WO2020135747A1 - Système de synthèse de protéine acélullaire in vitro optimisé et application correspondante - Google Patents

Système de synthèse de protéine acélullaire in vitro optimisé et application correspondante Download PDF

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WO2020135747A1
WO2020135747A1 PCT/CN2019/129289 CN2019129289W WO2020135747A1 WO 2020135747 A1 WO2020135747 A1 WO 2020135747A1 CN 2019129289 W CN2019129289 W CN 2019129289W WO 2020135747 A1 WO2020135747 A1 WO 2020135747A1
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protein synthesis
cell
synthesis system
vitro
free protein
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PCT/CN2019/129289
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Chinese (zh)
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郭敏
杨宁
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康码（上海）生物科技有限公司
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    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • 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
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material

Definitions

  • the invention belongs to the technical field of protein synthesis, and in particular relates to a cell-free protein synthesis system for in vitro protein synthesis.
  • Protein is an important molecule in a cell, and is involved in almost all the functions of the cell. The different sequence and structure of the protein determine its different functions (1). Within the cell, proteins can act as enzymes to catalyze various biochemical reactions, can act as signaling molecules to coordinate various activities of organisms, can support biological morphology, store energy, transport molecules, and move organisms (2). In the field of biomedicine, protein antibodies, as targeted drugs, are an important means of treating cancer and other diseases (1,2).
  • the traditional protein expression system refers to a molecular biological technique for expressing foreign genes through model organisms such as bacteria, fungi, plant cells or animal cells (3).
  • the cell-free expression system also known as the in vitro protein synthesis system, came into being. It is the foreign target mRNA or DNA as a protein synthesis template, and the substrate and transcription required for protein synthesis are supplemented by manual control. Translation-related protein factors and other substances can achieve the synthesis of the target protein (3, 4).
  • Expressing proteins in an in vitro translation system does not require plasmid construction, transformation, cell culture, cell collection, and disruption steps. It is a fast, time-saving, and convenient way of protein expression (5,6).
  • In vitro protein synthesis system generally refers to the addition of mRNA or DNA template, RNA polymerase, amino acids and ATP to the lysis system of bacteria, fungi, plant cells or animal cells to complete the rapid and efficient translation of foreign proteins (5, 7).
  • E. coli system E. coli
  • RRL rabbit reticulocyte
  • WGE wheat germ
  • Insect cell extract ICE
  • human source systems 5, 6
  • E. coli system E. coli system
  • RRL rabbit reticulocyte
  • WGE wheat germ
  • Insect cell extract ICE
  • human source systems 5, 6
  • the protein-free cell-free synthesis system has many advantages. For example, it can express special proteins that are toxic to cells or contain unnatural amino acids (such as D-amino acids).
  • PCR products are used as templates to synthesize multiple proteins in parallel, and to conduct high-throughput drug screening and proteomics research (7).
  • E. coli in vitro synthesis systems are widely used.
  • Escherichia coli is easy to culture and ferment, has low cost, simple cell disruption, and can synthesize higher-yield protein (6).
  • the cultivation of eukaryotic cells is more difficult and expensive, and the preparation process of their cell extracts is cumbersome. Therefore, their translation system costs more and is only suitable for special laboratory use (1,2). Therefore, eukaryotic in vitro protein expression systems suitable for industrial large-scale (ton scale) preparation and production do not currently exist.
  • the object of the present invention is to provide a low-cost and efficient in vitro protein synthesis reaction system. It mainly solves the technical problems of excessively high cost of protein synthesis system and insufficient reaction efficiency in the prior art.
  • the cell-free protein synthesis system includes:
  • the buffering agent is a trishydroxymethylaminomethane buffering agent
  • a DNA molecule template encoding a foreign protein is prepared using a nucleic acid isothermal amplification method, and the DNA molecule template is inserted with SEQ ID NO. 1 upstream of the encoding sequence of the foreign protein The sequence shown.
  • the protein synthesis system further includes one or more components of the following group:
  • the active enzyme is selected from amylase, phosphorylase, galactosidase, glucose phosphate mutase, or a combination thereof. Further preferably, the amylase is alpha amylase.
  • the phosphoric acid compound is selected from orthophosphate, dihydrogen phosphate, dihydrogen phosphate, metaphosphate, pyrophosphate, or a combination thereof.
