EP1341802A1 - In-vitroproteinsynthese unter verwendung von glykolytischen zwischenprodukten als energiequelle - Google Patents

In-vitroproteinsynthese unter verwendung von glykolytischen zwischenprodukten als energiequelle

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Publication number
EP1341802A1
EP1341802A1 EP00980413A EP00980413A EP1341802A1 EP 1341802 A1 EP1341802 A1 EP 1341802A1 EP 00980413 A EP00980413 A EP 00980413A EP 00980413 A EP00980413 A EP 00980413A EP 1341802 A1 EP1341802 A1 EP 1341802A1
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Prior art keywords
synthesis
pyruvate
reaction
atp
glucose
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French (fr)
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EP1341802A4 (de
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James Swartz
Dong-Myung Kim
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Leland Stanford Junior University
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Leland Stanford Junior University
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/02—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length in solution

Definitions

  • the directed synthesis of proteins and other biological macromolecules is one of the great achievements of biochemistry.
  • the development of recombinant DNA techniques has allowed the characterization and synthesis of highly purified coding sequences, which in turn can be used to produce highly purified proteins, even though in native cells the protein may be available only in trace amounts.
  • Polypeptide chains can be synthesized by chemical or biological processes.
  • the biological synthesis may be performed within the environment of a cell, or using cellular extracts and coding sequences to synthesize proteins in vitro.
  • in vitro protein synthesis has served as an effective tool for lab-scale expression of cloned or synthesized genetic materials.
  • in vitro protein synthesis system has been considered as an alternative to conventional recombinant DNA technology, because of disadvantages associated with cellular expression.
  • proteins can be degraded or modified by several enzymes synthesized with the growth of the cell, and after synthesis may be modified by post-translational processing, such as glycosylation, deamination or oxidation.
  • post-translational processing such as glycosylation, deamination or oxidation.
  • many products inhibit metabolic processes and their synthesis must compete with other cellular processes required to reproduce the cell and to protect its genetic information.
  • in vitro protein synthesis has advantages in the production of cytotoxic, unstable, or insoluble proteins.
  • the overproduction of protein beyond a predetermined concentration can be difficult to obtain in vivo, because the expression levels are regulated by the concentration of product.
  • the concentration of protein accumulated in the cell generally affects the viability of the cell, so that over-production of the desired protein is difficult to obtain.
  • many kinds of protein are insoluble or unstable, and are either degraded by intracellular proteases or aggregate in inclusion bodies, so that the loss rate is high.
  • compositions and methods are provided for the enhanced in vitro synthesis of protein molecules.
  • Glycolytic intermediates or glucose are used as an energy source, in combination with NADH or NAD + added in catalytic quantities.
  • Coenzyme A may also be included in the reaction mix.
  • inhibition of enzymes catalyzing undesirable reactions is achieved by: addition of inhibitory compounds to the reaction mix; modification of the reaction mixture to decrease or eliminate the responsible enzyme activities; or a combination of the two.
  • Figure 1 is a graph illustrating the synthesis of chloramphenicol acetyl transferase.
  • 33 mM sodium pyruvate, 0.33 mM NAD and 0.27 mM CoA were added in the indicated combinations to 15 ⁇ L reaction mixtures to regenerate ATP during the synthesis reaction. Reactions were carried out for 2 hours and TCA-insoluble radioactivities were measured. In the control reaction, 33 mM PEP was used instead of pyruvate and cofactors.
  • Figure 2 Proposed mechanism of ATP regeneration with pyruvate.
  • FIGS 3A and 3B Time course of protein synthesis and ATP concentration.
  • 120 ⁇ L standard reaction mixtures with 33 mM PEP were prepared and incubated in the presence of 0.33 mM NAD, 0.27 mM CoA, and 2.7 mM sodium oxalate.
  • 10 ⁇ L samples were withdrawn, mixed with the same volume of 10 % TCA solution, and centrifuged for 10 min. 10 ⁇ L of the supernatant was used for ATP analysis as described in the Materials and Methods. At the given time points, 5 ⁇ l samples were taken and TCA-insoluble radioactivities were counted to measure protein synthesis (B).
