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WO2025055767A1 - Procédé de purification de protéine - Google Patents

Procédé de purification de protéine Download PDF

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Publication number
WO2025055767A1
WO2025055767A1 PCT/CN2024/116250 CN2024116250W WO2025055767A1 WO 2025055767 A1 WO2025055767 A1 WO 2025055767A1 CN 2024116250 W CN2024116250 W CN 2024116250W WO 2025055767 A1 WO2025055767 A1 WO 2025055767A1
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Prior art keywords
protein
target protein
fusion protein
seq
amino acid
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Chinese (zh)
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林章凛
杨晓锋
黄源
张媛媛
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South China University of Technology SCUT
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South China University of Technology SCUT
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
    • C07K14/43595—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from coelenteratae, e.g. medusae
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    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/30—Extraction; Separation; Purification by precipitation
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    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/34—Extraction; Separation; Purification by filtration, ultrafiltration or reverse osmosis
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/475—Growth factors; Growth regulators
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/61—Growth hormone [GH], i.e. somatotropin
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/36—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against blood coagulation factors
    • C—CHEMISTRY; METALLURGY
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    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
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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
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
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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
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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
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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
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1085—Transferases (2.) transferring alkyl or aryl groups other than methyl groups (2.5)
    • C12N9/1088—Glutathione transferase (2.5.1.18)
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2477—Hemicellulases not provided in a preceding group
    • C12N9/248—Xylanases
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    • 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
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    • C12P21/00—Preparation of peptides or proteins
    • C12P21/06—Preparation of peptides or proteins produced by the hydrolysis of a peptide bond, e.g. hydrolysate products
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    • C12Y—ENZYMES
    • C12Y205/00—Transferases transferring alkyl or aryl groups, other than methyl groups (2.5)
    • C12Y205/01—Transferases transferring alkyl or aryl groups, other than methyl groups (2.5) transferring alkyl or aryl groups, other than methyl groups (2.5.1)
    • C12Y205/01018—Glutathione transferase (2.5.1.18)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/50—Fusion polypeptide containing protease site
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    • C12N2800/00—Nucleic acids vectors
    • C12N2800/10—Plasmid DNA
    • C12N2800/101—Plasmid DNA for bacteria
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    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/185—Escherichia
    • C12R2001/19—Escherichia coli

Definitions

  • the present invention relates to the field of genetic engineering, and more specifically, to a method for producing a target protein by using a fusion protein comprising a target protein, a self-aggregating peptide and a connecting peptide portion, and an application of the method in high-throughput production and purification.
  • Recombinant proteins are widely used in many fields including biomedicine and biocatalysis. In practical applications, there are usually high requirements for the activity and purity of proteins. However, the large-scale production and purification of many recombinant proteins are still challenging.
  • eukaryotic expression systems such as Chinese hamster ovary (CHO), yeast or insect cells can express most proteins, including proteins with complex structures, but these systems have long production cycles and high costs, or may cause immunogenicity due to incorrect modifications such as glycosylation; Escherichia coli is the most attractive expression system because of its clear genetic background and simple genetic manipulation.
  • the purity of the target protein must be high enough.
  • proteins for medical or pharmaceutical use usually require a purity of more than 99%; proteins used for X-ray crystal diffraction or biochemical analysis usually require a purity of more than 95%, and proteins used for antigen-antibody reaction and N-terminal sequencing analysis usually require a purity of more than 90%.
  • separating and purifying the target protein from the host cell protein background is the technical bottleneck for the preparation of recombinant proteins.
  • Traditional recombinant protein purification methods rely on column chromatography, including ion exchange chromatography, size exclusion chromatography, affinity chromatography, etc.
  • Npro elastin-like polypeptide
  • ELP elastin-like polypeptide
  • the multiple rounds of phase transitions required for the purification process of the ELP tag usually require 3-5 rounds of phase transitions to obtain a recombinant protein with a purity of 95%, which affects the activity of the protein and prolongs the purification cycle (Biomacromolecules 2007, 8, 1417-1424).
  • the molecular weight of the ELP tag is relatively large (generally more than 30kDa to ensure the purification effect), resulting in a low recovery rate of the target protein.
  • the cleavable self-aggregation tag method induces the fusion protein to form active protein aggregates through self-assembling peptides, and then releases the target protein into the supernatant by introducing the self-cleavage of the internal peptide, thereby achieving the expression and purification of the recombinant protein (Wu W. et al., Microbial Cell Factories, 2011, 10, 9; Xing L. et al., Microbial Cell Factories, 2011, 10, 42).
  • the existing cSAT technology can only obtain recombinant proteins with medium purity (60-80%), and two standard column purification steps are still required to obtain high-purity proteins with a purity of >95%.
  • the present invention provides a low-cost, simple, and efficient protein/polypeptide production and one-step purification method based on the cleavage tag of the self-aggregating peptide, in particular, a column-free one-step purification method, which can achieve low-cost, simple, and efficient production of high-purity target proteins. More importantly, the column-free one-step purification method can be used for high-throughput purification of proteins and high-throughput screening of proteins, especially in the screening of protein drugs.
