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WO2018161374A1 - Système de synthèse de protéines pour la synthèse de protéines in vitro, trousse et procédé de préparation associé - Google Patents

Système de synthèse de protéines pour la synthèse de protéines in vitro, trousse et procédé de préparation associé Download PDF

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WO2018161374A1
WO2018161374A1 PCT/CN2017/077814 CN2017077814W WO2018161374A1 WO 2018161374 A1 WO2018161374 A1 WO 2018161374A1 CN 2017077814 W CN2017077814 W CN 2017077814W WO 2018161374 A1 WO2018161374 A1 WO 2018161374A1
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protein synthesis
synthesis system
vitro
cell
protein
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PCT/CN2017/077814
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English (en)
Chinese (zh)
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郭敏
柴智
刘帅龙
符雷
王海鹏
于雪
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康码（上海）生物科技有限公司
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Priority to KR1020197026572A priority Critical patent/KR102126985B1/ko
Publication of WO2018161374A1 publication Critical patent/WO2018161374A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
    • G01N33/6806—Determination of free amino acids
    • G01N33/6812—Assays for specific amino acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/06—Lysis of microorganisms
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids

Definitions

  • the present invention relates to the field of biotechnology, and in particular to a protein synthesis system, a kit for preparing protein synthesis in vitro, and a preparation method thereof.
  • a conventional protein expression system refers to a molecular biological technique for expressing a foreign gene by a model organism, a fungus, a plant cell or an animal cell.
  • a cell-free expression system also known as an in vitro protein synthesis system, has emerged. It is an exogenous target mRNA or DNA that is a template for protein synthesis, and artificially controls the substrate and transcription required for protein synthesis. Translation of related protein factors and other substances, can achieve the synthesis of the target protein.
  • Protein expression in an in vitro translation system requires no plasmid construction, transformation, cell culture, cell collection and fragmentation steps, and is a fast, time-saving, and convenient way to express proteins.
  • Escherichia coli is easy to culture and ferment, low in cost, simple in breaking cells, and capable of synthesizing higher yield proteins.
  • eukaryotic cells are difficult to culture and costly, and the preparation process of their cell extracts is cumbersome. Therefore, their translation systems are costly and suitable for special laboratories. Therefore, eukaryotic in vitro protein expression systems suitable for industrial large-scale (ton-class) preparation and production are currently not available.
  • the present invention provides a high yield, low cost in vitro expression system.
  • the invention provides an establishment and optimization of a method for synthesizing proteins in vitro by using DNA as a template to overcome the defects and deficiencies of the prior art.
  • a first object of the present invention is to provide a method for preparing a yeast extract, and a second object of the present invention is to provide an in vitro protein synthesis kit.
  • a first aspect of the invention provides an in vitro cell-free protein synthesis system, the cell-free protein synthesis system comprising:
  • the cell-free protein synthesis system further comprises one or more components selected from the group consisting of:
  • the cell-free protein synthesis system further comprises one or more components selected from the group consisting of:
  • the yeast cell is selected from the group consisting of yeast of one or more sources: Saccharomyces cerevisiae, Pichia pastoris, Kluyveromyces, or a combination thereof; preferably, the yeast cell Including: Kluyveromyces, more preferably Kluyveromyces lactis.
  • the yeast cell extract is an aqueous extract of yeast cells.
  • the yeast cell extract is free of yeast endogenous long chain nucleic acid molecules.
  • the yeast cell extract is prepared by a method comprising the steps of:
  • the solid-liquid separation comprises centrifugation.
  • centrifugation is carried out in a liquid state.
  • the centrifugation conditions are from 5,000 to 100,000 x g, preferably from 8,000 to 30,000 x g.
  • the centrifugation time is from 0.5 to 2 h, preferably from 20 min to 50 min.
  • the centrifugation is carried out at 1-10 ° C, preferably at 2-6 ° C.
  • the washing treatment is carried out using a washing liquid at a pH of 7-8 (preferably, 7.4).
  • the washing liquid is selected from the group consisting of potassium 4-hydroxyethylpiperazine ethanesulfonate, potassium acetate, magnesium acetate, or a combination thereof.
  • the cell disruption treatment comprises high pressure disruption, freeze-thaw (eg, liquid nitrogen cryolysis) disruption.
  • the substrate for the synthetic RNA comprises: a nucleoside monophosphate, a nucleoside triphosphate, or a combination thereof.
  • the substrate of the synthetic protein comprises: 1-20 natural amino acids, and unnatural amino acids.
  • the magnesium ion is derived from a source of magnesium ions selected from the group consisting of magnesium acetate, magnesium glutamate, or a combination thereof.
  • the potassium ion is derived from a source of potassium ions selected from the group consisting of potassium acetate, potassium glutamate, or a combination thereof.
  • the energy regeneration system is selected from the group consisting of a phosphocreatine/phosphocreatase system, a glycolysis pathway and its intermediate energy system, or a combination thereof.
  • the cell-free protein synthesis system further comprises (f1) a synthetic tRNA.
  • the buffering agent is selected from the group consisting of 4-hydroxyethylpiperazineethanesulfonic acid, trishydroxymethylaminomethane, or a combination thereof.
  • the cell-free protein synthesis system further comprises (g1) a foreign DNA molecule for directing protein synthesis.
  • the DNA molecule is linear.
  • the DNA molecule is cyclic.
  • the DNA molecule contains a sequence encoding a foreign protein.
  • the sequence encoding the foreign protein comprises a genomic sequence, a cDNA sequence.