  • the cell source of the cell extract is one or more types of cells selected from the group consisting of E. coli, bacteria, mammalian cells, plant cells, yeast cells, or a combination thereof; preferably, The yeast cell is selected from Saccharomyces cerevisiae, Pichia pastoris, Kluyveromyces, or a combination thereof; more preferably, the Kluyveromyces is Kluyveromyces lactis.
  • the glucose concentration is 8.8-128 mmol/L.
  • the concentration of the maltodextrin is 84-500 mmol/L.
  • the concentration (v/v) of the cell extract is 20%-70%, preferably 30-60%, more preferably 40%-50%, based on the total volume of the protein synthesis system meter.
  • the present invention also provides a kit, which includes a container and the above-mentioned cell-free protein synthesis system components located in the container.
  • the invention also provides a method for synthesizing exogenous protein in vitro, including:
  • the method further comprises: (iii) optionally isolating or detecting the foreign protein from the in vitro cell-free protein synthesis system.
  • the reaction cost is greatly reduced without reducing the activity of the in vitro protein synthesis reaction system; through the optimization of the DNA template Increase the target output per unit time; use nucleic acid isothermal amplification technology to prepare DNA molecular templates encoding foreign proteins, and use a very small amount (nack-microgram) of DNA template to complete efficient, high-throughput, and extremely simple protein synthesis, etc. .
  • the invention can greatly reduce the cost of in vitro protein synthesis reaction, and at the same time increase the in vitro protein synthesis capacity by more than 30 times.
  • FIG. 1 is a graph of data results of RFU values of fluorescent proteins synthesized by two protein synthesis systems in Example 2 of the present invention; wherein System 1 is the optimized protein synthesis system of Example 2, and System 2 is the original protein synthesis system of Example 2, The negative control is that system 1 does not add DNA template, and the detection time is 3 hours and 20 hours, respectively.
  • NC is a protein synthesis system without adding DNA template.
  • FIG. 3 is a graph showing the data results of the RFU value of the fluorescent protein synthesized in the maltodextrin + glucose protein synthesis system in Example 1 of the present invention; wherein the glucose concentration is 0-200 mM, the maltodextrin concentration is 320 mM, and the detection time is 20 hours.
  • in vitro cell-free protein synthesis system in vitro expression system
  • in vitro protein synthesis system in vitro protein synthesis reaction system
  • in vitro protein synthesis system in vitro protein synthesis system
  • glucose, maltodextrin and phosphoric acid compounds as energy sources for in vitro biological reactions can slowly release ATP and reduce costs. It is a new energy regeneration system that can be industrialized.
  • the reaction system containing glucose + maltodextrin has an RFU value increased by more than 30 times compared to the phosphocreatine + phosphocreatine kinase system; the RFU value has increased compared to the reaction system with glucose as the energy source More than 5 times. Based on this, the present invention optimizes the in vitro cell-free protein synthesis system from various aspects, and provides an efficient and low-cost protein synthesis system.
  • the in vitro cell-free protein synthesis system is not particularly limited, and a preferred cell-free protein synthesis system is yeast in vitro protein synthesis system, preferably Kluyveromyces in vitro protein synthesis system (more preferably, lactic acid Kluyveromyces in vitro protein synthesis system).
  • Yeast has the advantages of simple culture, efficient protein folding, and post-translational modification. Among them, Saccharomyces cerevisiae and Pichia pastoris are model organisms that express complex eukaryotic and membrane proteins. Yeast can also be used as a raw material for the preparation of in vitro translation systems.
  • Kluyveromyces is an ascomycete yeast
  • Kluyveromyces marxianus and Kluyveromyces lactis are yeasts widely used in industry. Compared with other yeasts, Kluyveromyces lactis has many advantages, such as superb secretion ability, better large-scale fermentation characteristics, food safety level, and the ability to post-translational modification of proteins.
  • the in vitro cell-free protein synthesis system of the present invention includes: (a) cell extracts, which are yeast cell extracts inserted with the T7 RNA polymerase gene; (b) carbohydrates, which are glucose Mixture with maltodextrin; (c) phosphate compound; (d) buffer, the buffer is trishydroxymethylaminomethane buffer; (e) DNA molecule template encoding foreign protein, the DNA molecule template It is prepared by the nucleic acid isothermal amplification method, and the sequence shown in SEQ ID NO. 1 is inserted in the DNA molecule template upstream of the coding sequence of the foreign protein.
  • the protein synthesis system further includes one or more components of the following group: (f1) polyethylene glycol; (f2) substrate for synthesizing RNA; (f3) amino acid mixture; (f4) magnesium ion; (f5) potassium ions; (f6) dithiothreitol (DTT); (f7) active enzymes capable of catalyzing the metabolism of carbohydrates to produce ATP; (f8) optional water or aqueous solvents.
