Improved fluorescent protein

Abstract

Methods and compositions for on-device detection of protein synthesis using fluorescent proteins are provided herein. The methods are applicable for monitoring on microfluidic devices. Split ccGFP variants have, at either end of their coding sequences, ccGFP 11 v1 peptide and can be used as a general method for the detection of a protein of interest (POI). A drawback of using the previously described split ccGFP system is the low solubility of the protein, which requires purification of the protein from inclusion bodies and limits the working concentration available for detection assays.

Description

Technical Field

[0001] Field of the Invention Methods and compositions for on-device detection of protein synthesis using fluorescent proteins are provided herein. The methods are applicable to monitoring on microfluidic devices.

Background Art

[0002] Background of the Invention When performing cell-free protein synthesis on the microfluidic scale in a microfluidic device (e.g., a digital microfluidic device), it is useful to detect the protein synthesized from the cell-free protein synthesis reaction in real time. However, it is difficult to perform real-time detection of proteins in the cell-free protein synthesis reaction environment. The reaction contains many other proteins and biomolecules at high concentrations, making non-specific protein detection via standard protein staining methods (e.g., Coomassie Brilliant Blue G-250, SYPRO TM Ruby, silver staining) difficult. Immunostaining or affinity-based purification, followed by non-specific protein staining, is similarly not useful. This is because a significant amount of washing on the solid support must be performed to prevent background interference. Since it is known that washing is difficult in microfluidic and digital microfluidic devices, background interference can be reduced.

[0003] Current existing luminescent complementation approaches often cannot achieve long-term real-time detection in cell-free protein synthesis reactions that exceed 16 hours. This limitation is due to a combination of reasons including O2 consumption by the luminescence-generating enzyme competing with the cell-free protein synthesis O2 requirement and temporary or permanent luminescent substrate depletion over 3 to 24 hours for recombinant protein expression detection.

[0004] The protein of interest can also be expressed as a fusion to a fluorescent protein (e.g., green fluorescent protein (GFP)). However, GFP is a 26.9 kDa protein, which is a typical size for most fluorescent proteins. A tag of this size increases the overall size of the protein of interest, especially when the protein of interest has to be tagged with another large fusion protein such as maltose binding protein (MBP) (which is 42.5 kDa). Considering that the average size of human proteins is about 52 kDa and the average size of E. coli proteins is about 35 kDa (Kim, Y. E. et al. Annu. Rev. Biochem. 2013. 82:323-355), the addition of a fluorescent protein tag of comparable size can significantly alter the biological function and biophysical properties of the protein.

[0005] Many prior arts disclose the use of sub-component tags for monitoring expression in cell systems. For example, U.S. Patent No. 7,666,606 discloses a protein-protein interaction detection system using microdomains.

[0006] Schinn et al. Biotechnol. Bioeng 114 10 October 2017 2412-2417. (https: / / onlinelibrary.wiley.com / doi / 10.1002 / bit.26305) discloses a rapid in vitro screening for the position-dependent effects of unnatural amino acids on protein expression and activity - Schinn - 2017 - Biotechnology and Bioengineering - Wiley Online Library. Split green fluorescent protein (GFP) is used in various applications. ccGFP is a green fluorescent protein engineered from a tetrameric GFP found in Corynactis californica, a cnidarian that forms pale red colonies similar to those of anemones and scleractinian stony corals, as described in Nguyen et al.; Nature Scientific Reports volume 11, Article number: 18440 (2021). Here, the inventors optimized the use of ccGFP for use in cell-free protein synthesis.

Prior Art Documents

Non-Patent Documents

[0007]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

Summary of the Invention

Means for Solving the Problems

[0008] Abstract The split ccGFP variants have ccGFP at either end of their coding sequences 11It can be used as a general method for detecting a protein of interest (POI) containing the v1 peptide. The drawback of using the previously described split ccGFP system is the low solubility of the protein, which requires purification of the protein from inclusion bodies and limits the working concentration available for detection assays.

[0009] Herein, the present invention describes an improved ccGFP variant amino acid sequence and its components, nucleic acids for its expression, and the use of the improved fluorescent protein. Mutations in the strands adjacent to strand 11 can be modified to improve the properties of the protein. Beta-barrel strands 3 and 10 are within the H-bonding interaction with strand 11. The changes should not destabilize GFP 1-10 :GFP 11 complementation and can alter the exposed hydrophobic core of split GFP, which is likely to cause insoluble expression.

[0010] ccGFP 1-11 The wild-type amino acid sequence is 221 amino acids (strand 3 is underlined):

Chemical formula

[0011] ccGFP having a mutation in the strand 3 region GQKTLKLRV (residues 40 - 48) 1-11 sequences are disclosed. ccGFP having a mutation at position K45 (which can be K45E) 1-11 sequences are disclosed. Amino acid sequences having position 45E with at least 90% homology to the following sequences are disclosed:

Chemical formula

[0012] The above sequences, in split form, for example, ccGFP 11 and ccGFP 1-10It can be used as such. The described sequences can be shortened so that amino acids can be removed from the N-terminus or C-terminus without affecting the activity or fluorescence of the folded core. When the sequence is shortened, the modified positions may change in numbering, and the reference to K45 refers to the wild-type sequence of 221 amino acids. The reference to strand 3 refers to amino acids 40 - 48 of the wild-type sequence of 221 amino acids.

[0013] Amino acid changes in strand 3 gave rise to promising positions for amino acid changes. One example of an amino acid position is K45. This can be changed to K45E. The strand 3 sequence can include GEQELELRV.

[0014] A specific ccGFP 1-10 The variant K45E is described. This is ccGFP 11 Once mixed, ccGFP 11 completes the β-sheet 11 that is bound and lost in ccGFP, generating a fluorescent signal that can be detected and quantified. This enables the detection of tagged proteins both in vitro and in vivo. The ccGFP 11 K45E protein variant described herein exhibits improved solubility when compared to the original ccGFP 1-10 parent. Solubility can be further improved by the addition of an N-terminal MBP fusion protein. 1-10 The modifications herein can improve the physical properties (e.g., solubility; complementation rate) of the above proteins without affecting other things (e.g., the color of the fluor; the sequence of the split strands, etc.). According to one example; the parent ccGFP

[0015] 1-10 ​When expressed, it is 5 - 10% soluble. The mutations described herein increased this to approximately 55%. Addition of MBP as a fusion protein further increased the solubility. The above parent protein (27 KDa) caused precipitation when concentrated beyond 1 mg / mL. Using the variants described herein, the mutant protein in its elution buffer can be concentrated to 65 mg / mL without significant precipitation. This higher concentration enables direct use for in - situ (real - time and end - point) detection in a small volume of liquid without the need for a large final dilution to add sufficient protein for end - point readings.

[0016] Here, the inventors report the use of an improved split - peptide system for fluorescence - based monitoring of the expression of a protein of interest in a cell - free protein synthesis reaction. The above monitoring can be performed in real - time and also provides end - point measurements.

[0017] A ccGFP variant containing the K45E mutation is disclosed herein. The above variant is 1-10 a variant or ccGFP 1-11 and can be a variant. The above ccGFP 1-10 variant is 11 and can be complexed with the ccGFP variant.

[0018] The above ccGFP 1-10 The K45E variant sequence can include the following sequence:

Chemical formula

[0019] The underlined E is the site of the change from wild - type K.

Chemical formula

[0020] The above-mentioned protein ccGFP K45E variant sequence may include a sequence having a homology higher than 95% with the following fixed E sequence:

Chemical formula

[0021] The above-mentioned protein is highly soluble. The above-mentioned mutated protein can be used at a concentration higher than 10 mg / mL. The above-mentioned mutated protein can be used at a concentration higher than 50 mg / mL. The above-mentioned mutated protein can be used at a concentration higher than 65 mg / mL. The above-mentioned protein can be bound to an additional sequence such as maltose-binding protein (MBP) to further increase its solubility.

