WO2019024379A1 - 新型融合蛋白的制备及其在提高蛋白质合成的应用 - Google Patents

新型融合蛋白的制备及其在提高蛋白质合成的应用 Download PDF

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WO2019024379A1
WO2019024379A1 PCT/CN2017/115972 CN2017115972W WO2019024379A1 WO 2019024379 A1 WO2019024379 A1 WO 2019024379A1 CN 2017115972 W CN2017115972 W CN 2017115972W WO 2019024379 A1 WO2019024379 A1 WO 2019024379A1
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protein
fusion protein
vitro
yeast
synthesis system
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French (fr)
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郭敏
代田纯
王海鹏
薛银鸽
柴智
刘帅龙
于雪
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Kangma Healthcode Shanghai Biotech Co Ltd
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Kangma Healthcode Shanghai Biotech Co Ltd
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Priority to KR1020207006125A priority Critical patent/KR102384271B1/ko
Priority to US16/633,524 priority patent/US11946084B2/en
Priority to EP17920595.0A priority patent/EP3663319A4/en
Priority to JP2020506225A priority patent/JP7246100B2/ja
Publication of WO2019024379A1 publication Critical patent/WO2019024379A1/zh
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    • C12P21/00—Preparation of peptides or proteins
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    • C07K14/37—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi
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    • C12N15/09—Recombinant DNA-technology
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    • 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
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0069—Oxidoreductases (1.) acting on single donors with incorporation of molecular oxygen, i.e. oxygenases (1.13)
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    • C12Y113/12—Oxidoreductases acting on single donors with incorporation of molecular oxygen (oxygenases) (1.13) with incorporation of one atom of oxygen (internal monooxygenases or internal mixed function oxidases)(1.13.12)
    • C12Y113/12007—Photinus-luciferin 4-monooxygenase (ATP-hydrolysing) (1.13.12.7), i.e. firefly-luciferase
    • C—CHEMISTRY; METALLURGY
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    • C12Y306/00—Hydrolases acting on acid anhydrides (3.6)
    • C12Y306/04—Hydrolases acting on acid anhydrides (3.6) acting on acid anhydrides; involved in cellular and subcellular movement (3.6.4)
    • C12Y306/04013—RNA helicase (3.6.4.13)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence

Definitions

  • the present invention relates to the field of genetic engineering, and in particular to the preparation of novel fusion proteins and their use in enhancing protein synthesis.
  • Protein is an important molecule in cells and is involved in the execution of all functions of cells. The sequence and structure of the protein are different, which determines the difference in function. In cells, proteins can act as enzymes to catalyze various biochemical reactions. They can act as signal molecules to coordinate various activities of organisms, support biological forms, store energy, transport molecules, and make organisms move. In the field of biomedicine, protein antibodies are targeted drugs and are important means for treating diseases such as cancer.
  • the four processes of protein translation include translation initiation, translation extension, translation termination, and ribosome recycling, where translation initiation is the most regulated process.
  • the ribosomal small subunit (40S) binds (tRNA) i Met and recognizes the 5' end of the mRNA under the action of a translation initiation factor.
  • the small subunit moves downstream and binds to the ribosomal large subunit (60S) at the initiation codon (ATG) to form a complete ribosome and enters the translational extension phase.
  • the rate of protein synthesis is approximately 13,000 per second. In vivo, the rate of protein synthesis is limited by the number of ribosomes, the average number of ribosomes in cells is about 200,000, and the number of mRNA molecules is about 15,000-60,000.
  • E. coli extract E. coli extract
  • RRL Rabbit reticulocyte Lysate
  • WGE Wheat germ extract
  • Insect cell Extract, ICE
  • the yield of the prokaryotic system can reach ⁇ 0.5 mg/mL, and the cost is about ⁇ 10 RMB/ ⁇ g.
  • the yield of the CHO system in eukaryotic systems can reach ⁇ 0.7 mg/mL and the cost is about -20RMB/ ⁇ g. Therefore, both the intracellular and extracellular artificial protein synthesis systems in nature have the characteristics of low efficiency and slow speed, which greatly limits the application of protein synthesis.
  • a first aspect of the invention provides a fusion protein having the structure of Formula Ia or Formula Ib:
  • A is a PabI component
  • B is no or a linker peptide
  • C is an eIF4G component
  • S is an optional signal peptide
  • Each "-" is a peptide bond.
  • the Formula Ia or Ib is a structure from the N-terminus to the C-terminus.
  • the element A comprises a wild type and a mutant PabI sequence.
  • the PabI is PabI from yeast.
  • the element A has the sequence of SEQ ID NO.: 1 or an active fragment thereof, or has ⁇ 85% homology with the amino acid sequence shown in SEQ ID NO: 1 (preferably, ⁇ 90%) Homology; etc. preferably ⁇ 95% homology; most preferably, ⁇ 97% homology, such as 98% or more, 99% or more) and having the same activity as the SEQ ID NO.:1 sequence Peptide.
  • said element C comprises wild-type and mutant eIF4G sequences.
  • the eIF4G is eIF4G from yeast.
  • element C has the sequence set forth in SEQ ID NO.: 2 or an active fragment thereof, or has ⁇ 85% homology to the amino acid sequence set forth in SEQ ID NO: 2 (preferably, ⁇ 90%) Homology; etc. preferably ⁇ 95% homology; most preferably, ⁇ 97% homology, such as 98% or more, 99% or more) and having the same activity as the SEQ ID NO.: 2 sequence Peptide.
  • the fusion protein is a recombinant protein, preferably a recombinant protein expressed by yeast.
  • the yeast is selected from the group consisting of Kluyveromyces, Saccharomyces cerevisiae, or a combination thereof.
  • the yeast is selected from the group consisting of Kluyveromyces cerevisiae, K. marxianus, K. cerevisiae, or a combination thereof.
  • the element A is derived from a PabI protein of yeast.
  • the element C is derived from the eIF4G protein of yeast.
  • the peptide linker is 0-50 amino acids in length, preferably 10-40 amino acids, more preferably 15-25 amino acids.
  • the fusion protein is selected from the group consisting of:
  • A a polypeptide having the amino acid sequence of SEQ ID NO: 3;
  • B having ⁇ 80% homology to the amino acid sequence shown by SEQ ID NO: 3 (preferably, ⁇ 90% homology; ⁇ 95% homology; most preferably, ⁇ 97% homology, such as 98% or more, 99% or more) of the polypeptide, and the polypeptide has a function or activity to increase the efficiency of expression of the foreign protein;
  • (C) forming the amino acid sequence of any one of SEQ ID NO: 3 by substitution, deletion or addition of 1-15 (preferably, 2-10, more preferably, 3-8) amino acid residues And a derivative polypeptide having a function or activity for increasing the expression efficiency of a foreign protein.
  • amino acid sequence of the fusion protein is shown in SEQ ID NO.: 3.
  • the fusion protein has one or more properties selected from the group consisting of:
  • the exogenous protein is selected from the group consisting of luciferin, or luciferase (such as firefly luciferase), green fluorescent protein, yellow fluorescent protein, aminoacyl tRNA synthetase, glyceraldehyde- 3-phosphate dehydrogenase, catalase, actin, variable region of antibody, luciferase mutation, alpha-amylase, enteromycin A, hepatitis C virus E2 glycoprotein, insulin precursor Interferon alpha A, interleukin-1 beta, lysozyme, serum albumin, single chain antibody fragment (scFV), thyroxine transporter, tyrosinase, xylanase, or a combination thereof.
  • luciferin or luciferase (such as firefly luciferase)
  • green fluorescent protein yellow fluorescent protein
  • aminoacyl tRNA synthetase aminoacyl tRNA synthetase
  • a second aspect of the invention provides an isolated polynucleotide encoding the fusion protein of the first aspect of the invention.
  • the polynucleotide is selected from the group consisting of a DNA sequence, an RNA sequence.
  • the DNA sequence is selected from the group consisting of a genomic sequence and a cDNA sequence.
  • the polynucleotide is mRNA or cDNA, and the polynucleotide has the structure shown in Formula II:
  • A1 is a nucleotide sequence encoding the above A element
  • C1 is a nucleotide sequence encoding the above C element
  • the element A1 has the sequence shown in SEQ ID NO.: 4.
  • the element C1 has the sequence shown in SEQ ID NO.: 5.
  • a third aspect of the invention provides a vector comprising the polynucleotide of the second aspect of the invention.
  • a fourth aspect of the invention provides a host cell comprising the vector of the third aspect of the invention or a polynucleotide in which the second aspect of the invention is integrated.
  • the host cell is formed by transferring the expression vector of the third aspect of the invention or the polynucleotide of the second aspect of the invention and is homologously recombined, thereby A coding sequence for the fusion protein of the first aspect of the invention is integrated into the chromosome.
  • the host cell is selected from the group consisting of Kluyveromyces, Saccharomyces cerevisiae, or a combination thereof.
  • the host cell is selected from the group consisting of Kluyveromyces cerevisiae, K. marxianus, Kluyveromyces cerevisiae, or a combination thereof.
  • the host cell is Kluyveromyces lactis.
  • a fifth aspect of the invention provides an in vitro protein synthesis system for expressing a foreign protein, the reaction system comprising:
  • a yeast in vitro protein synthesis system comprising (a) a yeast cell extract; (b) an optional polyethylene glycol; (c) optionally exogenous sucrose; and (d) optionally a solvent which is water or an aqueous solvent;
  • reaction system further comprises: (iii) an additional added eIF4G protein.
  • the eIF4G protein is induced to be expressed by a constitutive or inducible promoter.
  • the constitutive or inducible promoter is derived from yeast.
  • the yeast is selected from the group consisting of Kluyveromyces, Saccharomyces cerevisiae, or a combination thereof.
  • the constitutive or inducible promoter is selected from the group consisting of pScTEF1, pScPGK1, pK1TEF1, pK1PGK1, pScADH1, pScTPI1, pScTDH3, pK1ADH1, pK1TPI1, pK1TDH3, or a combination thereof.
  • a sixth aspect of the invention provides a method of producing the fusion protein of the first aspect of the invention, comprising:
  • the seventh aspect of the present invention provides the use of the fusion protein of the first aspect of the present invention, for preparing an in vitro protein synthesis system for expressing a foreign protein, wherein the in vitro protein synthesis system is used for improving the expression efficiency of the foreign protein. .
  • reaction system further comprises an additional eIF4G protein.
  • An eighth aspect of the present invention provides the use of the fusion protein of the first aspect of the present invention for the preparation of a preparation for improving the in vitro protein synthesis ability of an in vitro protein synthesis system.
  • a ninth aspect of the invention provides a method of expressing a foreign protein to be expressed, comprising:
  • the fusion protein is additionally added.
  • the fusion protein and the other proteins in the yeast in vitro protein synthesis system are extracts from the same yeast.
  • the method is non-diagnostic and non-therapeutic.
  • the step (ii) further comprises the step (iii): detecting the expression activity Q1 of the activity of the foreign protein, and incubating the wild-type yeast strain under the same conditions as in the step (ii), detecting the foreign source The activity of protein Q2, if Q1 is significantly higher than Q2, indicates that the expression efficiency of the foreign protein is significantly increased.
  • the "significantly higher" means Q1/Q2 ⁇ 2, preferably ⁇ 3, more preferably ⁇ 4.
  • the yeast in vitro protein synthesis system is a genetically engineered Kluyveromyces in vitro protein synthesis system (preferably Kluyveromyces cerevisiae in vitro protein synthesis system).
  • the coding sequence of the foreign protein is from a prokaryote, a eukaryote.
  • the coding sequence of the foreign protein is from an animal, a plant, or a pathogen.
  • the coding sequence of the foreign protein is from a mammal, preferably a primate, a rodent, including a human, a mouse, a rat.
  • the coding sequence of the foreign protein is selected from the group consisting of a luciferin protein, or a luciferase (such as firefly luciferase), a green fluorescent protein, a yellow fluorescent protein, and an aminoacyl tRNA synthesis.
  • a luciferin protein or a luciferase (such as firefly luciferase)
  • a green fluorescent protein e.g., a green fluorescent protein
  • a yellow fluorescent protein e.glyceraldehyde-3-phosphate dehydrogenase, catalase, actin, exogenous DNA of a variable region of an antibody, DNA of a luciferase mutant, or a combination thereof.
  • the exogenous protein is selected from the group consisting of luciferin, or luciferase (such as firefly luciferase), green fluorescent protein, yellow fluorescent protein, aminoacyl tRNA synthetase, glyceraldehyde- 3-phosphate dehydrogenase, catalase, actin, variable region of antibody, luciferase mutation, alpha-amylase, enteromycin A, hepatitis C virus E2 glycoprotein, insulin precursor Interferon alpha A, interleukin-1 beta, lysozyme, serum albumin, single chain antibody fragment (scFV), thyroxine transporter, tyrosinase, xylanase, or a combination thereof.
  • luciferin or luciferase (such as firefly luciferase)
  • green fluorescent protein yellow fluorescent protein
  • aminoacyl tRNA synthetase aminoacyl tRNA synthetase
  • Figure 1 shows the plasmid map of pKM-CAS1.0-KleIF4G.
