix between residues 292-313.
• There is an N-terminal domain up to the transmembrane helix with structures available.
• The remaining sequence after the transmembrane domain has multiple disordered regions.
• The following truncations were selected for template synthesis: o Full length: 21-556 o N-terminal domain: 21-277 o C-terminal domain: 327-556
• Despite the transmembrane domain present in the protein, expression of the full length protein was attempted, however, it was expected that this would not have high expression.
• The N-terminal domain is the most likely candidate for obtainability. The X-ray structures including CD19 are of this domain suggesting it can be purified well and is stable.
• The N-terminal domain was not extended to the start of the transmembrane helix at residue 292. This was because the Alphafold structure suggested a small helix prior to the transmembrane helix. It was noted that this small helix contained an abundance of hydrophobic residues on the side towards the transmembrane helix. For this reason the truncation was stopped at residue 277, which was the end of the N-terminal domain.
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SUBSTITUTE SHEET (RULE 26) • The residue 327 was selected for the start of the C-terminal truncation as this was the residue at the end of the transmembrane helix. The C-terminal domain appears to be mostly disordered. It seemed unlikely that this protein would provide obtainable protein.
Templates for the 3 amino acid sequences for CD19 and each of seven solubility tags and a no solubility tag were prepared using the commercially available eGene Prep Kit NC3001 and associated protocol. Electrophoresis agarose gels indicated each of the 24 templates was obtained as a single band after purification to remove amplification primers.
Expression and purification screen was performed using Nuclera’s eProtein Discovery™ system.
As CD19 contains disulphide bonds in the N-terminal domain, the following additives were selected: o PDI+/GSSG - Chaperone and to support disulfide bond formation o GSSG - Redox modification to oxidizing environment o TRXB1 - acts as a reductase in redox regulation o 3C protease - cleavage of solubility tag o DnaK - Chaperone mix to support folding and prevent aggregation o Cofactor Mix - assist in folding, stability and activity o Calcium - C-terminal domain might have some interaction with Calcium.
• The following Core and additives pairs were selected: o Additive buffer + Additive buffer o GSSG + Calcium o PDI+/GSSG + 3C protease o PDI+/GSSG + Calcium o TRXB1 + GSSG o TRXB1 + Cofactor Mix o DnaK + GSSG o DnaK + Calcium
The expression system screens 192 sets of conditions to identify the highest expressing protein. A graph was created of the 192 expression screening results (Figure 13).
46
SUBSTITUTE SHEET (RULE 26) 30 conditions showing the highest level of expression (10 per protein variant) were selected for the purification screen. The predicted scale up results for purification are shown in Figure 14. Based on the purification screen, the following construct and expression pairs were selected for scale-up (Table 2):
Table 2: Predicted yields for a 200 l scale-up for CD19.
Scale-up
Scale-up purifications were performed using commercially available scale-up and additive kits from Nuclera (NC3004 & NC3005) and associated protocols for expression and bead based purification. After overnight incubation at 29°C, samples were purified using magnetic STREP beads. Samples of the total expressed material, bead flow through (FT) and bead elution were studied by SDS-PAGE (Figure 15).
The purity is shown in Figure 16a and the yield in Figure 16b. The 21-227 template had the best production and purification of the nucleic acid templates tested. However, the full length template was also purified despite there being a transmembrane domain present in the centre of the protein.
PARK7 expression screening and scale-up
PARK7 (also called DJ-1 or Parkinsonism associated deglycase) is a pleiotropic protein belonging to the peptidase C56 family. It acts as a positive regulator of androgen receptor-dependent transcription, redox-sensitive chaperone, sensor for oxidative stress, and apparently protects neurons against oxidative stress and cell death. Dysfunctions in PARK7 are related to autosomal recessive early-onset Parkinson disease 7 and cancer forms.
There are 2 potential isoforms mapped to the PARK7 as follows:
47
SUBSTITUTE SHEET (RULE 26) 1- K7EN27 (https://www.uniprot.Org/uniprotkb/K7EN27/entry#function)
2- K7ELW0 (https://www.uniprot.org/uniprotkb/K7ELW0/entry)
Amino acid sequences
PARK7_2-189
PARK7_lsoform_K7EN27_40-180
PARK7_lsoform_K7ELW0_2-64_85-180
Templates for the 3 amino acid sequences for CD19 and each of seven solubility tags and a no solubility tag were prepared using the commercially available eGene Prep Kit NC3001 and associated protocol. The PGR products for each sample were purified using NUCPure beads, then the purified eGene products were run on an agarose gel. Gel showed clean DNA constructs after PGR.
