High throughput protein expression screening

Abstract

Provided herein are methods of cell-free protein synthesis, optimised cell-free protein synthesis (CFPS) reagents, and methods for optimising CFPS reagents to increase protein expression yields via high throughput screening of a selection of expression compositions in parallel using low volumes of reagents. The methods comprise the steps of adding enzymes for CFPS to a plurality of isolated volumes, wherein the isolated volumes are covered with a hydrophobic fluid, and adding a nucleic acid template in aqueous solution to the plurality of isolated volumes such that the aqueous layer sinks through the hydrophobic fluid layer and mixes with the previously provided enzyme composition. The methods are applicable to protein expression in tubes or plates using a hydrophobic fluid layer to prevent evaporation.

Description

[0001] High throughput protein expression screening

[0002] FIELD OF THE INVENTION

[0003] Provided herein are methods of cell-free protein synthesis, optimised cell-free protein synthesis (CFPS) reagents, and methods for optimising CFPS reagents to increase protein expression yields via high throughput screening of a selection of expression compositions in parallel using low volumes of reagents. The methods are applicable to protein expression in tubes or plates using a hydrophobic fluid layer to prevent evaporation.

[0004] BACKGROUND TO THE INVENTION

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

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

[0007] Cell-free protein synthesis, also known as in vitro protein synthesis or CFPS, is the production of peptides or proteins using biological machinery in a cell-free system, that is, without the use of living cells. The in-vitro protein synthesis environment is not constrained within a cell wall or limited by conditions necessary to maintain cell viability, and enables the rapid production of any desired protein from a nucleic acid template, usually plasmid DNA or RNA from an in- vitro transcription. CFPS has been known for decades, and many commercial systems are available. Cell-free protein synthesis encompasses systems based on crude lysate (Cold Spring Harb Perspect Biol. 2016 Dec; 8(12): a023853) and systems based on reconstituted, purified molecular reagents, such as the PURE system for protein production (Methods Mol Biol. 2014; 1118: 275-284). CFPS requires significant concentrations of biomacromolecules, including DNA, RNA, proteins, polysaccharides, molecular crowding agents, and more (Febs Letters 2013, 2, 58, 261-268). Reconstituted reagents for cell-free protein synthesis are commercially available. For example PUREfrex® kit is a reconstituted in vitro Coupled Transcription / Translation Systems, completely different from an E.coli extract S30 system. By adding DNA or mRNA that encodes the target protein to the reaction solution, proteins can be synthesized easily and quickly without using living cells(https: / / purefrex.genefrontier.com / ).

[0008] Cell-free protein synthesis requires expression for several hours, typically at temperatures higher than 20 °C. Under such conditions the aqueous phase typically evaporates, especially under conditions where small volumes of reagents are used. Described herein are methods of preventing evaporation and improved liquid handling techniques to enable high-throughput screening of CFPS conditions.

[0009] References relating to the use of small volumes of reagents for CFPS include for example the technical resource note from New England Biolabs (https: / / www.neb.com / en-gb / - sealed with adhesive films, hence transfer of further reagents after sealing is not possible.

[0010] SUMMARY

[0011] Disclosed herein are methods to prevent evaporation of aqueous reagents during biological assays. Disclosed is the use of a layer of hydrophobic fluid to which aqueous reagents can be added. The aqueous reagents sink into the hydrophobic fluid, and thereby are not exposed to atmospheric evaporation. Any biological assay may benefit from the use of the hydrophobic fluid layer to prevent evaporation of the aqueous layer. The assay may be cell-free protein synthesis (CFPS). The hydrophobic fluid is less dense than the aqueous reagents such that the aqueous reagents sink when placed on or in the hydrophobic fluid.

[0012] Cell-free protein synthesis requires a reagent composition containing a variety of different enzymes, along with a nucleic acid template which codes for the protein of interest. The enzyme reagents typically contain a core set of essential components which is required in all reactions (although the core can be enhanced with additives). The nucleic acid templates typically vary as they code for different amino acid sequences. The components require mixing in order to initiate the reactions. The components can be mixed in either orientation, either the nucleic acid templates or the expression core can be under the hydrophobic layer prior to addition of the other component(s).

[0013] Disclosed is a method for performing cell-free protein synthesis comprising: a) taking a plurality of isolated volumes of an aqueous composition containing a nucleic acid template, wherein the isolated volumes are covered with a hydrophobic fluid to prevent evaporation; b) adding a composition containing enzymes for cell-free protein synthesis (CFPS) in aqueous solution to each of the isolated volumes such that the aqueous layer sinks through the hydrophobic fluid layer and mixes with the nucleic acid templates; and c) allowing the cell-free protein synthesis reaction to proceed in the covered aqueous layers. Disclosed is a method for performing cell-free protein synthesis comprising: a) taking a plurality of isolated volumes of an aqueous composition containing enzymes for cell-free protein synthesis (CFPS), wherein the isolated volumes are covered with a hydrophobic fluid to prevent evaporation; b) adding a nucleic acid template in aqueous solution to each of the isolated volumes such that the aqueous layer sinks through the hydrophobic fluid layer and mixes with the enzyme composition; and c) allowing the cell-free protein synthesis reaction to proceed in the covered aqueous layers.

[0014] The assay may be performed for an extended period of time. The assay may be performed for at least 2 hours. The assay may be performed for at least 4 hours. The assay may be performed for at least 6 hours. The assay may be performed for at least 8 hours. The assays, having an upper hydrophobic layer that is non-volatile, can be open to the atmosphere and do not require any type of sealing to prevent aqueous evaporation. The lack of a physical seal or cover means that further reagents can be added during or after the expression process, for example to detect or purify the expressed protein.

[0015] The assay may be performed at a temperature higher than 20 °C. The assay may be performed at a temperature higher than 25 °C. The assay may be performed at a temperature in the range of 20 °C to 40 °C.

[0016] The expressed protein may be detected in order to monitor the level of expression. The expressed protein may contain a detector tag. The detector tag may be visualised using a detector moiety. The detector moiety can be present during expression, enabling real-time measurement, or may be added after completion of the expression (end-point measurement). The detector moiety can be added after expression using an aqueous composition which sinks through the hydrophobic fluid layer, and generates a signal on binding to the detector tag.