[0022] A nucleic acid sequence encoding an amino acid having a K45E mutation is also disclosed. The above nucleic acid sequence may include:

Chemical formula

[0023] A method for detecting a target protein, the method including the steps of retrieving the target protein bound to ccGFP 11 , binding the above ccGFP 11 to the ccGFP 1-10 variant K45E, and monitoring the presence of the above target protein by detecting the fluorescence signal from the assembled ccGFP 1-11 is also disclosed. The use of the above ccGFP 1-10 variant K45E as a solubility tag to assist in the soluble expression of the target protein is also disclosed.

[0024] A method for monitoring cell-free protein synthesis in droplets on a digital microfluidic device, the method including a.ccGFP 11 Cell-free transcription and translation of a protein of interest fused to a peptide tag; and b. said ccGFP 11 A further ccGFP having a K45E amino acid change that generates a detectable signal in the presence of a peptide tag 1-10 Using the polypeptide to monitor the presence of said peptide tag, is disclosed herein.

[0025] The terms "in vitro" and "cell free" may be used interchangeably herein.

[0026] Any in vitro transcription and translation may be used (e.g., an extract-based system derived from rabbit reticulocyte lysate, human lysate, Chinese hamster ovary lysate, wheat germ, HEK293 lysate, E. coli lysate, insect lysate, yeast lysate).

[0027] Alternatively, said in vitro transcription and translation may be assembled from purified components, e.g., a purified recombinant elements (PURE) system.

[0028] Said in vitro transcription and translation may or may not be coupled.

[0029] The above sequences may have more than 90% homology with any of the sequences mentioned herein. The above sequences may have more than 95% homology with any of the sequences mentioned herein.

[0030] The above variant sequences may constitute more than 95% homology with the following sequences having one or more amino acid changes:

Chemical formula

[0031] The above sequences are the following fixed Ecan form a homology higher than 95% with the array:

Chem.

[0032] the above complementary GFP 11 The peptide amino acid sequence is as follows:

Chem.

[0033] GFP11 or GFP1-10 can be fused to the protein of interest through an amino acid linker. In one embodiment, the oligopeptide, peptide, or polypeptide linker can be 0 to 50 amino acids.

[0034] Nucleic acid sequences for expressing specific tags are also disclosed. The nucleic acid sequences include:

Chem.

[0035] These sequences can be repeated one or more times to generate proteins with multiple GFP 11 domains.

[0036] The above protein can be fused to multiple tags. For example, the above protein can be fused to multiple ccGFP 11 peptide tags, and the synthesis occurs in the presence of multiple ccGFP 1-10 polypeptides. The protein of interest can be fused to one or more sfCherry 11 peptide tags and one or more GFP 11 peptide tags, and the above synthesis can be carried out in the presence of one or more GFP 1-10 polypeptides and one or more sfCherry1-10 It occurs in the presence of a polypeptide.

[0037] Any protein of interest can be synthesized. The protein can be an enzyme, such as a terminal deoxynucleotidyl transferase (TdT) enzyme or a truncated version thereof or a homologous amino acid sequence of a terminal deoxynucleotidyl transferase (TdT) enzyme of another species, or a homologous amino acid sequence of any species of Polμ, Polβ, Polλ, and Polθ, or a homologous amino acid sequence of any species of X family polymerase.

[0038] The synthesis can be carried out in a microfluidic device, such as a dielectric electro-wetting (EWoD) device. Alternatively, the synthesis can be carried out in a microtiter plate format.

Brief Description of the Drawings

[0039]

Figure 1

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Figure 6

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Figure 10

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Figure 11

Mode for Carrying Out the Invention

[0050] Detailed Description of the Invention Here, the present inventors report the use of an improved split peptide system for fluorescence-based monitoring of the expression of a protein of interest in a cell-free protein synthesis reaction. The above monitoring can be performed on a device during the process of expression, and thus can be used in real-time measurement or as an endpoint measurement.

[0051] ccGFP variants containing one or more mutations in the β-strand adjacent to strand 11 are disclosed herein. ccGFP variants containing one or more mutations in β-strand 3 or 10 are disclosed. The amino acid positions thereof may include K45 located in strand 3. Strand 3 is amino acids 40 - 48, the sequence GQKTLKLRV. ccGFP having a mutation in the strand 3 region GQKTLKLRV (residues 40 - 48) 1-11 The sequence is disclosed.

[0052] ccGFP variants containing the K45E mutation are disclosed herein. Said variants are ccGFP 1-10 variants or ccGFP 1-11 and may be variants. Said ccGFP 1-10 variants may complex with ccGFP 11 variants.

[0053] A method for monitoring cell-free protein synthesis in droplets on a digital microfluidic device, said method comprising a. cell-free transcription and translation of a protein of interest fused to a peptide tag; and 11 b. using a further ccGFP polypeptide having a K45E amino acid change that generates a detectable signal in the presence of said peptide tag to monitor the presence of said peptide tag wherein said ccGFP 11 is disclosed herein. 1-10 1-10

[0054] Due to the increased solubility of said ccGFP 1-10 expression of the protein of interest fused to ccGFP 11 can be carried out in the presence of ccGFP 1-10 . Thus, monitoring of expression can be carried out in real time when GFP 1-11 assembles. Alternatively, said ccGFP 1-10 can be co-expressed.

[0055] Said peptide tag 11 can be a ccGFP

Chemical formula

[0056] Said tag should contain at least the region LQEHAVAK. Said tag should be at least 13 amino acids in length.

[0057] The nucleic acid sequences for the expression of the above tags can be the following shortened versions:

Chemical formula

[0058] The above tag sequence LQEHAVAK can be expressed using the following sequences:

Chemical formula

[0059] The protein sequences disclosed herein can be conjugated to additional elements to improve solubility. The above variants can be conjugated to one or more solubility enhancing sequences. The above solubility enhancing sequences can be peptide sequences or naturally occurring sequences. The above solubility enhancing sequences can be selected, for example, from maltose binding protein (MBP), small ubiquitin-like modifier (SUMO), glutathione S-transferase (GST) or thioredoxin (TRX). The above tags can be conjugated to either the C-terminus or the N-terminus or both the C-terminus and the N-terminus. Any example of a solubility enhancing factor can be used. A list of possible proteins is shown below. Any sequence selected from the following list can be selected:

Table 1-1

Table 1-2

[0060] For increasing the soluble yield of the protein expressed in the CFPS system, the use of soluble ccGFP as a solubility enhancing factor during the expression process is disclosed. The presence of a specific solubility enhancing factor during the above expression process increases the yield of soluble expression. The above protein can be expressed without the need for synthesis of a solubility factor as part of the amino acid sequence, the above ccGFP 1-10The detection polypeptide can be used as a solubility factor after the above protein is expressed. The binding of the solubility factor via the binding tag means that there is no need to wait for the expressed protein to dissolve immediately and for the expressed sequence on the POI to fold correctly to enhance solubility. Furthermore, the CFPS resources are not used to synthesize the solubility tag protein.

[0061] The applicant understands that the presence of a solubilizing factor that binds rapidly to a protein after the protein is expressed increases the soluble yield of a particular protein. A method for improving the soluble yield of a protein of interest (POI) expressed in a cell or cell-free expression system, the method comprising expressing the POI in the presence of a highly soluble ccGFP 1-10 moiety that binds to a binding sequence bound to the POI is disclosed. The above ccGFP 11 binding sequence can be bound to either the C or N terminus.

[0062] A method for cell-free expression of peptides or proteins in a digital microfluidic device is disclosed. Droplets having the components required for cell-free protein synthesis (CFPS, also known as in vitro protein synthesis) can be manipulated by electrokinetics to effect and improve protein expression.