  • Lanes M standard molecular weight markers
  • C control reaction without the template plasmid
  • 1 standard reaction
  • 2 reaction with 0.33 mM NAD and 0.27 mM CoA
  • 3 reaction with 0.33 mM NAD, 0.27 mM CoA, and 2.7 mM sodium oxalate
  • 4 reaction with 0.33 mM NAD, 0.27 mM CoA, 2.7 mM sodium oxalate, and 2 mM amino acids.
  • FIGS. 4A and 4B Supplementation of PEP, amino acids, and magnesium during protein synthesis.
  • a synthesis reaction was carried out in the presence of 2 mM amino acids, 33 mM PEP, 0.33 mM NAD, 0.27 mM CoA, and 2.7 mM sodium oxalate in a 120 ⁇ l volume.
  • the initial concentrations of PEP, amino acids, and magnesium acetate were added to the reaction every hour. 5 ⁇ l samples were taken at the given time points to measure the concentration of ATP ( Figure 4A) and the yield of CAT synthesis ( Figure 4B).
  • the same volumes of water were added to the single batch reaction. Open circles, single batch reaction; filled circles, reaction with the additions.
  • compositions and methods are provided for the enhanced in vitro synthesis of protein molecules, by the use of glycolytic pathways in the generation of ATP to drive the reaction.
  • NADJNADH is added to the reaction.
  • Exemplary is the use of glucose in combination with the enzyme hexokinase; pyruvate; or phosphoenol pyruvate (PEP) as the energy source.
  • acetyl CoA is also included in the reaction mixture.
  • the phosphate that is hydrolyzed from ATP is recycled during the glucose or pyruvate oxidation, thereby preventing a net accumulation of free phosphate, which can have an inhibitory effect on synthetic reactions.
  • Glucose or glycolytic intermediate energy source refers to compounds that provide energy for the synthesis of ATP from ADP, and which are part of the glycolytic pathway. These energy sources include glucose, glucose-1-phosphate, glucose-6-phosphate, fructose-6- phosphate, fructose-1,6-diphosphate, triose phosphate, 3-phosphoglycerate, 2-phosphoglycerate, phosphoenol pyruvate (PEP) and pyruvate. Preferred energy sources are PEP, pyruvate, and glucose-6-phosphate. The energy sources may also be homeostatic with respect to phosphate, that is they do not result in the accumulation of inorganic phosphate.
  • Such secondary sources of energy recycle the free phosphate generated by ATP hydrolysis.
  • the required high energy phosphate bonds are generated in situ, e.g. through coupling with an oxidation reaction.
  • a homeostatic energy source will typically lack high energy phosphate bonds itself, and will therefore utilize free phosphate present in the reaction mix during ATP regeneration. Since inorganic phosphate can be an inhibitory by-product of synthesis, the period of time when synthesis is maintained in vitro can be extended.
  • a homeostatic energy source may be provided in combination with an enzyme that catalyzes the creation of high energy phosphate bonds.
  • Exemplary glycolytic intermediates that are homeostatic for phosphate metabolism are pyruvate and glucose.
  • glucose it is desirable to include the enzyme hexokinase if not already present in the cell extract.
  • NADH NADH
  • the energy source may be supplied as a suitable biologically acceptable salt or as the free acid, e.g. pyruvic acid, where applicable.
  • the final concentration of energy source at initiation of synthesis will usually be at least about 1 mM, more usually at least about 10 mM, and not more than about 1000 mM, usually not more than about 100 mM. Additional amounts may be added to the reaction mix during the course of synthesis to provide for longer reaction times.
  • Cofactors exogenous cofactor NADH or NAD + ( ⁇ -nicotinamide adenine dinucleotide) is added to the reaction mixture at a concentration of at least about 0.1 mM, preferably 0.2 to 1 mM, and usually not more than about 10 mM.
  • NADH or NAD + ⁇ -nicotinamide adenine dinucleotide
  • acetyl CoA acetyl coenzyme A
  • coenzyme A is also included in the reaction mixture.
  • the useful concentrations are at least about 0.05 mM, usually at least about 0.1 mM, and not more than about 1 mM, usually not more than about 0.5 mM.