[0017] For example the expressed protein may contain a component of a fluorescent protein. The expressed protein may contain for example a ccGFPn or sfGFPn sequence. The detector moiety may be a sub-component of a fluorescent protein such as for example ccGFPi.10 or sfGFPi.10. The fluorescent protein could include sfGFP, ccGFP, GFP, eGFP, deGFP, frGFP, eYFP, eBFP, eCFP, Citrine, Venus, Cerulean, Dronpa, DsRED, mKate, mCherry, mRFP, FAST, SmURFP, miRFP670nano. For example the peptide tag may be GFPn and the further polypeptide GFP1-10. The peptide tag may be ccGFPn and the further polypeptide ccGFPi-10. The peptide tag may be one component of sfCherry. The peptide tag may be sfCherryn and the further polypeptide sfCherryi- . The peptide tag may be CFASTn or CFAST10 and the further polypeptide NFAST in the presence of a hydroxybenzylidene rhodanine analog.

[0018] A large number of screening conditions can be tested in parallel. Additives can be used to supplement the expression system. The aqueous composition containing enzymes for CFPS may be supplemented with additives to test expression in different conditions, for example one or more reagents selected from synthesized or isolated ribosomes, initiation factors, elongation factors, termination factors, aminoacyl-tRNA synthetases, methionyl tRNA transformylases, tRNAs, amino acids, ribonucleoside triphosphates, 10-formyl 5, 6,7,8- tetrahydrofolic acid (FD), salts, a polyethylene glycol, an allolactose, an aldohexose or a thiogalactopyranoside, buffers, surfactants, metal ions, chaperones, co-factors or additional protein components.

[0019] Additional protein components may be selected from chaperones, glycosylating enzymes, proteases, redox active enzymes, phosphorylases and kinases. Example may include a TEV or a 3C protease. Examples may include inorganic pyrophosphatase, nucleoside-diphosphate kinase or adenylate kinase.

[0020] The additives may be added by allowing a composition containing the additives to sink through the hydrophobic fluid layer. Where for example the assays are performed in tubes or wells, different additives or mixtures of additives or volumes of additives may be added to different tubes. Thus varying conditions can be screened in parallel.

[0021] The assays described herein typically require expensive reagents and therefore minimal usage of reagent volume is desirable. Assays may be performed in less than 10 pL of aqueous reagents. Assays may be performed in less than 1 pL of aqueous reagents. The assays may be performed in open plates without a physical cover. The presence of the hydrophobic fluid being sufficient to prevent evaporation. The assays may be performed for example in a microtitre plate, such as a 384 or 1536 well plate. The assays are performed in isolated enclosures. The isolated volumes containing the reagents may have tapered sides such that the aqueous reagents descend to the narrowest part of the enclosure.

[0022] The hydrophobic fluid should be immiscible with the aqueous layer and lighter (less dense) than the aqueous layer. The hydrophobic fluid may be a silicone oil. The hydrophobic fluid may be a fluorous layer. The hydrophobic fluid may be an organic hydrocarbon. The hydrophobic fluid may optionally contain a surfactant. The hydrophobic fluid may be dodecamethylpentasiloxane (DMPS), decane or dodecane.

[0023] CFPS conditions typically benefit from the presence of oxygen. The hydrophobic fluid may be oxygenated prior to or during the process of expression, providing that the oxygenation does not enhance evaporation.

[0024] After expression the protein may be purified, for example using beads or solid supports. The beads may be magnetic or paramagnetic beads. The expressed proteins may be purified using binding tags (i.e. the expressed protein has a binding tag which allows binding to a solid support). The binding / purification tag may be selected from:

[0025] Alfa-tag (SRLEEELRRRLTE) (Seq ID 1)

[0026] Avi-tag (GLNDIFEAQKIEWHE) (Seq ID 2)

[0027] C-tag (EPEA) (Seq ID 3)

[0028] Calmodulin-tag (KRRWKKNFIAVSAANRFKKISSSGAL) (Seq ID 4)

[0029] Dogtag (DIPATYEFTDGKHYITNEPIPPK) (Seq ID 5)

[0030] E-tag (GAPVPYPDPLEPR) (Seq ID 6)

[0031] FLAG (DYKDDDDK) (Seq ID 7)

[0032] G4T (EELLSKNYHLENEVARLKK) (Seq ID 8)

[0033] HA (YPYDVPDYA) (Seq ID 9)

[0034] His (HHHHHH) (Seq ID 10)

[0035] Isopeptag (TDKDMTITFTNKKDAE) (Seq ID 11) lanthanide binding tag (LBT) (FIDTNNDGWIEGDELLLEEG) (Seq ID 12)

[0036] Myc (EQKLISEEDL) (Seq ID 13)

[0037] NE-Tag (TKENPRSNQEESYDDNES) (Seq ID 14)

[0038] Poly Glutamate-tag (EEEEEEE) (Seq ID 15)

[0039] Poly Arginine-tag (RRRRRRR) (Seq ID 16) Rho1 D4-tag (TETSQVAPA) (Seq ID 17)

[0040] SBP-tag (MDEKTTGWRGGHVVEGLAGELEQLRARLEHHPQGQREP) (Seq ID 18) Spytag (DPIVMIDNDKPIT) (Seq ID 19) SH3 (STVPVAPPRRRRG) (Seq ID 20) SNAC (GSHHW) (Seq ID 21)

[0041] Snooptag (KLGDIEFIKVNK) (Seq ID 22)

[0042] Softag 1 (SLAELLNAGLGGS) (Seq ID 23)

[0043] Softag 3 (TQDPSRVG) (Seq ID 24)

[0044] Spot-tag (PDRVRAVSHWSS) (Seq ID 25)

[0045] Spytag (AHIVMVDAYKPTK) (Seq ID 26)

[0046] S-tag (KETAAAKFERQHMDS) (Seq ID 27)

[0047] Strep-tag (AWAHPQPGG) (Seq ID 28) (AWRHPQFGG) (Seq ID 29)

[0048] Strep-tag II (WSHPQFEK) (Seq ID 30)

[0049] T7tag (MASMTGGQQMG) (Seq ID 31)

[0050] TC-tag (EVHTNQDPLD) (Seq ID 32)

[0051] Ty-tag (CCPGCC) (Seq ID 33)

[0052] VSV-tag (YTDIEMNRLGK) (Seq ID 34)

[0053] Xpress-tag (DLYDDDDK) (Seq ID 35).

[0054] The expression reagents may be made using an expression system reconstituted from purified components. Alternatively the expression reagents may be cell lysates, for example derived from mammalian cells prokaryotic cells, yeast cells, plant cells or protozoa. The cell lysates may be derived from human embryonic kidney cells (HEK293), Chinese hamster ovary cells (CHO), HeLa, BHK21 , NSO, or Sp2 / 0 cells. The cell lysates may be derived from Escherichia coli cells, Saccharomyces cerevisiae or Pichia pastoris cells, tobacco or wheat cells, or Leishmania tarentolae. The expression mix may be a mix of reconstituted and lysate. The lysate may be obtained from HEK293, HeLa, BHK21 , NSO, Sp2 / 0, or CHO or Escherichia coli.