[0063] Electrowetting is the modification of the wetting properties of a surface (which is typically hydrophobic) by the application of an electric field. Microfluidic devices for manipulating droplets or magnetic beads based on electrowetting have been widely described. In the case of droplets in a channel, this can be achieved by moving the droplet through a microfluidic channel defined by wells in a cartridge or microfluidic tubing, for example, in the presence of an immiscible carrier fluid. Electrodes covered with a dielectric layer are embedded in the wells of the cartridge or tubing. Each of these electrodes is connected to an A / C bias circuit that can be rapidly switched on and off at intervals to modify the electrowetting field characteristic of the layer. This results in the ability to manipulate the droplet along a predefined path.

[0064] As an alternative to a microfluidic channel system, droplets can also be generated and manipulated on a flat surface using digital microfluidics (DMF). In contrast to channel-based microfluidics, DMF utilizes alternating current to the electrode array to move fluids on the surface of the array. Liquids can thus be moved on an open-plan device by electrowetting. Digital microfluidics enables precise control over droplet movement, including droplet fusion and separation.

[0065] Cell-free protein synthesis (also known as in vitro protein synthesis or CFPS) is the generation of peptides or proteins that uses biological machinery in a cell-free system, i.e., without using living cells. The in vitro protein synthesis environment is not confined within a cell wall and is not limited by the conditions necessary to maintain cell viability, enabling the rapid generation of any desired protein from a nucleic acid template (usually plasmid DNA or RNA from in vitro transcription). CFPS has been known for decades and many commercial systems are available. Cell-free protein synthesis encompasses systems based on crude lysates (Cold Spring Harb Perspect Biol. 2016 Dec; 8(12): a023853) and systems based on reconstituted purified molecular reagents (e.g., the PURE system for protein production (Methods Mol Biol. 2014; 1118: 275-284)). CFPS requires fairly high concentrations of biopolymers (including DNA, RNA, proteins, polysaccharides, molecular crowding agents, etc.) (Febs Letters 2013, 2, 58, 261-268).

[0066] To date, digital microfluidics, dielectrophoretic electro-wetting (EWoD), and electrokinetics have generally found limited use in cell-free biology-based applications. In those cases, mostly due to biofouling, biological components (e.g., proteins, nucleic acids, crude cell extracts, and other bioproducts) adsorb to and / or denature on hydrophobic surfaces. Biofouling is well known in the art to limit the ability of EWoD devices to manipulate droplets containing biopolymers. Wheeler and colleagues reported that the maximum operating time of droplets on an EWoD device containing a biological medium is 30 minutes, after which biofouling inhibits EWoD-based droplet actuation (Langmuir 2011, 27, 13, 8586-8594).

[0067] Digital microfluidics can be performed in an air-filled system where droplets of liquid are manipulated on their surfaces in air. However, at high temperatures or over long periods, volatile aqueous droplets simply dry on the surface by evaporation. This problem is exacerbated by the high surface area to volume ratio of nanoliter and microliter sized droplets. Thus, air-filled systems are generally not suitable for protein expression where the temperature of the system needs to be maintained at a temperature appropriate for enzyme activity and the duration of synthesis needs to be long enough for the synthesized protein levels to be detectable.

[0068] Protein expression typically requires a sufficient supply of oxygen. The most convenient and high-yield method for powering CFPS is via oxidative phosphorylation where O2 serves as the final electron acceptor; however, there are other methods that involve replenishing energy molecules not involved in oxidative phosphorylation. In droplet-confined microfluidic or digital microfluidic systems, only insufficient oxygen is available to enable efficient protein synthesis.

[0069] Improved methods for enabling cell-free expression of peptides or proteins in digital microfluidic devices are described herein. A method for cell-free expression of peptides or proteins in a microfluidic device, where the method includes a cell-free system including one or more droplets containing a nucleic acid template (i.e., DNA or RNA) and components for protein expression in an oil-filled environment, and the method includes the step of moving the droplets using electrokinetics. The components for the cell-free protein synthesis droplets can be premixed before being introduced into the digital microfluidic device or can be mixed on the device.

[0070] The above droplets can be repeatedly moved for at least 30 minutes during the expression of the above protein. The above droplets can be repeatedly moved for at least 2 hours during the expression of the above protein. The above droplets can be repeatedly moved for at least 12 hours during the expression of the above protein. The act of moving the above droplets enables oxygen to be supplied to the above droplets and distributed throughout the above droplets. The act of moving improves the level of protein expression beyond that of droplets remaining stationary.

[0071] The above droplets can be moved using any means of electrokinetics. The above droplets can be moved using dielectrophoretic electro-wetting (EWoD). The electrical signal to the above EWoD or optical EWoD device can be delivered via a split electrode, an active matrix thin film transistor, or a digital micromirror.

[0072] The oil in the above device can be any water-immiscible liquid. The above oil can be mineral oil, silicone oil (e.g., dodecamethylpentasiloxane (DMPS)), an alkyl-based solvent (e.g., decane or dodecane), or a fluorinated oil. The above oil can be oxygenated before or during the expression process. Alternatively, the above device can be an air-filled device in which droplets containing cell-free protein synthesis reagents are rapidly moved to a predetermined position under a humidified gas, fixed to an array, and evaporation is prevented. Humidification can be achieved by enclosing or sealing the above digital microfluidic device and providing an on-board reagent reservoir. Further, humidification can be achieved by connecting an aqueous reservoir to the enclosed or sealed digital microfluidic device. The above aqueous reservoir can have a predetermined temperature or solute concentration (e.g., a saturated potassium sulfate solution at 30°C) to provide a specific relative humidity.

[0073] An oxygen source can be supplied to the above-mentioned droplets. For example, gaseous oxygen or droplets or bubbles containing dissolved oxygen can be combined with the above-mentioned droplets during protein expression. Furthermore, the oxygen source can be formed by oxygenating the above-mentioned oil used as the filler medium. It is well known in the art that oils (e.g., hexadecane, HFE-7500, etc.) can be oxygenated to support the oxygen requirements for cell growth, particularly E. coli cell growth (RSC Adv., 2017, 7, 40990-40995). Oxygenation can be achieved by aerating the above-mentioned oil with pure oxygen or air. Alternatively, the oxygen supply source can be a molecular supply source that releases oxygen. Alternatively, the above-mentioned droplets can be moved to the air / liquid interface to allow for increased diffusion of oxygen from the gas environment. Alternatively, the above-mentioned droplets can be presented in a humid air-filled device.

[0074] The above-mentioned droplets can be formed before entering the microfluidic device and flow into the device. Alternatively, the above-mentioned droplets can be combined on the device. A method is included that involves combining a first droplet containing a nucleic acid template (e.g., a plasmid) and a second droplet containing a cell-free extract having components for protein expression to form a combined droplet capable of cell-free protein synthesis.

[0075] The above-mentioned droplets can be divided on the device either before or after expression. A method is included herein that further involves the step of dividing the above-mentioned aqueous droplets into a plurality of droplets. If desired, the divided droplets can be screened with additional additives. A method is included in which one or more of the divided droplets are combined with additional droplets for screening.

[0076] Cell-free expression of peptides or proteins can use a cell lysate with reagents to enable protein expression. General components of a cell-free reaction include an energy source, an amino acid supply, cofactors (e.g., magnesium), and related enzymes. The cell extract is obtained by lysing the target cells and removing cell walls, DNA genomes, and other debris by centrifugation. The remainder is the cellular machinery including ribosomes, aminoacyl-tRNA synthetases, translation initiation and elongation factors, nucleases, etc. Once an appropriate nucleic acid template is added, the nucleic acid template can be expressed as a peptide or protein using the expression machinery derived from the cells.

[0077] Any specific nucleic acid template can be expressed using the systems described herein. Three types of nucleic acid templates used in CFPS include plasmids, linear expression templates (LETs), and mRNAs. Plasmids are circular templates, which can be generated either in cells or synthetically. LETs can be made via PCR. LETs are easier and quicker to make, but plasmid yields are usually higher in CFPS. mRNAs can be generated via in vitro transcription systems. The above methods use one nucleic acid template per droplet. The above methods can use multiple droplets having different nucleic acid templates per droplet.