  • Glucose Where the homeostatic energy source is glucose, an enzyme will be included in the reaction mixture to catalyze the formation of glucose-6-phosphate from glucose. Hexokinase, EC 2.7.1.1 , is generally used for this purpose. Hexokinase is widely available commercially, and has been isolated and cloned from a number of species.
  • Examples include the enzymes corresponding to SwissProt P27595, HXK1_BOVIN; P19367, HXK1JHUMAN; P17710, HXK1_MOUSE; P05708, HXK1_RAT; Q09756, HXK1_SCHPO; P04806, HXKA_YEAST; Q42525, HXK_ARATH; P50506, HXK_DEBOC; P80581, HXK_EMENI; P33284, HXK_KLULA; Q02155, HXK_PLAFA; Q26609, HXK_SCHMA.
  • the reaction mix will comprise a concentration of hexokinase sufficient to maintain the ATP pool, usually at least about 0.1 U/ml, more usually at least about 1 U/ml, and preferably at least about 10 U/ml, where the unit definition is that 1 unit reduces 1 ⁇ mole of NAD per minute in a coupled assay system with glucose-6-phosphate dehydrogenase at 30°C, pH 8.0. It will be understood by one of skill in the art that higher concentrations may be present, although generally at less than about 1000 U/ml.
  • the hexokinase may be provided in the reaction mix in a variety of ways. Purified or semi- purified enzyme may be added to the reaction mix. Commercial preparations are available, or the enzyme may be purified from natural or recombinant sources according to conventional methods.
  • the genetic sequences of hexokinases may be used as a source of recombinant forms of the enzyme, for example S. cerevisiae hexokinase Pll gene, accession number M14410; or hexokinase Pll, accession number M14411 , both described in Kopetzki et al. (1985) Gene 39:95- 102, etc.
  • the enzyme may also be included in the extracts used for synthesis.
  • extracts can be derived from E. coli for protein synthesis.
  • the E. coli used for production of the extracts may be genetically modified to encode a suitable hexokinase.
  • a template e.g. mRNA encoding hexokinase, plasmid comprising a suitable expression construct of hexokinase, etc. may be spiked into the reaction mix, such that a suitable amount of hexokinase is produced during synthesis.
  • Aspartic acid and asparagine are formed from phosphoenol pyruvate.
  • the enzyme phosphoenol pyruvate synthetase (pps) converts pyruvate into PEP and consumes 2 equivalents of high-energy phosphate bonds (as ATP is converted to AMP) per molecule of PEP synthesized.
  • pps phosphoenol pyruvate synthetase
  • Oxalic acid is added at a concentration of at least about 0.5 mM, and not more than about 100 mM, usually at least about 1 mM, and preferably at a concentration of about 3 mM.
  • E. coli pyruvate oxidase which converts pyruvate into acetate consuming oxygen, and/or phosphoenol pyruvate synthetase (pps) can be disrupted or otherwise inactivated.
  • the coding sequence for E. coli phosphoenol pyruvate synthetase may be accessed in Genbank, no. X59381 ; and is also published in Niersbach et al. (1992) Mol. Gen. Genet. 231:332-336.
  • the coding sequence for E. coli pyruvate oxidase may be accessed in Genbank, no. X04105; and is also published in Grabau and Cronan (1986) Nucleic Acids Res. 14:5449-5460.
  • In vitro synthesis refers to the cell-free synthesis of polypeptides in a reaction mix comprising biological extracts and/or defined reagents.
  • the reaction mix will comprise at least ATP, an energy source; a template for production of the macromolecule, e.g. DNA, mRNA, etc.; amino acids, nucleotides and such co-factors, enzymes and other reagents that are necessary for the synthesis, e.g. ribosomes, tRNA, polymerases, transcriptional factors, etc.
  • ribosomes e.g. ribosomes, tRNA, polymerases, transcriptional factors, etc.
  • the cell free synthesis reaction may be performed as batch, continuous flow, or semi- continuous flow, as known in the art.
  • Reaction mix refers to a reaction mixture capable of catalyzing the synthesis of polypeptides from a nucleic acid template.
  • the mixture may comprise metabolic inhibitors that decrease undesirable enzymatic reactions.