  • Figure 2 shows the plasmid map of pKM-pScTEF1-KleIF4G-DD.
  • Figure 3 shows the plasmid map of pKM-pScPGK1-KleIF4G-DD.
  • Figure 4 shows the plasmid map of pKM-pKlTEF1-KleIF4G-DD.
  • Figure 5 shows the plasmid map of pKM-pKlPGK1-KleIF4G-DD.
  • Figure 6 shows the plasmid map of pKM-CAS1.0-KlTDH3-1.
  • Figure 7 shows the plasmid map of pKM-CAS1.0-KlTDH3-2.
  • Figure 8 shows the plasmid map of pKM-KlTDH3-1-F-KleIF4G-DD.
  • Figure 9 shows the plasmid map of pKM-KlTDH3-2-F-KleIF4G-DD.
  • Figure 10 shows the plasmid map of pKM-CAS1.0-K1Pab1.
  • Figure 11 shows the plasmid map of pKM-KlPab1-KleIF4G-DD.
  • Figure 12 shows a schematic representation of the in vitro translational activity assay of engineered strains.
  • the intensity of the fluorescent protein is used to indicate the protein expression ability of the system.
  • a fusion protein having the structure of Formula Ia or Formula Ib was unexpectedly discovered, and the fusion protein of the present invention can greatly improve the translation efficiency in vitro.
  • the present inventors have also found that insertion of a constitutive or inducible promoter (e.g., pScTEF1, pScPGK1, pKlTEF1, pKlPGK1, pScADH1, pScTPI1, pScTDH3, pK1ADH1, pK1TPI1, pK1TDH3, etc.) before eIF4G can significantly enhance the ability of protein synthesis in vitro.
  • a constitutive or inducible promoter e.g., pScTEF1, pScPGK1, pKlTEF1, pKlPGK1, pScADH1, pScTPI1, pScTDH3, pK1ADH1, pK1TPI1, pK1TDH3, etc.
  • the inventors have also found that the fusion protein of the present invention does not increase the expression level of the element eIF4G of the fusion protein of the present invention when the in vitro translation efficiency is improved.
  • the relative luminescence enzyme activity of the synthesized luciferase activity is as high as 1.50 ⁇ 10 9 , and is inserted into the constitutive or inducible promoter before eIF4G.
  • the relative light unit value of luciferase activity is as high as 1.57 ⁇ 10 9 , which is much higher than the relative light unit value (4.11 ⁇ 10 8 ) of the luciferase synthesized by the wild type yeast strain (such as Y1140).
  • the inventors completed the present invention.
  • eIF4F In eukaryotes, a variety of translation initiation factors are involved in the protein translation initiation process (Table 1). Among them, eIF4F is responsible for the identification of "hat structure” and the recruitment of downstream translation initiation factors and ribosomes. eIF4F consists of three protein subunits: eIF4E, eIF4G and eIF4A. eIF4E specifically binds to the "hat structure", anchoring eIF4F to the 5' untranslated region of mRNA; eIF4A is an RNA helicase; eIF4G is almost the scaffold protein for the entire translation initiation process, and can be translated from a variety of translations. The initiation factor interaction plays an important role in the downstream factor recruitment process.
  • a constitutive or inducible promoter such as pScTEF1, pScPGK1, pKlTEF1, pKlPGK1, pScADH1, pScTPI 1, pScTDH3, pKlADH1
  • a yeast source such as Saccharomyces cerevisiae, Kluyveromyces, etc.
  • eIF4G pScTPI 1, pScTDH3, etc.
  • nucleotide sequence of the eIF4G is set forth in SEQ ID NO.: 5; the protein sequence of the eIF4G is set forth in SEQ ID NO.: 2.
  • Pab1 element (Pab1 protein)
  • Pab1 is a 71 kDa RNA binding protein consisting of four RRM (RNA recognition motif 1-4) domains and one MLLE domain. Each RRM domain contains two conserved RNP structures (RNP1/2) responsible for binding to RNA.
  • nucleotide sequence of the Pabl is set forth in SEQ ID NO.: 4; the protein sequence of the Pabl is set forth in SEQ ID NO.: 1.
  • fusion protein of the invention As used herein, the terms "fusion protein of the invention", “PabI-eIF4G fusion protein of the invention” and “PabI-eIF4G fusion protein” are used interchangeably and refer to a fusion protein formed by fusion of a PabI element with an eIF4G element.
  • the PabI element and the eIF4G element may or may not contain a linker peptide or a flexible linker.
  • the fusion protein may or may not contain an initial Met; it may or may not contain a signal peptide; and may or may not contain a tag sequence (eg, 6His, etc.).
  • the fusion protein of the invention has the structure of formula Ia or formula Ib described above.
  • the amino acid sequence of the fusion protein of the present invention is shown in SEQ ID NO.: 3.
  • the fusion protein of the present invention can significantly enhance the in vitro protein synthesis ability of cell-free, in vitro protein synthesis systems, especially yeast in vitro protein synthesis systems.
  • the fusion protein of the present invention can be used to enhance the protein synthesis ability of an in vitro protein synthesis system.
  • a typical in vitro protein synthesis system is a yeast in vitro protein synthesis system.
  • 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 is a yeast that uses lactic acid as its sole source of carbon and energy. Compared with other yeasts, Kluyveromyces cerevisiae has many advantages, such as superior secretion capacity, good large-scale fermentation characteristics, food safety grade, and the ability to simultaneously modify the protein, as a host system expression drug. The use of protein has also shown great potential.
  • the yeast in vitro protein synthesis system is not particularly limited, and a preferred yeast in vitro protein synthesis system is the Kluyveromyces expression system (more preferably, the K. lactis expression system).
  • Kluyveromyces cerevisiae e.g., Kluyveromyces lactis
  • Kluyveromyces lactis is not particularly limited, and includes any Kluvi (e.g., Kluyveromyces lactis) strain capable of improving the efficiency of synthetic proteins.
  • the yeast in vitro protein synthesis system of the invention is a genetically engineered Kluyveromyces cerevisiae expression system.
  • the invention provides an in vitro cell-free protein synthesis system, the synthesis system comprising:
  • 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 from 0.03 to 40% by weight, preferably from 0.08 to 10% by weight, more preferably from 0.1 to 5% by weight, based on the protein synthesis The total weight of the system.
  • the in vitro protein synthesis system comprises: 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), creatine phosphate Kinase, RNase inhibitor, fluorescein, luciferase DNA, RNA polymerase.
  • yeast cell extract 4-hydroxyethylpiperazineethanesulfonic acid
  • potassium acetate magnesium acetate
  • adenosine triphosphate (ATP) adenosine triphosphate
  • GTP guanosine triphosphate
  • CTP cytosine triphosphate
  • TTP thymidine triphosphate
  • amino acid mixture amino acid mixture
  • creatine phosphate dithio
  • 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 an in vitro protein synthesis system is not particularly
  • the yeast cell extract typically comprises 20-70%, preferably 30-60%, more preferably 40-50% of the system in the in vitro protein synthesis protein synthesis system.
  • 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 g, preferably 8,000 to 30,000 g.
  • the centrifugation time is not particularly limited, and a preferred centrifugation time is from 0.5 min to 2 h, preferably from 20 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 of sucrose is from 0.03 to 40% by weight, preferably from 0.08 to 10% by weight, more preferably from 0.1 to 5% by weight, based on the total weight of the protein synthesis system.
  • a particularly preferred in vitro protein synthesis system in addition to the yeast extract, contains the following components: 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 Alcohol, 0.5% to 2% sucrose, 8-20 ng/ ⁇ l of firefly luciferase DNA, 0.027-0.054 mg/mL T7 RNA polymerase.
  • the yeast in vitro protein synthesis system of the present invention further comprises: (a) a fusion protein of the present invention, namely a PabI-eIF4G fusion protein.
  • the yeast in vitro protein synthesis system of the present invention further comprises an eIF4G protein; wherein the eIF4G protein of the present invention is constitutive or inducible by a yeast (such as Saccharomyces cerevisiae, Kluyveromyces, etc.) (For example, pScTEF1, pScPGK1, pKlTEF1, pKlPGK1, pScADH1, pScTPI1, pScTDH3, pK1ADH1, pKlTPI1, pKlTDH3, etc.) were induced to express.
  • a yeast such as Saccharomyces cerevisiae, Kluyveromyces, etc.
  • the yeast in vitro protein synthesis system containing the fusion protein of the present invention can significantly enhance the ability of protein synthesis in vitro. Furthermore, the yeast in vitro protein synthesis system using the fusion protein of the present invention in combination with the eIF4G protein has a higher in vitro protein synthesis ability.
  • yeast in vitro protein synthesis systems A preferred class of yeast in vitro protein synthesis systems is described in the inventor's prior application CN201710125619.9. This patent document is incorporated herein by reference in its entirety. The yeast in vitro protein synthesis system in this document does not employ the fusion protein of the present invention.
  • the yeast in vitro protein synthesis system of the invention comprises (a) a yeast cell extract; (b) an optional polyethylene glycol; (c) optionally exogenous sucrose; and (d) an optional solvent,
  • the solvent is water or an aqueous solvent; and (ii) a fusion protein of the invention.
  • the cell-free protein synthesis system further comprises one or more components selected from the group consisting of:
  • 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 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 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 non-days 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 present invention improves the protein synthesis efficiency of the translation system by genetically modifying the technology and modifying the intracellular genes by means of a highly efficient cell transformation platform.
  • the present invention first discovered a fusion protein, and the fusion protein of the present invention can significantly enhance the ability of protein synthesis in vitro.
  • the present inventors have found for the first time that insertion of a constitutive or inducible promoter (such as pScTEF1, pScPGK1, pKlTEF1, pKlPGK1, pScADH1, pScTPI1, pScTDH3, pK1ADH1, pKlTPI1, pKlTDH3, etc.) before eIF4G can significantly enhance protein synthesis in vitro. ability.
  • a constitutive or inducible promoter such as pScTEF1, pScPGK1, pKlTEF1, pKlPGK1, pScADH1, pScTPI1, pScTDH3, pK1ADH1, pKlTPI1, pKlTDH3, etc.
  • the present invention is the first to transform eIF4G by the CRISPR-Cas9 gene editing technology, thereby enhancing the ability of protein synthesis in vitro.
  • the present inventors have found for the first time that the expression level of the eIF4G element of the fusion protein of the present invention is not increased when the fusion protein of the present invention increases the efficiency of the in vitro translation system.
  • Example 1 Theoretical model for improving protein synthesis by genetic modification
  • the present invention optimizes the translation initiation factors eIF4G and Pab1 in K. lactis by the CRISPR-Cas9 gene editing technology to improve the efficiency of the cell-free in vitro translation system.
  • Example 2 Modification of translation initiation factors by CRISPR-Cas9 to improve the efficiency of in vitro translation systems
  • eIF4G is an important factor in the process of translation initiation.
  • the expression of endogenous eIF4G is optimized by gene editing technology to enhance the in vitro translation activity.
  • the invention is based on implementation The theoretical model in Example 1 was modified by the CRISPR-Cas9 gene editing technique to improve the expression of the translation initiation factor KleIF4G to improve the efficiency of the cell-free in vitro translation system.
  • the present invention inserts the pScTEF1, pScPGK1, pK1TEF1 and pKlPGK1 promoters in advance of the KleIF4G gene by CRISPR-Cas technology.
  • the plasmid construction and transformation methods are as follows:
  • PCR amplification was carried out using the pCAS plasmid as a template. 17 ⁇ L of the amplified product was mixed, and 1 ⁇ L of DpnI, 2 ⁇ L of 10 ⁇ digestion buffer was added, and the mixture was incubated at 37° C. for 3 hours. 10 ⁇ L of DpnI-treated product was added to 100 ⁇ L of DH5 ⁇ competent cells, placed on ice for 30 min, and heat-shocked at 42 ° C for 45 s.
  • the present invention first inserts donor DNA into a pMD18 plasmid, and then obtains a linear donor DNA sequence by PCR amplification.
  • PCR amplification was carried out using the genomic DNA of Kluyveromyces cerevisiae as a template, primers PF2:GAGCTCGGTACCCGGGGATCCTCTAGAGATAATAAAATTTCAACCTTTAAGCCATTGAATTTTACCATTACG (SEQ ID NO.:9) and PR2:GCCAAGCTTGCATGCCTGCAGGTCGACGATCTTGTTAGTAATCTCAACCTTCGCTGG (SEQ ID NO.:10); pMD18 plasmid as template and primer pMD18 -F: ATCGTCGACCTGCAGGCATG (SEQ ID NO.: 11) and pMD18-R: ATCTCTAGAGGATCCCCGGG (SEQ ID NO.: 12) were subjected to PCR amplification.
  • primer PF3 ATGGGCGAACCTACATCCGATC (SEQ ID NO.: 13) and PR3: ATCTGCTTTGAAAAACCGCTCTTTCTCTC (SEQ ID NO.: 14) for PCR amplification; using S.