  • the present invention provides a fusion protein, which comprises from N to C terminus (a) a self-aggregating peptide, a connecting peptide, a spacer and a target protein portion or (b) a target protein, a spacer, a connecting peptide and a self-aggregating peptide portion, wherein the target protein is connected to the connecting peptide via a spacer, and the spacer comprises a cleavage site.
  • the target protein is selected from insulin-like growth factors such as insulin-like growth factor-1 (IGF-1), growth hormones such as human growth hormone (hGH), nanobodies such as the anti-new crown nanobody NB21, xylanase, caplacizumab and glutathione sulfhydryltransferase (GST).
  • IGF-1 insulin-like growth factor-1
  • hGH human growth hormone
  • nanobodies such as the anti-new crown nanobody NB21, xylanase, caplacizumab and glutathione sulfhydryltransferase (GST).
  • the connecting peptide comprises an amino acid sequence selected from elastin-like polypeptide (ELP) and protease propeptide (ProP), in particular selected from Deinococcus gobiensis protease propeptide PEP or a homologue thereof.
  • ELP elastin-like polypeptide
  • ProP protease propeptide
  • the self-aggregating peptide comprises a self-assembling peptide, including a surfactant-like short peptide, such as L6KD (SEQ ID NO: 5), or one or more tandemly repeated amphipathic ⁇ -folded short peptides, such as ELK16 (SEQ ID NO: 6).
  • the self-aggregating peptide comprises a salt concentration-responsive self-aggregating peptide, including an ⁇ -helical MpA or a ⁇ -folded short peptide EIK8 (SEQ ID NO: 58) and EFR8 (SEQ ID NO: 59).
  • an isolated polynucleotide which comprises a nucleotide sequence encoding the fusion protein of the first aspect or a complementary sequence thereof.
  • an isolated polynucleotide which comprises a nucleotide sequence encoding a self-aggregating peptide, a connecting peptide and a spacer, and a multiple cloning site, wherein the multiple cloning site is used to clone a nucleotide sequence for expressing a target protein, whereby the polynucleotide can produce the fusion protein of the first aspect after expression.
  • a construct such as an expression construct, comprising the polynucleotide of the second aspect or the third aspect.
  • a host cell which comprises the polynucleotide of the second aspect or is transformed by the construct of the fourth aspect, wherein the host cell is capable of expressing the fusion protein.
  • the host cell is selected from prokaryotes, yeast and higher eukaryotic cells, wherein the prokaryotes include bacteria of the genera Escherichia, Bacillus, Salmonella, and Pseudomonas and Streptomyces, preferably Escherichia coli.
  • a method for producing and purifying a target protein is provided:
  • step (a) of the method comprises: culturing the host cell of the fifth aspect to express the fusion protein.
  • step (b) of the method comprises: lysing the host cell, and then obtaining an insoluble portion containing the fusion protein by aggregation of the self-aggregating peptide, for example, inducing the fusion protein to form an insoluble portion under a certain salt concentration.
  • a method for high-throughput production and purification of multiple target proteins comprising the following steps:
  • Figure 1 shows the expression and purification strategy of the target protein based on self-aggregating peptide (SAP) and linker peptide (linker) and the structure diagram of the expression vector used.
  • SAP self-aggregating peptide
  • linker linker peptide
  • FIG. 2 shows the results of SDS-PAGE analysis of the expression and purification of insulin-like growth factor-1 (IGF-1), human growth hormone (hGH), anti-new crown nanoantibody NB21, xylanase, capecitabine, and glutathione sulfhydryltransferase (GST) fusion proteins.
  • IGF-1 insulin-like growth factor-1
  • hGH human growth hormone
  • anti-new crown nanoantibody NB21 anti-new crown nanoantibody NB21
  • xylanase xylanase
  • capecitabine glutathione sulfhydryltransferase
  • A IGF-1 expression and purification SDS-PAGE quantitative analysis results
  • B hGH expression and purification SDS-PAGE quantitative analysis results
  • C NB21 expression and purification SDS-PAGE quantitative analysis results
  • D xylanase expression and purification SDS-PAGE quantitative analysis results
  • E capecitabine expression and purification SDS-PAGE quantitative analysis results
  • F GST expression and purification SDS-PAGE quantitative analysis results.
  • Figure 3 shows the high-throughput expression and purification strategy of the target protein and the SDS-PAGE analysis results.
  • A Schematic diagram of the expression and purification strategy;
  • B SDS-PAGE analysis results of the target protein,
  • Xyl xylanase,
  • Cap capecitabine.
  • Figure 4 shows the results of fermentation production, expression and purification of capecitabine.
  • A OD and glucose control process of fermentation production process;
  • B SDS-PAGE analysis results of capecitabine expression and purification.
  • Figure 5 shows a protein purification method based on salt concentration responsive self-aggregating peptides and linker peptides and a structure diagram of the expression vector used.
  • A expression and purification strategy;
  • B SAP-Linker-Mtu-POI vector structure diagram.