  • the enhanced reaction mix will be engineered through genetic or other processes to decrease the enzymatic activity responsible for undesirable side-reactions, that result in amino acid depletion or accumulation.
  • the reaction mixture comprises extracts from bacterial cells, e.g. E. coli S30 extracts, as is known in the art.
  • the organism used as a source of extracts may be referred to as the source organism. While such extracts are a useful source of ribosomes and other factors necessary for protein synthesis, they can also contain small amounts of endogenous enzymes responsible for undesirable side-reactions that are unrelated to protein synthesis, but which deplete ATP, pyruvate or other reagents.
  • endogenous is used to refer to enzymes, factors, etc. present in the extracts.
  • Exogenous components are those that are introduced into the extracts through addition, and may be added at the time of synthesis, or may be added through genetic or other manipulation of the cells used as the starting material for extracts.
  • plasmids encoding an exogenous enzyme of interest may be added to the bacterial cells priorto preparation of the extracts.
  • the extracts may be optimized for expression of genes under control of a specific promoter, (for example see Nevin and Pratt (1991) FEBS Lett 291(2):259-63, which system consists of an E. coli crude extract (prepared from cells containing endogenous T7 RNA polymerase) and rifampicin (an E. coli RNA polymerase inhibitor)). Kim et al. (1996) Eur. J. Biochem. 239: 881-886 further enhance protein production by optimizing reagent concentrations.
  • the reaction mix may comprise metabolic inhibitors of the undesirable enzyme activity. Frequently such inhibitors will be end-products of the reaction, that then inhibit by a feedback mechanism.
  • the specific inhibitors are determined based on the metabolic pathways of the source organism. These pathways are well-known in the art for many bacterial and eukaryotic species, e.g. E. coli, S. cerevisiae, H. sapiens, etc.
  • the inhibitor is added at a concentration sufficient to inhibit the undesirable enzymatic activity while increasing protein synthesis. Pathways of particular interest relate to the metabolism of pyruvate in E. coli cells, including the synthesis of aspartate from oxalacetate.
  • the undesirable enzymes may be removed or otherwise deleted from the reaction mix.
  • the coding sequence for the enzyme is "knocked-out" or otherwise inactivated in the chromosome of the source organism, by deletion of all or a part of the coding sequence; frame-shift insertion; dominant negative mutations, etc.
  • the genomes of a number of organisms, including E. coli, have been completely sequenced, thereby facilitating the genetic modifications.
  • a markerless knockout strategy method is described by Arigoni et al. (1998) Nat Biotechnol 16(9):851-6.
  • a preferred method for inactivating targeted genes is described by Hoang et al. (1998) Gene 212:77-86.
  • gene replacement vectors are employed that contain a tetracycline resistance gene and a gene encoding levan sucrase (sacB) as selection markers for recombination.
  • the target gene is first cloned and mutagenized, preferably by deleting a significant portion of the gene. This gene is then inserted by ligation into a vector designed for facilitating chromosomal gene replacement.
  • the E. coli cells are then transformed with those vectors.
  • Cells that have incorporated the plasmid into the chromosome at the site of the target gene are selected, then the plasmid is forced to leave the chromosome by growing the cells on sucrose.
  • Sucrose is toxic when the sacB gene resides in the chromosome.
  • the properly mutated strain is selected based on its phenotype of tetracycline sensitivity and sucrose resistance. PCR analysis or DNA sequencing then confirms the desired genetic change.
  • the enzyme reducing the duration and yield of the protein synthesis reaction may be essential for the growth of the source organism.
  • a conditional knock-out may be used.
  • anti-sense sequences corresponding to the targeted gene are introduced into the source organism on an inducible promoter.
  • the cells are grown for a period of time, and then the anti-sense construct induced, in order to deplete the cell of the targeted enzyme.
  • the enzyme can be removed from the cell extract after cell disruption and before use.
  • Any of the several means known in the art of protein purification may be used, including affinity purification techniques such as the use of antibodies or antibody fragments with specific affinity for the target enzymes; use of affinity tags expressed as part of the target enzymes to facilitate their removal from the cell extract; and conventional purification methods.