  • primers PF4 AGAGAGAAAGAGCGGTTTTTCAAAGCAGATCCACACACCATAGCTTCAAAATGTTTCTAC (SEQ ID NO.: 15) and PR4: TGGTTGCTGATCGGATGTAGGTTCGCCCATCTTAGATTAGATTGCTATGCTCTCTCTAATGAGC (SEQ ID NO.: 16) PCR amplification (pScTEF1 promoter amplification); S.
  • primers PF5 AGAGAGAAAGAGCGGTTTTTCAAAGCAGATAGACGCGAATTTTTCGAAGAAGTACC (SEQ ID NO.: 17) and PR5: AGCTTCAACAGCTGGTTGCTGATCGGATGTAGGTTCGCCCATTGTTTTATATTTGTTGTAAAAAGTAGATAATTACTTCCTTGATGATC (SEQ ID NO.: 18) PCR amplification (pScPGK1 promoter amplification); using Kluyveromyces cerevisiae genomic DNA as a template, primers PF6: AGAGAAAGAGCGGTTTTTCAAAGCAGATGAGCCTGTCCAAGCAAATGCC (SEQ ID NO.: 19) and PR6: TGGTTGCTGATCGGATGTAGGTTCGCCCATTTTTAATGTTACTTCTCTTGCAGTTAGGGAAC (SEQ ID NO.
  • PCR amplification (pKlTEF1 promoter amplification); using Kluyveromyces cerevisiae genomic DNA as a template, primer PF7: AGAGAGAAAGAGCGGTTTTTCAAAGCAGATG PCR amplification (pKlPGK1 promoter amplification) was performed by TTCCTCATCACTAGAAGCCGAACTG (SEQ ID NO.: 21) and PR7: AGCTTCAACAGCTGGTTGCTGATCGGATGTAGGTTCGCCCATTTTTATTAATTCTTGATCGATTTTTTTGTTATTTCTGAAGTAACTCT (SEQ ID NO.: 22).
  • the PF3/PR3 amplification products were mixed with PF4/PR4, PF5/PR5, PF6/PR6 and PF7/PR7 amplification products, respectively, to construct pKM-pScTEF1-KleIF4G-DD, pKM-pScPGK1-KleIF4G-DD, pKM-pKlTEF1, respectively.
  • - KleIF4G-DD and pKM-pKlPGK1-KleIF4G-DD Figures 2, 3, 4, 5.
  • the specific steps were as follows: 8.5 ⁇ L of each of the two PCR products were mixed, and 1 ⁇ L of DpnI, 2 ⁇ L of 10 ⁇ digestion buffer was added, and the mixture was incubated at 37° C.
  • Kluyveromyces cerevisiae solution was streaked on YPD solid medium and single cloned, and cultured overnight in 25 mL 2 ⁇ YPD liquid medium, and 2 mL of bacterial solution was taken in 50 mL of liquid 2 ⁇ YPD medium.
  • the yeast cells were collected by centrifugation at 3000 g for 5 min at 20 ° C, resuspended in 500 ⁇ L of sterile water, and the cells were collected by centrifugation under the same conditions.
  • a competent cell solution (5% v/v glycerol, 10% v/v DMSO) was prepared and the yeast cells were dissolved in 500 ⁇ L of this solution. Dispense 50 ⁇ L into a 1.5 mL centrifuge tube and store at -80 °C.
  • the competent cells were thawed at 37 ° C for 15-30 s, centrifuged at 13,000 g for 2 min and the supernatant was removed.
  • TDH3 exists as a tetramer and participates in the catalytic reaction in the glycolytic pathway. Its promoter pTDH3 is a persistent strong promoter widely used in genetic engineering.
  • the present invention ligates the KleIF4G gene to the 3' end of the ORF of the K. lactis TDH3 gene.
  • TDH3 homologous gene sequence in K. lactis was determined by BLAST alignment analysis with the TDH3 gene in the NCBI database. It was found by comparison that there are two TDH3 homologous genes in the K. lactis genome, which are named KlTDH3-1 (1024297...1025292 located on chromosome A) and KlTDH3-2 (located on chromosome F) in the present invention. 1960417...1961406).
  • KlTDH3-1 1024297...1025292 located on chromosome A
  • KlTDH3-2 located on chromosome F
  • KlTDH3-1 CTTGTTGCTAAGAACTAAAG (SEQ ID NO.: 23)
  • KlTDH3-2 CTCTGAAAGAGTTGTCGATT (SEQ ID NO.: 24) is located at chromosome 1960378...1960397).
  • PCR amplification was carried out using the primer PF8: CTTGTTGCTAAGAACTAAAGGTTTTAGAGCTAGAAATAGCAAGTTAAAAT (SEQ ID NO.: 25), PR8: GCTCTAAAACCTTTAGTTCTTAGCAACAAGAAAGTCCCATTCGCCACCCG (SEQ ID NO.: 26), using the pCAS plasmid as a template. 17 ⁇ L of the amplification product was mixed, and 1 ⁇ L of DpnI, 2 ⁇ L of 10 ⁇ digestion buffer was added, and the mixture was incubated at 37° C. for 3 hours.
  • PCR amplification was carried out using the primer PF9: CTCTGAAAGAGTTGTCGATTGTTTTAGAGCTAGAAATAGCAAGTTAAAAT (SEQ ID NO.: 27), PR9: GCTCTAAAACAATCGACAACTCTTTCAGAGAAAGTCCCATTCGCCACCCG (SEQ ID NO.: 28), using the pCAS plasmid as a template. 17 ⁇ L of the amplified product was mixed, and 1 ⁇ L of DpnI, 2 ⁇ L of 10 ⁇ digestion buffer was added, and the mixture was incubated at 37° C. for 3 hours.
  • the present invention first inserts donor DNA into a pMD18 plasmid, and then obtains a linear donor DNA sequence by PCR amplification.
  • KlTDH3-1 For KlTDH3-1, using K. lactis genomic DNA as a template, PCR amplification with primers PF10:GAGCTCGGTACCCGGGGATCCTCTAGAGATCATCCACTCCATCACCGCTACCCAA (SEQ ID NO.:29) and PR10:GCCAAGCTTGCATGCCTGCAGGTCGACGATCAACGTCCCCATCTACAAGAGC (SEQ ID NO.:30); pMD18 The plasmid was used as a template and PCR amplification was carried out with primers pMD18-F: ATCGTCGACCTGCAGGCATG (SEQ ID NO.: 31) and pMD18-R: ATCTCTAGAGGATCCCCGGG (SEQ ID NO.: 32).
  • primers PF11:GATGCATTGATGGATGCCGAAGATGATTAAAGAGGTTGATGTAATTGATATTTTCCTGATAAAATTACTATTG SEQ ID NO.:33
  • PR11:AGCTGGTTGCTGATCGGATGTAGGTTCGCCAGATCCACCTCCTTCCACGTTTGTTGGTCTTGATCCACCTCCACCGTTCTTAGCAACAAGTTCGACCAAATCG SEQ ID NO.:34
  • Primers PF12: GGCGAACCTACATCCGATCAGC SEQ ID NO.: 35
  • PR12: TTAATCATCTTCGGCATCCATCAATGC SEQ ID NO.: 36
  • the two amplification products were each 8.5 ⁇ L, 1 ⁇ L of DpnI, 2 ⁇ L of 10 ⁇ digestion buffer, and incubated at 37° C. for 3 h.
  • 10 ⁇ L of DpnI-treated product was added to 100 ⁇ L of DH5 ⁇ competent cells, placed on ice for 30 min, and heat-shocked at 42 ° C for 45 s.
  • 1 mL of LB liquid medium was added and shaken at 37 ° C for 1 h, and applied to Amp-resistant LB solid culture at 37 ° C. Inverted to a monoclonal growth. Five monoclonal clones were picked and shaken in LB liquid medium. After PCR detection and sequencing confirmed, the plasmid was preserved and named pKM-KlTDH3-1-F-KleIF4G-DD (Fig. 8).
  • PCR amplification of KlTDH3-2 with K. lactis genomic DNA as template primers PF13:GAGCTCGGTACCCGGGGATCCTCTAGAGATGAAGCTTTGATGACTACCGTTC (SEQ ID NO.:39) and PR13:GCCAAGCTTGCATGCCTGCAGGTCGACGATGTCTATTGTATCGGAAGAACTGTCA (SEQ ID NO.:40); pMD18
  • the plasmid was used as a template, and PCR amplification was carried out with primers pMD18-F: ATCGTCGACCTGCAGGCATG (SEQ ID NO.: 41) and pMD18-R: ATCTCTAGAGGATCCCCGGG (SEQ ID NO.: 42).
  • primers PF14 GATGCATTGATGGATGCCGAAGATGATTAAATTACTCTTTTAAGTTAACGAACGCTTTTGATGAG (SEQ ID NO.:43) and PR14: AGCTGGTTGCTGATCGGATGTAGGTTCGCCAGATCCACCTCCTTCCACGTTTGTTGGTCTTGATCCACCTCCACCAGCAACGTGCTCAACtAAgTCaACgACcCTTTCAGAGTAACCGTATTCGTTATCG (SEQ ID NO.:44) was amplified; in K.
  • lactis DNA as template, Amplification was carried out with primers PF15: GGCGAACCTACATCCGATCAGC (SEQ ID NO.: 45) and PR15: TTAATCATCTTCGGCATCCATCAATGC (SEQ ID NO.: 46).
  • the two amplification products were each 8.5 ⁇ L, 1 ⁇ L of DpnI, 2 ⁇ L of 10 ⁇ digestion buffer, and incubated at 37° C. for 3 h.
  • 10 ⁇ L of DpnI-treated product was added to 100 ⁇ L of DH5 ⁇ competent cells, placed on ice for 30 min, and heat-shocked at 42 ° C for 45 s.
  • the competent cells were thawed at 37 ° C for 15-30 s, centrifuged at 13,000 g for 2 min and the supernatant was removed.
  • KlTDH3-1-CICF1 (KlTDH3-1 sequence primer): CTTCTACTGCTCCAATGTTCGTCGTT (SEQ ID NO.: 49) and primer K1TDH3-2-CICF1 (KlTDH3-2 sequence primer): TTAACGAAGACAAGTACAACGGTGA (SEQ ID NO.: 50), respectively PCR amplification, paired with KleIF4G-CICR2 (KleIF4G sequence primer): TTCTCTTCGACAGCCTTCTTAGCAG (SEQ ID NO.: 51) for PCR, KleIF4G insertion of KlTDH3-1 and KlTDH3-2 sites, positive PCR results The strain identified by sequencing was identified as a positive strain.
  • the Pab1 protein interacts with the eIF4G protein during translation initiation.
  • the invention combines KlPab1 and KleIF4G by CRISPR-Cas9 gene editing technology to promote the interaction between the two to improve the translation efficiency in vitro.
  • the K1Pab1 gene sequence in Kluyveromyces cerevisiae was obtained.
  • the PAM sequence (NGG) was searched for near the KlPab1 gene stop codon and the gRNA sequence was determined.
  • the principle of gRNA selection is: GC content is moderate, the standard of the invention is GC content of 40%-60%; avoid the existence of poly T structure.
  • the K1Pab1gRNA sequence identified in the present invention is TGCTTACGAAAACTTCAAGA (SEQ ID NO.: 52), located at chromosome 1555058...1555077 of chromosome C.
  • PCR amplification was carried out using the primer PF16: TGCTTACGAAAACTTCAAGAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCG (SEQ ID NO.: 53), PR16: GCTCTAAAACTCTTGAAGTTTTCGTAAGCAAAAGTCCCATTCGCCACCCG (SEQ ID NO.: 54), using the pCAS plasmid as a template. 17 ⁇ L of the amplified product was mixed, and 1 ⁇ L of DpnI, 2 ⁇ L of 10 ⁇ digestion buffer was added, and the mixture was incubated at 37° C. for 3 hours.
  • the donor DNA is first inserted into the pMD18 plasmid, and then amplified by PCR to obtain a linear donor DNA sequence.
  • PCR amplification was carried out using the genomic DNA of Kluyveromyces cerevisiae as a template, primers PF17:GAGCTCGGTACCCGGGGATCCTCTAGAGATCCGGTAAGCCATTGTACGTTGCCAT (SEQ ID NO.:55) and PR17:GCCAAGCTTGCATGCCTGCAGGTCGACGATCAGTATACCGTCCATGTTGATGACT (SEQ ID NO.:56); pMD18 plasmid as template and primer pMD18 -F: ATCGTCGACCTGCAGGCATG (SEQ ID NO.: 57) and pMD18-R: ATCTCTAGAGGATCCCCGGG (SEQ ID NO.: 58) were subjected to PCR amplification.
  • primers PF18 GATGCATTGATGGATGCCGAAGATGATTAAACTTGATTTTTTGACCTTGATCTTCATCTTGTC (SEQ ID NO.: 59) and PR18: CTTGAACTTCATCTTGAGTTGAACCTCCACCTCCAGATCCACCTCCACCAGCTTGAGCTTCTTGTTCtTTtTTaAAaTTcTCGTAAGCAGCTAAGGCTTC (SEQ ID NO.:60) was amplified; in K.