[0055] Also disclosed is a microtitre plate wherein each well of the plate contains reagents for cell- free protein synthesis and a layer of hydrophobic fluid. Upon addition of a nucleic acid template, the cell-free protein expression is initiated once the template mixes with the enzymes for CFPS. The microtitre plate can be prepared and stored without the nucleic acid template as a ready to use expression screening system which starts once the template is added.

[0056] FIGURES

[0057] Figure 1 shows loading a single well of a plate. The wells would all be loaded as shown, either in series or parallel. The wells may be loaded using a manual pipette or using an automated liquid handling system. The wells may be loaded with the hydrophobic layer first, followed by the first aqueous composition, or may be loaded first with the first aqueous composition followed by the hydrophobic fluid layer. After the first aqueous compositions under the hydrophobic fluid layer are obtained the second aqueous compositions are added. The second aqueous composition sinks through the hydrophobic fluid to mix with the first composition.

[0058] Figure 2 shows results from cell-free protein expression using reactions were set up using different orders of addition of oil, Core, and DNA.

[0059] DETAILED DESCRIPTION OF THE INVENTION

[0060] Disclosed herein is a composition for the cell-free synthesis of proteins and use thereof.

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

[0062] Disclosed herein is a method using a hydrophobic fluid layer to prevent evaporation. The hydrophobic fluid layer can be used as an open system to which aqueous reagents are added. The aqueous reagents sink into the hydrophobic fluid and are thus isolated from atmospheric evaporation.

[0063] Disclosed is a method for performing cell-free protein synthesis comprising: a) taking a plurality of isolated volumes of an aqueous composition containing enzymes for cell-free protein synthesis (CFPS), wherein the isolated volumes are covered with a hydrophobic fluid to prevent evaporation; b) adding a nucleic acid template in aqueous solution to each of the isolated volumes such that the aqueous layer sinks through the hydrophobic fluid layer and mixes with the enzyme composition; and c) allowing the cell-free protein synthesis reaction to proceed in the covered aqueous layers. The loading may use a manual pipette, either single channel or multi-channel or using an automated liquid handling system. The volumes may be loaded with the hydrophobic fluid layer first, followed by the first aqueous composition, or may be loaded first with the first aqueous composition followed by the hydrophobic fluid layer. After the first aqueous compositions under the hydrophobic fluid layer are obtained the second aqueous compositions are added. The second aqueous composition sinks through the hydrophobic fluid to mix with the first composition.

[0064] Protein expression, particularly using cell lysates, typically requires an ample supply of oxygen. The most convenient and high yielding way to power CFPS is via oxidative phosphorylation where O2 serves as the final electron acceptor; however, there are other ways that involve replenishing with energy molecules not involved in oxidative phosphorylation. In a confined microfluidic system of droplets, insufficient oxygen is available to enable efficient protein synthesis. The hydrophobic fluid layer can contain sufficient oxygen to enable protein expression. The hydrophobic fluid is exposed to the atmosphere, and thus should enable oxygen permeation via the hydrophobic fluid whilst minimising evaporation.

[0065] The hydrophobic liquid may be a hydrophobic or non-ionic liquid. For example the hydrophobic liquid may be decane or dodecane. The hydrophobic liquid may be a silicone oil such as dodecamethylpentasiloxane (DMPS). The hydrophobic liquid may contain a surfactant, for example a sorbitan ester such as Span 85. The oil can be oxygenated prior to or during the expression process.

[0066] The cell-free expression of peptides or proteins can use a cell lysate having the reagents to enable protein expression. Common components of a cell-free reaction include an energy source, a supply of amino acids, cofactors such as magnesium, and the relevant enzymes. A cell extract is obtained by lysing the cell of interest and removing the cell walls, DNA genome, and other debris by centrifugation. The remains are the cell machinery including ribosomes, aminoacyl-tRNA synthetases, translation initiation and elongation factors, nucleases, etc. Once a suitable nucleic acid template is added, the nucleic acid template can be expressed as a peptide or protein using the cell derived expression machinery.

[0067] 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 additional protein components may be involved in any post-translational modification process, for example methylation, ubiquitinylation, sumoylation, isoprenylation or glycosylation.

[0068] Common post-translation modifications include phosphorylation, methylation, sulfation, acetylation, ubiquitylation, prenylation, myristoylation, SUMOylation, palmitoylation, different types of glycosylation (N-glycosylation, O-glycosylation, C-glycosylation and S-glycosylation), phosphoglycosylation and glycosylphosphatidylinositol (GPI anchored).

[0069] Protein phosphorylation is an important reversible regulatory mechanism that plays a key role in the activities of many enzymes, membrane channels and many other proteins in prokaryotic and eukaryotic organisms. Phosphorylation target sites are Ser, Thr, Tyr, His, Pro, Arg, Asp and Cys amino acid residues, but mainly happens on Ser, Thr, Tyr and His residues. Phosphorylation involves transferring a phosphate group from adenosine triphosphate to the receptor residues by kinase enzymes. Conversely, dephosphorylating or removal of a phosphate group is an enzymatic reaction catalyzed by phosphatases. Phosphorylation can change the function of proteins via one of the two principal ways: by allostery or by binding to interaction domains.

[0070] Acetylation is typically catalyzed via lysine acetyltransferase (KAT) and histone acetyltransferase (HAT) enzymes. Acetyltransferases use acetyl CoA as a cofactor for adding an acetyl group (COCH3) to the c-amino group of lysine side chains, whereas deacetylases (HDACs) remove an acetyl group on lysine side chains. Forms of acetylation include Na- acetylation, Nc-acetylation and O-acetylation, and may occur on Lys, Ala, Arg, Asp, Cys, Gly, Glu, Met, Pro, Ser, Thr and Vai residues with different frequencies, although the acetylation is more reported on Lysine residue. Ne-acetylation is more biologically significant compared to the other types of acetylation. Acetylation has an essential role in biological processes such as chromatin stability, protein-protein interaction, cell cycle control, cell metabolism, nuclear transport and actin nucleation.