  • sequence encoding the foreign protein further comprises a promoter sequence, a 5' untranslated sequence, and a 3' untranslated sequence.
  • the cell-free protein synthesis system comprises a component selected from the group consisting of 4-hydroxyethylpiperazineethanesulfonic acid, potassium acetate, magnesium acetate, nucleoside triphosphate, amino acid, creatine phosphate Dithiothreitol (DTT), phosphocreatine kinase, RNA polymerase, or a combination thereof.
  • the polyethylene glycol is selected from the group consisting of PEG3000, PEG 8000, PEG 6000, PEG 3350, or a combination thereof.
  • the polyethylene glycol comprises polyethylene glycol having a molecular weight (Da) of from 200 to 10,000, preferably polyethylene glycol having a molecular weight of from 3,000 to 10,000.
  • the concentration (v/v) of the component (a) in the protein synthesis system is from 20% to 70%, preferably from 30% to 60%, more preferably from 40% to 50%. %, based on the total volume of the protein synthesis system.
  • the concentration (w/v, for example, g/ml) of the component (b) in the protein synthesis system is 0.1 to 8%, preferably 0.5 to 4%, more preferably, 1-2%.
  • the concentration of component (c) in the protein synthesis system is 0.2 to 4%, preferably 0.5 to 4%, more preferably 0.5 to 1%, to synthesize the protein.
  • the total volume of the system is 0.2 to 4%, preferably 0.5 to 4%, more preferably 0.5 to 1%, to synthesize the protein.
  • the nucleoside triphosphate is selected from the group consisting of adenosine triphosphate, guanosine triphosphate, cytidine triphosphate, uridine nucleoside triphosphate, or a combination thereof.
  • the concentration of the component (e1) in the protein synthesis system is from 0.1 to 5 mM, preferably from 0.5 to 3 mM, more preferably from 1 to 1.5 mM.
  • the amino acid is selected from the group consisting of glycine, alanine, valine, leucine, isoleucine, phenylalanine, valine, tryptophan, serine, Tyrosine, cysteine, methionine, asparagine, glutamine, threonine, aspartic acid, glutamic acid, lysine, arginine, histidine, or a combination thereof.
  • the amino acid comprises a D-form amino acid and/or an L-form amino acid.
  • the concentration of the component (e2) in the protein synthesis system is 0.01 to 0.48 mM, preferably 0.04 to 0.24 mM, more preferably 0.04 to 0.2 mM, optimally , 0.08 mM.
  • the concentration of the component (e3) in the protein synthesis system is 1-10 mM, preferably 1-5 mM, more preferably 2-4 mM.
  • the concentration of the component (e4) in the protein synthesis system is 30-210 mM, preferably 30-150 mM, more preferably 30-60 mM.
  • the concentration of the component (e6) in the protein synthesis system is 0. 01-0.3 mg/ml, preferably 0.02-0.1 mg/ml, more preferably 0.027-0.054 mg/ml.
  • the concentration of 4-hydroxyethylpiperazineethanesulfonic acid in the protein synthesis system is 5 to 50 mM, preferably 10 to 50 mM, preferably 15 to 30 mM, more preferably , 20-25 mM.
  • the concentration of the potassium acetate in the protein synthesis system is 20-210 mM, preferably 30-210 mM, preferably 30-150 mM, more preferably 30-60 mM.
  • the magnesium acetate has a concentration of 1-10 mM, preferably 1-5 mM, more preferably 2-4 mM.
  • the concentration of creatine phosphate is 10-50 mM, preferably 20-30 mM, more preferably 25 mM.
  • the concentration of the heme in the protein synthesis system is 0.01 to 0.1 mM, preferably 0.02 to 0.08 mM, more preferably 0.03 to 0.05 mM, most preferably 0.04 mM. .
  • the spermidine concentration in the protein synthesis system is 0.05-1 mM, preferably 0.1-0.8 mM, more preferably, more preferably 0.2-0.5 mM, more preferably Ground, 0.3-0.4 mM, optimally, 0.4 mM.
  • the concentration of the dithiothreitol (DTT) in the protein synthesis system is from 0.2 to 15 mM, preferably from 0.2 to 7 mM, more preferably from 1 to 2 mM.
  • the concentration of the phosphocreatine kinase in the protein synthesis system is 0.1 to 1 mg/ml, preferably 0.2 to 0.5 mg/ml, more preferably 0.27 mg/ml.
  • the concentration of the T7 RNA polymerase in the protein synthesis system is 0.01-0.3 mg/ml, preferably 0.02-0.1 mg/ml, more preferably 0.027-0.054 mg/ml. .
  • the cell-free in vitro synthesis system has the following properties:
  • composition of the cell-free protein synthesis system comprises:
  • composition of the cell-free protein synthesis system further comprises:
  • the PEG is selected from the group consisting of PEG 3350, PEG 3000, and/or PEG 8000.
  • the RNA polymerase is T7 RNA polymerase.
  • a second aspect of the invention provides a method of synthesizing a protein in vitro comprising the steps of:
  • step (ii) incubating the protein synthesis system of step (i) for a period of time T1 under suitable conditions to synthesize the protein encoded by the exogenous DNA.
  • the method further comprises: (iii) isolating or detecting the protein encoded by the exogenous DNA, optionally from the protein synthesis system.
  • the exogenous DNA is from a prokaryote, a eukaryote.
  • the exogenous DNA is from an animal, a plant, or a pathogen.
  • the exogenous DNA is from a mammal, preferably a primate, a rodent, including a human, a mouse, a rat.