  • Two amplification products of 8.5 ⁇ L each, 1 ⁇ L of DpnI, 2 ⁇ L of 10 ⁇ digestion buffer were mixed, and the mixture was incubated at 37° C. for 3 hours.
  • the pKM-KlPab1-KleIF4G-DD plasmid was used as a template, and amplified with primers M13-F: GTAAAACGACGGCCAGT (SEQ ID NO.: 63) and M13-R: CAGGAAACAGCTATGAC (SEQ ID NO.: 64) to obtain linear donor DNA. .
  • the competent cells were thawed at 37 ° C for 15-30 s, centrifuged at 13,000 g for 2 min and the supernatant was removed.
  • the genetically modified Kluyveromyces lactis strain was prepared into an in vitro protein synthesis system, and a firefly Luciferase (Fluc) gene DNA template was added to determine the protein translation ability of the engineered strain.
  • the above reaction system was placed in an environment of 25-30 ° C, and allowed to stand for about 2-6 h. After the reaction, add an equal volume of Fluc substrate luciferin to a 96-well white plate or a 384-well white plate, and immediately place it on the Envision 2120 Multiplate Reader (Perkin Elmer). The number is measured for Fluc activity, and the Relative Light Unit (RLU) is used as the activity unit.
  • RLU Relative Light Unit
  • KleIF4G pre-inserted promoter pKlPGK1 structure pKlPGK1::KleIF4G, and KleIF4G linked to KlPab1 C-terminal structure KlPab1-KleIF4G showed stronger in vitro protein synthesis ability than wild-type yeast strain Y1140.
  • the relative light unit values of the encoded synthetic Lucc protein reached 1.57 ⁇ 10 9 and 1.50 ⁇ 10 9 , respectively, while the relative light unit value of the Fluc protein synthesized by the wild-type yeast strain Y1140 was only 4.11 ⁇ 10 8 . This suggests that the modification of KleIF4G can effectively enhance the efficiency of protein synthesis in yeast in vitro protein synthesis systems ( Figure 12).
  • NC indicates negative control, negative control.
  • the above experimental results indicate that the fusion protein of the present invention can significantly enhance the efficiency of protein production by the yeast in vitro protein synthesis system by the related modification of the KleIF4G gene of K. lactis.

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Abstract

提供了一种融合蛋白及其制备方法，所述融合蛋白可提高体外翻译效率，在融合蛋白中的eIF4G前插入组成型或诱导型启动子(如pKlPGK1)，可增强体外蛋白质的合成能力。

Description

新型融合蛋白的制备及其在提高蛋白质合成的应用 技术领域
本发明涉及基因工程领域，具体地，涉及新型融合蛋白的制备及其在提高蛋白质合成的应用。
背景技术
蛋白质是细胞中的重要分子，几乎参与了细胞所有功能的执行。蛋白的序列和结构不同，决定了其功能的不同。在细胞内，蛋白可以作为酶类催化各种生化反应，可以作为信号分子协调生物体的各种活动，可以支持生物形态，储存能量，运输分子，并使生物体运动。在生物医学领域，蛋白质抗体作为靶向药物，是治疗癌症等疾病的重要手段。
在细胞中，蛋白质翻译的调节在应对营养缺失等外界压力，细胞发育与分化等很多过程中发挥重要作用。蛋白质翻译的四个过程包括翻译起始、翻译延伸、翻译终止和核糖体再循环，其中翻译起始是受调控最多的一个过程。在翻译起始阶段，核糖体小亚基(40S)结合(tRNA)i Met，并在翻译起始因子的作用下识别mRNA 5'末端。小亚基向下游移动，并在起始密码子(ATG)位置与核糖体大亚基(60S)结合，形成完整核糖体，并进入翻译延伸阶段。
在快速分裂的酵母细胞中，蛋白的合成速率大约为13,000个/秒。在体内，蛋白的合成速率受到核糖体数目的限制，细胞的平均核糖体数目约为200,000个，mRNA分子的数目约为15,000-60,000个。
目前，经常实验的商业化体外蛋白表达系统包括大肠杆菌系统(E.coli extract，ECE)、兔网织红细胞(Rabbit reticulocyte Lysate，RRL)、麦胚(Wheat germ extract,WGE)、昆虫(Insect cell extract,ICE)和人源系统。
现有的商业化蛋白质体外合成体系中，原核系统的产量可以达到～0.5mg/mL，费用约为～10RMB/μg。真核系统中CHO系统的产量可以达到～0.7mg/mL，费用约为～20RMB/μg。因此无论自然界中细胞内还是细胞外的人造蛋白质合成体系都具有效率低，速度慢的特点，极大的限制了蛋白质合成的应用。
因此，本领域迫切需要开发一种可以有效增强体外蛋白质合成效率的体外蛋白质合成体系。
发明内容
本发明的目的在于提供一种可以有效增强体外蛋白质合成效率的体外蛋白质合成体系。
本发明第一方面提供了一种融合蛋白，所述融合蛋白具有式Ia或式Ib结构：
S-A-B-C     (Ia)
S-C-B-A     (Ib)；
式中，
A为PabI元件；
B为无或连接肽；
C为eIF4G元件；
S为任选的信号肽；以及
各“-”为肽键。
在另一优选例中，所述式Ia或Ib为从N端至C端的结构。
在另一优选例中，所述元件A包括野生型和突变型的PabI序列。
在另一优选例中，所述的PabI为来自酵母的PabI。
在另一优选例中，元件A具有SEQ ID NO.:1所示的序列或其活性片段，或者具有与SEQ ID NO:1所示氨基酸序列≥85％同源性(优选地，≥90％的同源性；等优选地≥95％的同源性；最优选地，≥97％的同源性，如98％以上，99％以上)且具有与SEQ ID NO.:1序列相同活性的多肽。
在另一优选例中，所述元件C包括野生型和突变型的eIF4G序列。
在另一优选例中，所述的eIF4G为来自酵母的eIF4G。
在另一优选例中，元件C具有SEQ ID NO.:2所示的序列或其活性片段，或者具有与SEQ ID NO:2所示氨基酸序列≥85％同源性(优选地，≥90％的同源性；等优选地≥95％的同源性；最优选地，≥97％的同源性，如98％以上，99％以上)且具有与SEQ ID NO.:2序列相同活性的多肽。
在另一优选例中，所述融合蛋白是重组蛋白，较佳地为酵母表达的重组蛋白。
在另一优选例中，所述酵母选自下组：克鲁维酵母、酿酒酵母、或其组合。
在另一优选例中，所述酵母选自下组：乳酸克鲁维酵母、马克斯克鲁维酵母、多布克鲁维酵母、或其组合。
在另一优选例中，所述元件A衍生自酵母的PabI蛋白。
在另一优选例中，所述元件C衍生自酵母的eIF4G蛋白。
在另一优选例中，所述的肽接头的长度为0-50氨基酸，较佳地为10-40个氨基酸，更佳地为15-25个氨基酸。
在另一优选例中，所述融合蛋白选自下组：
(A)具有SEQ ID NO:3所示氨基酸序列的多肽；(B)具有与SEQ ID NO:3所示氨基酸序列≥80％同源性(优选地，≥90％的同源性；等优选地≥95％的同源性；最优选地，≥97％的同源性，如98％以上，99％以上)的多肽，且所述多肽具有提高外源蛋白表达效率的功能或活性；
(C)将SEQ ID NO:3中任一所示氨基酸序列经过1-15(较佳地，2-10，更佳地，3-8)个氨基酸残基的取代、缺失或添加而形成的，且具有提高外源蛋白表达效率的功能或活性的衍生多肽。
在另一优选例中，所述融合蛋白的氨基酸序列如SEQ ID NO.:3所示。
在另一优选例中，所述融合蛋白具有选自下组的一个或多个特性：
(a)提高外源蛋白表达效率；
(b)提高体外翻译效率。
在另一优选例中，所述外源蛋白选自下组：荧光素蛋白、或荧光素酶(如萤火虫荧光素酶)、绿色荧光蛋白、黄色荧光蛋白、氨酰tRNA合成酶、甘油醛-3-磷酸脱氢酶、过氧化氢酶、肌动蛋白、抗体的可变区域、萤光素酶突变、α-淀粉酶、肠道菌素A、丙型肝炎病毒E2糖蛋白、胰岛素前体、干扰素αA、白细胞介素-1β、溶菌酶素、血清白蛋白、单链抗体段(scFV)、甲状腺素运载蛋白、酪氨酸酶、木聚糖酶、或其组合。
本发明第二方面提供了一种分离的多核苷酸，所述的多核苷酸编码本发明第一方面所述的融合蛋白。
在另一优选例中，所述的多核苷酸选自下组：DNA序列、RNA序列。
在另一优选例中，所述的DNA序列选自下组：基因组序列、cDNA序列。
在另一优选例中，所述的多核苷酸为mRNA或cDNA，并且所述多核苷酸具有式II所示结构：
A1-C1     (式II)
式中，
A1为编码上述A元件的核苷酸序列；
C1为编码上述C元件的核苷酸序列；
“-”为元件A1和元件C1之间的连接键。
在另一优选例中，所述元件A1具有SEQ ID NO.:4所示的序列。
在另一优选例中，所述元件C1具有SEQ ID NO.:5所示的序列。
本发明第三方面提供了一种载体，所述载体含有本发明第二方面所述的多核苷酸。
本发明第四方面提供了一种宿主细胞，所述宿主细胞含有本发明第三方面所述的载体或基因组中整合有本发明第二方面所述的多核苷酸。
在另一优选例中，所述宿主细胞是通过转入本发明第三方面所述的表达载体或本发明第二方面所述的多核苷酸并经同源重组而形成的，从而在基因组或染色体中整合有本发明第一方面所述融合蛋白的编码序列。
在另一优选例中，所述宿主细胞选自下组：克鲁维酵母、酿酒酵母、或其组合。
在另一优选例中，所述宿主细胞选自下组：乳酸克鲁维酵母、马克斯克鲁维酵母、多布克鲁维酵母、或其组合。
在另一优选例中，所述宿主细胞是乳酸克鲁维酵母。
本发明第五方面提供了一种用于表达外源蛋白的体外蛋白质合成体系，所述反应体系包括：
(i)酵母体外蛋白质合成体系，所述的合成体系含有(a)酵母细胞提取物；(b)任选的聚乙二醇；(c)任选的外源蔗糖；和(d)任选的溶剂，所述溶剂为水或水性溶剂；以及
(ii)本发明第一方面所述的融合蛋白。
在另一优选例中，所述反应体系还包括：(iii)额外添加的eIF4G蛋白。
在另一优选例中，所述eIF4G蛋白由组成型或诱导性启动子诱导表达。
在另一优选例中，所述组成型或诱导性启动子来源于酵母。
在另一优选例中，所述酵母选自下组：克鲁维酵母、酿酒酵母、或其组合。
在另一优选例中，所述组成型或诱导型启动子选自下组：pScTEF1、pScPGK1、pKlTEF1、pKlPGK1、pScADH1、pScTPI1、pScTDH3、pKlADH1、pKlTPI1、pKlTDH3、或其组合。