  • Figure 6 shows the expression and salt purification of human growth hormone (hGH, SEQ ID NO: 10) fusion protein mediated by 0.7M (NH 4 ) 2 SO 4 , as well as the SDS-PAGE results of quantitative analysis.
  • the self-aggregating peptides are A: MpA (SEQ ID NO: 7); B: MpA-PEP; C: EIK8 (SEQ ID NO: 58); D: EIK8-PEP; E: EFR8 (SEQ ID NO: 59); F: EFR8-PEP.
  • Figure 7 shows the expression and purification strategy of the target protein based on self-aggregating peptides, linker peptides and intein (Mxe) and the structure diagram of the expression vector used.
  • A expression and purification strategy;
  • B POI-Mxe-Linker-ELK16 vector structure diagram.
  • Figure 8 shows the SDS-PAGE analysis results of the intein peptide Mxe (SEQ ID NO: 4) used for the expression and purification of xylanase fusion protein.
  • Figure 9 shows the SDS-PAGE analysis results of the linker peptides HD (SEQ ID NO: 44) and HFBII (SEQ ID NO: 45) for fusion protein expression and purification.
  • A SDS-PAGE analysis results of insulin-like growth factor-1 (IGF-1) expression and purification;
  • B SDS-PAGE analysis results of capecitabine expression and purification.
  • IGF-1 insulin-like growth factor-1
  • Figure 10 shows the SDS-PAGE analysis results of the linker peptides P1-P10 for fusion protein expression and purification.
  • A SDS-PAGE analysis results of insulin-like growth factor-1 (IGF-1) expression and purification;
  • B SDS-PAGE analysis results of capecitabine expression and purification.
  • IGF-1 insulin-like growth factor-1
  • Figure 11 shows the SDS-PAGE analysis results of the linker peptide PEP truncated forms N1 (SEQ ID NO: 56) and N2 (SEQ ID NO: 57) for fusion protein expression and purification.
  • A SDS-PAGE analysis results of insulin-like growth factor-1 (IGF-1) expression and purification
  • B SDS-PAGE analysis results of capecitabine expression and purification
  • C SDS-PAGE analysis results of xylanase expression and purification.
  • IGF-1 insulin-like growth factor-1
  • the present invention is not limited to the specific methods, schemes, reagents, etc. described herein, because these can change.
  • the terms used herein are only used for the purpose of describing specific embodiments rather than for limiting the scope of the present invention. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art.
  • the protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, immunology-related terms and laboratory operation steps used herein are terms and routine steps widely used in the corresponding fields.
  • the experimental methods not specifically described in the present invention are all carried out according to the specific methods in the book "Molecular Cloning Experiment Guide” (4th Edition) J. Sambrook, or according to the relevant product instructions.
  • the biological reagents used in the present invention, without special instructions, can all be obtained from commercial sources. Those skilled in the art can make various changes, modifications and substitutions without departing from the spirit of the present invention.
  • the term “and/or” encompasses all combinations of items connected by the term, and each combination should be considered to have been listed separately herein.
  • “A and/or B” encompasses “A,” “A and B,” and “B.”
  • “A, B, and/or C” encompasses “A,” “B,” “C,” “A and B,” “A and C,” “B and C,” and “A and B and C.”
  • nucleic acid sequences are referred to herein in a 5' to 3' direction from left to right; amino acid sequences are referred to herein in a 5' to 3' direction from left (upstream) to right (downstream).
  • nucleotide sequence As used herein, the terms “nucleotide sequence”, “polynucleotide”, “nucleic acid” and “nucleic acid sequence” are used interchangeably and refer to a macromolecule composed of multiple nucleotides connected by 3'-5'-phosphodiester bonds, wherein the nucleotides include ribonucleotides and deoxyribonucleotides.
  • the sequence of the polynucleotide of the present invention can be codon-optimized for different host cells (such as Escherichia coli) to improve the expression of the fusion protein. Methods for codon optimization are known in the art.
  • peptide As used herein, the terms “peptide,” “polypeptide,” and “protein” are used interchangeably, and “enzyme” and “antibody” are both considered “proteins,” and are defined as biological molecules composed of amino acid residues linked by peptide bonds.
  • self-aggregation refers to a property of a polypeptide, that is, polypeptide monomers assemble into polymers under the induction of certain physical and/or chemical conditions.
  • the present invention provides a fusion protein, which comprises from N to C terminus (a) a self-aggregating peptide, a connecting peptide, a spacer and a target protein portion or (b) a target protein, a spacer, a connecting peptide and a self-aggregating peptide portion, wherein the target protein portion is connected to the connecting peptide portion via a spacer, and wherein the spacer portion comprises a cleavage site.
  • target protein refers to any polypeptide or protein that can be purified by the method of the present invention, non-limiting examples of which include enzymes, hormones, immunoglobulin chains, therapeutic polypeptides such as anti-cancer polypeptides, diagnostic polypeptides, or polypeptides that can be used for immunization purposes or biologically active fragments thereof, etc.