  • the exogenous DNA is selected from the group consisting of luciferin, or luciferase (such as firefly luciferase), green fluorescent protein, yellow fluorescent protein, aminoacyl tRNA synthetase, glycerol An aldehyde-3-phosphate dehydrogenase, a catalase, an actin, an exogenous DNA of a variable region of an antibody, a DNA of a luciferase mutant, or a combination thereof.
  • luciferin or luciferase (such as firefly luciferase)
  • green fluorescent protein yellow fluorescent protein
  • aminoacyl tRNA synthetase aminoacyl tRNA synthetase
  • glycerol an aldehyde-3-phosphate dehydrogenase
  • an actin an exogenous DNA of a variable region of an antibody
  • DNA of a luciferase mutant or a combination thereof.
  • nucleotide sequence of the exogenous DNA is as shown in any one of SEQ ID NO.: 1-7.
  • the reaction temperature is 20 to 37 ° C, preferably 20 to 25 ° C.
  • the reaction time is from 1 to 6 h, preferably from 2 to 4 h.
  • a third aspect of the invention provides a kit for in vitro cell-free synthesis of a protein comprising:
  • first container, the second container, and the third container are the same container or different containers.
  • the kit further comprises an optional one or more containers selected from the group consisting of:
  • Figure 1 is a schematic diagram showing the comparison of a protein synthesis system and a control reaction for direct protein synthesis in vitro from a DNA template.
  • A is Buffer itself
  • B is an in vitro protein synthesis protein synthesis system without added Firefly luciferase (Fluc) DNA
  • C is an in vitro protein synthesis protein synthesis system supplemented with Firefly luciferase (Fluc) DNA.
  • the reaction conditions were 20 ° C for 4 h.
  • the activities of negative controls A and B were 77 RLU and 160 RLU, respectively.
  • the activity of the reaction sample was 1,303,884 RLU. All errors are the standard deviation of three replicates.
  • FIG. 2 is a schematic diagram of the effect of different reaction solutions on the in vitro protein synthesis system.
  • A is Buffer itself
  • B is an in vitro protein synthesis protein synthesis system without added Firefly luciferase (Fluc) DNA
  • C is added with Firefly luciferase (Fluc) DNA in acetic acid.
  • D is an in vitro protein synthesis protein synthesis system added with firefly luciferase (Fluc) DNA in magnesium glutamate and potassium glutamate reaction solution .
  • the reaction conditions were 20 ° C and the reaction was carried out for 2 h.
  • the activities of negative controls A and B were 77 RLU and 160 RLU, respectively.
  • the activity of protein synthesis in vitro was 1,303,884 RLU; in the glutamic acid protein synthesis system, the activity of protein synthesis in vitro was 1,469,472 RLU.
  • Figure 3 is a graphical representation of the effect of different bacterial concentration and reaction time on the in vitro protein synthesis system.
  • the reaction temperature was 20 ° C and the reaction time was 2-6 h.
  • the reaction time is 2-4 h, and the difference is not large.
  • the activity of the negative control without mRNA and buffer itself was 520 RLU and 400 RLU, respectively.
  • Figure 4 is a schematic diagram showing the effect of different centrifugal force treatment of yeast extract on the in vitro protein synthesis system.
  • the reaction conditions were 20 ° C and the reaction was carried out for 2 h.
  • the reaction buffer is a system of magnesium acetate and potassium acetate.
  • A is Buffer itself
  • B is an in vitro protein synthesis reaction without adding Firefly luciferase (Fluc) DNA
  • C is an in vitro protein synthesis reaction of 30,000 ⁇ g centrifuged yeast extract
  • D is 18,000 ⁇ g centrifugation.
  • E is an in vitro protein synthesis reaction of 15,000 x g centrifuged yeast extract
  • F is an in vitro protein synthesis reaction of 12,000 x g centrifuged yeast extract.
  • the activity of the negative control without mRNA and buffer itself was 200 RLU and 320 RLU, respectively.
  • Figure 5 is a graphical representation of the effect of magnesium acetate concentration on in vitro protein synthesis systems.
  • the reaction conditions were 20 ° C for 2 h, and the reaction liquid was a magnesium acetate and potassium acetate system.
  • the concentration of magnesium acetate in the protein synthesis system ranged from 1 to 8 mM, and the activity of the negative control without mRNA and buffer itself was 80 RLU and 40 RLU, respectively.
  • Figure 6 is a graphical representation of the effect of potassium acetate concentration on in vitro protein synthesis systems.
  • the reaction conditions were 20 ° C for 2 h, and the reaction liquid was a magnesium acetate and potassium acetate system.
  • the concentration of potassium acetate in the protein synthesis system ranged from 30 to 180 mM, and the activity of the negative control without mRNA and buffer itself was 80 RLU and 40 RLU, respectively.
  • Figure 7 is a graphical representation of the effect of amino acid concentration on an in vitro protein synthesis system.
  • the reaction conditions were 20 ° C for 2 h, and the reaction liquid was a magnesium acetate and potassium acetate system.
  • the concentration of amino acids in the protein synthesis system ranged from 0.04 to 0.24 mM, and the activity of the negative control without mRNA and buffer itself was 52 RLU and 90 RLU, respectively.
  • Figure 8 is a graphical representation of the effect of ATP concentration on in vitro protein synthesis systems.
  • the reaction conditions were 20 ° C for 2 h, and the reaction liquid was a magnesium acetate and potassium acetate system.