本发明第六方面提供了一种生产本发明第一方面所述融合蛋白的方法，包括：
(i)在适合表达的条件下，培养本发明第四方面所述的宿主细胞，从而表达出本发明第一方面所述的融合蛋白；和
(ii)分离所述融合蛋白。
本发明第七方面提供了一种本发明第一方面所述融合蛋白的用途，用于制备一表达外源蛋白的体外蛋白质合成体系，所述体外蛋白质合成体系用于提高外源蛋白的表达效率。
在另一优选例中，所述反应体系还包括额外的eIF4G蛋白。
本发明第八方面提供了一种本发明第一方面所述的融合蛋白的用途，用于制备提高体外蛋白质合成体系的体外蛋白合成能力的制剂。
本发明第九方面提供了一种表达待表达的外源蛋白的方法，包括：
(i)提供一酵母体外蛋白质合成体系，其中所述的合成体系中含有本发明第一方面所述的融合蛋白；和
(ii)在适合表达蛋白的条件下，在所述外源蛋白的模板存在下，孵育所述酵母体外蛋白质合成体系，从而表达所述的外源蛋白。
在另一优选例中，所述的融合蛋白为额外添加的。
在另一优选例中，所述的融合蛋白与所述的酵母体外蛋白质合成体系中的其他蛋白为来自相同酵母的提取物。
在另一优选例中，所述的方法是非诊断性和非治疗性的。
在另一优选例中，所述步骤(ii)还包括步骤(iii)：检测外源蛋白活性的表达活性Q1，并且在步骤(ii)相同条件下孵育野生型酵母菌株，检测所述外源蛋白的活性Q2，如果Q1显著高于Q2，则表明外源蛋白的表达效率显著提高。
在另一优选例中，所述“显著高于”指Q1/Q2≥2，较佳地，≥3，更佳地，≥4。
在另一优选例中，所述酵母体外蛋白合成体系为基因改造的克鲁维酵母体外蛋白合成体系(优选乳酸克鲁维酵母体外蛋白合成体系)。
在另一优选例中，所述外源蛋白的编码序列来自原核生物、真核生物。
在另一优选例中，所述外源蛋白的编码序列来自动物、植物、病原体。
在另一优选例中，所述外源蛋白的编码序列来自哺乳动物，较佳地灵长动物，啮齿动物，包括人、小鼠、大鼠。
在另一优选例中，所述的外源蛋白的编码序列选自下组：编码荧光素蛋白、或荧光素酶(如萤火虫荧光素酶)、绿色荧光蛋白、黄色荧光蛋白、氨酰tRNA合成酶、甘油醛-3-磷酸脱氢酶、过氧化氢酶、肌动蛋白、抗体的可变区域的外源DNA、萤光素酶突变体的DNA、或其组合。
在另一优选例中，所述外源蛋白选自下组：荧光素蛋白、或荧光素酶(如萤火虫荧光素酶)、绿色荧光蛋白、黄色荧光蛋白、氨酰tRNA合成酶、甘油醛-3-磷酸脱氢酶、过氧化氢酶、肌动蛋白、抗体的可变区域、萤光素酶突变、α-淀粉酶、肠道菌素A、丙型肝炎病毒E2糖蛋白、胰岛素前体、干扰素αA、白细胞介素-1β、溶菌酶素、血清白蛋白、单链抗体段(scFV)、甲状腺素运载蛋白、酪氨酸酶、木聚糖酶、或其组合。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
附图说明
图1显示了pKM-CAS1.0-KleIF4G的质粒图谱。
图2显示了pKM-pScTEF1-KleIF4G-DD的质粒图谱。
图3显示了pKM-pScPGK1-KleIF4G-DD的质粒图谱。
图4显示了pKM-pKlTEF1-KleIF4G-DD的质粒图谱。
图5显示了pKM-pKlPGK1-KleIF4G-DD的质粒图谱。
图6显示了pKM-CAS1.0-KlTDH3-1的质粒图谱。
图7显示了pKM-CAS1.0-KlTDH3-2的质粒图谱。
图8显示了pKM-KlTDH3-1-F-KleIF4G-DD的质粒图谱。
图9显示了pKM-KlTDH3-2-F-KleIF4G-DD的质粒图谱。
图10显示了pKM-CAS1.0-KlPab1的质粒图谱。
图11显示了pKM-KlPab1-KleIF4G-DD的质粒图谱。
图12显示了改造菌株体外翻译活性测定示意图。利用荧光蛋白的强度指示系统的蛋白表达能力。
具体实施方式
经过广泛而深入的研究，通过大量筛选和摸索，首次意外地发现了一种具有式Ia或式Ib结构的融合蛋白，本发明的融合蛋白可大幅度的提高体外翻译效率。此外，本发明还发现，在eIF4G前插入组成型或诱导型启动子(如pScTEF1、pScPGK1、pKlTEF1、pKlPGK1、pScADH1、pScTPI1、pScTDH3、pKlADH1、pKlTPI1、pKlTDH3等)可显著增强体外蛋白质的合成能力。
并且，本发明人还发现，本发明的融合蛋白在提高体外翻译效率时，本发明的融合蛋白的元件eIF4G的表达量并未增加。
具体地，在含有本发明的融合蛋白的酵母体外蛋白质合成体系中，所合成的荧光素酶活性的相对光单位值高达1.50×109，在eIF4G前插入组成型或诱导型启动子所合成的荧光素酶活性的相对光单位值高达1.57×109，远远高于野生型酵母菌株(如Y1140)合成的荧光素酶的相对光单位值(4.11×108)。在此基础上，本发明人完成了本发明。
eIF4F元件
真核生物中，多种翻译起始因子参与蛋白质翻译起始过程(表1)。其中eIF4F负责“帽子结构”的识别以及下游翻译起始因子和核糖体的招募。eIF4F由三个蛋白质亚基组成：eIF4E、eIF4G和eIF4A。eIF4E特异性结合“帽子结构”，将eIF4F锚定在mRNA 5'端非翻译区；eIF4A是一种RNA解旋酶；eIF4G则几乎是整个翻译起始过程的支架蛋白，能与多种翻译起始因子相互作用，在下游因子招募过程中具有重要作用。
表1酵母中翻译起始因子
Figure PCTCN2017115972-appb-000001
Figure PCTCN2017115972-appb-000002
在本发明中，通过在eIF4G前插入酵母来源(如酿酒酵母、克鲁维酵母等)的组成型或诱导型启动子(如pScTEF1、pScPGK1、pKlTEF1、pKlPGK1、pScADH1、pScTPI 1、pScTDH3、pKlADH1、pKlTPI 1、pKlTDH3等)，从而增强体外蛋白质的合成能力。
在一优选实施方式中，所述eIF4G的核苷酸序列如SEQ ID NO.:5所示；所述eIF4G的蛋白序列如SEQ ID NO.:2所示。
Figure PCTCN2017115972-appb-000003
Figure PCTCN2017115972-appb-000004
Figure PCTCN2017115972-appb-000005
Pab1元件(Pab1蛋白)
Pab1是一个71kDa的RNA结合蛋白，由4个RRM(RNA recognition motif1-4)结构域和1个MLLE结构域组成。每个RRM结构域中都包含2个保守的RNP结构(RNP1/2)，负责与RNA的结合。
在一优选实施方式中，所述Pab1的核苷酸序列如SEQ ID NO.:4所示；所述Pab1的蛋白序列如SEQ ID NO.:1所示。
Figure PCTCN2017115972-appb-000006
Figure PCTCN2017115972-appb-000007
融合蛋白
如本文所用，术语“本发明融合蛋白”、“本发明PabI-eIF4G融合蛋白”以及“PabI-eIF4G融合蛋白”可互换使用，指PabI元件与eIF4G元件融合形成的融合蛋白。在本发明的融合蛋白中，PabI元件与eIF4G元件之间可以含有或不含有连接肽或柔性接头。此外，所述融合蛋白可以含有或不含有起始的Met；可以含有或不含有信号肽；以及含有或不含有标签序列(如6His等)。
在一优选实施方式中，本发明所述的融合蛋白具有上述的式Ia或式Ib结构。优选地，本发明的融合蛋白的氨基酸序列如SEQ ID NO.:3所示。
Figure PCTCN2017115972-appb-000008
在本发明中，本发明的融合蛋白可显著提高无细胞的、体外蛋白质合成体系(尤其是酵母体外蛋白质合成体系)的体外蛋白质合成能力。
体外蛋白质合成体系
本发明融合蛋白可用于提高体外蛋白质合成体系的蛋白质合成能力。
一种典型的体外蛋白质合成体系是酵母体外蛋白质合成体系。
酵母(yeast)兼具培养简单、高效蛋白质折叠、和翻译后修饰的优势。其中酿酒酵母(Saccharomyces cerevisiae)和毕氏酵母(Pichia pastoris)是表达复杂真核蛋白质和膜蛋白的模式生物，酵母也可作为制备体外翻译系统的原料。
克鲁维酵母(Kluyveromyces)是一种子囊孢子酵母，其中的马克斯克鲁维酵母(Kluyveromyces marxianus)和乳酸克鲁维酵母(Kluyveromyces lactis)是工业上广泛使用的酵母。例如乳酸克鲁维酵母是一种能够以乳酸作为其唯一的碳源和能源的酵母。与其他酵母相比，乳酸克鲁维酵母具有许多优点，如超强的分泌能力，良好的大规模发酵特性、食品安全的级别及同时具有蛋白翻译后修饰的能力等，其作为宿主系统表达药用蛋白也已显示出巨大的潜力。
在本发明中，酵母体外蛋白质合成体系不受特别限制，一种优选的酵母体外蛋白质合成体系为克鲁维酵母表达系统(更佳地，乳酸克鲁维酵母表达系统)。
在本发明中，克鲁维酵母(如乳酸克鲁维酵母)不受特别限制，包括任何一种能够提高合成蛋白效率的克鲁维(如乳酸克鲁维酵母)菌株。
在一优选实施方式中，本发明的酵母体外蛋白质合成体系为基因改造后的酸克鲁维酵母表达系统。
在一优选实施方式中，本发明提供了一种体外的无细胞的蛋白合成体系，所述合成体系包括：
(a)酵母细胞提取物；
(b)聚乙二醇；
(c)任选的外源蔗糖；和
(d)任选的溶剂，所述溶剂为水或水性溶剂。
在另一优选例中，所述聚乙二醇选自下组：PEG3000、PEG8000、PEG6000、PEG3350、或其组合。
在另一优选例中，所述聚乙二醇包括分子量(Da)为200-10000的聚乙二醇，较佳地，分子量为3000-10000的聚乙二醇。
在另一优选例中，所述蛋白合成体系中，组分(a)的浓度(v/v)为20％-70％，较佳地，30-60％，更佳地，40％-50％，以所述蛋白合成体系的总体积计。
在另一优选例中，所述蛋白合成体系中，组分(b)的浓度(w/v，例如g/ml)为0.1-8％，较佳地，0.5-4％，更佳地，1-2％。
在另一优选例中，所述蛋白合成体系中，组分(c)的浓度为0.03-40wt％，较佳地，0.08-10wt％，更佳地，0.1-5wt％，以所述蛋白合成体系的总重量计。
在一特别优选的实施方式中，本发明提供的体外蛋白合成体系包括：酵母细胞提取物，4-羟乙基哌嗪乙磺酸，醋酸钾，醋酸镁，腺嘌呤核苷三磷酸(ATP)，鸟嘌呤核苷三磷酸(GTP)，胞嘧啶核苷三磷酸(CTP)，胸腺嘧啶核苷三磷酸(TTP)，氨基酸混合物，磷酸肌酸，二硫苏糖醇(DTT)，磷酸肌酸激酶，RNA酶抑制剂，荧光素，萤光素酶DNA，RNA聚合酶。
在本发明中，RNA聚合酶没有特别限制，可以选自一种或多种RNA聚合酶，典型的RNA聚合酶为T7 RNA聚合酶。
在本发明中，所述酵母细胞提取物在体外蛋白合成体系中的比例不受特别 限制，通常所述酵母细胞提取物在体外蛋白质合成蛋白合成体系中所占体系为20-70％，较佳地，30-60％，更佳地，40-50％。
在本发明中，所述的酵母细胞提取物不含完整的细胞，典型的酵母细胞提取物包括用于蛋白翻译的核糖体、转运RNA、氨酰tRNA合成酶、蛋白质合成需要的起始因子和延伸因子以及终止释放因子。此外，酵母提取物中还含有一些源自酵母细胞的细胞质中的其他蛋白，尤其是可溶性蛋白。
在本发明中，所述的酵母细胞提取物所含蛋白含量为20-100mg/mL,较佳为50-100mg/mL。所述的测定蛋白含量方法为考马斯亮蓝测定方法。
在本发明中，所述的酵母细胞提取物的制备方法不受限制，一种优选的制备方法包括以下步骤：
(i)提供酵母细胞；
(ii)对酵母细胞进行洗涤处理，获得经洗涤的酵母细胞；
(iii)对经洗涤的酵母细胞进行破细胞处理，从而获得酵母粗提物；
(iv)对所述酵母粗提物进行固液分离，获得液体部分，即为酵母细胞提取物。
在本发明中，所述的固液分离方式不受特别限制，一种优选的方式为离心。
在一优选实施方式中，所述离心在液态下进行。
在本发明中，所述离心条件不受特别限制，一种优选的离心条件为5000-100000g，较佳地，8000-30000g。
在本发明中，所述离心时间不受特别限制，一种优选的离心时间为0.5min-2h，较佳地，20-50min。
在本发明中，所述离心的温度不受特别限制，优选的，所述离心在1-10℃下进行，较佳地，在2-6℃下进行。
在本发明中，所述的洗涤处理方式不受特别限制，一种优选的洗涤处理方式为采用洗涤液在pH为7-8(较佳地，7.4)下进行处理，所述洗涤液没有特别限制，典型的所述洗涤液选自下组：4-羟乙基哌嗪乙磺酸钾、醋酸钾、醋酸镁、或其组合。
在本发明中，所述破细胞处理的方式不受特别限制，一种优选的所述的破细胞处理包括高压破碎、冻融(如液氮低温)破碎。
所述体外蛋白质合成体系中的核苷三磷酸混合物为腺嘌呤核苷三磷酸、鸟嘌呤核苷三磷酸、胞嘧啶核苷三磷酸和尿嘧啶核苷三磷酸。在本发明中，各种单核苷酸的浓度没有特别限制，通常每种单核苷酸的浓度为0.5-5mM，较佳地为1.0-2.0mM。