[0078] The energy source is an important part of the cell-free reaction. Usually, a separate mixture containing the required energy source along with the amino acid supply 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 additional reagents to the droplets during the expression process.

[0079] Cell-free extracts having the components for protein expression contain, separate from the nucleic acid template, everything required for protein expression. Thus, the term includes all 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 the need for further reagents.

[0080] Thus, the cell lysate can be supplemented with further reagents before the template is added. Cell-free extracts having the components for protein expression are typically generated as bulk reagents or “master mix” that can be formulated into many identical droplets before a separate template is added separately to a separate droplet. Common cell extracts used today are made from E. coli (ECE), rabbit reticulocytes (RRL), wheat germ (WGE), insect cells (ICE), and yeast Kluyveromyces (D2P system). All of these extracts are commercially available.

[0081] Rather than being derived from cell extracts, the cell-free system can be assembled from the required reagents. Systems based on reconstituted purified molecular reagents are commercially available (e.g., the PURE system for protein production) and can be used as supplied. The PURE system is composed of all the enzymes involved in transcription and translation, and highly purified 70S ribosomes. The protein synthesis reaction of the PURE system lacks proteases and ribonucleases, which are often present as unwanted molecules in cell extracts.

[0082] The term, digital microfluidic device, refers to a device having a two-dimensional array of flat microelectrodes. The term excludes any device having simply droplets in a flow of oil in the channels. The droplets are moved across the surface by electrokinetic forces by the actuation of specific electrodes. Upon actuation of the electrodes, the dielectric layer becomes more hydrophobic and spreads the droplets on the surface. Digital microfluidic (DMF) device setups are known in the art and depend on the substrate used, the electrodes, the configuration of those electrodes, the use of dielectric materials, the thickness of the dielectric materials, the hydrophobic layer, and the voltage applied.

[0083] Once the CFPS reagent is encapsulated in the droplets, further reagent can be supplied by combining the original droplet with a second droplet. The second droplet can carry any desired further reagent (e.g., oxygen or an “electrical” source, or a test reagent desired to be exposed to the expressed protein).

[0084] The droplets can be aqueous droplets. The droplets can contain an oil-immiscible organic solvent (such as DMSO). The droplets can be a mixture of water and solvent, providing that the droplets are insoluble in bulk oil.

[0085] The droplets can be in a bulk oil layer. A dry gas environment simply dries the bubbles on the surface during the expression process, leaving a comet-type smear of the dried material by evaporation. Thus, the device is filled with liquid for the expression process. Alternatively, the aqueous droplets can be in a humid gas environment. A device filled with air can be sealed and humidified to provide an environment that reduces evaporation of the CFPS droplets.

[0086] Droplets containing a cell-free extract with components for protein expression are thus typically in an oil-filled environment before a nucleic acid template is added to the droplets. The template can be added by bringing droplets together on a microfluidic device. Alternatively, the template can be added to the droplets from outside the device and then flowed into the device for the expression process. For example, the expression process can be initiated on the device by raising the temperature. Expression systems typically function optimally at temperatures above standard room temperature (e.g., at or above 29 °C).

[0087] The expression process typically takes a long time. Thus, the process should be allowed to stand for at least 30 minutes or 1 hour, typically at least 2 hours. Expression can be allowed to stand for at least 12 hours. During the expression process, the droplets should be moved within the device. This moving step improves the process by mixing the reagents and ensuring that sufficient oxygen is available within the droplets. The moving step can be continuous or can be repeated with intervening periods of non-movement.

[0088] Thus, the aqueous droplets can be repeatedly moved for at least a period of 30 minutes or 1 hour while protein is being expressed. The aqueous droplets can be repeatedly moved for at least a period of 2 hours while protein is being expressed. The aqueous droplets can be repeatedly moved for at least a period of at least 12 hours while protein is being expressed. The act of moving the droplets allows for mixing within the droplets and allows oxygen or other reagents to be supplied to the droplets. The act of moving improves the level of protein expression beyond that of droplets that remain static.

[0089] A method is disclosed that includes the transcription of a nucleic acid sequence comprising the following sequence:

Chemical formula

[0090] The above array can be repeated to express a number of ccGFP 11 amino acid tags. Transcription can be performed in droplets on a digital microfluidic device. The fluorescence signal is obtained when the expressed protein containing the above ccGFP 11 tag complements the ccGFP 1-10 protein.

[0091] Digital microfluidics (DMF) refers to a two-dimensional flat surface platform for lab-on-a-chip systems based on the manipulation of microdroplets. Droplets can be dispensed, moved, stored, mixed, reacted, or analyzed on a platform having a set of insulated electrodes. Digital microfluidics can be used with analytical procedures such as mass spectrometry, colorimetry, electrochemistry, and electrochemiluminescence.

[0092] The above droplets can be moved using any means of electrokinetics. The above aqueous droplets can be moved using dielectrophoretic electro-wetting (EWoD). Dielectrophoretic electro-wetting (EWoD) is a variant of the electro-wetting phenomenon based on dielectric materials. During EWoD, droplets of a conductive liquid are placed on a dielectric layer having insulating and hydrophobic properties. Upon activation of the electrodes, the dielectric layer becomes non-hydrophobic, causing the droplets to spread on the surface.

[0093] Electrical signals to EWoD or optically activated amorphous silicon (a-Si) EWoD devices can be delivered via split 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).

[0094] The oil in the device can be any water-immiscible or hydrophobic liquid. The oil can be mineral oil, silicone oil (e.g., dodecamethylpentasiloxane (DMPS)), an alkyl-based solvent (e.g., decane or dodecane), or a fluorinated oil. The air in the device can be any humidified gas.

[0095] Through affinity tags (e.g., FLAG-tag, HIS-tag, GST-tag, MBP-tag, STREP-tag, or other forms of affinity tags), the CFPS-expressed protein can be immobilized on a solid support affinity resin, and a fresh batch of the CFPS reagent can be delivered onto the resin. Thus, the regenerated reagent can be used for protein synthesis, closely mimicking the industrial methods of continuous flow (CF) and continuous exchange (CE) CFPS. By mimicking CF- and CE-CFPS, the user can scale up those CFPS production methods.

[0096] The droplets can be actuated on the hydrophobic surface of a digital microfluidic device (ACS Nano 2018, 12, 6, 6050 - 6058). The hydrophobic surface is a hydrophobic surface (e.g., polytetrafluoroethylene (PTFE), Teflon® It can be AF (DuPont Inc), CYTOP (AGC Chemicals Inc), or FluoroPel (Cytonix LLC). The hydrophobic surface can be modified in a way that reduces biofouling, particularly biofouling resulting from exposure to CFPS reagents or nucleic acid reagents. The hydrophobic surface can also be superhydrophobic (e.g., NeverWet (NeverWet LLC) or Ultra-Ever Dry (Flotech Performance Systems Ltd)). Superhydrophobic surfaces prevent biofouling compared to typical fluorocarbon-based hydrophobic surfaces. Superhydrophobic surfaces thus extend the ability of digital microfluidic devices to move common solutions containing CFPS droplets and biopolymers (RSC Adv., 2017, 7, 49633-49648). The hydrophobic surface can also be a slippery liquid-infused porous surface (SLIPS). This can be formed by immersing a porous PTFE film in Krytox-103 oil (DuPont) (Lab Chip, 2019, 19, 2275).

[0097] The droplets can also contain additives to reduce the effect of biofouling on the digital microfluidic surface. Specifically, droplets containing CFPS components can also contain additives such as surfactants or detergents to reduce the effect of biofouling on the hydrophobic or superhydrophobic surface of the digital microfluidic device (Langmuir 2011, 27, 13, 8586-8594). Such droplets can use antifouling additives such as TWEEN® 20, Triton® X-100, and / or Pluronic® F127. Specifically, droplets containing CFPS components can contain 0.1% v / v of TWEEN® 20, 0.1% v / v of Triton® X-100, and / or 0.05% w / v of Pluronic® F127.