  • the target polypeptide can be from any source, including microbial-derived polypeptides, mammalian-derived polypeptides, and artificial proteins (e.g., fusion proteins or mutant proteins), etc.
  • the target protein is selected from a therapeutic molecule, a detectable molecule or a targeting molecule.
  • the therapeutic molecule includes, but is not limited to, nucleic acid drugs, protein drugs (including therapeutic polypeptides, therapeutic antibodies, etc.), etc.
  • Exemplary therapeutic molecules include, but are not limited to, toxins, immunomodulators, antagonists, apoptosis inducers, hormones, radiopharmaceuticals, anti-angiogenic agents, gene drug cytokines, chemokines, prodrugs, chemotherapeutic drugs, etc.
  • the detectable molecules include, but are not limited to, fluorescent proteins, enzymes, labels, such as red fluorescent protein (RFP), glutathione sulfhydryl transferase, xylanase, etc.
  • the targeting molecules include, but are not limited to, targeting antibodies, specific receptor ligands, etc.
  • the targeting molecules can be antibodies that specifically target tumor antigens.
  • the target protein of the present invention is soluble under certain conditions, for example, it is soluble under conditions where cleavage is induced at the cleavage site, or after cleavage, it becomes soluble by changing solution conditions such as pH, salt concentration, etc., while the remaining part of the fusion protein after cleavage remains an insoluble aggregate due to the aggregation effect of the self-aggregating peptide.
  • the amino acid sequence of the target protein of the present invention contains at least two cysteines, which can form an intramolecular disulfide bond, such as one intramolecular disulfide bond, two intramolecular disulfide bonds or more intramolecular disulfide bonds.
  • the target protein of the present invention is a nanobody.
  • the target protein of the present invention is a nanobody.
  • the target protein is an enzyme.
  • the target protein can be 20-500 amino acids in length, for example, about 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500 amino acids in length.
  • the target protein or polypeptide is selected from insulin-like growth factors such as insulin-like growth factor-1 (IGF-1), growth hormones such as human growth hormone (hGH), nanobodies such as the anti-new crown nanobody NB21, xylanase, capecitabine and glutathione sulfhydryltransferase (GST).
  • IGF-1 insulin-like growth factor-1
  • hGH human growth hormone
  • nanobodies such as the anti-new crown nanobody NB21, xylanase, capecitabine and glutathione sulfhydryltransferase (GST).
  • the target protein comprises an amino acid sequence selected from any one of SEQ ID NOs: 10-11 or an amino acid sequence encoded by a nucleotide sequence selected from any one of SEQ ID NOs: 38-43.
  • human growth hormone contains 191 amino acids and has two intramolecular disulfide bonds. Human growth hormone can act on almost all tissues and cells, such as maintaining positive nitrogen balance and initiating protein synthesis in muscle cells, increasing amino acid uptake in skeletal muscle, regulating longitudinal growth of bones, protecting myocardial cells and lymphocytes from apoptosis, etc. (Levarski et al., Protein Expression and Purification, 2014, 100: 40-47; Zamani et al., Molecular Biotechnology, 2015, 57: 241-250). Therefore, human growth hormone has been widely used in the treatment of many diseases, and there are 11 growth hormone indications approved by the US FDA. Currently, the global sales of growth hormone exceed US$3 billion.
  • insulin-like growth factor contains 70 amino acids and three intramolecular disulfide bonds. It is a group of polypeptide substances with growth-promoting effects. Its secretory cells are widely distributed in human liver, kidney, lung, heart, brain and intestine.
  • the IGF family includes IGF-I and IGF-II. The production of IGF-I is more dependent on GH, and its growth-promoting effect is strong. It is an important growth factor in childhood.
  • Insulin-like growth factor has been identified as a key factor in many biological systems, including regulating cell development and DNA synthesis, improving blood circulation and participating in cell proliferation and differentiation. IGF-I has also been used in oxidative folding models, which has promoted the study of disulfide bond formation and rearrangement processes (Jonathan S. et al., Science, 253(5026):1386-1393).
  • nanobodies are single-domain antibodies (VHH) containing only one heavy chain variable domain. Compared with general chemical drugs and common antibody drugs IgG, they have many advantages such as high stability, strong penetration, simple structure, and low immunogenicity. They are antiviral drugs with great potential. Experiments have shown that a class of nanobodies can bind to the spike protein of SARS-CoV-2 with high affinity, preventing cells from being infected by the virus (Schoof et al., Science, 2020, 370(6523):1473-1479).
  • EMA approved Sanofi's nano-antibody drug Cablivi for the treatment of acquired thrombotic thrombocytopenic purpura (aTTP) in adults.
  • Acquired or immune-mediated thrombotic thrombocytopenic purpura is a rare thrombotic microangiopathy (TMA) characterized by thrombocytopenia and hemolytic anemia.
  • TTA thrombotic microangiopathy
  • the pathogenesis of acquired TTP is that the patient's own antibodies inhibit the activity of von Willebrand factor (vWF) cleavage protease ADAMTS13, causing vWF to adhere to platelets and form microvascular thrombi.