The DNA was quantified and the concentrations of each construct in pg/mL were converted to nM. Each sample was diluted to 5 nM using UMAMI elution buffer (HEPES 10 mM pH8, 0.05% F127) to make it compatible with the eProtein Discovery workflow. The constructs were stored at -20°C until use.
Expression and purification screen was performed using Nuclera’s eProtein Discovery™ system. The additives were selected based on the data from the reported literatures as shown in the screen image for PARK7 (Figure 17).
The expression system screens 192 sets of conditions to identify the highest expressing protein. A graph was created of the 192 expression screening results (Figure 18). The dots highlighted were selected for purification.
30 conditions showing the highest level of expression (10 per protein variant) were selected for the purification screen. The predicted scale up results for purification are shown in Figure 19. Based on the purification screen, the following construct and expression pairs were selected for scale-up (Table 3):
48
SUBSTITUTE SHEET (RULE 26) Table 3: Predicted yields for a 200 l scale-up for PARK7.
Droplet handling is shown in Figures 22-25.
Figure 22 (top panel) shows the expression reaction occurring in 12 columns of droplets in 16 rows. Each of the central 192 droplets is a different combination of nucleic acid template and expression blend (the edge droplets are controls and reagents for later steps in the process. The lower panel shows the 192 droplets split in half, and one portion of each droplet being mixed with the detector reagent.
Figure 23 shows the expression screening result. The top panel is white light, and shows all the droplets. The 192 droplets having the detector are fluorescent (shown in the fluorescent blue light image in the lower panel). The droplets without the detector are available for subsequent steps.
Figure 24 shows the processing step of droplet selection. 32 of the droplets are chosen for purification. The screened droplets, and the unselected droplets are all removed to leave only 32 droplets behind. These chosen droplets are then evenly spaced out on the device for a purification screen.
Figure 25 shows a purification screen. The droplets are bound to magnetic beads, which are repeatedly immobilised and washed with fresh wash buffer. The top panel shows the 32 bead locations and wash droplets. Two of the locations are purification and elution controls, thus enabling screening of 30 of the expression reactions. The bound material is eluted with an elution buffer to remove the protein from the beads. Detector droplets are added and the fluorescence measured. The blue light image is shown in the lower panel of Figure 25. Thus the expressed proteins which can best be purified and eluted are identified.
Scale-up:
Scale-up purifications were performed using commercially available scale-up and additive kits from Nuclera (NC3004 & NC3005) and associated protocols for expression and bead based purification. After overnight incubation at 29°C, samples were purified using magnetic STREP
49
SUBSTITUTE SHEET (RULE 26) beads. Samples of the total expressed material, bead flow through (FT) and bead elution were studied by SDS-PAGE (Figure 20). Arrows indicate the target proteins based on the absence of the band from the no DNA control and the expected molecular mass of the protein. The expected molecular mass for the purified samples were 30, 27, and 34 kDa respectively. As seen from the SDS-PAGE, the purity was good for the scaled-up proteins and was >80% by the Gelanalyzer.
Figure 21 shows the scale-up purifications of PARK7 as pg of obtained proteins.
The tested constructs were successfully obtained using the eProtein Discovery protocol, which could successfully predict the best conditions to obtain the protein in the chosen cell-free blends.
50
SUBSTITUTE SHEET (RULE 26)

Claims

1. A method for synthesising, characterising and purifying one or more proteins having detection tags and binding tags, the method comprising the steps of: i mixing a cell-free protein expression system with one or more nucleic acid templates to form a plurality of merged liquid volumes; either iia splitting the merged volumes into at least two aliquots and allowing protein expression in at least two aliquots; or iib allowing protein expression then splitting into at least two aliquots; iii adding a detector reagent which binds to the detection tags to at least one of the aliquots in order to determine the level of protein expression and determining the conditions where protein is expressed; iv selecting one or more of the volumes where protein has been expressed for purification using a solid support which binds to the binding tags; v separating the expressed proteins from the expression reagents using the binding tags; vi eluting the expressed protein from the solid support by disrupting the binding to the binding tags; and vii adding a detector reagent which binds to the detection tags to at least one of the eluted aliquots in order to determine the level of protein purification and determining the conditions where purified protein is obtained.
2. The method according to claim 1 , wherein the solid support is in the form of beads which are present during the expression step.