[0098] Regarding dielectrophoretic electro-wetting (EWoD), the change in the contact angle of a reagent upon application of a potential is an inverse function of the surface tension. Thus, for low-voltage EWoD operation, reduction of the surface tension is achieved by adding a surfactant to the reagent, which means for CFPS reactions, both in the lysate and for DNA. This results in dilution of the lysate, and in experiments, it has been observed that by diluting or otherwise contaminating the lysate, a decrease in the expression level of the target protein occurs. Thus, performing CFPS with DMF added to the solution in which the surfactant is being moved necessarily results in dilution and contamination of the lysate, and thus a decrease in the protein expression level. In addition to being a problem in itself, this further complicates the extrapolation of the on-DMF results to the estimation of protein yield in the tube. A further cause of loss due to the requirement to add surfactant to the sample is that this increases the time required for sample preparation and the potential for inconsistent results due to "user error", as there is more handling of the reagent. A further cause of loss due to the requirement to add surfactant to the sample is that certain downstream operations are hindered. For example, if the target protein is expressed in a cell-free system with a GFP 11 (or similar) peptide tag, its downstream complementation with a GFP1-10 (or similar) detection polypeptide is hindered in the presence of the surfactant. This is shown in Figures 8 and 9. Thus, removal of the surfactant from the aqueous phase is advantageous.

[0099] Rather than adding a surfactant to an aqueous sample, it is possible instead to add a surfactant (e.g., a sorbitan ester such as Span® 85 (e.g., sorbitan trioleate, Sigma Aldrich, SKU 8401240025)) to the oil. This has the advantage of allowing the CFPS reaction to proceed on DMF without dilution or contamination. Furthermore, it simplifies the sample preparation procedure in order to set up the reaction and increase ease of use and result consistency. Using 1% w / w Span® 85 in dodecane allows for CFPS reactions without dilution on DMF and detection of expressed non-fluorescent proteins without dilution. Other surfactants other than Span® 85 and oils other than dodecane can be used. A certain concentration range of Span® 85 can be used. The surfactant can be nonionic, anionic, cationic, amphoteric, or a mixture thereof. The oil can be a mineral oil or a synthetic oil (including silicone oil, petroleum oil, and perfluorinated oil). The surfactant can have a detrimental effect on (1) the above CFPS reaction and (2) the efficiency of the above detection system (when the above detection system includes complementation of a tag and a detection polypeptide). For example, by performing the CFPS reaction using an oil-surfactant mix on DMF, detection of the expressed protein can also proceed without dilution and without adding an aqueous surfactant. Since surfactants have been shown to reduce the efficiency of some detection systems (including, but not limited to, the Split ccGFP (e.g., ccGFP11 / ccGFP1-10) system), it can be beneficial to remove the surfactants from the reagent mix and add them to the oil instead.

[0100] The peptide tag can be attached to the C-terminus or N-terminus of the protein. The protein can be fused to a number of tags. For example, the protein can be fused to a number of ccGFP 11 and can be synthesized with a number of ccGFP 1-10It occurs in the presence of a polypeptide.

[0101] After expression, the protein can be purified on the device. A method is disclosed that includes the following: a. Removing a digital microfluidic device having a flat array of electrodes; b. Synthesizing a protein of interest having a binding tag in a droplet on the device; c. Capturing the protein via the binding tag, thereby immobilizing the protein; d. Using the electrodes to move the droplet, thereby removing the synthesized protein from the droplet; e. Optionally, washing the immobilized protein; and f. Optionally, releasing the protein into a further droplet.

[0102] The binding sub-tag can be a sub-component of a fluorescent protein. Thus, the fully assembled protein is fluorescent. For example, if the immobilized substance contains ccGFP 1-10 and the tag contains a ccGFP 11 peptide, complementation forms immobilized fluorescent ccGFP, enabling simultaneous monitoring and purification. The immobilized substance can be washed and then eluted by disrupting the complemented split ccGFP, for example, via the use of salt or temperature. Alternatively, the ccGFP 11 tag can be removed using a protease (e.g., TEV or 3C protease).

[0103] The protease can be selected from the following: TEV, C3, enterokinase (EK) light chain, factor Xa (FXA), furin (FN) or thrombin. Enterokinase (EK) cleaves the NNNNL motif. Factor Xa cleaves the I(E / D)GR motif. Furin cleaves the RXXR motif. Thrombin cleaves the LVPRGS motif. TEV protease is a cysteine protease that recognizes the sequence Glu-Asn-Leu-Tyr-Phe-Gln-(Gly / Ser) and cleaves between the Gln and Gly / Ser residues. C3 protease is a cysteine protease that recognizes Leu-Glu-Val-Leu-Phe-Gln / Gly-Pro (LEVLFQ / GP) and cleavage occurs between the Gln and Gly-Pro residues.

[0104] A method is disclosed that includes the following steps: a. Removing a digital microfluidic device having a flat array of electrodes; b. Synthesizing a protein of interest having one or more ccGFP 11 tags in a droplet on the device; c. Capturing the protein via the ccGFP 11 tag, thereby immobilizing the protein; d. Moving the droplet using the electrodes, thereby removing the synthesized protein from the droplet; e. Optionally, washing the immobilized protein; and f. Optionally, releasing the protein into a further droplet.

[0105] Immobilization can be performed using the immobilized ccGFP 1-10 Once immobilized via complementation, the protein becomes fluorescent. Thus, correctly expressed protein can be monitored and purified by complexing with the immobilized ccGFP 1-10 .

[0106] Affinity tags can be added to proteins so that the proteins can be purified from their crude biological sources using affinity techniques. The purification tag can be selected, for example, from a FLAG-tag, His-tag, GST-tag, MBP-tag, STREP-tag. The Flag® tag (also known as the DYKDDDDK-tag) is a common protein tag commonly used in affinity chromatography and protein research. The His tag is a polyhistidine string of amino acids and typically has a length of between 6 and 9 histidine amino acids.

[0107] The binding moiety for purification can contain 4 or more amino acids. The binding sequence can contain 4 to 30 amino acids. The binding moiety can be selected from the following: Alfa-tag (SRLEEELRRRLTE) Avi-tag (GLNDIFEAQKIEWHE) C-tag (EPEA) Calmodulin-tag (KRRWKKNFIAVSAANRFKKISSSGAL) Dogtag (DIPATYEFTDGKHYITNEPIPPK) E-tag (GAPVPYPDPLEPR) FLAG (DYKDDDDK) G4T (EELLSKNYHLENEVARLKK) HA (YPYDVPDYA) His (HHHHHH) Isopeptag (TDKDMTITFTNKKDAE) Lanthanide binding tag (LBT) (FIDTNNDGWIEGDELLLEEG) Myc (EQKLISEEDL) NE-tag (TKENPRSNQEESYDDNES) Polyglutamic acid-tag (EEEEEEE) Polyarginine-tag (RRRRRRR) Rho1D4-tag (TETSQVAPA) SBP-tag (MDEKTTGWRGGHVVEGLAGELEQLRARLEHHPQGQREP) Sdytag (DPIVMIDNDKPIT) SH3 (STVPVAPPRRRRG) SNAC (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).