  • Capacilizumab contains 259 amino acids, consisting of AAA amino acid linkers connecting two 128 amino acid anti-vWF humanized nano-antibodies, which are usually expressed by yeast fermentation.
  • xylanase is a commonly used industrial enzyme that helps hydrolyze D-xylan into D-xylose.
  • Hemicellulose, composed of xylan, is the main component of most plant cell walls. Xylanase will hydrolyze this component and can therefore be used in a variety of applications, including viscosity reduction of wheat, barley, corn cobs and other difficult to digest foods.
  • GST glutathione S-transferase
  • self-aggregating peptide refers to a polypeptide that is fused to a protein portion and can mediate the formation of insoluble active aggregates of the fusion protein in the cell after expression in a host cell.
  • amphiphilic polypeptides are known in the art. Due to the interaction between their hydrophilic and hydrophobic regions and other driving forces, they can spontaneously form specific self-assembly structures under appropriate conditions (Zhao Q et al., Microbial Cell Factories, 2016, 15: 136).
  • the self-aggregating peptides used in the present invention can be selected from amphiphilic ⁇ -folded short peptides, amphiphilic ⁇ -helical short peptides and surfactant-like short peptides.
  • Surfactant-like short peptides are composed of hydrophobic amino acid tails and hydrophilic amino acid heads, and can form assembly structures such as micelles and nanotubes in aqueous solution.
  • Surfactant-like short peptides suitable for use as the self-aggregating peptides of the present invention include L6KD, whose amino acid sequence is shown in SEQ ID NO: 5.
  • surfactant-like peptides with similar structures such as L6KD, L6K2 (SEQ ID NO: 60), L6D2 (SEQ ID NO: 61), etc.
  • L6KD L6K2
  • L6D2 SEQ ID NO: 61
  • have similar activities and can promote the formation of insoluble active aggregates of fusion proteins in cells Zhou et al., Microbial Cell Factories, 2012, 11, 10).
  • Amphiphilic ⁇ -folded short peptides refer to short peptides composed of 4-30 amino acid residues, in which hydrophobic amino acids and charged hydrophilic amino acids are arranged alternately to form a ⁇ -folded structure. In this structure, the hydrophobic amino acid residues are located on one side, while the alternating positively and negatively charged hydrophilic amino acid residues are located on the other side. These short peptides can form self-assembly structures through hydrophobic interactions, electrostatic interactions and hydrogen bonding. Generally speaking, the longer the length of the amphiphilic ⁇ -folded structure or the stronger the hydrophobicity, the easier it is for self-assembly to occur, and the higher the mechanical strength of the self-aggregate formed. Specific examples of amphiphilic ⁇ -folded short peptides suitable for the present invention include EIK8 (SEQ ID NO: 58), EFR8 (SEQ ID NO: 59), ELK16 (SEQ ID NO: 6), etc.
  • MpA is an amphipathic ⁇ -helical short peptide, which has a separation of hydrophilic and hydrophobic regions.
  • the state of MpA is regulated by salt concentration. In a saline solution, under the action of salt ion-induced hydrophobic interactions and other driving forces, MpA can spontaneously form a specific self-aggregation structure. Within a certain range, as the salt ion concentration increases, the strength of the aggregate increases and the volume increases (Daniel E.W. et al., Proceedings of the National Academy of Sciences, 2005, 102: 12656-12661; Lin Zhanglin et al., WO2022253266A1).
  • the salt is selected from monovalent metal salts such as potassium salts or sodium salts, etc., divalent metal salts such as magnesium salts, calcium salts, manganese salts or copper salts, etc., or ammonium salts, preferably ammonium salts, potassium salts or sodium salts.
  • the anion of the salt is selected from sulfate, hydrogen phosphate, acetate, halide ions such as fluoride ions, chloride ions, bromide ions or iodide ions, etc., nitrate, perchlorate, or thiocyanate ions, preferably sulfate, hydrogen phosphate, chloride ions or acetate.
  • the salt is selected from sodium chloride, sodium sulfate, sodium nitrate, disodium hydrogen phosphate, sodium dihydrogen phosphate, sodium carbonate, potassium chloride, potassium sulfate, potassium nitrate, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, potassium carbonate, ammonium nitrate, ammonium sulfate or ammonium chloride, preferably sodium chloride, sodium sulfate or ammonium sulfate.
  • spacer refers to a polypeptide consisting of an amino acid sequence of a certain length, which includes a sequence for achieving cleavage and connects the parts of the fusion protein without affecting the structure and activity of each part.
  • cleavage sequences include enzymatic cleavage sequences that can be used for protease recognition, or intein sequences for self-cleavage, etc.
  • the polypeptide produced by recombinant production needs to have a consistent sequence with the target polypeptide, that is, there are no additional amino acid residues at both ends.
  • the spacer in the fusion protein of the present invention also includes a cleavage site.
  • the target polypeptide can be released from the fusion protein.
  • the cleavage site used to release the soluble target protein part from the insoluble part (precipitation) in the present invention can be selected from a temperature-dependent cleavage site, a pH-dependent cleavage site, an ion-dependent cleavage site, an enzyme cleavage site or a self-cleavage site, or any other known cleavage site.