3. The method according to claim 1 for synthesising, characterising and purifying one or more proteins having detection tags and binding tags, the method comprising the steps of: i mixing a cell-free protein expression system with one or more nucleic acid templates to form a plurality of merged liquid volumes; either iia splitting the merged volumes into at least two aliquots and allowing protein expression in at least two aliquots; or iib allowing protein expression then splitting into at least two aliquots; iii adding a detector reagent which binds to the detection tags to at least one of the aliquots in order to determine the level of protein expression and determining the conditions where protein is expressed;
51
SUBSTITUTE SHEET (RULE 26) iv selecting one or more of the volumes where protein has been expressed and adding magnetic beads to a sub-population of the aliquots, wherein the beads bind to the binding tags; v separating the magnetic beads from the expression reagents; vi optionally adding wash reagents to the magnetic beads to remove unbound material; vii eluting the bound protein from the magnetic beads by disrupting the binding to the binding tag; and viii adding a detector reagent which binds to the detection tags to at least one of the eluted aliquots in order to determine the level of protein purification and determining the conditions where purified protein is obtained.
4. The method according to any one of claims 1 to 3, wherein the ratio of soluble and insoluble POI is determined in volumes where the detector reagent is added.
5. The method according to any one of claims 1 to 4, wherein the degree of aggregation is measured by counting the number of aggregates, the area of the aggregates, the intensity of the aggregates or by using a measurement of dispersion.
6. The method according to any one of claims 1 to 5, wherein the method is performed in droplets on a digital microfluidic device.
7. The method according to claim 6, wherein the digital microfluidic device comprises activematrix thin-film transistors.
8. The method according to any one of claims 1 to 7, wherein the solid support takes the form of discreet locations on the surface of the device over which the droplets are moved.
9. The method according to any one of claims 1 to 8, wherein each of the binding sequences contains four or more amino acids.
10. The method according to any one of claims 1 to 9, wherein the detector reagent is a component of a fluorescent protein.
11 . The method according to claim 10, wherein the protein of interest has a ccGFPn tag and the detector reagent comprises ccGFPi-i0.
52
SUBSTITUTE SHEET (RULE 26)
12. The method according to claim 11 , wherein the POI contains a ccGFPn peptide amino sequence tag selected from:
KRDHMVLLEFVTAAGITGT KRDHMVLHEFVTAAGITGT
KRDHMVLHESVNAAGIT
RDHMVLHEYVNAAGIT
GDAVQIQEHAVAKYFTV
GDTVQLQEHAVAKYFTV GETIQLQEHAVAKYFTE or a truncated version thereof.
13. The method according to any one of claims 1 to 12, wherein the POI is expressed with a solubility enhancer selected from:
53
SUBSTITUTE SHEET (RULE 26)
54
SUBSTITUTE SHEET (RULE 26)
14. The method according to any one of claims 1 to 13, wherein the expression is performed using cell-free lysates.
15. The method according to any one of claims 1 to 13, wherein the expression is performed using assembled components for transcription and translation in a system of purified recombinant elements (PURE).
16. The method according to any one preceding claim, wherein the expression is performed for at least 3 hours before the detector reagent is added to the droplets.
17. The method according to any one of claims 6 to 16, 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 onboard reagent reservoirs.
18. The method according to any one of claims 1 to 17, wherein the binding moiety is selected from:
Alfa-tag (SRLEEELRRRLTE)
Avi-tag (GLNDIFEAQKIEWHE)
55
SUBSTITUTE SHEET (RULE 26) 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)
Rho1 D4-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).
56
SUBSTITUTE SHEET (RULE 26)
19. The method according to claim 18, wherein the binding tag is Strep-tag II (WSHPQFEK) or poly His.
20. 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, splitting the droplets into at least two volumes, merging droplets containing ccGFPi-io into one of the droplets following the expression process and measuring the intensity of the fluorescent signal to determine the amount of the expressed protein, taking corresponding droplets without ccGFPi-io which contain protein having been expressed and adding magnetic beads which bind to the strep-tag, immobilizing and washing the beads to remove unbound material; eluting the POI by disrupting the strep binding, adding droplets containing ccGFPi-io following the elution process and measuring the fluorescent signal in order to determine the level of protein purification and determining the conditions where purified protein is obtained.
21 . 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.
22. The method according to any one preceding claim, wherein the device can separately manipulate at least 192 droplets.
23. The method according to any one preceding claim, wherein the device can manipulate magnetic beads in at least 30 locations.
57
SUBSTITUTE SHEET (RULE 26)
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