[0108] After expression, the protein can be purified by binding to particles such as magnetic beads, for example. The above ccGFP 1-10 The species can be bound to a purification tag to enable purification of the expressed protein. The above ccGFP 1-10 The species can be bound to a solubility tag to further enhance the solubility of the expressed protein. ccGFP having one or more mutations in strand 3 bound to an affinity purification tag and a solubility tag 1-10 species are disclosed herein. The solubility tag can be selected from the following: [Table 2-1] [Table 2-2]

[0109] The above purification tags can be selected from the following: Alfa-tag (SRLEEELRRRLTE) Avi-tag (GLNDIFEAQKIEWHE) C-tag (EPEA) Calmodulin-tag (KRRWKKNFIAVSAANRFKKISSSGAL) Dogtag (DIPATYEFTDGKHYITNEPIPPK) E-tag (GAPVPYPDPLEPR) FLAG (DYKDDDDK) G4T (EELLSKNYHLENEVARLKK) HA (YPYDVPDYA) His (HHHHHH) Isopeptag (TDKDMTITFTNKKDAE) Lanthanide binding tag (LBT) (FIDTNNDGWIEGDELLLEEG) Myc (EQKLISEEDL) NE-tag (TKENPRSNQEESYDDNES) Polyglutamic acid-tag (EEEEEEE) Polyarginine-tag (RRRRRRR) Rho1D4-tag (TETSQVAPA) SBP-tag (MDEKTTGWRGGHVVEGLAGELEQLRARLEHHPQGQREP) Sdytag (DPIVMIDNDKPIT) SH3 (STVPVAPPRRRRG) SNAC (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).

[0110] ccGFP having one or more mutations in chain 3 conjugated to an affinity polyHis purification tag 1-10 Species are disclosed herein. ccGFP having one or more mutations in chain 3 conjugated to a P17 solubility tag 1-10 Species are disclosed herein. ccGFP having one or more mutations in chain 3 conjugated to a NEXT solubility tag 1-10 Species are disclosed herein. ccGFP having one or more mutations in chain 3 conjugated to an Fh8 solubility tag 1-10 Species are disclosed herein. ccGFP having one or more mutations in chain 3 conjugated to a Trx solubility tag 1-10 Species are disclosed herein. ccGFP having one or more mutations in chain 3 conjugated to a Mocr solubility tag 1-10 Species are disclosed herein. ccGFP having one or more mutations in chain 3 conjugated to a SNUT solubility tag 1-10 Species are disclosed herein. ccGFP having one or more mutations in chain 3 conjugated to a GST solubility tag 1-10 Species are disclosed herein. ccGFP having one or more mutations in chain 3 conjugated to a CusF solubility tag 1-10 Species are disclosed herein. ccGFP having one or more mutations in chain 3 conjugated to a MBP solubility tag 1-10 Species are disclosed herein. ccGFP having one or more mutations in chain 3 conjugated to a ZZ solubility tag 1-10Species are disclosed herein. ccGFP having one or more mutations in chain 3 linked to a polyHis purification tag and a solubility tag 1-10 Species are disclosed herein. In each case, the ccGFP species may have a modification (e.g., K45E) at K45.

[0111] ccGFP comprising the following sequence 11 Detection polypeptide species that bind thereto are disclosed herein:

Chemical formula

[0112] NEXT

Chemical formula

[0113] Fh8

Chemical formula

[0114] Trx

Chemical formula

[0115] Mocr

Chemical formula

Chemical formula

[0116] SNUT

Chemical formula

[0117] GST

Chemical formula

[0118] MBP

Chem.

[0119] CusF

Chem.

Chem.

[0120] ZZ

Chem.

[0121] Nucleic acid sequences for expressing the above-mentioned detection polypeptide species containing the following nucleic acid sequences are disclosed herein: MBP

Chem.

Chem.

[0122] P17

Chem.

[0123] NEXT

Chem.

[0124] Fh8

Chem.

[0125] MBP [Chemistry] [Chemistry]

[0126] TRX [Chemistry]

[0127] MOCR [Chemistry] [Chemistry]

[0128] SNUT [Chemistry]

[0129] GST [Chemistry] [Chemistry]

[0130] CusF [Chemistry]

[0131] ZZ [Chemistry] [Chemistry]

[0132] Device Manipulation of droplets by application of a potential can be achieved on an insulator or dielectric, or on an electrode covered with a series of insulators or dielectrics. Droplet manipulation as a result of the applied potential is known as electro-wetting. Electrokinetics occurs as a result of a non-uniform electric field that affects the hydrostatic equilibrium of a dielectric liquid (dielectrophoresis or DEP) or the change in the contact angle of a liquid on a solid surface (dielectric electro-wetting or EWoD). DEP can also be used to generate a force on polymerizable particles to induce their movement. Electrical signals can be transmitted to separated electrodes, transistors, an array of transistors, or a sheet of semiconductor film, the electrical properties of which can be adjusted by an optical signal.

[0133] The EWoD phenomenon occurs when a droplet is actuated between two parallel electrodes covered with a hydrophobic insulator or dielectric. The electric field at the electrode–electrolyte interface induces a change in surface tension, which results in movement of the droplet as a result of a change in the droplet contact angle. The electro-wetting effect can be quantitatively treated using Young–Lippmann's equation:

Equation

[0134] When a droplet is actuated by EWoD, there exists a set of two opposing forces acting on the droplet: the electro-wetting force induced by the electric field and the resistance force resulting from the interaction between the droplet and the filling medium and the friction of the contact line (reference), including the resistance force. The minimum voltage (threshold voltage) applied to balance the electro-wetting force and the sum of all resistance forces is variably the insulator / dielectric, (t / ε r ) 1 / 2 determined by the thickness-to-dielectric contact ratio. Therefore, in order to reduce the actuation voltage, it is required to reduce (t / ε r ) 1 / 2 (that is, increase the dielectric constant or decrease the thickness of the insulator / dielectric). In order to achieve low-voltage actuation, a thin insulator / dielectric layer 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 electro-wetting contact angle is large enough to drive the droplet. Therefore, most academic research reports the use of much higher voltages >100V for thick dielectric films (>3μm) that are easily fabricated to bring about electro-wetting.

[0135] However, high-voltage EWoD-based devices with thick dielectric films have greatly limited industrial applicability due to their limited droplet multiplexing capabilities. By using low-voltage devices including thin-film transistors (TFTs) and optically activated amorphous silicon layers (a-Si), a way has been opened for the industrial adoption of EWoD-based devices due to their greater flexibility in addressing electrical signals in a high multiplexing mode. The drive voltages of TFTs or optically activated a-Si are low (typically, <15V). For the adoption of low-voltage devices, there were technical challenges in depositing high-quality thin-film insulators / dielectrics, and thus, there are special needs to improve the fabrication and composition of thin-film insulator / dielectric devices.

[0136] Typically, the above electrodes (or 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 are composed of fluoropolymers (e.g., Teflon® AF 1600 or CYTOP). The thickness of this material as a hydrophobic coating on the dielectric is typically <100 nm and may have drawbacks in the form of pinholes or a porous structure; thus, it is particularly important that the insulator / dielectric be free of pinholes to avoid electrical short circuits. Teflon® has also been used as an insulator / dielectric, but has higher voltage requirements due to its low dielectric constant and the thickness required to make it pinhole-free. Other hydrophobic insulator / dielectric materials may include those based on polymer dielectrics (e.g., siloxanes, epoxies (e.g., SU-8), or parylenes (e.g., parylene N, parylene C, parylene D, or parylene HT)). Due to the minimum contact angle hysteresis and higher contact angle with aqueous solutions, Teflon® is still used as a hydrophobic top coat on these insulator / dielectric polymers. However, it is difficult to reliably produce a pinhole-free coating of <1 micron of parylene or SU-8; thus, the thickness of these materials is typically maintained at 2 - 5 microns at the expense of increased voltage requirements for electro-wetting. Conventional EWoD devices with parylene C have also been reported to be fragile and unstable to repeated droplet manipulation with cell culture media. Multilayer insulator devices with deposited metal oxide and parylene C films have been used to produce a stronger insulator / dielectric and enable operation at lower applied voltages. Inorganic materials (e.g., metal oxides and semiconductor oxides commonly used as "gate dielectrics" in the CMOS industry) have been used as the insulator / dielectric of EWoD devices. They offer the advantage of utilizing standard clean-room processes for thin film deposition (<100 nm).These materials are inherently hydrophilic, require additional hydrophobic coatings, and are susceptible to pinhole formation as a result of the thin film layer deposition process. When combined with the need for lower voltage operation of EWoD, recent development efforts have focused on (1) using materials with improved dielectric properties (e.g., using high permittivity insulators / dielectrics), and (2) optimizing the fabrication process to eliminate insulator / dielectric pinholes and avoid dielectric breakdown.