[0071] Ubiquitylation is an important reversible PTM and can occur on all 20 amino acids, however, it occurs on lysine more frequently. This PTM has a major role in the degradation of intracellular proteins via the ubiquitin (Ub)-proteasome pathway. Ubiquitylation is catalyzed by an enzyme complex that contains ubiquitin-activating (E1), ubiquitin-conjugating (E2) and ubiquitin ligase (E3) enzymes. Ubiquitinated proteins may be acetylated on Lys, or phosphorylated on Ser, Thr or Tyr residues. Ubiquitylation modification in substrate proteins can be removed by several specialized families of proteases called deubiquitinases. Ubiquitination plays important roles in stem cell preservation and differentiation by regulation of the pluripotency, and plays a role in many various cell activities such as proliferation, regulation of transcription, DNA repair, replication, intracellular trafficking and virus budding, the control of signal transduction, degradation of the protein, innate immune signaling, autophagy and apoptosis.

[0072] Methylation is a reversible PTM, which often occurs in the cell nucleus and on the nuclear proteins such as histone proteins. Methylation occurs on the Lys, Arg, Ala, Asn, Asp, Cys, Gly, Glu, Gin, His, Leu, Met, Phe and Pro residues in target proteins, with lysine and arginine the two main target residues, at least in eukaryotic cells. One important role of methylation is in histone modification. Histone proteins, after synthesis of their polypeptide chains, are methylated at Lys, Arg, His, Ala or Asn residues. NMysine methylation is one of the most abundant histone modifications in eukaryotic chromatin, involves transferring the methyl groups from S-adenosylmethionine to histone proteins via methyltransferase enzyme. In eukaryotes, methylated arginine has been observed in histone and non-histone proteins. Recent studies have shown that methylation is associated with fine tuning of various biological processes ranging from transcriptional regulation to epigenetic silencing via heterochromatin assembly.

[0073] Glycosylation occurs in multiple subcellular locations, such as endoplasmic reticulum, the Golgi apparatus, cytosol and the sarcolemma membrane. Glycosylation occurs in eukaryotic and prokaryotic membranes and secreted proteins, and nearly 50% of the plasma proteins are glycosylated. In this modification, oligosaccharide chains are linked to specific residues by a covalent bond. This enzymatic process, which is catalyzed by a glycosyltransferase enzyme, usually occurs in the side chain of residues such as Trp, Ala, Arg, Asn, Asp, lie, Lys, Ser, Thr, Vai, Glu, Pro, Tyr, Cys and Gly; however, it occurs more frequently on Ser, Thr, Asn and Trp residues in proteins and lipoproteins. According to the target residues, glycosylation can be classified into six groups: N-glycosylation, O-glycosylation, C-glycosylation, S-glycosylation, phosphoglycosylation and glypiation (GPI-anchored). N-glycosylation and O-glycosylation are two major types of glycosylation and have important roles in the maintenance of protein conformation and activity. Glycosylation has a great role in many important biological processes such as cell adhesion, cell-cell and cell-matrix interactions, molecular trafficking, receptor activation, protein solubility effects, protein folding and signal transduction, protein degradation, and protein intracellular trafficking and secretion.

[0074] Small Ubiquitin-Related Modifier (SUMO) ylation has been discovered in a wide range of eukaryotic organisms. SUMOylation can occur in both cytoplasm and nucleus on lysine residues. SUMOylation occurs as a modifier in e-amino group of lysine residues in target protein through a multi-enzymatic cascade. In this reaction, SUMO is connected to a lysine residue in substrate protein by covalent linkage via three enzymes, namely activating (E1), conjugating (E2) and ligase (E3). Often, SUMOylation modifications occur at a consensus motif WKxE (where W represents Lys, He, Vai or Phe and X any amino acid). SUMOylation plays a major role in many basic cellular processes like transcription control, chromatin organization, accumulation of macromolecules in cells, regulation of gene expression and signal transduction and is necessary for the conservation of genome integrity.

[0075] Lipidation involves the covalent attachment of lipids to proteins. These PTMs may use a variety of lipids, including octanoic acid, myristic acid, palmitic acid, palmitoleic acid, stearic acid, cholesterol, etc. Myristoylation, palmitoylation and prenylation can be considered as the three main types of these lipid modifications. Palmitoylation is the covalent attachment of fatty acids, like palmitic acid on the Cys, Gly, Ser, Thr and Lys. S-palmitoylation contains a reversible covalent addition of a 16-carbon fatty acid chains, palmitate, to a cysteine via a thioester linkage. Palmitoyl-CoA (as the lipid substrate) is attached to the target protein. Mostly, S-palmitoylation occurs in eukaryotic cells and plays critical roles in many different biological processes including protein function regulation, protein-protein interaction, membrane-protein associations, neuronal development, signal transduction, apoptosis and mitosis.

[0076] Myristoylation (N-myristoylation) is an irreversible PTM that occurs mainly on cytoplasmic eukaryotic proteins. Myristoylation happens approximately in 0.5-1.5% of eukaryotic proteins. In myristoylation, after removal of the initiating Met, a 14-carbon saturated fatty acid, called myristic acid, is attached to the N-terminal glycine residue via a covalent bond. This attachment is often observed in Met-Gly-X-X-X- Ser / Thr motif and is catalyzed by an N- myristoyl transferase (NMT) (there are at least two types of NMT enzymes, NMT1 and NMT2, in humans). Proteins that undergo this PTM play critical roles in regulating the cellular structure and many biological processes such as stabilizing the protein structure maturation, signalling, extracellular communication, metabolism and regulation of the catalytic activity of the enzymes.

[0077] Prenylation is another important lipid-based PTM, which occurs after translation as an irreversible covalent linkage mainly in the cytosol. This reaction occurs on cysteine and near the carboxyl-terminal end of the substrate protein. Prenylation has two main forms: farnesylation and geranylation. These two forms contain the addition of two different types of isoprenoids to cysteine residues: farnesyl pyrophosphate (15-carbon) and geranylgeranyl pyrophosphates (20-carbon), respectively. In prenylated proteins, one can find a consensus motif at the C-terminal; the motif is CAAX where C is cysteine, A is an aliphatic amino acid and X is any amino acid. This process is catalyzed by three prenyltransferase enzymes: farnesyltransferase (FT) and two geranyl transferases (GT1 and GT2). Prenylation is known as a crucial physiological process for facilitating many cellular processes such as proteinprotein interactions, endocytosis regulation, cell growth, differentiation, proliferation and protein trafficking.

[0078] N-sulfation or O-sulfation includes the addition of a negatively charged sulfate group by nitrogen or oxygen to an exposed residue on the target protein. Currently, PTS is observed mainly in secreted and transmembrane proteins in multicellular eukaryotes. This reaction is catalyzed by two transmembrane enzymes, tyrosyl protein sulfotransferases 1 and 2 (TPST1 and TPST2). TPSTs govern the transfer of an activated sulfate from 3-phospho adenosine 5- phosphosulfate to residues within acidic motifs of polypeptides.