  • the concentration of ATP in the protein synthesis system ranged from 0.04 to 0.24 mM, and the activity of the negative control without mRNA and buffer itself was 52 RLU and 90 RLU, respectively.
  • Figure 9 is a schematic illustration of the names and sequence structures of different sequences of the luciferase gene. These include the sequence of the 5' and 3' ends of the gene sequence, the number of polyadenylation nucleotides, and the like. Omega sequence is derived from tobacco mosaic The virus, the CrPV sequence is derived from the ricin virus, and the SITS2 sequence is derived from an independent translation initiation sequence. Meanwhile, the number of polyadenosine nucleotides includes 48A, 70A and 90A.
  • Figure 10 is a schematic diagram showing the effect of the polyadenylation deoxynucleotide of the 3'-untranslated region of the luciferase gene on the in vitro protein synthesis system.
  • the reaction conditions were 25 ° C for 2 h, and the reaction liquid was a magnesium acetate and potassium acetate system.
  • PC1 represents a commercial rabbit reticulocyte in vitro protein synthesis system as a positive control.
  • NC1 is a negative control in vitro protein synthesis system without a DNA template, and NC2 is a negative control buffer system.
  • the positive control PC1 activity was 1,334,396 RLU.
  • the activity of the negative control without DNA and buffer itself was 66 RLU and 69 RLU, respectively.
  • Figure 11 is a schematic diagram showing the effect of the 5' end sequence of the luciferase gene on the in vitro protein synthesis system.
  • the reaction conditions were 25 ° C for 2 h, and the reaction liquid was a magnesium acetate and potassium acetate system.
  • NC represents an in vitro protein synthesis system with a negative control DNA-free template with an activity of 44 RLU.
  • Figure 12 is a graphical representation of the effect of different PEG and different concentrations on in vitro protein synthesis systems.
  • the reaction conditions were 25 ° C for 2 h, and the reaction liquid was a magnesium acetate and potassium acetate system.
  • PEG contains three kinds, PEG3350, PEG8000 and PEG3000. Each PEG contains three to four concentrations of 0.5%, 1%, 2%, and 4% in the protein synthesis system.
  • NC represents an in vitro protein synthesis protein synthesis system with a negative control DNA-free template with an activity of 44 RLU.
  • Figure 13 is a graphical representation of the effect of sucrose concentration on an in vitro protein synthesis system.
  • the reaction conditions were 25 ° C for 2 h, and the reaction liquid was a magnesium acetate and potassium acetate system.
  • the sucrose concentrations contained in the protein synthesis system were three concentrations of 0.5%, 1%, and 2%.
  • NC represents an in vitro protein synthesis protein synthesis system with a negative control DNA-free template and an activity of 190 RLU.
  • Figure 14 is a graphical representation of the effect of heme concentration on an in vitro protein synthesis system.
  • the reaction conditions were 25 ° C for 2 h, and the reaction liquid was a magnesium acetate and potassium acetate system.
  • the heme concentration contained in the protein synthesis system was 0, 0.01, 0.02, 0.03, 0.04 mM.
  • Figure 15 is a graphical representation of the effect of spermidine concentration on an in vitro protein synthesis system.
  • the reaction conditions were 25 ° C for 2 h, and the reaction liquid was a magnesium acetate and potassium acetate system.
  • the concentration of spermidine contained in the protein synthesis system was 0, 0.1, 0.2, 0.3, 0.4 mM.
  • the in vitro cell-free expression system of the present invention not only can synthesize proteins extremely efficiently, but also can synthesize complex proteins such as glycosylated proteins, as compared to rabbit reticulocyte in vitro expression systems. On the basis of this, the inventors completed the present invention.
  • the relative light unit value of the synthesized luciferase activity can be up to about 60 times that of current commercial systems, such as rabbit reticulocyte in vitro expression systems.
  • expression system of the invention As used herein, the terms "expression system of the invention”, “in vitro expression system of the invention”, “in vitro cell-free expression system”, “in vitro cell-free expression system” are used interchangeably and refer to the yeast-based, An in vitro protein expression system that does not contain living cells.
  • Yeast combines the advantages of simple, efficient protein folding, and post-translational modification. Among them, Saccharomyces cerevisiae and Pichia pastoris are model organisms that express complex eukaryotic proteins and membrane proteins. Yeast can also be used as a raw material for the preparation of in vitro translation systems.
  • Kluyveromyces is an ascomycete, in which Kluyveromyces marxianus and Kluyveromyces lactis are industrially widely used yeasts.
  • Kluyveromyces cerevisiae has many advantages over other yeasts, such as superior secretion capacity, better large-scale fermentation characteristics, food safety levels, and the ability to simultaneously modify post-translational proteins.
  • the yeast in vitro expression system is not particularly limited, and a preferred yeast in vitro expression system is the Kluyveromyces expression system (more preferably, the K. lactis expression system).
  • the invention provides an in vitro cell-free protein synthesis system, the synthesis system comprising:
  • the in vitro protein synthesis system comprises a group selected from the group consisting of One or more or all of the components: yeast cell extract, 4-hydroxyethylpiperazineethanesulfonic acid, potassium acetate, magnesium acetate, adenosine triphosphate (ATP), guanosine triphosphate (GTP) ), cytosine triphosphate (CTP), thymidine triphosphate (TTP), amino acid mixture, creatine phosphate, dithiothreitol (DTT), phosphocreatine kinase, RNase inhibitor, fluorescein , luciferase DNA, RNA polymerase, spermidine, heme.