  • the active enzyme is selected from amylase, phosphorylase, galactosidase, glucose phosphate mutase, or a combination thereof. Further preferably, the amylase is alpha amylase.
  • the phosphoric acid compound is selected from orthophosphate, dihydrogen phosphate, dihydrogen phosphate, metaphosphate, pyrophosphate, or a combination thereof.
  • the content and purity of the cell extract are not particularly limited.
  • the concentration (v/v) of the cell extract is 20%-70%, preferably 30-60%, more preferably 40%-50%, based on the total volume of the protein synthesis system meter.
  • the cell extract is an aqueous extract of yeast cells.
  • the cell extract does not contain yeast endogenous long-chain nucleic acid molecules.
  • the substrate for synthesizing RNA includes one or a combination of nucleoside monophosphate and nucleoside triphosphate.
  • the amino acid mixture includes: 20 kinds of natural amino acids and unnatural amino acids.
  • the magnesium ion is derived from a magnesium ion source, and the magnesium ion source is selected from the group consisting of one of magnesium acetate and magnesium glutamate or a combination thereof.
  • the potassium ion is derived from a potassium ion source, and the potassium ion source is selected from the group consisting of one of potassium acetate and potassium glutamate or a combination thereof.
  • the concentration of the nucleoside triphosphate mixture and the amino acid mixture described in each embodiment refers to the concentration of a single substance in the mixture, not the total substance concentration of the mixture.
  • Fluorescent protein activity measurement Immediately after the reaction, it was placed in the Envision 2120 multifunctional microplate reader (Perkin Elmer) to detect the intensity of the fluorescent signal, and the relative fluorescence unit value (Relative Fluorescence Unit, RFU) was used as the active unit.
  • RFU Relative Fluorescence Unit
  • the relative light unit value RFU of the cell-free protein synthesis system in vitro was 60.
  • the yield of enhanced green fluorescent protein was 1.50 ⁇ g/mL. After 3 hours of reaction, the RFU value began to decrease slowly.
  • Example 1 In vitro cell-free protein synthesis system containing glucose + maltodextrin
  • Fluorescent protein activity measurement Immediately after the reaction, it was placed in the Envision 2120 multifunctional microplate reader (Perkin Elmer) to detect the intensity of the fluorescent signal, and the relative fluorescence unit value (Relative Fluorescence Unit, RFU) was used as the active unit.
  • RFU Relative Fluorescence Unit
  • NC refers to a protein synthesis system without adding a DNA template. It can be seen from Figure 2 that when the maltodextrin concentration is in the range of 256-400 mM, the fluorescent protein yield is the highest. When 320 mM maltodextrin was added and reacted at 20° C. for 20 h, the relative light unit value RFU of the in vitro protein synthesis reaction system was about 1900. The yield of enhanced green fluorescent protein was 121.10 ⁇ g/mL.
  • alpha amylase is conducive to the hydrolysis of maltodextrin.
  • the relative light unit value RFU of the in vitro protein synthesis reaction system is about 1750, and the yield of enhanced green fluorescent protein is 111.06 ⁇ g /mL.
  • FIG. 3 for a fixed 320 mM maltodextrin concentration, test the effect of different concentrations of glucose, and obtain a graph of data results of fluorescent protein RFU values; wherein the glucose concentration is 0-200 mM, the maltodextrin concentration is 320 mM, and the detection time is 20 hours. It can be seen from Figure 3 that when the glucose concentration is around 20 mM, the fluorescent protein RFU value is the highest.
  • Fluorescent protein activity measurement Immediately after the reaction, it was placed in the Envision 2120 multifunctional microplate reader (Perkin Elmer) to detect the intensity of the fluorescent signal, and the relative fluorescence unit value (Relative Fluorescence Unit, RFU) was used as the active unit.
  • RFU Relative Fluorescence Unit
  • Fluorescent protein activity measurement Immediately after the reaction, it was placed in the Envision 2120 multifunctional microplate reader (Perkin Elmer) to detect the intensity of the fluorescent signal, and the relative fluorescence unit value (Relative Fluorescence Unit, RFU) was used as the active unit.
  • RFU Relative Fluorescence Unit
  • Tris-HCl buffer at pH 8.0 was used instead of 4-Hydroxyethylpiperazine ethanesulfonic acid (Hepes-KOH) buffer at pH 7.4.
  • Hepes-KOH 4-Hydroxyethylpiperazine ethanesulfonic acid
  • nucleoside triphosphates including adenine nucleoside triphosphate (ATP), guanine nucleoside triphosphate (GTP), cytosine nucleoside triphosphate (CTP) and uracil nucleoside triphosphate (UTP) are commercial nucleoside triphosphate mixtures, and are the highest unit cost of all reaction components.
  • ATP adenine nucleoside triphosphate
  • GTP guanine nucleoside triphosphate
  • CTP cytosine nucleoside triphosphate
  • UDP uracil nucleoside triphosphate
  • nucleoside triphosphate was prepared using trishydroxymethylaminomethane at pH 8.0 and four nucleoside triphosphate powders, instead of the commercially purchased nucleoside triphosphate mixture, in an in vitro protein synthesis system Shows a higher RFU value.