[0137] The operation of EWoD devices is impaired by contact angle saturation and hysteresis, which are thought to be caused by any one or combination of these phenomena: (1) charge trapping at the hydrophobic film or insulator / dielectric interface, (2) ion adsorption, (3) thermodynamic contact angle instability, (4) dielectric breakdown of the dielectric layer, (5) capacitance at the electrode - electrode - insulator interface (resulting from the double layer effect), and (6) fouling of the surface (e.g., by biopolymers). One of the detrimental effects of this hysteresis is the reduction of the operating life of EWoD - based devices.

[0138] Contact angle hysteresis is thought to be the result of charge accumulation at the interface or within the hydrophobic insulator after some operations. The required operating voltage increases due to this charging phenomenon, resulting in final catastrophic dielectric breakdown. The most likely explanation is that pinholes in the insulator / dielectric can bring the liquid into contact with the electrode and cause electrolysis. Electrolysis is further promoted by hydrophobic insulators that are prone to pinholes or porous.

[0139] Most of the studies to understand the contact angle hysteresis related to EWOD have been conducted in a short time frame and with low-conductivity solutions. Long-duration operation (e.g., >1 hour) and high-conductivity solutions (e.g., 1M NaCl) can produce several effects other than electrolysis. Ions in the solution can permeate through the hydrophobic coating (under the applied electric field) and interact with the underlying insulator / dielectric. Ion permeation can result in (1) a change in dielectric constant due to charge trapping (which is different from interfacial charging) and (2) a change in the surface potential of pH-sensitive metal oxides. Both can cause a reduction in the electro-wetting force for manipulating aqueous droplets, resulting in contact angle hysteresis. The inventors have previously found that damage from high-conductivity solutions reduces or renders electro-wetting on the electrodes ineffective by inhibiting the adjustment of the contact angle when an electric field is applied.

[0140] The electrokinetic device includes a first substrate having a matrix of electrodes, where each of the matrix electrodes is connected to a thin-film transistor, the matrix electrodes are covered at the top with a functional coating including a dielectric 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 including a top electrode; a spacer disposed between the first substrate and the second substrate to define an electrokinetic working space; and a voltage source operably connected to the matrix electrodes.

[0141] The dielectric layer includes 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 strontium barium titanate. The dielectric layer can be between 10 nm and 100 μm in thickness. Combinations of more than one material can be used, and the dielectric layer can include more than one sublayer of different materials.

[0142] The conformal layer may include parylene, siloxane, or epoxy. It can be a thin protective parylene coating between an insulating dielectric and a hydrophobic coating. Typically, parylene is used as a dielectric layer on simple devices. In the present invention, the theoretical basis for the deposition of parylene is not to improve the insulating / dielectric properties (e.g., reduction of pinholes), but rather to act as a conformal layer between the dielectric layer and the hydrophobic layer. The inventors have found that parylene prevents contact angle hysteresis caused by highly conductive solutions or solutions that deviate from neutral pH over a long period of time, in contrast to other similar insulating coatings of the same thickness such as PDMS (polydimethylsiloxane). The conformal layer can be between 10 nm and 100 μm in thickness.

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

[0144] The above element may include one or more of a plurality of array elements (each element includes an element circuit; a separate electrode; a thin-film semiconductor whose electrical properties can be adjusted by incident light; and a thin-film photoconductor whose properties can be adjusted by incident light).

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

[0146] The above electrokinetic device may include a controller for adjusting the voltage provided to the individual matrix electrodes. The electrokinetic device may include a plurality of scan lines and a plurality of gate lines, where each of the thin film transistors is connected to a scan line and a gate line, and the plurality of gate lines are operably connected to the controller. This enables each of the individual elements to be controlled individually.

[0147] The second substrate may also include a second hydrophobic layer disposed on the second electrode. The first and second substrates may be arranged such that the hydrophobic layer and the second hydrophobic layer face each other, thereby defining an electrokinetic working space between the hydrophobic layers.

[0148] The method is particularly suitable for aqueous droplets having a volume of 1 μl or less.

[0149] The EWoD-based devices shown and described below are active matrix thin film transistor devices that include a thin film dielectric coating having a Teflon® hydrophobic top coating. These devices are based on the devices described in the E Ink Corp patent of U.S. Patent Application 2019 / 0111433, filed under "Digital microfluidic devices including dual substrate with thin-film transistors and capacitive sensing" and incorporated herein by reference.

[0150] An electrokinetic device, A first substrate having a matrix of electrodes, where each of the matrix electrodes is connected to a thin film transistor, and the matrix electrodes are as follows: A dielectric layer in contact with the matrix electrode, A conformal layer in contact with the dielectric layer, and A hydrophobic layer in contact with the conformal layer, A first substrate having a top covered with a functional coating including ; A second substrate including a top electrode; A spacer disposed between the first substrate and the second substrate and defining an electrokinetic working space; and A voltage source operably connected to the matrix electrode, An electrokinetic device including is described herein.

[0151] An electrokinetic device, A first substrate having a matrix of electrodes, where each of the matrix electrodes is connected to a thin film transistor, and the matrix electrodes are as follows: One or more dielectric layers including silicon nitride, hafnium oxide, or aluminum oxide, in contact with the matrix electrodes, A conformal layer including parylene, in contact with the dielectric layer, and A hydrophobic layer in contact with the conformal layer, A first substrate having a top covered with a functional coating including ; A second substrate including a top electrode; A spacer disposed between the first substrate and the second substrate and defining an electrokinetic working space; and A voltage source operably connected to the matrix electrode, An electrokinetic device including is described herein.

[0152] The electrokinetic device as described can be used with other elements (e.g., a device for heating and cooling a reagent cartridge for introducing devices or reagents if needed).

Example

[0153] Example Preparation and analysis of amino acid variants ccGFP 1-10 v3 K45E and ccGFP1-10 A starting culture of v3(WT) ± MBP was generated. Colonies were selected and incubated overnight at 37°C. The above substances were dissolved and centrifuged at 21000 rcf for 10 minutes at 20°C. Its supernatant (SN) with soluble proteins was removed. The pellet (P) was resuspended with a needle and syringe and transferred to a new tube (insoluble protein). Protein gels were performed to measure the soluble (SN) and insoluble fractions (P): · Mix 5 μL of each of the soluble and insoluble samples and mix with 5 μL of protein loading buffer + DTT. · Heat the samples to 95°C for 5 minutes. · Transfer 4 μL of each sample to PAGE. · Run PAGE at 250 V for 20 minutes. · Stain the gel in Coomassie for 20 - 30 minutes.

[0154] The sample bands of PAGE under the above four general conditions were quantified with GelAnalyzer. The supernatant and pellet of each sample were added until it reached 100%, and then the relative ratio was calculated. The gel images are shown in Figure 2.

Table 3

Table 4

[0155] The above ccGFP 1-10 The v3 K45E variant is more soluble than v3 「WT」. C-terminal MBP fusion proteins with a rigid α-helical linker are also generally more soluble than the free protein alone. ccGFP 1-10 The soluble fraction of v3 K45E was 30 - 40%, but the MBP fusion protein of the above variant had the advantage of a detergent fraction and was 60 - 75%. The change from K45 to E45 gave an approximately 40% increase in solubility. The above MBP fusion ccGFP 1-10v3 K45E (NDET06) was prepared up to 130 mg / mL without any signs of precipitation.

[0156] Use of variants for CFPS on an electro-wetting device Method Variant MBP-K45E ccGFP 1-10 (NDET06) To analyze the detection characteristics, an electro-wetting device (eDrop) experiment was set up to express the protein with the ccGFP 11 tag.