  • yeast cell extract 4-hydroxyethylpiperazineethanesulfonic acid
  • potassium acetate magnesium acetate
  • adenosine triphosphate (ATP), guanosine triphosphate (GTP) cytosine triphosphate (CTP), thymidine triphosphate (T
  • the RNA polymerase is not particularly limited and may be selected from one or more RNA polymerases, and a typical RNA polymerase is T7 RNA polymerase.
  • the ratio of the yeast cell extract in the in vitro protein synthesis system is not particularly limited, and generally the yeast cell extract accounts for 20-70% of the in vitro protein synthesis protein synthesis system, preferably Ground, 30-60%, more preferably, 40-50%.
  • the yeast cell extract does not contain intact cells, and typical yeast cell extracts include ribosomes for protein translation, transfer RNA, aminoacyl tRNA synthetase, initiation factors required for protein synthesis, and The elongation factor and the termination release factor.
  • the yeast extract contains some other proteins in the cytoplasm derived from yeast cells, especially soluble proteins.
  • the yeast cell extract contains a protein content of 20 to 100 mg/ml, preferably 50 to 100 mg/ml.
  • the method for determining protein content is a Coomassie Brilliant Blue assay.
  • the preparation method of the yeast cell extract is not limited, and a preferred preparation method comprises the following steps:
  • the solid-liquid separation method is not particularly limited, and a preferred mode is centrifugation.
  • the centrifugation is carried out in a liquid state.
  • the centrifugation conditions are not particularly limited, and a preferred centrifugation condition is 5,000 to 100,000 x g, preferably 8,000 to 30,000 x g.
  • the centrifugation time is not particularly limited, and a preferred centrifugation time is from 0.5 min to 2 h, preferably from 20 min to 50 min.
  • the temperature of the centrifugation is not particularly limited.
  • the centrifugation is carried out at 1-10 ° C, preferably at 2-6 ° C.
  • the washing treatment method is not particularly limited, and a preferred washing treatment method is treatment with a washing liquid at a pH of 7-8 (preferably, 7.4), and the washing liquid is not particularly Typically, the wash liquor is typically selected from the group consisting of potassium 4-hydroxyethylpiperazine ethanesulfonate, potassium acetate, magnesium acetate, or combinations thereof.
  • the manner of the cell disruption treatment is not particularly limited, and a preferred cell disruption treatment includes high pressure disruption, freeze-thaw (e.g., liquid nitrogen low temperature) disruption.
  • the mixture of nucleoside triphosphates in the in vitro protein synthesis system is adenine nucleoside triphosphate, guanosine triphosphate, cytidine triphosphate, and uridine nucleoside triphosphate.
  • the concentration of each of the single nucleotides is not particularly limited, and usually the concentration of each single nucleotide is from 0.5 to 5 mM, preferably from 1.0 to 2.0 mM.
  • the mixture of amino acids in the in vitro protein synthesis system can include natural or unnatural amino acids, and can include D-form or L-form amino acids.
  • Representative amino acids include, but are not limited to, 20 natural amino acids: glycine, alanine, valine, leucine, isoleucine, phenylalanine, valine, tryptophan, serine, Tyrosine, cysteine, methionine, asparagine, glutamine, threonine, aspartic acid, glutamic acid, lysine, arginine and histidine.
  • the concentration of each amino acid is usually from 0.01 to 0.5 mM, preferably from 0.02 to 0.2 mM, such as 0.05, 0.06, 0.07, 0.08 mM.
  • the in vitro protein synthesis system further comprises polyethylene glycol or an analog thereof.
  • concentration of polyethylene glycol or the like is not particularly limited, and usually, the concentration (w/v) of polyethylene glycol or the like is from 0.1 to 8%, preferably from 0.5 to 4%, more preferably, 1-2%, based on the total weight of the protein synthesis system.
  • Representative examples of PEG include, but are not limited to, PEG3000, PEG 8000, PEG 6000, and PEG 3350. It should be understood that the system of the present invention may also include other various molecular weight polyethylene glycols (e.g., PEG 200, 400, 1500, 2000, 4000, 6000, 8000, 10000, etc.).
  • the in vitro protein synthesis system further comprises sucrose.
  • concentration of sucrose is not particularly limited, and usually, the concentration (w/v) of sucrose is 0.2 to 4%, preferably 0.5 to 4%, more preferably 0.5 to 1%, based on the total volume of the protein synthesis system. meter.
  • the in vitro protein synthesis system further comprises heme.
  • concentration of hemoglobin is not particularly limited, and usually, the concentration of heme is 0.01 to 0.1 mM, preferably 0.02 to 0.08 mM, more preferably 0.03 to 0.05 mM, most preferably 0.04 mM.
  • the in vitro protein synthesis system further comprises spermidine.
  • concentration of spermidine is not particularly limited, and usually, the concentration of spermidine is 0.05 to 1 mM, preferably 0.1 to 0.8 mM, and more preferably, More preferably, 0.2-0.5 mM, more preferably, 0.3-0.4 mM, optimally, 0.4 mM.
  • the in vitro protein synthesis system further contains a buffer, the composition of which is not particularly limited, and a preferred buffer contains 4-hydroxyethylpiperazineethanesulfonic acid, and/or Tris buffer. liquid.
  • the buffer may further contain other buffer components such as potassium acetate or magnesium acetate to form a reaction solution or a reaction buffer having a pH of 6.5 to 8.5 (preferably 7.0 to 8.0).
  • the type and content of the buffer are not particularly limited.
  • the buffer is present at a concentration of 1-200 mM or 1-100 mM, preferably 5-50 mM.