[0079] The expressed protein may contain an AVI tag. The Avi-tag peptide (GLNDIFEAQKIEWHE) is recognized by BirA ligase which enzymatically attaches a biotin molecule to a single lysine residue within the Avi-tag sequence. The enzyme can be present during expression to attach the biotin to the expressed tag after expression of the POI.

[0080] 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.

[0081] By way of example, the screening reagents may include,

[0082] ■ Chaperone mix (e.g., PUREfrex GrpE mix)

[0083] ■ Kinase 1 (e.g., NEB CK2)

[0084] ■ Kinase 2 (e.g., NEB PKA)

[0085] ■ Protease 1 (e.g., NEB TEV)

[0086] ■ Protease 2 (e.g., Merck HRV 3C)

[0087] ■ N-acetyl transferase

[0088] ■ Common metal ions cocktail

[0089] ■ Common co-factors cocktail

[0090] 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. Metal ions may include one or more of the following: MgCh, CuCh, ZnCh, CaCh, MnCl2, NiCh, C0CI2.

[0091] Co-factors may include one or more of Nicotinamide adenine dinucleotide (NAD), Flavin adenine dinucleotide (FAD), S-adenosyl methionine (SAM), pyridoxal phosphate (PLP) Coenzyme A (CoA), thiamine pyrophosphate (TPP) or haem.

[0092] Chaperones may include one or more of DnaK, DnaJ, GroE, GrpE, heat shock proteins, protein disulfide isomerase (PDI), human protein disulfide isomerase (hPDI), disulfide bond C (DsbC), a thioredoxin, such as TRXB1 , Caseinolytic peptidase B protein homolog (CLPB) or FK506 binding proteins (FKBPs).

[0093] The additive may be for example one or more reducing agents. The additive may be selected from DTT, glutathione (GSH) or glutathione disulfide (GSSG).

[0094] The added protease may cleave the protein of interest from flanking regions. The flanking regions mat include tags used for detection or solubility or other buffer regions. The flanking regions may be cleaved by any protease. The protease may be a TEV or 3C protease.

[0095] The TEV protease may act upon the amino acid sequence ENLYFQS (Seq ID 36). The template for expression of the POI may include the nucleic acid sequence GAGAACCTGTACTTCCAGAGC (Seq ID 37).

[0096] The 3C protease may act upon the amino acid sequence LEVLFQGP (Seq ID 38). The template for expression of the POI may include the nucleic acid sequence CTCGAGGTTCTGTTCCAAGGACCT (Seq ID 39).

[0097] The method may be used to perform a surfactant screen to identify the best surfactant for expression of a particular protein. The surfactant may be ionic, nonionic, or zwitterionic.

[0098] Surfactant molecules are composed of a hydrophobic region and a hydrophilic region. This amphiphilic structure enables surfactants to obtain a discoidal conformation in the solution, known as micelles. Micelles solubilize membrane proteins by encompassing the transmembrane domains of integral membrane proteins, with the loops and hydrophilic regions exposed to solvent. The minimum concentration of a surfactant necessary to form micelles and extract membrane proteins is called critical micelle concentration or CMC. Depending on the charge of hydrophilic group, surfactants are classified into three groups: ionic, nonionic, and zwitterionic surfactants. Ionic surfactants carry a charged group, either negative (anionic) or positive (cationic), and historically have been the most efficient group of detergents in extracting membrane proteins from lipid bilayers. However, ionic detergents can have deleterious effects on protein-protein interactions and often lead to protein denaturation. Sodium dodecyl sulfate (SDS) and sodium cholate are two common examples of ionic detergents.

[0099] Nonionic surfactants are currently the most popular and successful group of surfactants in solubilizing membrane proteins for both functional and structure determination purposes. This is due to their nondisruptive nature, which enables them to preserve the native structure of the target protein by breaking protein-lipid interactions instead of protein-protein interactions. Alkyl glycoside surfactants such as n-dodecyl-B-D-maltoside (DDM), n-decyl-B-D-maltoside (DM), n-Octyl-B-D-Glucopyranoside (OG), and n-Nonyl-B-D-Glucopyranoside (NG) by contributing to the purification and crystallization of about 70% of membrane proteins are the most common nonionic surfactants for protein studies. Another advantage of nonionic surfactants is that they do not interfere with optical measurements, which enables fluorescence-based experiments on expressed proteins.

[0100] Zwitterionic surfactants typically have an intermediate level of harshness between ionic and nonionic detergents. They carry both positive and negative charged groups in their polar regions with an overall net charge of zero. An example of a Zwitterionic surfactant is lauryldimethylamine-N-oxide or LDAO.

[0101] Membrane mimetic systems, such as nanodiscs and styrene malic acid lipid particles (SMALPs), provide an alternative platform for stabilization of membrane proteins and hence eliminate the deleterious effects of detergents on these macromolecules. Nanodiscs are composed of phospholipid patches surrounded by two copies of membrane scaffold protein (MSP), a genetically engineered version of human serum apolipoprotein A-l.

[0102] Any particular nucleic acid template can be expressed using the system described herein. Three types of nucleic acid templates used in CFPS include plasmids, linear expression templates (LETs), and mRNA. Plasmids are circular templates, which can be produced either in cells or synthetically. LETs can be made via PCR. While LETs are easier and faster to make, plasmid yields are usually higher in CFPS. mRNA can be produced through in vitro transcription systems. The methods use a single nucleic acid template per droplet. The methods can use multiple droplets having a different nucleic acid template per droplet. 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.

[0103] Thus the cell-lysate can be supplemented with additional reagents prior to the template being added. The cell-free extract having the components for protein expression would typically be produced as a bulk reagent or ‘master mix’ which can be formulated into many identical droplets prior to the distinct template being separately added to separate droplets. Common cell extracts in use today are made from E. coli (ECE), rabbit reticulocytes (RRL), wheat germ (WGE), insect cells (ICE) and Yeast Kluyveromyces (the D2P system). All of these extracts are commercially available.

[0104] Rather than originating from a cell extract, the cell-free system can be assembled from the required reagents. Systems based on reconstituted, purified molecular reagents are commercially available, for example the PURE system for protein production, and can be used as supplied (e.g. PURE-FREX). The PURE system is composed of all the enzymes that are involved in transcription and translation, as well as highly purified 70S ribosomes. The protein synthesis reaction of the PURE system lacks proteases and ribonucleases, which are often present as undesired molecules in cell extracts.