  • an antibody or antibody fragment (e.g., Fab orscFv) is selected for specific affinity for the target enzyme using phage display or other well developed techniques. That antibody or antibody fragment is then immobilized on any of several purification beads or resins or membranes using any of several immobilization techniques. The immobilized antibody is contacted with the cell extract to bind to the target enzyme, and the immobilized antibody/enzyme complex then removed by filtration or gentle centrifugation.
  • the coding sequence of the targeted protein may be modified to include a tag, such as the Flag® extension (developed by Immunex Corp. and sold by Stratagene), or a poly- histidine tail. Many other examples have been published and are known to those skilled in the art.
  • the tagged proteins are then removed by passage over the appropriate affinity matrix or column.
  • the amino acid extension and binding partner are chosen so that only specific binding occurs under conditions compatible with the stability of the cell extract, and without significantly altering the chemical composition of the cell extract.
  • the target enzyme or enzymes are separated by any of several methods commonly used for protein purification, such as substrate affinity chromatography, ion exchange chromatography, hydrophobic interaction chromatography, electrophoretic separation, or other methods practiced in the art of protein purification.
  • the subject system is useful for in vitro protein synthesis, which may include the transcription of RNA from DNA or RNA templates.
  • the reactions may utilize a large scale reactor, small scale, or may be multiplexed to perform a plurality of simultaneous syntheses.
  • Continuous reactions will use a feed mechanism to introduce a flow of reagents, and may isolate the end- product as part of the process.
  • Batch systems are also of interest, where additional reagents may be introduced to prolong the period of time for active synthesis.
  • a reactor may be run in any mode such as batch, extended batch, semi-batch, semi-continuous, fed-batch and continuous, and which will be selected in accordance with the application purpose.
  • mRNA RNA to produce proteins
  • translation may be coupled to in vitro synthesis of mRNA from a DNA template.
  • a cell-free system will contain all factors required for the translation of mRNA, for example ribosomes, amino acids, tRNAs, aminoacyl synthetases, elongation factors and initiation factors.
  • Cell-free systems known in the art include wheat germ extracts (Roberts et al. (1973) P.N.A.S. 70:2330), reticulocyte extracts (Pelham etal. (1976) Eur. J. Biochem. 67:247), E. coli extracts, etc., which can be treated with a suitable nuclease to eliminate active endogenous mRNA.
  • materials specifically required for protein synthesis may be added to the reaction. These materials include salt, polymeric compounds, cyclic AMP, inhibitors for protein or nucleic acid degrading enzymes, inhibitor or regulator of protein synthesis, oxidation/reduction adjuster, non-denaturing surfactant, buffer component, spermine, spermidine, etc.
  • the salts preferably include potassium, magnesium, ammonium and manganese salt of acetic acid or sulfuric acid, and some of these may have amino acids as a counter anion.
  • the polymeric compounds may be polyethylene glycol, dextran, diethyl aminoethyl, quaternary aminoethyl and aminoethyl.
  • the oxidation/reduction adjuster may be dithiothreitol, ascorbic acid, glutathione and/or their oxides.
  • a non-denaturing surfactant such as Triton X-100 may be used at a concentration of 0-0.5 M.
  • Spermine and spermidine may be used for improving protein synthetic ability, and cAMP may be used as a gene expression regulator.
  • concentration of a particular component of the reaction medium that of another component may be changed accordingly.
  • concentrations of several components such as nucleotides and energy source compounds may be simultaneously controlled in accordance with the change in those of other components.
  • concentration levels of components in the reactor may be varied overtime.
  • the reaction is maintained in the range of pH 5-10 and a temperature of 20°-50° C, and more preferably, in the range of pH 6-9 and a temperature of 25°-40° C.
  • the product output from the reactor flows through a membrane into the protein isolating means.
  • a semi-continuous operation mode the outside or outer surface of the membrane is put into contact with predetermined solutions that are cyclically changed in a predetermined order. These solutions contain substrates such as amino acids and nucleotides.
  • the reactor is operated in dialysis, diafiltration batch or fed-batch mode.
  • a feed solution may be supplied to the reactor through the same membrane or a separate injection unit. Synthesized protein is accumulated in the reactor, and then is isolated and purified according to the usual method for protein purification after completion of the system operation.