  • a particularly preferred in vitro protein synthesis system comprising, in addition to the yeast extract, one or more or all of the components selected from the group consisting of 22 mM 4-hydroxyethylpiperazineethanesulfonic acid having a pH of 7.4, 30-150 mM potassium acetate, 1.0-5.0 mM magnesium acetate, 1.5-4 mM nucleoside triphosphate mixture, 0.08-0.24 mM amino acid mixture, 25 mM phosphocreatine, 1.7 mM dithiothreitol, 0.27 mg/mL phosphocreatine Kinase, 1%-4% polyethylene glycol, 0.5%-2% sucrose, 8-20 ng/ ⁇ l of firefly luciferase DNA, 0.027-0.054 mg/mL T7 RNA polymerase, 0.03-0.04 mM heme, 0.3 - 0.4 mM spermidine.
  • the cell-free protein synthesis system When used for in vitro protein synthesis, the cell-free protein synthesis system also includes (g1) exogenous DNA molecules for directing protein synthesis.
  • the DNA molecule is linear or circular.
  • the DNA molecule contains a sequence encoding a foreign protein.
  • examples of the sequence encoding the foreign protein include, but are not limited to, a genomic sequence, a cDNA sequence.
  • the sequence encoding the foreign protein further comprises a promoter sequence, a 5' untranslated sequence, and a 3' untranslated sequence.
  • the selection of the exogenous DNA is not particularly limited.
  • the exogenous DNA is selected from the group consisting of a luciferin protein, or a luciferase (such as firefly luciferase), a green fluorescent protein, and a yellow fluorescent protein. , aminoacyl tRNA synthetase, glyceraldehyde-3-phosphate dehydrogenase, catalase, actin, exogenous DNA of a variable region of an antibody, DNA of a luciferase mutant, or a combination thereof.
  • a representative sequence of exogenous DNA is selected from the group consisting of: SEQ ID NO.: 1-SEQ ID NO.: 7.
  • the invention provides a kit for in vitro cell-free synthesis of proteins, comprising:
  • the first container, the second container and the third container are the same container or different containers.
  • a particularly preferred kit for in vitro protein synthesis comprises an in vitro protein synthesis protein synthesis system comprising one or more or all of the components selected from the group consisting of yeast cell extracts, 4-hydroxyethyl piperidine Acetone ethanesulfonic acid, potassium acetate, magnesium acetate, adenine nucleoside triphosphate (ATP), guanosine triphosphate (GTP), cytosine triphosphate (CTP), thymidine triphosphate (TTP) , amino acid mixture, creatine phosphate, dithiothreitol (DTT), phosphocreatine kinase, RNase inhibitor, fluorescein, luciferase DNA, T7 RNA polymerase, spermidine, heme.
  • yeast cell extracts 4-hydroxyethyl piperidine Acetone ethanesulfonic acid
  • potassium acetate magnesium acetate
  • ATP adenine nucleoside triphosphate
  • GTP guanosine
  • the kit of the present invention can be used for in vitro protein synthesis of DNA as a template, which is simpler and faster than expression of an in vitro protein using RNA as a template.
  • the yeast in vitro expression system of the present invention omits time-consuming and labor-intensive steps such as plasmid transformation, cell culture, collection, fragmentation and centrifugation, greatly improving work efficiency, and synthetic Proteins are easier to purify, saving users a lot of time and cost.
  • the yeast in vitro expression system of the invention expresses more active proteins, especially in the expression membrane proteins, cytotoxic proteins, molecular chaperones, macromolecular protein complexes, etc. obvious advantage.
  • the yeast extract prepared by high pressure crushing or liquid nitrogen disruption has the ability to directly synthesize proteins using a DNA template and is optimized by reaction conditions such as magnesium acetate, potassium acetate, amino acids, ATP, DNA templates of different sequences. , polyethylene glycol, sucrose optimization, etc., the relative activity of the synthesized luciferase has reached 60,000,000 RLU, while the commercial cell-free expression system, such as rabbit reticulocyte in vitro expression system, the activity of the synthesized luciferase is only 1,000,000 RLU.
  • the relative light unit value of the luciferase activity synthesized by the present invention under optimal conditions is 60 times that of the commercial system.
  • the preparation of the yeast extract and the kit prepared in the present invention not only overcomes the defects of the prior art, but also It has greater advantages and prospects than the prior art.
  • the raw material yeast cells used in the present invention are simple in culture, convenient in operation, rapid in propagation, low in cost, suitable for large-scale preparation, have advantages in industrial production, and the crushing method used in the present invention: high-pressure homogenizer
  • the crushing method and the liquid nitrogen mechanical crushing method are simple, efficient and easy to enlarge, and are suitable for large-scale preparation of industrial production.
  • Example 1 Preparation of yeast cell extract by high pressure crushing
  • yeast seed solution Single colony of Kluyveromyces cerevisiae was picked from the plate and inoculated into 50 mL of YPD medium (the composition of YPD medium was: 1% yeast extract, 2% peptone, 2% glucose, In a 250 mL Erlenmeyer flask of pH 5.5) (the amount of liquid was 20%, the same applies hereinafter), the inoculated flask was placed in a shaker and cultured at a temperature of 30 ° C, a rotation speed of 200 rpm, and cultured for 24 hours. The seed is obtained;
  • the seed solution prepared in 1.1 was inoculated into a 2 L Erlenmeyer flask containing 400 mL of YPD medium at a dose of 0.1-1%, and placed in a shaker for cultivation.