  • the cleavage site is a self-cleavage site, for example, an amino acid sequence comprising an intein that can cleave by itself.
  • the intein-based cleavage method does not require external enzymes or the use of harmful substances such as hydrogen bromide. It can simply induce cleavage by changing the buffer environment in which the aggregate is located (Wood et al., Nature Biotechnology, 1999, 17: 889-892).
  • a variety of inteins with self-cleavage properties are known in the field, such as a series of inteins with different self-cleavage properties developed by NEB.
  • the cleavage site is a pH-dependent cleavage site.
  • the spacer is connected to the N-terminus or C-terminus of the target polypeptide portion. It should be understood that those skilled in the art can select a suitable spacer as needed and select a suitable connection position for the spacer.
  • the length of the spacer is 10-250 amino acid residues, such as 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240 or 250 amino acid residues.
  • the spacer consists of a cleavage site described herein, such as a self-cleavage site.
  • the spacer is directly connected to the target polypeptide portion and/or the salt concentration responsive self-aggregating peptide portion.
  • the spacer further comprises an amino acid linker at its N-terminus and/or C-terminus.
  • the spacer is connected to the target polypeptide portion and/or the salt concentration responsive self-aggregating peptide portion through an amino acid linker.
  • the cleavage site is located at the C-terminus of the spacer, and the cleavage site is adjacent to the N-terminus of the target polypeptide portion.
  • the cleavage site is located at the N-terminus of the spacer, and the cleavage site is adjacent to the C-terminus of the target polypeptide portion.
  • the spacer is connected to the target polypeptide portion through the cleavage site. In some embodiments, the spacer is directly connected to the N-terminus or C-terminus of the target polypeptide portion through the cleavage site.
  • the spacer comprises an intein, which comprises a self-cleavage site.
  • an intein is a special sequence polypeptide with protease activity, which can be cleaved at a specific amino acid residue at a designed site after inducing its protease activity, so that the target polypeptide is separated from the fusion polypeptide and released into the solution, and a high-purity target polypeptide can be obtained.
  • the intein-based cleavage method does not require the addition of an enzyme or the use of harmful substances such as hydrogen bromide used in chemical methods, but only requires changing the buffer environment in which the aggregate is located to simply induce cleavage (Wu et al., 1998; TELENTI et al., 1997).
  • a variety of self-cleaving inteins are known in the art, such as a series of inteins with different self-cleavage properties from NEB.
  • the intein is selected from Mxe GyrA, Ssp DnaB or Mtu ⁇ I-CM.
  • the "Mtu ⁇ I-CM” is derived from the wild-type intein of Mtu recA, which is obtained by deleting the nuclease domain of the Mtu recA super-large intein, retaining 110 amino acids at the N-terminus and 58 amino acids at the C-terminus, obtaining a very small intein, and then introducing four mutations C1A, V67L, D24G, and D422G (Wood et al., Nature Biotechnology, 1999, 17: 889-892).
  • the Mtu ⁇ I-CM comprises the amino acid sequence shown in SEQ ID NO: 3.
  • the nucleotide sequence encoding the Mtu ⁇ I-CM has the nucleotide sequence shown in SEQ ID NO: 36.
  • the Mtu ⁇ I-CM is connected to the C-terminus of the target polypeptide portion.
  • the intein Mtu ⁇ I-CM can induce self-cleavage of the intein at its carboxyl terminus in a buffer system at pH 5.5-6.8, such as 6.2.
  • the spacer is Mtu ⁇ I-CM or a mutant thereof.
  • the intein is Mxe GyrA, which has a sequence shown in SEQ ID NO:4.
  • Mxe GyrA is connected to the C-terminus of the target protein.
  • DTT dithiothreitol
  • self-cleavage of Mxe GyrA at its amino terminus can be induced.
  • the technician can determine the concentration and reaction time of DTT.
  • DTT can also be removed in subsequent operations.
  • the spacer is Mxe GyrA or a mutant thereof.
  • connector peptide is a peptide segment located between the self-aggregating peptide and the spacer. As found by the present inventors, due to the presence of the connector peptide, after the cleavage site is cut and the target protein is released, the remaining portion (comprising the self-aggregating peptide, the connector peptide and the optional spacer or a portion thereof) maintains a tendency or tendency to aggregate, reducing the disaggregation of the self-aggregating peptide, so that the purity, cutting efficiency and/or yield of the target protein can be improved.
  • the connecting peptide comprises an amino acid sequence selected from an elastin-like polypeptide (ELP) or a protease propeptide (ProP), in particular selected from the gobi anisoglobulin protease propeptide PEP or a homologue thereof (e.g., at least 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to the PEP).
  • ELP elastin-like polypeptide
  • ProP protease propeptide
  • the term "identity” is used to refer to the match between two polypeptides.
  • the "percent identity” between two sequences is determined by the number of matching positions shared by the two sequences divided by the number of positions compared ⁇ 100.