所述体外蛋白质合成体系中的氨基酸混合物可包括天然或非天然氨基酸，可包括D型或L型氨基酸。代表性的氨基酸包括(但并不限于)20种天然氨基酸：甘氨酸、丙氨酸、缬氨酸、亮氨酸、异亮氨酸、苯丙氨酸、脯氨酸、色氨酸、丝氨酸、酪氨酸、半胱氨酸、蛋氨酸、天冬酰胺、谷氨酰胺、苏氨酸、天冬氨酸、谷氨酸、赖氨酸、精氨酸和组氨酸。每种氨基酸的浓度通常为0.01-0.5mM，较佳地0.02-0.2mM，如0.05、0.06、0.07、0.08mM。
在优选例中，所述体外蛋白质合成体系还含有聚乙二醇或其类似物。聚乙二醇或其类似物的浓度没有特别限制，通常，聚乙二醇或其类似物的浓度(w/v)为0.1-8％，较佳地，0.5-4％，更佳地，1-2％，以所述蛋白合成体系的总重量计。代表性的PEG例子包括(但并不限于)：PEG3000，PEG8000，PEG6000和PEG3350。 应理解，本发明的体系还可包括其他各种分子量的聚乙二醇(如PEG200、400、1500、2000、4000、6000、8000、10000等)。
在优选例中，所述体外蛋白质合成体系还含有蔗糖。蔗糖的浓度没有特别限制，通常，蔗糖的浓度为0.03-40wt％，较佳地，0.08-10wt％，更佳地，0.1-5wt％，以所述蛋白合成体系的总重量计。
一种特别优选的体外蛋白质合成体系，除了酵母提取物之外，还含有以下组分：22mM，pH为7.4的4-羟乙基哌嗪乙磺酸，30-150mM醋酸钾，1.0-5.0mM醋酸镁，1.5-4mM核苷三磷酸混合物，0.08-0.24mM的氨基酸混合物，25mM磷酸肌酸，1.7mM二硫苏糖醇，0.27mg/mL磷酸肌酸激酶，1％-4％聚乙二醇，0.5％-2％蔗糖，8-20ng/μl萤火虫荧光素酶的DNA，0.027-0.054mg/mL T7 RNA聚合酶。
在一优选实施方式中，本发明的酵母体外蛋白质合成体系还含有：(a)本发明所述的融合蛋白，即PabI-eIF4G融合蛋白。
在一优选实施方式中，本发明的酵母体外蛋白质合成体系还包括eIF4G蛋白；其中，本发明的eIF4G蛋白通过来源于酵母(如酿酒酵母、克鲁维酵母等)组成型或诱导型的启动子(如pScTEF1、pScPGK1、pKlTEF1、pKlPGK1、pScADH1、pScTPI1、pScTDH3、pKlADH1、pKlTPI1、pKlTDH3等)进行诱导表达。
在本发明中，含有本发明融合蛋白的酵母体外蛋白质合成体系可显著增强体外蛋白质的合成能力。此外，将本发明的融合蛋白和eIF4G蛋白联合使用的酵母体外蛋白质合成体系具有更高的体外蛋白质的合成能力。
一类优选的酵母体外蛋白质合成体系描述于本发明人的在先申请CN201710125619.9中。在本文中，该专利文献通过引用方式全部纳入本文。该文献中的酵母体外蛋白质合成体系未采用本发明所述的融合蛋白。
典型地，本发明的酵母体外蛋白质合成体系含有(a)酵母细胞提取物；(b)任选的聚乙二醇；(c)任选的外源蔗糖；和(d)任选的溶剂，所述溶剂为水或水性溶剂；以及(ii)本发明所述的融合蛋白。
在另一优选例中，所述无细胞的蛋白合成体系还包括选自下组的一种或多种组分：
(e1)用于合成RNA的底物；
(e2)用于合成蛋白的底物；
(e3)镁离子；
(e4)钾离子；
(e5)缓冲剂；
(e6)RNA聚合酶；
(e7)能量再生系统。
在另一优选例中，所述蛋白合成体系中，组分(e1)的浓度为0.1-5mM，较佳地，0.5-3mM，更佳地，1-1.5mM。
在另一优选例中，所述的酵母细胞提取物为对酵母细胞的水性提取物。
在另一优选例中，所述酵母细胞提取物不含酵母内源性的长链核酸分子。
在另一优选例中，所述的合成RNA的底物包括：核苷单磷酸、核苷三磷酸、或其组合。
在另一优选例中，所述的合成蛋白的底物包括：1-20种天然氨基酸、以及非天 然氨基酸。
在另一优选例中，所述镁离子来源于镁离子源，所述镁离子源选自下组：醋酸镁、谷氨酸镁、或其组合。
在另一优选例中，所述钾离子来源于钾离子源，所述钾离子源选自下组：醋酸钾、谷氨酸钾、或其组合。
在另一优选例中，所述能量再生系统选自下组：磷酸肌酸/磷酸肌酸酶系统、糖酵解途径及其中间产物能量系统、或其组合。
在另一优选例中，所述无细胞的蛋白合成体系还包括(f1)人工合成的tRNA。
在另一优选例中，所述缓冲剂选自下组：4-羟乙基哌嗪乙磺酸、三羟甲基氨基甲烷、或其组合。
在另一优选例中，所述无细胞的蛋白合成体系还包括(g1)外源的用于指导蛋白质合成的DNA分子。
在另一优选例中，所述的DNA分子为线性的。
在另一优选例中，所述的DNA分子为环状的。
在另一优选例中，所述的DNA分子含有编码外源蛋白的序列。
在另一优选例中，所述的编码外源蛋白的序列包括基因组序列、cDNA序列。
在另一优选例中，所述的编码外源蛋白的序列还含有启动子序列、5'非翻译序列、3'非翻译序列。
本发明的主要优点包括：
(a)本发明首次通过基因改造技术，借助高效的细胞转化平台，对细胞内基因进行改造，从而提高了翻译系统的蛋白合成效率。
(b)本发明首次发现一种融合蛋白，本发明的融合蛋白可显著增强体外蛋白质的合成能力。
(c)本发明首次发现，在eIF4G前插入组成型或诱导型的启动子(如pScTEF1、pScPGK1、pKlTEF1、pKlPGK1、pScADH1、pScTPI1、pScTDH3、pKlADH1、pKlTPI1、pKlTDH3等)可显著增强体外蛋白质的合成能力。
(d)本发明首次通过CRISPR-Cas9基因编辑技术改造eIF4G，从而著增强体外蛋白质的合成能力。
(e)本发明首次发现，本发明的融合蛋白在提高体外翻译系统的效率时，本发明的融合蛋白的元件eIF4G的表达量并未增加。
实施例1通过基因改造提高蛋白质合成的理论模型
本发明通过CRISPR-Cas9基因编辑技术，对K.lactis中翻译起始因子eIF4G和Pab1进行优化，以提高无细胞体外翻译系统的效率。
实施例2通过CRISPR-Cas9对翻译起始因子进行改造，提高体外翻译系统效率
2.1通过CRISPR-Cas9技术在翻译起始因子KleIF4G前加入强启动子
2.1.1 KleIF4G序列检索及CRISPR gRNA序列确定
eIF4G是翻译起始过程中的重要因子。目前的报导中，未见有通过基因编辑技术优化内源eIF4G表达，从而提高体外翻译活性的案例。本发明依据实施 例1中的理论模型，通过CRISPR-Cas9基因编辑技术，对翻译起始因子KleIF4G的表达进行改造，以提高无细胞体外翻译系统的效率。
i.基于S.cerevisiae酵母中eIF4G基因序列。在NCBI数据库中以eIF4G基因进行BLAST比对分析，确定乳酸克鲁维酵母中eIF4G同源基因序列，命名为KleIF4G(位于染色体A的421863...424928)。在这里以此基因尾部插入一段标记DNA为例，其他目标基因或插入位置、序列均可采用类似方法操作。
ii.在KleIF4G基因起始密码子，搜索临近的PAM序列(NGG)，并确定gRNA序列。gRNA选择的原则为：GC含量适中，本发明的标准为GC含量为40％-60％；避免poly T结构的存在。最终，本发明确定的优化的KleIF4G gRNA序列为CGGTTTTTCAAAGCAGATAT(SEQ ID NO.:6)，位于染色体A的424927...424936位点。
2.1.2 CRISPR-Cas9介导的在KleIF4G前插入强启动子质粒构建
为了实现KleIF4G的过量表达，本发明通过CRISPR-Cas技术在KleIF4G基因前分别插入pScTEF1、pScPGK1、pKlTEF1和pKlPGK1启动子。质粒构建及转化方法如下：
i.CRISPR质粒构建：
使用引物PF1：CGGTTTTTCAAAGCAGATATGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCG
(SEQ ID NO.:7)，PR1：GCTCTAAAACATATCTGCTTTGAAAAACCGAAAGTCCCATTCGCCACCCG(SEQ ID NO.:8)，以pCAS质粒为模板，进行PCR扩增。将扩增产物17μL混合，加入1μL DpnI，2μL10×digestion buffer，37℃温浴3h。将DpnI处理后产物10μL加入100μL DH5α感受态细胞中，冰上放置30min，42℃热激45s后，加入1mL LB液体培养基37℃振荡培养1h，涂布于Kan抗性LB固体培养，37℃倒置培养至单克隆长出。挑取5个单克隆在LB液体培养基中振荡培养，PCR检测阳性并测序确认后，提取质粒保存，命名为pKM-CAS1.0-KleIF4G(图1)。
ii.供体DNA质粒构建及扩增
为了便于线性供体DNA的保存及扩增，本发明首先将供体DNA插入到pMD18质粒中，然后通过PCR扩增得到线性供体DNA序列。
以乳酸克鲁维酵母基因组DNA为模板，以引物PF2：GAGCTCGGTACCCGGGGATCCTCTAGAGATAATAAAATTTCAACCTTTAAGCCATTGAATTTTACCATTACG(SEQ ID NO.:9)和PR2：GCCAAGCTTGCATGCCTGCAGGTCGACGATCTTGTTAGTAATCTCAACCTTCGCTGG(SEQ ID NO.:10)进行PCR扩增；以pMD18质粒为模板，以引物pMD18-F：ATCGTCGACCTGCAGGCATG(SEQ ID NO.:11)和pMD18-R：ATCTCTAGAGGATCCCCGGG(SEQ ID NO.:12)进行PCR扩增。将两次扩增产物各8.5μL混合，加入1μL DpnI，2μL 10×消化缓冲液，37℃温浴3h。将DpnI处理后产物10μL加入100μL DH5α感受态细胞中，冰上放置30min，42℃热激45s后，加入1mL LB液体培养基37℃振荡培养1h，涂布于Amp抗性LB固体培养，37℃倒置培养至单克隆长出。挑取5个单克隆在LB液体培养基中振荡培养，PCR检测阳性并测序确认后，提取质粒保存，命名为pKM-KleIF4G-DD。
以pKM-KleIF4G-DD质粒为模板，以引物PF3：ATGGGCGAACCTACATCCGATC(SEQ  ID NO.:13)和PR3：ATCTGCTTTGAAAAACCGCTCTTTCTCTC(SEQ ID NO.:14)进行PCR扩增；以S.cerevisiae酵母基因组DNA为模板，以引物PF4：AGAGAGAAAGAGCGGTTTTTCAAAGCAGATCCACACACCATAGCTTCAAAATGTTTCTAC(SEQ ID NO.:15)和PR4：TGGTTGCTGATCGGATGTAGGTTCGCCCATCTTAGATTAGATTGCTATGCTTTCTTTCTAATGAGC(SEQ ID NO.:16)进行PCR扩增(pScTEF1启动子扩增)；以S.cerevisiae酵母基因组DNA为模板，以引物PF5：AGAGAGAAAGAGCGGTTTTTCAAAGCAGATAGACGCGAATTTTTCGAAGAAGTACC(SEQ ID NO.:17)和PR5：AGCTTCAACAGCTGGTTGCTGATCGGATGTAGGTTCGCCCATTGTTTTATATTTGTTGTAAAAAGTAGATAATTACTTCCTTGATGATC(SEQ ID NO.:18)进行PCR扩增(pScPGK1启动子扩增)；以乳酸克鲁维酵母基因组DNA为模板，以引物PF6：AGAGAGAAAGAGCGGTTTTTCAAAGCAGATGAGCCTGTCCAAGCAAATGCC(SEQ ID NO.:19)和PR6：TGGTTGCTGATCGGATGTAGGTTCGCCCATTTTTAATGTTACTTCTCTTGCAGTTAGGGAAC(SEQ ID NO.:20)进行PCR扩增(pKlTEF1启动子扩增)；以乳酸克鲁维酵母基因组DNA为模板，以引物PF7：AGAGAGAAAGAGCGGTTTTTCAAAGCAGATGTTCCTCATCACTAGAAGCCGAACTG(SEQ ID NO.:21)和PR7：AGCTTCAACAGCTGGTTGCTGATCGGATGTAGGTTCGCCCATTTTTATTAATTCTTGATCGATTTTTTTGTTATTTCTGAAGTAACTCT(SEQ ID NO.:22)进行PCR扩增(pKlPGK1启动子扩增)。将PF3/PR3扩增产物分别与PF4/PR4、PF5/PR5、PF6/PR6和PF7/PR7扩增产物混合，分别构建pKM-pScTEF1-KleIF4G-DD、pKM-pScPGK1-KleIF4G-DD、pKM-pKlTEF1-KleIF4G-DD和pKM-pKlPGK1-KleIF4G-DD(如图2，3，4，5)。具体步骤为：两种PCR产物各8.5μL混合，加入1μL DpnI，2μL 10×消化缓冲液，37℃温浴3h。将DpnI处理后产物10μL加入100μL DH5α感受态细胞中，冰上放置30min，42℃热激45s后，加入1mL LB液体培养基37℃振荡培养1h，涂布于Amp抗性LB固体培养，37℃倒置培养至单克隆长出。挑取5个单克隆在LB液体培养基中振荡培养，PCR检测阳性并测序确认后，提取质粒保存。