  • the direction of liquid flow can be perpendicular and/or tangential to a membrane. Tangential flow is effective for recycling ATP and for preventing membrane plugging and may be superimposed on perpendicular flow.
  • Flow perpendicular to the membrane may be caused or effected by a positive pressure pump or a vacuum suction pump.
  • the solution in contact with the outside surface of the membrane may be cyclically changed, and may be in a steady tangential flow with respect to the membrane.
  • the reactor may be stirred internally or externally by proper agitation means.
  • the protein isolating means for selectively isolating the desired protein may include a unit packed with particles coated with antibody molecules or other molecules immobilized with a component for adsorbing the synthesized, desired protein, and a membrane with pores of proper sizes.
  • the protein isolating means comprises two columns for alternating use.
  • the protein product may be absorbed using expanded bed chromatography, in which case a membrane may or may not be used.
  • the amount of protein produced in a translation reaction can be measured in various fashions.
  • One method relies on the availability of an assay which measures the activity of the particular protein being translated.
  • An example of an assay for measuring protein activity is a luciferase assay system, or chloramphenical acetyl transferase assay system. These assays measure the amount of functionally active protein produced from the translation reaction. Activity assays will not measure full length protein that is inactive due to improper protein folding or lack of other post translational modifications necessary for protein activity.
  • Another method of measuring the amount of protein produced in coupled in vitro transcription and translation reactions is to perform the reactions using a known quantity of radiolabeled amino acid such as 35 S-methionine or 3 H-leucine and subsequently measuring the amount of radiolabeled amino acid incorporated into the newly translated protein. Incorporation assays will measure the amount of radiolabeled amino acids in all proteins produced in an in vitro translation reaction including truncated protein products.
  • the radiolabeled protein may be further separated on a protein gel, and by autoradiography confirmed that the product is the proper size and that secondary protein products have not been produced.
  • Pyruvate glucose or glycolytic intermediates can provide the energy for ATP regeneration, even in the absence of any exogenous enzymes.
  • two pathways may be proposed for the mechanism of action, whereby pyruvate provides ATP regeneration potential to the synthesis reaction.
  • ATP regeneration is accomplished through an electron transport phosphorylation reaction. Since the extract is prepared from a total cell lysate, it is likely that the extract contains inverted membrane vesicles with the respiratory chain components properly embedded.
  • the pyruvate After its conversion into acetyl-CoA by the endogenous pyruvate dehydrogenase complex, the pyruvate enters the TCA cycle to regenerate NADH, which in turn regenerates ATP using the respiratory chain and the FIFO ATPase.
  • the generated acetyl-CoA is converted to acetyl phosphate by phosphotransacetylase.
  • the resulting acetyl phosphate is then used for ATP regeneration.
  • the oxidation of pyruvate provides the energy for ATP generation without accumulating any harmful by-products, and exogenous enzyme is not required.
  • phosphoenol pyruvate can be used as the energy source, combining both the energy obtained by glycolysis and energy obtained from in situ ATP generation.
  • Phosphoenolpyruvate (PEP) and E.coli total tRNA mixture were purchased from Roche
  • T7 RNA polymerase was prepared from E.coli strain BL21 (pAR1219) according to the procedures of Davanloo et a/.(1984). Plasmid pK7CAT which contains the bacterial chloramphenicol acetyltransferase (CAT) sequence between the T7 promoter and T7 terminator was used as a template for protein synthesis. The plasmid was purified using the Maxi kit from Qiagen (Valencia, CA).
  • S30 extract was prepared from E.coli K12 (strain A19) as described earlier (Kim et al., 1996, supra.; Kim and Swartz (1999), supra.)
  • the standard reaction mixture consists of the following components: 57 mM Hepes-KOH (pH7.5), 1.2 mM ATP, 0.85 mM each of GTP, UTP and CTP, 1 mM DTT, 0.64 mM cAMP, 200 mM potassium glutamate, 80 mM ammonium acetate, 12 mM magnesium acetate, 34 ⁇ g/ml folinic acid, 6.7 ⁇ g/ml plasmid, 33 ⁇ g/ml T7RNA polymerase, 500 ⁇ M each of 20 unlabeled amino acids, 11 ⁇ M [ 14 C]leucine, 2 % PEG 8000, 32 mM PEP, and 0.24 volume of S30 extract.