  • the cultured cell culture in 1.2 was pre-cooled in an ice-water mixture for 10-30 min.
  • Washing buffer consists of: 10-40 mM potassium 4-hydroxyethylpiperazine sulfonate pH 7.4, 50-150 mM potassium acetate, 1-4 mM magnesium acetate;
  • step 1.6 The yeast cells obtained in step 1.6 were directly subjected to subsequent operations, or frozen at -80 ° C after liquid freezing.
  • yeast cells were disrupted by a high-pressure homogenizer: resuspended in 0.2-0.5 mL of buffer A per gram of yeast cells, and the resuspension was disrupted by a high-pressure homogenizer to obtain a crude cell extract.
  • Conditions for high-pressure crushing pressure is 1000-1400 bar, temperature is 4 ° C, and the number of crushing is one or more times.
  • step 1.8 The crude yeast cell extract obtained in step 1.8 is centrifuged 1-2 times, the centrifugal force is 12000-30000 ⁇ g, the time is 30 min, and the temperature is 4 ° C;
  • the prepared yeast cell extract was dispensed, frozen in liquid nitrogen, and stored at -80 °C.
  • the protein content of the different batches of yeast cell extracts was determined to be about 20-100 mg/ml, with an average of about 60-70 mg/ml, as determined by the Coomassie Brilliant Blue assay.
  • yeast seed solution Single colony of Kluyveromyces cerevisiae was picked from the plate and inoculated into 50 mL YPD medium (the composition of YPD medium was: 1% yeast extract, 2% peptone, 2% glucose, In a 250 mL Erlenmeyer flask of pH 5.5) (liquid content: 20%, the same applies hereinafter), the inoculated flask was placed in a shaker and cultured under the conditions of a temperature of 30 ° C and a rotation speed of 200 rpm. The seed liquid obtained after culturing for 24 hours;
  • the seed solution prepared in 2.1 was inoculated into a 2 L Erlenmeyer flask containing 400 mL of YPD medium at a seeding rate of 0.1-1%, and placed in a shaker for cultivation.
  • the culture condition was 30 °C.
  • the rotation speed is 200 rpm.
  • the culture is terminated to obtain a cell culture solution;
  • the cultured cell culture in 2.2 was pre-cooled in an ice-water mixture for 10-30 min.
  • the pre-cooled cell culture in 2.3 was centrifuged in a cryogenic centrifuge, and centrifuged conditions: 3,000 ⁇ g, 10 min, 4 ° C, to obtain yeast cells.
  • the yeast cells were resuspended in 2.4 with pre-cooled Washing buffer, and the amount of Washing buffer was 50-100 ml/L.
  • the obtained resuspension was centrifuged in a low temperature centrifuge, and centrifuged conditions: 3000 ⁇ g, 10 min, 4 ° C, to obtain yeast cells.
  • the composition of Washing buffer is: 20-30 mM potassium 4-hydroxyethylpiperazine sulfonate, pH 7.4, 100-150 mM potassium acetate, 1-4 mM magnesium acetate;
  • step 2.6 The yeast cells obtained in step 2.6 were directly subjected to subsequent operations, or were frozen at -80 ° C after rapid freezing with liquid nitrogen.
  • Lysisbuffer consists of 10-40 mM potassium 4-hydroxyethylpiperazine sulfonate pH 7.4, 50-150 mM potassium acetate, 1-4 mM magnesium acetate, 2-7 mM dithiothreitol, 0.5-2 mM phenylmethylsulfonyl Fluorine composition.
  • step 2.9 The crude yeast cell extract obtained in step 2.9 is centrifuged 1-2 times, the centrifugal force is 12000-30000 ⁇ g time is 30 min, and the temperature is 4 ° C;
  • the prepared yeast cell extract was dispensed and stored in liquid nitrogen and stored at -80 °C.
  • the protein content of different batches of yeast cell extracts was determined to be about 25-100 mg/ml, with an average of about 60-70 mg/ml, as determined by the Coomassie Brilliant Blue assay.
  • Example 3 Cell-free in vitro protein synthesis system
  • luciferase activity After the reaction, add an equal volume of substrate luciferine to a 96-well white plate or a 384-well white plate, and immediately place it on the Envision 2120 multi-plate reader (PerkinElmer), read and test. Firefly luciferase activity, relative light unit value (RLU) as the unit of activity, as shown in Figures 1-8, Figure 10-13.
  • RLU relative light unit value
  • the relative light unit value of the in vitro protein synthesis reaction system is 1,303,884 (Relative Light Unit, RLU) under the reaction condition of 20 ° C for 2 h, and the activity of the non-DNA and buffer itself negative control are respectively 77RLU and 160RLU. It can be seen that the yeast extract has strong in vitro protein synthesis ability.
  • the yeast extract with an OD600 of 6.9 has a protein synthesis activity of 125,346 RLU.
  • the reaction liquid is magnesium acetate and potassium acetate system, and the centrifugal force is 30,000 ⁇ g (C), 18,000 ⁇ g (D), 15,000 ⁇ g (E), respectively.
  • the yeast extract obtained by the treatment of 12,000 ⁇ g (F) did not have a large difference in the in vitro protein synthesis reaction, and the activity was above 1,000,000 RLU.
  • the activity of the negative control without DNA and buffer itself was 200 RLU and 320 RLU, respectively.
  • the reaction conditions were 20 ° C for 2 h, and the reaction solution was a magnesium acetate and potassium acetate system.
  • the concentration of magnesium acetate in the reaction system ranged from 1 to 8 mM.