[0157] A mixture of different nucleic acid templates was expressed in four different protein expression systems to test the levels of expression and detection. NDET06 is present in the expression buffer. A comparable experiment with unmodified ccGFP 1-10 is not possible because the detection protein is not even sufficiently soluble as an MBP conjugate.

[0158] The results can be shown in Figure 6. Each droplet contains the same amount of NDET06. The difference in fluorescence measures the variation in the expression level of the protein with the ccGFP 11 tag. Thus, the polypeptide for NDET06 detection can function as a real-time and endpoint measurement of protein expression.

[0159] To analyze the optimal NDET06 concentration in the expression system, further experiments were set up at four known concentrations of recombinant MBP-ccGFP 11 . The amount of the detection polypeptide was varied. The results of the fluorescence intensity from the droplets are shown in Figure 7. The signal from the detection polypeptide is dependent on the amount of the expressed protein. When the detection polypeptide is in excess, the signal is dependent on the amount of ccGFP 11 expression. The four bars on the left are the same as when the amount of ccGFP 11 is limited. When ccGFP 11 is in excess, the signal is limited by the amount of the detection protein. The four bars on the right are different and the ccGFP present11 depending on the amount of

[0160] Summary of findings · NDET06 can detect soluble proteins at a 1.5-fold molar excess of the ccGFP11-tagged protein. · NDET is very soluble and can be used directly in the expression system at concentrations up to at least 200 μM. The optimal concentration detected when NDET06 is added in the lysate is 50 μM. · Considering that the optimal NDET06 concentration has been established, the detection polypeptide can quantify soluble proteins up to at least 30 μM.

[0161] Conclusion Adding the modified K45E (NDET06) with an MBP fusion in the expression system enables the following: Since NDET06 detects soluble proteins from the start of expression, it reduces the time to results; As higher sensitivity is achieved as a CPFS reaction, the fluorescence signal is not diluted by the addition of NDET06; Since NDET06 and the expression system are loaded as one reagent, device loading is easy; Since NDET06 does not occupy space on the device as a separate reagent, it gives more space on the device.

[0162] ccGFP 11 Tag shortening Full-length and 18 truncated MBP-ccGFP 11 (v1) The purified DNA of the variant was used in a reconstituted in vitro transcription / translation reaction together with a negative (-DNA) control and a positive (ccGFP 11 (v1) purified protein) control. Aliquots of the samples were analyzed and the relative MBP-ccGFP 11 (v1) expression was determined relative to ccGFP 1-10Prior to complementation in PAGE, it was quantified by PAGE. Figures 9 and 10 show the above ccGFP 11 Show the effect of shortening the GETIQLQEHAVAKYFTE tag. The region GETIQ can be removed, leaving LQEHAVAKYFTE. The region YFTE can be removed, leaving GETIQLQEHAVAK. Up to three amino acids can be removed from each end, leaving IQLQEHAVAKY.

[0163] Expression and endpoint detection on an EWoD device Figure 11 shows the experimental results from 24 different proteins expressed in a cell-free protein synthesis system reconstituted in droplets on a dielectric electro-wetting (EWoD) device. Each construct contains a GFP 11 tag. In the row labeled Screen, the GFP 1-10 detection polypeptide species is present from the start of expression. The row labeled Endpoint is the fluorescence signal from 10 hours of expression in the absence of the GFP 1-10 detection polypeptide species, followed by 5 hours of complementation with the GFP 1-10 detection polypeptide species. This experiment showed a significant difference between expression / complementation in Screen BioInk compared to Endpoint detection. Only in endpoint detection, the detected protein clusters formed after expression mean that the protein aggregated after expression, thereby reducing the soluble yield. From the images, it is clear that there are spots for some constructs. This indicates that there may be protein aggregation. From the level of aggregation, it becomes possible to identify conditions worth further testing and conditions with high levels of aggregated protein that are likely to give little material from further purification. Such experiments are made possible by the high solubility of the modified ccGFP protein, meaning that concentrated droplets of the detection polypeptide can be integrated with the expressed protein droplets and the level of the expressed protein can be measured.

Claims

1. A ccGFP variant containing one or more mutations to the β strand 3.

2. The ccGFP variant according to Claim 1, containing the K45 mutation.

3. The ccGFP variant according to Claim 1, containing the K45E mutation.

4. ccGFP １－１０ The ccGFP variant according to any one of claims 1 to 3, which is a

5. The ccGFP １－１０ is the ccGFP １１ complexed with the ccGFP variant according to any one of claims 1 to 3.

6. The ccGFP K45E variant sequence has a sequence with a fixed E: 【Chemical 44】 An array having a homology higher than 95% or ccGFP １１ The variant according to claim 4, which binds to １１ and becomes fluorescent, and includes a C-terminal or N-terminal truncation thereof.

7. The ccGFP K45E variant sequence has the sequence: 【Chemical Formula 45】 The variant according to any one of Claims 1 to 6, containing the sequence.

8. The variant according to any one of Claims 1 to 7, further containing a solubility-enhancing sequence.

9. The solubility-enhancing sequence is selected from glutathione S-transferase (GST), small ubiquitin-like modifier (SUMO), maltose-binding protein (MBP), Fasciola hepatica 8 kDa antigen (FH8), thioredoxin (TRX), solubility-enhancing ubiquitas tag (SNUT), 17-kilodalton protein (SKP), monomeric bacteriophage T7 orc protein (MOC), E. coli secreted protein A (ESPA), T7 phage tail (P17), metal-binding protein (CUSF), or 53-amino acid long N-terminal extension sequence (NEXT). The variant according to Claim 8.

10. The variant according to Claim 9, containing a sequence selected from the following: 【Chemical 46】 【Chemical 47】 【Chemical 48】

11. The variant according to any one of Claims 1 to 10, where the protein is at a concentration greater than 10 mg / mL.

12. The variant according to any one of Claims 1 to 11, where the protein is at a concentration greater than 50 mg / mL.

13. A nucleic acid sequence encoding the amino acid sequence according to any one of Claims 1 to 10.

14. The nucleic acid sequence according to Claim 13, containing the following: 【Chemical 49】

15. A method for detecting a target protein, the method comprising the steps of extracting the target protein bound to ccGFP １１ , binding the ccGFP １１ to the ccGFP variant according to any one of claims 4 or 6 to 10, and monitoring the presence of the target protein by detecting the fluorescence signal from the assembled ccGFP １－１０ . １－１１ A method comprising the steps of:

16. A method for improving the soluble yield of a target protein (POI) to be expressed, the method comprising expressing the POI in the presence of ccGFP bound to the POI １１ The ccGFP according to any one of claims 4 or 6 to 10, which binds to the binding sequence １－１０ by expression in the presence thereof.

17. ccGFP １１ The expression of the protein of interest bound to １１ is performed in droplets on a digital microfluidic device, according to the method of claim 15 or claim 16.

18. ccGFP according to any one of claims 4 or 6 to 10 １－１０ expressing the protein bound to ccGFP in droplets on a digital microfluidic device in the presence of １１ and monitoring the presence of the target protein by detecting the fluorescence signal from the assembled ccGFP １－１１ The method according to claim 17, comprising the step of

19. The method according to Claim 17 or Claim 18, where the digital microfluidic device is an active matrix thin film transistor (AM-TFT)-based device.

20. The method according to any one of Claims 17 to 19, where the digital microfluidic device contains two parallel plates separated by spacers to define a fluid volume filled with a hydrophobic or non-ionic liquid.

21. The method according to claim 20, wherein the protein is present in an aqueous droplet in an oil layer and the hydrophobic or nonionic liquid contains a surfactant. **Claim 22** The method according to claim 21, wherein the surfactant is a sorbitan ester. **Claim 23** The method according to any one of claims 20 to 23, wherein the hydrophobic or nonionic liquid is decane, dodecane or dodecamethylpentasiloxane.

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