  • the amount of synthesized protein was estimated from the measured TCA-insoluble radioactivities as described by Kim et al., 1996, supra.) using a liquid scintillation counter (Beckman LS3801). To determine the amount of soluble product, samples were centrifuged at 12,000 g for 10 min and TCA-precipitable radioactivities in the supernatants were measured. To estimate the molecular weight of synthesized protein, samples were loaded on a 16 % SDS-PAGE gel (Invitrogen, CA) with standard molecular weight markers (See Blue, Invitrogen, CA). Resulting gels were stained with Coomassie Brilliant Blue following standard procedures. The protein concentrations of cell-extracts were measured following the procedures of Bradford using a commercial assay reagent (Pierce, Rockford, IL).
  • ATP concentration diluted samples were added to an opaque microtiter plate containing luciferase solution (0.1 ⁇ g/mL luciferase and 125 ⁇ M luciferin) and the intensity of luminescence was measured in a plate luminometer (ML 3000, Dynatech Laboratories, Chantilly, VA). ATP concentrations in the samples were determined from the calibration curve obtained with ATP standards. Enzymatic activity of synthesized CAT was determined by spectrophotometric procedures.
  • pyruvate can serve as a secondary energy source to regenerate ATP during a cell-free protein synthesis reaction.
  • the enzyme, pyruvate oxidase converts pyruvate into acetyl phosphate which is then used to regenerate ATP with endogenous acetate kinase.
  • This pyruvate oxidase-dependent system substantially reduces the cost for energy source, leads to a stable maintenance of ATP during protein synthesis and avoids phosphate accumulation.
  • pyruvate oxidase (E.C.1.2.3.3) from Lactobacillus or Pediococcus sp. because the E. coli enzyme (E.G.1.2.2) cannot catalyze the formation of acetyl phosphate (it converts pyruvate to acetate instead of acetyl phosphate).
  • E.G.1.2.2 the E. coli enzyme
  • ATP is not regenerated and the level of protein synthesis is negligible.
  • pyruvate For pyruvate to be used for ATP regeneration in the cell-free system, it first needs to be converted to acetyl phosphate. In E.coli cells, pyruvate is not directly converted to acetyl phosphate. Instead, two different enzymes can convert pyruvate into acetyl-CoA, which can be used to produce acetyl phosphate by phosphotransacetylase. First, pyruvate dehydrogenase catalyzes the condensation of CoA and pyruvate to make acetyl-CoA in the presence of NAD as a cofactor. NAD is reduced to NADH during this reaction.
  • pyruvate-formate lyase can also make acetyl-CoA from pyruvate producing formate as the by-product. All of the enzymes required for these reactions were assumed to be present in the cell-extract. We thus tested the addition of the cofactors, NAD and CoA, to stimulate the regeneration of ATP from pyruvate in support of cell-free protein synthesis.
  • Oxalate a potent inhibitor of phosphoenolpyruvate synthetase enhances ATP concentration in the synthesis reactions with pyruvate or PEP.
  • the effect of oxalate was still observed in the presence of NAD/CoA.
  • the yield of CAT synthesis also increased upon the addition of 2.7 mM oxalate ( Figure 3B).
  • protein synthesis was further stimulated by increasing the initial concentrations of amino acids. When the concentration of ATP was elevated by the additions of the cofactors and oxalate, both the rate and duration of protein synthesis was significantly improved by increasing the initial concentrations of amino acids to 2 mM ( Figure 3B). Higher concentrations were not as effective.
  • Table 1 CAT yields and specific activities after 3 hr incubations with different ATP regeneration systems
  • Glucose-6-Phospate as an alternative secondary energy source.
  • pyruvate could be used for ATP regeneration
  • glycolytic intermediates could be used to regenerate ATP in our cell-free system.
  • the use of glucose-6-phosphate was examined as it is the first intermediate of the glycolytic pathway.
  • glucose-6-phosphate was used under the same reaction conditions as in the pyruvate/NAD system, it did support protein synthesis.