  • the relative light unit value of the yeast cell extract for synthesizing luciferase is not less than 1,000,000 RLU, wherein 2 mM magnesium acetate has the highest protein synthesis ability.
  • the relative light unit value can reach 2,884,286 RLU; and 6-8 mM magnesium acetate reduces the relative light unit value of the synthesized luciferase.
  • the reaction conditions were 20 ° C for 2 h, and the reaction liquid was a magnesium acetate and potassium acetate system.
  • the concentration of the amino acid in the protein synthesis reaction system ranges from 0.04 to 0.24 mM. It can be seen from Fig. 7 that when the concentration of the amino acid is in the range of 0.08-0.24 mM, the relative light unit value is not lower than 1,000,000 RLU, and the difference in activity between different amino acid concentrations is small.
  • the reaction conditions were 20 ° C for 2 h, and the reaction liquid was a magnesium acetate and potassium acetate system.
  • the concentration of ATP in the protein synthesis reaction system ranges from 1.5 to 4.5 mM. As can be seen from Fig. 8, when the concentration of ATP is 2.5, 3 and 4 mM, the relative light unit value is not less than 1,000,000 RLU, and the difference in activity is small. ATP below 1.5 mM or above 4.0 mM has an effect on the ability of protein synthesis in vitro.
  • Figure 9 includes seven different luciferase gene templates applied to this protein in vitro synthesis system, wherein the sequences include different 5' ends, such as omega sequences, SITS2 sequences and CrPV sequences.
  • the sequence at the 3' end mainly includes the terminator sequence from lacZ, and the number of different polyadenylation nucleotides.
  • PC1 represents a commercial rabbit reticulocyte in vitro protein synthesis system.
  • 50A, 70A, 90A, and the luciferase gene containing the lacZ terminator sequence were all translated in yeast extract, with 90A having the highest activity, relative to the positive control.
  • the unit of light value is 6,844,583 RLU, and the relative light unit of commercial rabbit reticulocyte synthesis luciferase in vitro is only 1,000,000 RLU.
  • the activity of the negative control without DNA and buffer itself was 66 RLU and 69 RLU, respectively.
  • the reaction conditions were 25 ° C for 2 h, and the reaction liquid was a magnesium acetate and potassium acetate system.
  • the luciferase gene was expressed in the yeast extract by the omega sequence, CrPV sequence and SITS2 sequence at the 5' end of the luciferase gene, and the highest activity was the SITS2 sequence.
  • the relative light unit value is 5,816,496 RLU.
  • the relative light unit value of the in vitro protein synthesis system of the omega sequence is 3,458,701 RLU.
  • the reaction conditions were 25 ° C for 2 h, and the reaction liquid was a magnesium acetate and potassium acetate system. It can be seen from Fig. 12 that compared with the reaction system without adding PEG, all three PEGs significantly improve the ability of yeast to extract protein synthesis, and 2% PEG3350, 2% PEG8000 and 4% PEG8000 are particularly prominent.
  • the light unit value can reach 60,000,000 RLU.
  • Figure 13 shows the effect of sucrose concentration on in vitro protein synthesis system
  • the reaction conditions were 25 ° C for 2 h, and the reaction liquid was a magnesium acetate and potassium acetate system. It can be seen from Fig. 13 that the concentrations of sucrose concentrations of 0.5%, 1% and 2% increase the ability of yeast to extract protein from outside the body compared with the reaction system without sucrose addition, and the 0.5% concentration is particularly prominent.
  • NC indicates an in vitro protein synthesis reaction system with a negative control DNA-free template and an activity of 190 RLU.
  • Figure 14 shows the effect of heme concentration on protein synthesis system in vitro.
  • the reaction conditions were 25 ° C for 2 h, and the reaction liquid was a magnesium acetate and potassium acetate system. It can be seen from Fig. 14 that the four concentrations of heme concentration of 0.01, 0.02, 0.03, and 0.04 mM all increase the ability of yeast to extract protein synthesis, compared with the reaction system without hemoglobin addition, and the concentration of 0.04 mM is particularly prominent. .
  • Figure 15 shows the effect of spermidine concentration on protein synthesis system in vitro.
  • the reaction conditions were 25 ° C for 2 h, and the reaction liquid was a magnesium acetate and potassium acetate system.
  • the spermidine concentration of 0.2, 0.3 and 0.4 mM increased the ability of yeast to extract protein synthesis outside the reaction system compared with the reaction system without the addition of spermidine, of which 0.4 mM was particularly prominent.
  • the results of the present invention indicate that the yeast extract prepared by the high pressure crushing method or the liquid nitrogen crushing method has direct utilization.
  • various reaction conditions such as magnesium acetate, potassium acetate, amino acid, ATP, DNA template with different sequence composition, polyethylene glycol, sucrose optimization, etc.
  • the relative activity of the synthesized luciferase has reached 60,000,000 RLU.
  • Commercially available cell-free expression systems such as rabbit reticulocyte in vitro expression systems have an activity of only 1,000,000 RLU.
  • the relative light unit value of the luciferase activity synthesized by the present invention under optimal conditions is 60 times that of the commercial system, which embodies the great advantage of the invention.

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Abstract

L'invention concerne un système de synthèse de protéines acellulaire destiné à la synthèse de protéines in vitro sur la base d'extraits de cellules de levure, une trousse et un procédé de préparation associé.
PCT/CN2017/077814 2017-03-04 2017-03-23 Système de synthèse de protéines pour la synthèse de protéines in vitro, trousse et procédé de préparation associé WO2018161374A1 (fr)

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