  • Phosphocreatine and phosphocreatine kinase are used as energy sources to provide ATP for in vitro reactions. Although ATP can be released through the corresponding kinase reaction to produce ATP, they often only provide a large amount of energy at the beginning stage, and these high-energy compounds are In vitro cell synthesis has an inhibitory effect, can not provide energy for a long time, and the cost is higher, which is not conducive to the improvement of the efficiency of in vitro protein synthesis system and industrial application.
  • glucose, maltodextrin and phosphate compounds as energy sources for in vitro biological reactions can slowly release ATP and reduce costs. It is a new energy regeneration system that can be industrialized. After optimization, using the final concentration of 20 mM glucose, 320 mM maltodextrin and 20 mM potassium phosphate as the energy source of the in vitro protein synthesis system showed a higher RFU value in the in vitro protein synthesis system.
  • RNA polymerase requires the addition of RNA polymerase.
  • the commercialized and laboratory-made RNA polymerase has the problems of high cost and low purity.
  • yeast cell extracts inserted with the T7 RNA polymerase gene for the insertion method of the T7 RNA polymerase gene, please refer to the patent content of the application number 2017107685501), so that no additional expensive biological enzymes are needed in the reaction system ( Such as RNA polymerase), without significant changes in RFU value, reducing the cost of protein synthesis in vitro.
  • PCR technology is dependent on temperature cycling, and often requires a higher temperature to denature the DNA template and amplify and extend the newly synthesized DNA molecule, and high temperature can cause denaturation and inactivation of protein factors in the in vitro synthesis system, so it is not suitable for use.
  • In vitro synthesis system Compared with PCR technology, the characteristic of isothermal nucleic acid amplification is to achieve nucleic acid amplification under specific and relatively mild temperature conditions, so that DNA replication, mRNA transcription and protein synthesis can be coupled in vitro.
  • DNA polymerases for isothermal nucleic acid amplification including phi29 DNA polymerase, T7 DNA polymerase, etc. have greater advantages in temperature and amplification efficiency, so that there is no need to prepare a large number of DNA molecules in advance, only a small amount
  • the DNA template can achieve in vitro protein synthesis.
  • the DNA replication, transcription, and translation coupling systems listed in Table 1 have achieved RFU values in the in vitro protein synthesis system that have reached the original system’s DNA template using PCR to amplify a large number of target protein DNA templates. Preparation method.
  • the GAA sequence and IRES (KLNCE102) sequence were inserted in sequence from the 5'end to the 3'end before the T7 promoter and the omega sequence of the 5'untranslated region.
  • the target protein is inserted with a leader peptide sequence and a His tag sequence in sequence from the 5'end to the 3'end.
  • the modified DNA template sequence is inserted into the sequence shown in SEQ ID NO.1 upstream of the coding sequence of the foreign protein.
  • the optimized protein synthesis system (System 1) and the original protein synthesis system (System 2) were placed in an environment of 20-30°C to react.
  • Fluorescent protein activity measurement different fluorescence can be observed during the reaction, and the color gradually becomes darker within a certain period of time. Immediately after the reaction, place it in the Envision 2120 Multifunctional Microplate Reader (Perkin Elmer), read it, select different filters, detect the intensity of each fluorescent signal, and take the relative fluorescence unit value (Relative Fluorescence Unit, RFU) as the active unit.
  • RFU Relative Fluorescence Unit
  • system 1 is the optimized protein synthesis system
  • system 2 is the original protein synthesis system
  • the negative control is that system 1 does not add a DNA template.

Abstract

L'invention concerne un système de synthèse de protéine acellulaire in vitro optimisé et une application correspondante, ledit système comprenant : un extrait cellulaire, étant un extrait de cellule de levure comportant un gène d'ARN polymérase de T7 inséré; un glucide, étant un mélange de glucose et de maltodextrine; un composé de phosphate; un agent tampon, étant un agent tampon de trishydroxyméthylaminométhane; et un modèle de molécule d'ADN codant pour une protéine hétérologue préparée à l'aide d'un procédé d'amplification isotherme d'acide nucléique. Dans le modèle de molécule d'ADN, une séquence représentée dans SEQ ID NO 1 est insérée en amont de la séquence codante de la protéine hétérologue. Au moyen d'une optimisation, le coût de la synthèse de protéines in vitro est réduit, et le rendement de la protéine cible est augmenté.
PCT/CN2019/129289 2018-12-28 2019-12-27 Système de synthèse de protéine acélullaire in vitro optimisé et application correspondante WO2020135747A1 (fr)

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