[0105] The expression system may be assembled from a mixture of sources, such as for example a mixture of eukaryotic lysates, or a mix of eukaryotic and prokaryotic lysates. The system may be a mixture of a lysate system and a reconstituted system (such as PUREFrex).

[0106] The aqueous layer typically sinks through the hydrophobic layer in order to reach the bottom of the well in which the reactions are performed. The cell-free extract having the components for protein expression will therefore typically be in the hydrophobic fluid layer before the nucleic acid templates are added. The templates can be added by any liquid handling device, including pipettes or automated systems. The expression system typically operates optimally at temperatures above standard room temperatures, for example at or above 29 °C.

[0107] The expression process typically takes many hours. Thus the process should be left for at least 30 minutes or 1 hour, typically at least 2 hours. Expression can be left for at least 12 hours. During the process of expression the aqueous fluids may be moved within the well. The moving improves the process of expression by mixing the reagents and ensuring sufficient oxygen is available within the aqueous expression system. The moving can be continuous, or can be repeated with intervening periods of non-movement.

[0108] Described herein is a microtitre plate containing the enzymes required for cell-free protein synthesis with a hydrophobic fluid layer which prevent evaporation.

[0109] The inventors herein have appreciated that during storage of the cell-free expression system, the enzymes required for protein expression are sufficiently active to metabolise the energy systems and nucleoside triphosphates (NTP’s) needed for protein expression despite the absence of any nucleic acid template. Thus when the template is added, the performance of the expression system is sub-optimal due to the lack of energy sources and / or NTP’s. The addition of energy sources and / or NTP’s along with the nucleic acid templates improves the efficiency of expression of the protein of interest.

[0110] Disclosed is a method for the cell-free synthesis of proteins, the method comprising mixing a first composition containing reagents for cell-free protein expression, and a second composition containing a nucleic acid template and NTP’s and / or an energy source for the cell-free protein expression reagents. The nucleoside triphosphates may be selected from one or more of ATP, CTP, GTP or UTP. The NTP may be ATP. The NTP may be CTP. The NTP may be GTP. The NTP may be UTP. Two or more NTP’s may be added, for example ATP and GTP. Four NTP’s may be added.

[0111] Disclosed is a method for the cell-free synthesis of proteins, the method comprising mixing a first composition containing reagents for cell-free protein expression, and a second composition containing a nucleic acid template and an energy source for the cell-free protein expression reagents. The second composition may comprise both an energy source and one or more NTP’s, for example ATP and / or GTP.

[0112] The energy source may be one or more of phosphoenolpyruvate, acetyl phosphate, and creatine phosphate. The energy source may be creatine phosphate.

[0113] Disclosed is a composition comprising a nucleic acid template and an energy source for the cell-free protein expression reagents selected from phosphoenolpyruvate, acetyl phosphate, and creatine phosphate, wherein the composition does not contain any enzymes that metabolise the energy sources. The composition may contain a nucleic acid template and at least 50 mM creatine phosphate. The composition may contain a nucleic acid template and at least 100 mM creatine phosphate. The composition may contain a nucleic acid template and 120 mM to 360 mM creatine phosphate.

[0114] The composition may contain a nucleic acid template and at least 3 mM ATP. The composition may contain a nucleic acid template and at least 3 mM GTP. The composition may contain a nucleic acid template and at least 3 mM ATP and at least 3 mM GTP. The composition may contain a nucleic acid template and at least 50 mM creatine phosphate and at least 3 mM ATP. The composition may contain a nucleic acid template and at least 50 mM creatine phosphate and at least 3 mM GTP. The composition may contain a nucleic acid template and at least 50 mM creatine phosphate and at least 3 mM ATP and at least 3mM GTP.

[0115] 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.

[0116] The level of protein may be detected after expression. The expressed protein may be fused to a peptide tag. The peptide tag may be one component of a fluorescent protein and the further polypeptide a complementary portion of the fluorescent protein. The fluorescent protein could include sfGFP, ccGFP, GFP, eGFP, deGFP, frGFP, eYFP, eBFP, eCFP, Citrine, Venus, Cerulean, Dronpa, DsRED, mKate, mCherry, mRFP, FAST, 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- . The peptide tag may be CFASTn or CFAST10 and the further polypeptide NFAST in the presence of a hydroxybenzylidene rhodanine analog. The detector reagent can be added after the expression process has completed to enable end-point detection. Alternatively the detector reagents can be present through the expression to provide real-time information. The detector can be added with the template nucleic acid, or can be present in the CFPS reagent composition.

[0117] The expressed protein may contain a binding / purification tag to allow purification of the expressed protein. The binding moiety may be selected from:

[0118] Alfa-tag (SRLEEELRRRLTE) (Seq ID 1) Avi-tag (GLNDIFEAQKIEWHE) (Seq ID 2)

[0119] C-tag (EPEA) (Seq ID 3)

[0120] Calmodulin-tag (KRRWKKNFIAVSAANRFKKISSSGAL) (Seq ID 4)

[0121] Dogtag (DIPATYEFTDGKHYITNEPIPPK) (Seq ID 5)

[0122] E-tag (GAPVPYPDPLEPR) (Seq ID 6)

[0123] FLAG (DYKDDDDK) (Seq ID 7)

[0124] G4T (EELLSKNYHLENEVARLKK) (Seq ID 8)

[0125] HA (YPYDVPDYA) (Seq ID 9)

[0126] His (HHHHHH) (Seq ID 10)

[0127] Isopeptag (TDKDMTITFTNKKDAE) (Seq ID 11) lanthanide binding tag (LBT) (FIDTNNDGWIEGDELLLEEG) (Seq ID 12)

[0128] Myc (EQKLISEEDL) (Seq ID 13)

[0129] NE-Tag (TKENPRSNQEESYDDNES) (Seq ID 14)

[0130] Poly Glutamate-tag (EEEEEEE) (Seq ID 15)

[0131] Poly Arginine-tag (RRRRRRR) (Seq ID 16)

[0132] Rho1 D4-tag (TETSQVAPA) (Seq ID 17)

[0133] SBP-tag (MDEKTTGWRGGHVVEGLAGELEQLRARLEHHPQGQREP) (Seq ID 18)

[0134] Spytag (DPIVMIDNDKPIT) (Seq ID 19)

[0135] SH3 (STVPVAPPRRRRG) (Seq ID 20)

[0136] SNAC (GSHHW) (Seq ID 21)

[0137] Snooptag (KLGDIEFIKVNK) (Seq ID 22)

[0138] Softag 1 (SLAELLNAGLGGS) (Seq ID 23)

[0139] Softag 3 (TQDPSRVG) (Seq ID 24)

[0140] Spot-tag (PDRVRAVSHWSS) (Seq ID 25)

[0141] Spytag (AHIVMVDAYKPTK) (Seq ID 26)

[0142] S-tag (KETAAAKFERQHMDS) (Seq ID 27)

[0143] Strep-tag (AWAHPQPGG) (Seq ID 28) (AWRHPQFGG) (Seq ID 29)

[0144] Strep-tag II (WSHPQFEK) (Seq ID 30)

[0145] T7tag (MASMTGGQQMG) (Seq ID 31)

[0146] TC-tag (EVHTNQDPLD) (Seq ID 32)

[0147] Ty-tag (CCPGCC) (Seq ID 33)

[0148] VSV-tag (YTDIEMNRLGK) (Seq ID 34)

[0149] Xpress-tag (DLYDDDDK) (Seq ID 35).