2.1.3乳酸克鲁维酵母转化及阳性鉴定
i.将乳酸克鲁维酵母菌液在YPD固体培养基上划线并挑取单克隆，于25mL 2×YPD液体培养基中振荡培养过夜，取2mL菌液于50mL液体2×YPD培养基中继续振荡培养2-8h。20℃条件下3000g离心5min收集酵母细胞，加入500μL无菌水重悬，同样条件下离心收集细胞。配制感受态细胞溶液(5％v/v甘油，10％v/v DMSO)并将酵母细胞溶解于500μL该溶液中。分装50μL至1.5mL离心管中，-80℃保存。
将感受态细胞置于37℃融化15-30s，13000g离心2min并去除上清。配制转化缓冲液：PEG 3350(50％(w/v))260μL，LiAc(1.0M)36μL，carrier DNA(5.0m g/mL)20μL，Cas9/gRNA质粒15μL，供体DNA 10μL，加入无菌水至最终体积360μL。热激后，13000g离心30s去除上清。加入1mL YPD液体培养基，培养2-3h，吸取200μL涂布于固体YPD(200μg/mL G418)培养 基，培养2-3天至单菌落出现。
ii.在乳酸克鲁维酵母转化后的平板上挑取10-20个单克隆，置于1mL YPD(200μg/mL G418)液体培养基中振荡培养过夜，以菌液为模板，以CRISPR Insertion Check引物对，对相应样品进行PCR检测。PCR结果阳性并经测序鉴定的菌株，确定为阳性菌株。
2.2通过CRISPR-Cas9技术将KleIF4G与高表达基因融合
2.2.1 KlTDH3序列检索及CRISPR gRNA序列确定
在酿酒酵母(S.cerevisiae)中，TDH3以四聚体形式存在，参与糖酵解途径中的催化反应。其启动子pTDH3是基因工程中广泛使用的一种持续型强启动子。为了实现乳酸克鲁维酵母中KleIF4G的足量表达，并在执行翻译起始功能时形成局部高浓度，本发明将KleIF4G基因连接到乳酸克鲁维酵母TDH3基因ORF 3'端。
i.基于S.cerevisiae酵母中TDH3基因序列。在NCBI数据库中以TDH3基因进行BLAST比对分析，确定乳酸克鲁维酵母中TDH3同源基因序列。经比对发现，在乳酸克鲁维酵母基因组中存在两个TDH3同源基因，在本发明中分别命名为KlTDH3-1(位于染色体A的1024297...1025292)和KlTDH3-2(位于染色体F的1960417...1961406)。在这里以此基因尾部插入一段标记DNA为例，其他目标基因或插入位置、序列均可采用类似方法操作。
ii.在KlTDH3基因终止密码子附近搜索PAM序列(NGG)，并确定KlTDH3gRNA序列(KlTDH3-1：CTTGTTGCTAAGAACTAAAG(SEQ ID NO.:23)，位于染色体A的1024272...1024291位点，KlTDH3-2：CTCTGAAAGAGTTGTCGATT(SEQ ID NO.:24)位于染色体F的1960378...1960397位点)。
2.2.2 CRISPR-Cas9介导的将KleIF4G整合到KlTDH3位点质粒构建
CRISPR质粒构建
i.对KlTDH3-1，使用引物PF8：CTTGTTGCTAAGAACTAAAGGTTTTAGAGCTAGAAATAGCAAGTTAAAAT(SEQ ID NO.:25)，PR8：GCTCTAAAACCTTTAGTTCTTAGCAACAAGAAAGTCCCATTCGCCACCCG(SEQ ID NO.:26)，以pCAS质粒为模板，进行PCR扩增。将扩增产物17μL混合，加入1μL DpnI，2μL10×消化缓冲液，37℃温浴3h。将DpnI处理后产物10μL加入100μL DH5α感受态细胞中，冰上放置30min，42℃热激45s后，加入1mL LB液体培养基37℃振荡培养1h，涂布于Kan抗性LB固体培养，37℃倒置培养至单克隆长出。挑取5个单克隆在LB液体培养基中振荡培养，PCR检测阳性并测序确认后，提取质粒保存，命名为pKM-CAS1.0-KlTDH3-1(如图6)。
ii.对KlTDH3-2，使用引物PF9：CTCTGAAAGAGTTGTCGATTGTTTTAGAGCTAGAAATAGCAAGTTAAAAT(SEQ ID NO.:27)，PR9：GCTCTAAAACAATCGACAACTCTTTCAGAGAAAGTCCCATTCGCCACCCG(SEQ ID NO.:28)，以pCAS质粒为模板，进行PCR扩增。将扩增产物17μL混合，加入1μL DpnI，2μL 10×digestion buffer，37℃温浴3h。将DpnI处理后产物10μL加入100μL DH5α感受态细胞中，冰上放置30min，42℃热激45s后，加入1mL LB液体培养基37℃振荡培养1h，涂布于Kan抗性LB固体培养，37℃倒置培养至单克隆长出。挑取5个单克隆在LB液体培养基中振荡培 养，PCR检测阳性并测序确认后，提取质粒保存，命名为pKM-CAS1.0-KlTDH3-2(如图7)。
2.2.3供体DNA质粒构建及扩增
为了便于线性供体DNA的保存及扩增，本发明首先将供体DNA插入到pMD18质粒中，然后通过PCR扩增得到线性供体DNA序列。
i.对KlTDH3-1，以乳酸克鲁维酵母基因组DNA为模板，以引物PF10：GAGCTCGGTACCCGGGGATCCTCTAGAGATCATCCACTCCATCACCGCTACCCAA(SEQ ID NO.:29)和PR10：GCCAAGCTTGCATGCCTGCAGGTCGACGATCAACGTCCCCATCTACAAGAGC(SEQ ID NO.:30)进行PCR扩增；以pMD18质粒为模板，以引物pMD18-F：ATCGTCGACCTGCAGGCATG(SEQ ID NO.:31)和pMD18-R：ATCTCTAGAGGATCCCCGGG(SEQ ID NO.:32)进行PCR扩增。将两次扩增产物各8.5μL混合，加入1μL DpnI，2μL 10×消化缓冲液，37℃温浴3h。将DpnI处理后产物10μL加入100μL DH5α感受态细胞中，冰上放置30min，42℃热激45s后，加入1mL LB液体培养基37℃振荡培养1h，涂布于Amp抗性LB固体培养，37℃倒置培养至单克隆长出。挑取5个单克隆在LB液体培养基中振荡培养，PCR检测阳性并测序确认后，提取质粒保存，命名为pKM-KlTDH3-1-DD。
以pKM-KlTDH3-1-DD为模板，以引物PF11:GATGCATTGATGGATGCCGAAGATGATTAAAGAGGTTGATGTAATTGATATTTTCCTGATAAAATTACTATTG(SEQ ID NO.:33)和PR11:AGCTGGTTGCTGATCGGATGTAGGTTCGCCAGATCCACCTCCTTCCACGTTTGTTGGTCTTGATCCACCTCCACCGTTCTTAGCAACAAGTTCGACCAAATCG(SEQ ID NO.:34)进行扩增；以K.lactis基因组DNA为模板，以引物PF12:GGCGAACCTACATCCGATCAGC(SEQ ID NO.:35)和PR12:TTAATCATCTTCGGCATCCATCAATGC(SEQ ID NO.:36)进行扩增。将两次扩增产物各8.5μL，1μL DpnI，2μL10×digestion buffer混合，37℃温浴3h。将DpnI处理后产物10μL加入100μL DH5α感受态细胞中，冰上放置30min，42℃热激45s后，加入1mL LB液体培养基37℃振荡培养1h，涂布于Amp抗性LB固体培养，37℃倒置培养至单克隆长出。挑取5个单克隆在LB液体培养基中振荡培养，PCR检测阳性并测序确认后，提取质粒保存，命名为pKM-KlTDH3-1-F-KleIF4G-DD(图8)。
以pKM-KlTDH3-1-F-KleIF4G-DD质粒为模板，以引物M13-F:GTAAAACGACGGCCAGT(SEQ ID NO.:37)和M13-R:CAGGAAACAGCTATGAC(SEQ ID NO.:38)进行扩增，得到线性供体DNA。
ii.对KlTDH3-2，以乳酸克鲁维酵母基因组DNA为模板，以引物PF13：GAGCTCGGTACCCGGGGATCCTCTAGAGATGAAGCTTTGATGACTACCGTTC(SEQ ID NO.:39)和PR13：GCCAAGCTTGCATGCCTGCAGGTCGACGATGTCTATTGTATCGGAAGAACTGTCA(SEQ ID NO.:40)进行PCR扩增；以pMD18质粒为模板，以引物pMD18-F：ATCGTCGACCTGCAGGCATG(SEQ ID NO.:41)和pMD18-R：ATCTCTAGAGGATCCCCGGG(SEQ ID NO.:42)进行PCR扩增。将两次扩增产物各8.5μL混合，加入1μL DpnI，2μL 10×消化缓冲液，37℃温浴3h。将DpnI处理后产物10μL加入100μL DH5α感受态细胞中，冰上放置30min，42℃热激45s后，加入1mL LB液体培养基37℃振荡培养1h，涂布于Amp抗性 LB固体培养，37℃倒置培养至单克隆长出。挑取5个单克隆在LB液体培养基中振荡培养，PCR检测阳性并测序确认后，提取质粒保存，命名为pKM-KlTDH3-2-DD。
以pKM-KlTDH3-2-DD为模板，以引物PF14:GATGCATTGATGGATGCCGAAGATGATTAAATTACTCTTTTAAGTTAACGAACGCTTTTGATGAG(SEQ ID NO.:43)和PR14:AGCTGGTTGCTGATCGGATGTAGGTTCGCCAGATCCACCTCCTTCCACGTTTGTTGGTCTTGATCCACCTCCACCAGCAACGTGCTCAACtAAgTCaACgACcCTTTCAGAGTAACCGTATTCGTTATCG(SEQ ID NO.:44)进行扩增；以乳酸克鲁维酵母DNA为模板，以引物PF15:GGCGAACCTACATCCGATCAGC(SEQ ID NO.:45)和PR15:TTAATCATCTTCGGCATCCATCAATGC(SEQ ID NO.:46)进行扩增。将两次扩增产物各8.5μL，1μL DpnI，2μL 10×消化缓冲液混合，37℃温浴3h。将DpnI处理后产物10μL加入100μL DH5α感受态细胞中，冰上放置30min，42℃热激45s后，加入1mL LB液体培养基37℃振荡培养1h，涂布于Amp抗性LB固体培养，37℃倒置培养至单克隆长出。挑取5个单克隆在LB液体培养基中振荡培养，PCR检测阳性并测序确认后，提取质粒保存，命名为pKM-KlTDH3-2-F-KleIF4G-DD(图9)。
以pKM-KlTDH3-2-F-KleIF4G-DD质粒为模板，以引物M13-F:GTAAAACGACGGCCAGT(SEQ ID NO.:47)和M13-R:CAGGAAACAGCTATGAC(SEQ ID NO.:48)进行扩增，得到线性供体DNA。
2.2.4乳酸克鲁维酵母转化及阳性鉴定
i.将乳酸克鲁维酵母菌液在YPD固体培养基上划线并挑取单克隆，于25mL2×YPD液体培养基中振荡培养过夜，取2mL菌液于50mL液体2×YPD培养基中继续振荡培养2-8h。20℃条件下3000g离心5min收集酵母细胞，加入500μL无菌水重悬，同样条件下离心收集细胞。配制感受态细胞溶液(5％v/v甘油，10％v/v DMSO)并将酵母细胞溶解于500μL该溶液中。分装50μL至1.5mL离心管中，-80℃保存。
将感受态细胞置于37℃融化15-30s，13000g离心2min并去除上清。配制转化缓冲液：PEG 3350(50％(w/v))260μL，LiAc(1.0M)36μL，carrier DNA(5.0mg/mL)20μL，Cas9/gRNA质粒15μL，供体DNA 10μL，加入无菌水至最终体积360μL。热激后，13000g离心30s去除上清。加入1mL YPD液体培养基，培养2-3h，吸取200μL涂布于固体YPD(200μg/mL G418)培养基，培养2-3天至单菌落出现。
ii.在乳酸克鲁维酵母转化后的平板上挑取10-20个单克隆，置于1mL YPD(200μg/mL G418)液体培养基中振荡培养过夜，以菌液为模板，以CRISPR Insertion Check引物KlTDH3-1-CICF1(KlTDH3-1序列内引物)：CTTCTACTGCTCCAATGTTCGTCGTT(SEQ ID NO.:49)和引物KlTDH3-2-CICF1(KlTDH3-2序列内引物)：TTAACGAAGACAAGTACAACGGTGA(SEQ ID NO.:50)分别与进行PCR扩增，分别与KleIF4G-CICR2(KleIF4G序列内引物)：TTCTCTTCGACAGCCTTCTTAGCAG(SEQ ID NO.:51)配对进行PCR，对KlTDH3-1和KlTDH3-2位点的KleIF4G插入进行检测，PCR结果阳性并经测序鉴定的菌株确定为阳性菌株。