  • the percent identity can be determined by a computer program such as the Needleman and Wunsch (J MoI Biol. 48:444-453 (1970)) algorithm that has been integrated into the GAP program of the GCG software package (available at www.gcg.com) or the algorithm of E. Meyers and W. Miller (Comput. Appl. Biosci., 4: 11-17 (1988)).
  • ELP elastin-like polypeptide
  • ELP is a synthetic polypeptide polymer derived from natural elastin.
  • ELP can undergo a reversible phase transition process, called inverse temperature transition. When the temperature is below the transition temperature (Tt), ELP is highly soluble in the liquid phase. However, when the temperature is higher than Tt, the hydrophilic ELP will dehydrate and agglutinate. ELP fusion protein also has this characteristic.
  • the ELP tag can be used to purify the ELP fusion protein conveniently and quickly, and the purification process does not require chromatographic separation, thereby avoiding expensive affinity resins and purification equipment, and the concentration and replacement of the ELP fusion protein buffer also become very simple.
  • the aggregation of the ELP fusion protein is triggered by heating or increasing the salt ion concentration, and the ELP fusion protein is separated from the expression host protein by centrifugation.
  • the precipitated ELP fusion protein is re-dissolved with a low-salt and low-temperature solution, and then centrifuged to remove the insoluble protein to obtain the ELP fusion protein.
  • a purer ELP fusion protein can be obtained by repeating the steps of triggering precipitation, centrifugation and re-dissolution. This purification process is called Inverse Transition Cycling (ITC).
  • ITC Inverse Transition Cycling
  • the inventors have found that the above ELP sequence can improve the solubility of the protein when expressed in fusion. In particular, it is likely that the repulsive force increases the distance between molecules, and an increase in the yield and recovery rate of the target protein is observed.
  • the propeptide of a protease is the part removed during the activation of a protease into a mature protease.
  • the propeptide can not only inhibit the activity of the protease, but also play the role of a molecular chaperone to promote the correct folding of the protein.
  • the propeptide is widely present in the proteases of prokaryotes, eukaryotes and viruses. It was originally discovered and studied in the in vitro renaturation process of a secretory protease, subtilisin E (Yabuta et al., Journal of Biological Chemistry, 2001, 276 (48): 44427-44434).
  • subtilisin E The propeptide is located at the N-terminus of the polypeptide chain and is essential in the folding process of subtilisin E.
  • subtilisin E becomes an unstructured polypeptide chain.
  • the denaturant is slowly removed, the mature peptide cannot fold correctly and still does not have a higher-order structure, indicating that the mature peptide of subtilisin E does not have all the information required for its own folding.
  • subtilisin E when the mature peptide of subtilisin E is co-expressed with a peptide segment at the N-terminus, the mature peptide can be folded into a protein with a spatial structure through renaturation, so this N-terminal peptide segment is named IMC (intramolecular chaperone). (Jia Y.
  • subtilisin E not only assists its own protease folding, but also helps its highly homologous and three-dimensionally similar subtilisin BPN and subtilisin Carlsberg to refold (Ujwal Shinde et al., PNAS, 1993, 90: 6924-6928).
  • some mutants lost the function of assisting protease folding, but their role as inhibitors was more significant.
  • it can spontaneously form a secondary structure without the presence of protease (Cynthia et al., Journal of Biological Chemistry, 2001, 305, 1: 151-165).
  • the chaperone activity and protease inhibitor activity of the propeptide may be separated.
  • propeptides are almost always less conservative than their homologous catalytic domains and contain a large number of charged amino acids.
  • the propeptide has two conservative domains N1, N2 (motifs N1 and N2), including hydrophobic amino acid residues Val 12 , Phe 14 , Ile 30 , Val 37 , Leu 51 , Val 56, Leu 59 , Val 65 and Val 68 (Cynthia et al., Journal of Biological Chemistry, 2001, 305, 1 : 151-165).
  • the two sides of the conservative domain are charged residues, which are presumably able to initiate the folding of the protease through "hydrophobic collapse".
  • propeptide PEP Peptide for Expression-Promoting
  • S8 protein family protease of Deinococcus gobiensis can enable the soluble expression of four milk proteins that were originally not expressed, including ⁇ S2-casein, ⁇ -casein, ⁇ -lactalbumin and bovine serum albumin, in Escherichia coli.
  • the connecting peptide comprises a (VPGXG)n motif, wherein n is an integer, preferably 10-20, such as 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20, more preferably 15. It is known in the art that the strength of the aggregation can be adjusted by adjusting the hydrophilicity of the peptide sequence (e.g., using AGVE and adjusting its ratio), which is within the knowledge of those skilled in the art.
  • the X is independently selected from A, G, V and E. In one embodiment, the X is independently selected from A, G and V. In one embodiment, the X is independently selected from V and E, preferably V:E is about 4:1.