[0150] After expression a solid support may be added to allow purification of the expressed protein.

[0151] The purification may use beads, for example magnetic beads.

[0152] Disclosed is a method for performing cell-free protein synthesis comprising: a) taking a microtitre plate wherein each well contains an aqueous composition containing enzymes for cell-free protein synthesis (CFPS) and a hydrophobic fluid to prevent evaporation; b) adding a nucleic acid template in aqueous solution to wells of the microtitre plate such that the aqueous layer sinks through the hydrophobic fluid layer and mixes with the enzyme composition, wherein the nucleic acid template contains at least 50 mM creatine phosphate; c) allowing the cell-free protein synthesis reaction to proceed in the covered aqueous layers, wherein the expressed protein contains a detector tag and a purification tag; d) adding a detector in an aqueous composition which sinks through the hydrophobic fluid layer, and generates a signal on binding to the detector tag; and e) identifying the wells of the plate in which protein has been expressed and adding beads to those wells, wherein the beads bind to the purification tag and removing the beads with bound protein from the wells of the plate.

[0153] Disclosed is a method for performing cell-free protein synthesis comprising: a) taking a microtitre plate wherein each well contains an aqueous composition containing a nucleic acid template and at least 50 mM creatine phosphate and a hydrophobic fluid to prevent evaporation; b) adding an aqueous composition containing enzymes for cell-free protein synthesis (CFPS) such that the aqueous layer sinks through the hydrophobic fluid layer and mixes with the nucleic acid template; c) allowing the cell-free protein synthesis reaction to proceed in the covered aqueous layers, wherein the expressed protein contains a detector tag and a purification tag; d) adding a detector in an aqueous composition which sinks through the hydrophobic fluid layer, and generates a signal on binding to the detector tag; and e) identifying the wells of the plate in which protein has been expressed and adding beads to those wells, wherein the beads bind to the purification tag and removing the beads with bound protein from the wells of the plate.

[0154] Disclosed is a microtitre plate containing an aqueous composition containing enzymes for cell- free protein synthesis (CFPS) and a hydrophobic fluid to prevent evaporation. The plate may be part of a kit also containing nucleic acid templates to be added to the wells of the plate. The nucleic acid templates may also contain at least 50 mM creatine phosphate. The kit may also comprise a detector moiety, for example a sub-component of a fluorescent protein. The kit may also comprise magnetic or paramagnetic beads to enable protein purification, washing buffers, elution buffers etc.

[0155] Examples To confirm that all possible orders of addition of oil (base fluid), enzyme core reagent, and nucleic acid template generate the same protein expression yield.

[0156] 10 |iL reactions (7.5 pL reconstituted protein expression system, 2.5 .L template nucleic acid for expressing fluorescent protein ccGFP) were assembled in triplicate the order indicated below. 20 pL of oil (hydrophobic fluid) was used in each reaction. Oils tested were dodecamethylpentasiloxane (DM PS) containing a surfactant Span 85 and dodecamethylpentasiloxane (DMPS) containing a surfactant Span 85 and Brij L4. Reactions were set up directly in a black, low volume, clear bottom, 384-well plate.

[0157] Table 1. Order of addition of each component to each reaction.

[0158] The reaction plate was sealed to avoid spillage / contamination, vortexed, spun at 500 xg for 30 seconds, and incubated at 29°C overnight. The plate was retrieved, centrifuged at 500 xg for 30 seconds, and scanned using a fluorescent plate reader along with a dilution control of the ccGFP protein sample to quantitate the level of expression. Results are shown in Figure 2. No significant differences in expression levels were seen when reactions were set up using different orders of addition of oil, Core, and DNA. The type of oil or surfactant did not impact the level of droplet mixing or subsequent expression. This confirms that there is no detriment to using oil (base fluid) as a septum for reaction assembly, and that the oil layer prevents evaporation of the microlitre volumes of aqueous reagents during the expression process.

Claims

Claims1. A method for performing cell-free protein synthesis comprising either: a) taking a plurality of isolated volumes of an aqueous composition containing enzymes for cell-free protein synthesis (CFPS), wherein the isolated volumes are covered with a hydrophobic fluid to prevent evaporation; b) adding a nucleic acid template in aqueous solution to each of the isolated volumes such that the aqueous layer sinks through the hydrophobic fluid layer and mixes with the enzyme composition; and c) allowing the cell-free protein synthesis reaction to proceed in the covered aqueous layers or a) taking a plurality of isolated volumes of an aqueous composition containing a nucleic acid template, wherein the isolated volumes are covered with a hydrophobic fluid to prevent evaporation; b) adding a composition containing enzymes for cell-free protein synthesis (CFPS) in aqueous solution to each of the isolated volumes such that the aqueous layer sinks through the hydrophobic fluid layer and mixes with the nucleic acid templates; and c) allowing the cell-free protein synthesis reaction to proceed in the covered aqueous layers.

2. The method according to claim 1 , wherein the expression is performed for at least 2 hours, the hydrophobic fluid layer preventing evaporation of the aqueous layer.

3. The method according to claim 1 , wherein the expression is performed for at least 4 hours, the hydrophobic fluid layer preventing evaporation of the aqueous layer.

4. The method according to any one of claims 1 to 3, wherein the expressed protein contains a detector tag.

5. The method according to claim 4, wherein after expression a detector moiety is added in an aqueous composition which sinks through the hydrophobic fluid layer, and generates a signal on binding to the detector tag.

6. The method according to any one of claims 1 to 5, wherein the expressed protein contains a component of a fluorescent protein.

7. The method according to claim 6, wherein the expressed protein contains a ccGFPn sequence.

8. The method according to any one preceding claim, wherein the aqueous composition containing enzymes for CFPS is supplemented with additives to test expression in different conditions.