2.3通过CRISPR-Cas9技术将KleIF4G与其互作蛋白融合
2.3.1 KlPab1序列检索及CRISPR gRNA序列确定
如前文所述，Pab1蛋白与eIF4G蛋白在翻译起始过程中存在相互作用。本发明通过CRISPR-Cas9基因编辑技术，将KlPab1和KleIF4G融合，促进两者的相互作用，以提高体外翻译效率。
基于Pab1序列，得到乳酸克鲁维酵母中KlPab1基因序列(位于染色体C的1553322...1555100)。在KlPab1基因终止密码子附近搜索PAM序列(NGG)，并确定gRNA序列。gRNA选择的原则为：GC含量适中，本发明的标准为GC含量为40％-60％；避免poly T结构的存在。最终，本发明确定的KlPab1gRNA序列为TGCTTACGAAAACTTCAAGA(SEQ ID NO.:52)，位于染色体C的1555058...1555077位点。
2.3.2 CRISPR-Cas9介导的将KleIF4G整合到KlPab1位点质粒构建
i.CRISPR质粒构建
使用引物PF16：TGCTTACGAAAACTTCAAGAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCG(SEQ ID NO.:53)，PR16：GCTCTAAAACTCTTGAAGTTTTCGTAAGCAAAAGTCCCATTCGCCACCCG(SEQ ID NO.:54)，以pCAS质粒为模板，进行PCR扩增。将扩增产物17μL混合，加入1μL DpnI，2μL 10×digestion buffer，37℃温浴3h。将DpnI处理后产物10μL加入100μL DH5α感受态细胞中，冰上放置30min，42℃热激45s后，加入1mL LB液体培养基37℃振荡培养1h，涂布于Kan抗性LB固体培养，37℃倒置培养至单克隆长出。挑取5个单克隆在LB液体培养基中振荡培养，PCR检测阳性并测序确认后，提取质粒保存，命名为pKM-CAS1.0-KlPab1(图10)。
ii.KlPab1-KleIF4G供体DNA质粒构建及扩增
为了便于线性供体DNA的保存及扩增，首先将供体DNA插入到pMD18质粒中，然后通过PCR扩增得到线性供体DNA序列。
以乳酸克鲁维酵母基因组DNA为模板，以引物PF17：GAGCTCGGTACCCGGGGATCCTCTAGAGATCCGGTAAGCCATTGTACGTTGCCAT(SEQ ID NO.:55)和PR17：GCCAAGCTTGCATGCCTGCAGGTCGACGATCAGTATACCGTCCATGTTGATGACT(SEQ ID NO.:56)进行PCR扩增；以pMD18质粒为模板，以引物pMD18-F：ATCGTCGACCTGCAGGCATG(SEQ ID NO.:57)和pMD18-R：ATCTCTAGAGGATCCCCGGG(SEQ ID NO.:58)进行PCR扩增。将两次扩增产物各8.5μL混合，加入1μL DpnI，2μL 10×消化缓冲液，37℃温浴3h。将DpnI处理后产物10μL加入100μL DH5α感受态细胞中，冰上放置30min，42℃热激45s后，加入1mL LB液体培养基37℃振荡培养1h，涂布于Amp抗性LB固体培养，37℃倒置培养至单克隆长出。挑取5个单克隆在LB液体培养基中振荡培养，PCR检测阳性并测序确认后，提取质粒保存，命名为pKM-KlPab1-DD。
以pKM-KlPab1-DD为模板，以引物PF18:GATGCATTGATGGATGCCGAAGATGATTAAACTTGATTTTTTGACCTTGATCTTCATCTTGTC(SEQID NO.:59)和PR18: CTTGAACTTCATCTTGAGTTGAACCTCCACCTCCAGATCCACCTCCACCAGCTTGAGCTTCTTGTTCtTTtTTaAAaTTcTCGTAAGCAGCTAAGGCTTC(SEQ ID NO.:60)进行扩增；以乳酸克鲁维酵母DNA为模板，以引物PF19:GTGGAGGTTCAACTCAAGATGAAGTTCAAGGTCCACATGCTGGTAAGTCTACTGTTGGTGGAGGTGGATCTGGCGAACCTACATCCGATCAGC(SEQ ID NO.:61)和PR19:TTAATCATCTTCGGCATCCATCAATGC(SEQ ID NO.:62)进行扩增。将两次扩增产物各8.5μL，1μL DpnI，2μL 10×digestion buffer混合，37℃温浴3h。将DpnI处理后产物10μL加入100μL DH5α感受态细胞中，冰上放置30min，42℃热激45s后，加入1mL LB液体培养基37℃振荡培养1h，涂布于Amp抗性LB固体培养，37℃倒置培养至单克隆长出。挑取5个单克隆在LB液体培养基中振荡培养，PCR检测阳性并测序确认后，提取质粒保存，命名为pKM-KlPab1-KleIF4G-DD(图11)。
以pKM-KlPab1-KleIF4G-DD质粒为模板，以引物M13-F:GTAAAACGACGGCCAGT(SEQ ID NO.:63)和M13-R:CAGGAAACAGCTATGAC(SEQ ID NO.:64)进行扩增，得到线性供体DNA。
2.3.3乳酸克鲁维酵母转化及阳性鉴定
i.将乳酸克鲁维酵母菌液在YPD固体培养基上划线并挑取单克隆，于25mL2×YPD液体培养基中振荡培养过夜，取2mL菌液于50mL液体2×YPD培养基中继续振荡培养2-8h。20℃条件下3000g离心5min收集酵母细胞，加入500μL无菌水重悬，同样条件下离心收集细胞。配制感受态细胞溶液(5％v/v甘油，10％v/v DMSO)并将酵母细胞溶解于500μL该溶液中。分装50μL至1.5mL离心管中，-80℃保存。
将感受态细胞置于37℃融化15-30s，13000g离心2min并去除上清。配制转化缓冲液：PEG3350(50％(w/v))260μL，LiAc(1.0M)36μL，carrier DNA(5.0m g/mL)20μL，Cas9/gRNA质粒15μL，供体DNA 10μL，加入无菌水至最终体积360μL。热激后，13000g离心30s去除上清。加入1mL YPD液体培养基，培养2-3h，吸取200μL涂布于固体YPD(200μg/mL G418)培养基，培养2-3天至单菌落出现。
ii.在乳酸克鲁维酵母转化后的平板上挑取10-20个单克隆，置于1mL YPD(200μg/mL G418)液体培养基中振荡培养过夜，以菌液为模板，以引物KlPAB1-CICF1(KlPAB1序列内引物)：TCTCCAGAAGAAGCTACCAAGGCTA(SEQ ID NO.:65)和引物KleIF4G-CICR2(KleIF4G序列内引物)：TTCTCTTCGACAGCCTTCTTAGCAG(SEQ ID NO.:66)进行PCR扩增，对KlPAB1位点KleIF4G插入进行PCR检测，PCR结果阳性并经测序鉴定的菌株，确定为阳性菌株。
实施例3改造菌株体外翻译活性测定
将基因改造后的乳酸克鲁维酵母菌株制备成体外蛋白质合成体系，并加入萤火虫荧光素酶(Firefly Luciferase,Fluc)基因DNA模板以测定改造菌株的蛋白翻译能力。将上述反应体系置于25-30℃的环境中，静置孵育约2-6h。反应结束后，在96孔白板或者384孔白板中加入等体积的Fluc底物荧光素(luciferin)，立即放置于Envision 2120多功能酶标仪(Perkin Elmer)，读 数，检测Fluc活性，相对光单位值(Relative Light Unit,RLU)作为活性单位。
在改造的结构当中，KleIF4G前插入启动子pKlPGK1的结构pKlPGK1::KleIF4G，及KleIF4G连接到KlPab1C端的结构KlPab1-KleIF4G，都表现出比野生型酵母菌株Y1140更强的体外蛋白质合成能力。其编码合成的Fluc蛋白放出的相对光单位值分别达到1.57×109和1.50×109，而野生型酵母菌株Y1140合成的Fluc蛋白的相对光单位值仅有4.11×108。这表明对KleIF4G的改造能够有效增强酵母体外蛋白质合成体系合成蛋白质的效率(图12)。
具体的效果如表1所示。
表1
NO. Data1 Data2 Data3 活性 稀释倍数 最终活性(RLU)
pKlPGK1_KleIF4G 33216610 28584890 32598650 31466717 50 1.57×109
pKlTEF1_KleIF4G 6685609 10189150 8594529 8489763、 50 4.24×108
pScPGK1_KleIF4G 4130719 8605461 4555399 5763860 50 2.88×108
pScTEF1_KleIF4G 8230202 6415045 7578242 7407830 50 3.70×108
KlTDH3_1_KleIF4G 788821 751243 941381 827148.3 50 4.13×107
KlTDH3_2_KleIF4G 8676941 6496592 7904461 7692665 50 3.85×108
KlPAB1_KleIF4G 22155330 33507550 34075530 29912803 50 1.50×109
Y1140 10925600 6729764 6997436 8217600 50 4.11×108
             
NC 707 965        
NC：表示negative control，阴性对照。
上述实验结果表明：通过对乳酸克鲁维酵母KleIF4G基因的相关改造，本发明的融合蛋白能够显著增强酵母体外蛋白质合成体系产生蛋白质的效率。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (15)

  1. 一种融合蛋白，其特征在于，所述融合蛋白具有式Ia或式Ib结构：
    S-A-B-C           (Ia)
    S-C-B-A           (Ib)；
    式中，
    A为PabI元件；
    B为无或连接肽；
    C为eIF4G元件；
    S为任选的信号肽；以及
    各“-”为肽键。
  2. 如权利要求1所述的融合蛋白，其特征在于，所述元件A衍生自酵母的PabI蛋白。
  3. 如权利要求1所述的融合蛋白，其特征在于，所述元件C衍生自酵母的eIF4G蛋白。
  4. 一种分离的多核苷酸，其特征在于，所述的多核苷酸编码权利要求1所述的融合蛋白。
  5. 一种载体，其特征在于，所述载体含有权利要求4所述的多核苷酸。
  6. 一种宿主细胞，其特征在于，所述宿主细胞含有权利要求5所述的载体或基因组中整合有权利要求4所述的多核苷酸。
  7. 一种用于表达外源蛋白的体外蛋白质合成体系，其特征在于，所述合成体系包括：
    (i)酵母体外蛋白质合成体系，所述的合成体系含有(a)酵母细胞提取物；(b)任选的聚乙二醇；(c)任选的外源蔗糖；和(d)任选的溶剂，所述溶剂为水或水性溶剂；以及
    (ii)权利要求1所述的融合蛋白。
  8. 如权利要求7所述的体外蛋白质合成体系，其特征在于，所述合成体系还包括：(iii)额外添加的eIF4G蛋白。
  9. 如权利要求8所述的体外蛋白质合成体系，其特征在于，所述eIF4G蛋白由组成型或诱导性启动子诱导表达。
  10. 一种生产权利要求1所述融合蛋白的方法，其特征在于，包括：
    (i)在适合表达的条件下，培养权利要求6所述的宿主细胞，从而表达出权利要求1所述的融合蛋白；和
    (ii)分离所述融合蛋白。
  11. 一种权利要求1所述的融合蛋白的用途，其特征在于，用于制备提高体外蛋白质合成体系的体外蛋白合成能力的制剂。
  12. 一种表达待表达的外源蛋白的方法，其特征在于，包括：
    (i)提供一酵母体外蛋白质合成体系，其中所述的合成体系中含有权利要求1所述的融合蛋白；和
    (ii)在适合表达蛋白的条件下，在所述外源蛋白的模板存在下，孵育所 述酵母体外蛋白质合成体系，从而表达所述的外源蛋白。
  13. 如权利要求12所述的方法，其特征在于，所述的融合蛋白为额外添加的。
  14. 如权利要求12所述的方法，其特征在于，所述步骤(ii)还包括步骤(iii)：检测外源蛋白活性的表达活性Q1，并且在步骤(ii)相同条件下孵育野生型酵母菌株，检测所述外源蛋白的活性Q2，如果Q1显著高于Q2，则表明外源蛋白的表达效率显著提高。
  15. 一种权利要求1所述融合蛋白的用途，其特征在于，用于制备一表达外源蛋白的体外蛋白质合成体系，所述体外蛋白质合成体系用于提高外源蛋白的表达效率。
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