  • the initial rate was substantially lower than in the reaction with PEP, protein synthesis continued for over 2 hours ( Figure 5A).
  • Figure 5A As a result, approximately 30 % more CAT was produced at the end of incubation. Most likely, this is due to the remarkably extended maintenance of ATP concentrations.
  • the ATP regeneration with glucose-6-phosphate indicates that all of the glycolytic enzymes required to convert glucose-6-phosphate into pyruvate are active under the present reaction conditions. This provides a great flexibility in choosing a secondary energy source for protein synthesis. Any of the glycolytic intermediates between glucose-6-phosphate and pyruvate can be used for ATP regeneration.
  • the reactions of ATP regeneration using pyruvate also can be used to improve the utilization efficiency of the conventional energy source, PEP.
  • PEP After being used for ATP regeneration or being degraded by phosphatase activities of cell-extract, PEP produces an equimolar amount of pyruvate. If the cofactors NAD and CoA are present in the reaction mixture, half of the newly generated pyruvate is available for ATP regeneration (the other half is used for the regeneration of NAD). As a result, through the two-stage utilization of the energy source, the overall concentration of ATP is elevated and prolonged and the productivity of protein synthesis is improved.
  • glucose-6-phosphate the first intermediate of glycolytic pathway
  • PEP the use of glucose-6-phosphate substantially increased the synthesis yield mainly by prolonging the reaction period. This seems to be due to the enhanced supply of ATP, which can be explained since glucose-6-phosphate offers a greater potential to regenerate ATP compared to PEP or pyruvate. While PEP or pyruvate can regenerate, at best, only the equivalent number of ATP molecules, 3 molecules of ATP can be generated during the oxidation of glucose- 6-phosphate into pyruvate.
  • glucose would provide a cell-free system that is highly competitive with traditional technologies for protein expression in terms of economic efficiency. Our initial results suggest such a possibility.
  • the use of glucose along with hexokinase did support protein synthesis.
  • the present cell-extract may contain active membrane vesicles, that could be used in oxidative phosphorylation to provide an extremely efficient method for ATP supply to the protein synthesis by mimicking the function of living cells.

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  • Proteomics, Peptides & Aminoacids (AREA)
  • Analytical Chemistry (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
EP00980413A 2000-11-14 2000-11-14 In-vitroproteinsynthese unter verwendung von glykolytischen zwischenprodukten als energiequelle Withdrawn EP1341802A4 (de)

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JP5259046B2 (ja) * 2002-08-19 2013-08-07 ボード オブ トラスティーズ オブ ザ レランド スタンフォード ジュニア ユニバーシティ 改良されたインビトロ・タンパク質合成の方法
BRPI0507233B8 (pt) 2004-03-11 2021-05-25 Genentech Inc processo para a produção de um polipeptídeo heterólogo em e. coli
AU2012203817B2 (en) * 2004-03-11 2015-05-28 Genentech, Inc. Process for producing polypeptides
WO2015025865A1 (ja) * 2013-08-21 2015-02-26 独立行政法人科学技術振興機構 酵素の保存液およびそれを用いた再構成型無細胞翻訳システム用反応液
EP3189162A1 (de) * 2014-09-02 2017-07-12 Pharmozyme, Inc. Plattform zur genetischen detektion
CN110366428B (zh) 2016-12-30 2024-05-17 Vaxcyte公司 具有非天然氨基酸的多肽-抗原缀合物
US11951165B2 (en) 2016-12-30 2024-04-09 Vaxcyte, Inc. Conjugated vaccine carrier proteins

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US4199498A (en) * 1975-07-15 1980-04-22 Snamprogetti S.P.A. Macromolecularized adenine derivatives
JPS5564599A (en) * 1978-11-08 1980-05-15 Unitika Ltd Adenosine triphosphate derivative
US5801006A (en) * 1997-02-04 1998-09-01 Specialty Assays, Inc. Use of NADPH and NADH analogs in the measurement of enzyme activities and metabolites
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AU2001217678A1 (en) 2002-05-27
WO2002040497A1 (en) 2002-05-23
JP2004513652A (ja) 2004-05-13
CA2428693A1 (en) 2002-05-23

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