9. The method according to claim 8, wherein the expression reaction is supplemented with one or more reagents selected from synthesized or isolated ribosomes, initiation factors, elongation factors, termination factors, aminoacyl-tRNA synthetases, methionyl tRNA transformylases, tRNAs, amino acids, ribonucleoside triphosphates, 10-formyl 5, 6,7,8- tetrahydrofolic acid (FD), salts, a polyethylene glycol, an allolactose, an aldohexose or a thiogalactopyranoside, buffers, surfactants, metal ions, chaperones, co-factors or additional protein components.

10. The method according to claim 9, wherein the additional protein components are selected from chaperones, glycosylating enzymes, proteases, redox active enzymes, phosphorylases and kinases.11 The method according to any one of claims 8 to 10, wherein the additives are added by allowing a composition containing the additives to sink through the hydrophobic fluid layer.

12. The method according to any one preceding claim, wherein each expression is performed in less than 10 pL of aqueous reagents.

13. The method according to any one preceding claim, wherein each expression is performed in less than 1 pL of aqueous reagents.

14. The method according to any one preceding claim, wherein the expression is performed in a microtitre plate, such as a 384 or 1536 well plate.

15. The method according to any one preceding claim, wherein the isolated volumes containing the reagents have tapered sides.

16. The method according to any one preceding claim, wherein the hydrophobic fluid is a silicone oil.

17. The method according to any one preceding claim, wherein the hydrophobic fluid is dodecamethylpentasiloxane, decane or dodecane.

18. The method according to any one preceding claim, wherein the hydrophobic fluid is oxygenated prior to or during the process of expression.

19. The method according to any one preceding claim, wherein the expressed proteins are purified using binding tags.

20. The method according to claim 19, wherein the binding moiety is selected from:Alfa-tag (SRLEEELRRRLTE) (Seq ID 1)Avi-tag (GLNDIFEAQKIEWHE) (Seq ID 2)C-tag (EPEA) (Seq ID 3)Calmodulin-tag (KRRWKKNFIAVSAANRFKKISSSGAL) (Seq ID 4)Dogtag (DIPATYEFTDGKHYITNEPIPPK) (Seq ID 5)E-tag (GAPVPYPDPLEPR) (Seq ID 6)FLAG (DYKDDDDK) (Seq ID 7)G4T (EELLSKNYHLENEVARLKK) (Seq ID 8)HA (YPYDVPDYA) (Seq ID 9)His (HHHHHH) (Seq ID 10)Isopeptag (TDKDMTITFTNKKDAE) (Seq ID 11) lanthanide binding tag (LBT) (FIDTNNDGWIEGDELLLEEG) (Seq ID 12)Myc (EQKLISEEDL) (Seq ID 13)NE-Tag (TKENPRSNQEESYDDNES) (Seq ID 14)Poly Glutamate-tag (EEEEEEE) (Seq ID 15)Poly Arginine-tag (RRRRRRR) (Seq ID 16)Rho1 D4-tag (TETSQVAPA) (Seq ID 17)SBP-tag (MDEKTTGWRGGHVVEGLAGELEQLRARLEHHPQGQREP) (Seq ID 18)Spytag (DPIVMIDNDKPIT) (Seq ID 19)SH3 (STVPVAPPRRRRG) (Seq ID 20)SNAC (GSHHW) (Seq ID 21)Snooptag (KLGDIEFIKVNK) (Seq ID 22)Softag 1 (SLAELLNAGLGGS) (Seq ID 23)Softag 3 (TQDPSRVG) (Seq ID 24)Spot-tag (PDRVRAVSHWSS) (Seq ID 25)Spytag (AHIVMVDAYKPTK) (Seq ID 26)S-tag (KETAAAKFERQHMDS) (Seq ID 27)Strep-tag (AWAHPQPGG) (Seq ID 28) (AWRHPQFGG) (Seq ID 29)Strep-tag II (WSHPQFEK) (Seq ID 30)T7tag (MASMTGGQQMG) (Seq ID 31)TC-tag (EVHTNQDPLD) (Seq ID 32)Ty-tag (CCPGCC) (Seq ID 33)VSV-tag (YTDIEMNRLGK) (Seq ID 34)Xpress-tag (DLYDDDDK) (Seq ID 35).21 . The method according to claim 19 or claim 20, wherein the purification uses magnetic beads.

22. The method according to any one preceding claim, wherein the expression reagents are made using an expression system reconstituted from purified components.

23. The method according to any one of claims 1 to 21 , wherein the expression reagents are cell lysates derived from mammalian cells prokaryotic cells, yeast cells, plant cells or protozoa such as HEK293, HeLa, BHK21 , NSO, Sp2 / 0, CHO or Escherichia coli.

24. A method for performing cell-free protein synthesis comprising either: a) taking a microtitre plate wherein each well contains an aqueous composition containing enzymes for cell-free protein synthesis (CFPS) and a hydrophobic fluid to prevent evaporation; b) adding a nucleic acid template in aqueous solution to wells of the microtitre plate such that the aqueous layer sinks through the hydrophobic fluid layer and mixes with the enzyme composition, wherein the nucleic acid template contains at least 50 mM creatine phosphate; c) allowing the cell-free protein synthesis reaction to proceed in the covered aqueous layers, wherein the expressed protein contains a detector tag and a purification tag; d) adding a detector in an aqueous composition which sinks through the hydrophobic fluid layer, and generates a signal on binding to the detector tag; and e) identifying the wells of the plate in which protein has been expressed and adding beads to those wells, wherein the beads bind to the purification tag and removing the beads with bound protein from the wells of the plate; or a) taking a microtitre plate wherein each well contains an aqueous composition containing a nucleic acid template and at least 50 mM creatine phosphate and a hydrophobic fluid to prevent evaporation; b) adding an aqueous composition containing enzymes for cell-free protein synthesis (CFPS) such that the aqueous layer sinks through the hydrophobic fluid layer and mixes with the nucleic acid template;c) allowing the cell-free protein synthesis reaction to proceed in the covered aqueous layers, wherein the expressed protein contains a detector tag and a purification tag; d) adding a detector in an aqueous composition which sinks through the hydrophobic fluid layer, and generates a signal on binding to the detector tag; and e) identifying the wells of the plate in which protein has been expressed and adding beads to those wells, wherein the beads bind to the purification tag and removing the beads with bound protein from the wells of the plate.

25. A microtitre plate containing an aqueous composition containing enzymes for cell-free protein synthesis (CFPS) and a hydrophobic fluid to prevent evaporation.

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