  • the connecting peptide comprises 30-100 amino acid residues, e.g., 30-95, 30-90, 30-85, 30-80, 30-75, 30-70, 30-65, 30-60, 30-55, 30-50, 30-40, 40-95, 40-90, 40-85, 40-80, 40-75, 40-70, 40-65, 40-60, 40-55, 40-50, 50-95, 50-90, 50-85, 50-80, 50-75, 50-70, 50-65, 50-60, 60-95, 60-90, 60-85, 60-80, 60-75, 60-70, 60-65, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 amino acid residues.
  • the connecting peptide comprises an amino acid sequence selected from any one of SEQ ID NOs: 1-2 and 46-55. In some embodiments, the connecting peptide comprises an amino acid sequence as shown in any one of SEQ ID NOs: 52-55, or an amino acid sequence having at least 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity with SEQ ID NO: 2.
  • the connecting peptide is from Deinococcus gobiensis, preferably the propeptide of a protease of the S8 protein family.
  • the PEP homolog is selected from P1-P10, wherein P1 (SEQ ID NO:46) is derived from yeast protease propeptide, P2 (SEQ ID NO:47) is derived from cucumber protease propeptide, P3 (SEQ ID NO:48) is derived from cyanobacterial protease propeptide, P4 (SEQ ID NO:49) is derived from subtilisin propeptide, P5 (SEQ ID NO:50) is derived from human PCSK9 protease propeptide, and P6-P10 (SEQ ID NO:51-55) are derived from bacterial protease propeptides.
  • an amino acid linker may be included between a self-aggregating peptide and a connecting peptide, a connecting peptide and a spacer, and/or a spacer and a target protein. In some embodiments, an amino acid linker is included between a self-aggregating peptide and a connecting peptide. In some embodiments, an amino acid linker is included between a connecting peptide and a spacer. In some embodiments, an amino acid linker is included between a spacer and a target protein.
  • amino acid linker refers to a polypeptide of a certain length composed of amino acids with low hydrophobicity and low charge effect, which can fully unfold the connected parts and fold into their respective natural conformations without interfering with each other when fusion protein.
  • Commonly used amino acid linker types include flexible GS type amino acid linkers, rich in glycine (G) and serine (S), and rigid PT type amino acid linkers, rich in proline (P) and threonine (T).
  • the amino acid linker is selected from a GS type amino acid linker and a PT type amino acid linker.
  • the length of the amino acid linker may be about 3-30 amino acids, such as about 3-25, 3-20, 3-15, 3-10, 3-5, 5-25, 5-20, 5-15, 5-10, 10-25, 10-25, 10-20, 10-15, 15-25, 15-20, 20-25 amino acids, such as about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 amino acids.
  • the amino acid sequence of the GS type amino acid linker used in the present invention is shown in SEQ ID NO: 8.
  • the amino acid sequence of the PT type amino acid linker used in the present invention is shown in SEQ ID NO: 9.
  • PEP or its homologous polypeptide sequence can promote the expression and aggregation of fusion proteins, and solve the problem of self-aggregation tag depolymerization that is common in previous purification methods based on cleavable self-aggregation tags, thereby increasing the purity of the target protein to 95%, 96%, 97% or even above 98%.
  • This is a new method that truly realizes the coupling of high-activity expression and high-purity purification of recombinant proteins. This method can achieve low-cost, simple and efficient production of high-purity and high-activity recombinant proteins. More importantly, this one-step method for achieving high-purity protein preparation can be used for high-throughput purification of proteins and high-throughput screening of proteins, especially in the screening of protein drugs.
  • the preferred screening method is surface display technology, such as phage surface display, which can quickly screen out potential target sequences within a few weeks, with a library capacity of >10 6 CFU (Lu et al., Journal of Biomedical Science, 2020, 27, 1).
  • the protein sequences obtained by surface display are likely to have false positives or insufficient affinity.
  • the current gold standard for antibody detection is still to detect the affinity between antigen and antibody through SPR or BLI, and both detection technologies require the purity of the test object to be >90%.
  • the purification technology provided by the present invention can not only obtain high-purity proteins in one step, but also the purification process is easy to standardize, without the need to optimize the purification conditions for each target protein separately, nor the need to use expensive reagents or materials, and is very suitable for development into high-throughput screening technology.
  • it can be used for the screening of nano-antibody drugs or antimicrobial peptides, or combined with AI-assisted protein design for protein evolution engineering.
  • the present invention provides an isolated polynucleotide comprising a nucleotide sequence encoding the fusion protein of the first aspect or a complementary sequence thereof.
  • the present invention provides an isolated polynucleotide comprising a nucleotide sequence encoding a self-aggregating peptide, a connecting peptide and a spacer, and a multiple cloning site, wherein the multiple cloning site is used to clone a nucleotide sequence for expressing a target protein, whereby the polynucleotide, after expression, can produce the fusion protein described in the first aspect.
  • expression generally refers to the process of producing polypeptides by transcription and translation of polynucleotides.
  • expression can be understood as “heterologous expression”, that is, it refers to expressing a polypeptide encoded by a heterologous nucleic acid in a host cell or in vitro.
  • the present invention provides a construct, such as an expression construct, comprising the polynucleotide of the second aspect or the third aspect.
  • expression construct refers to a vector such as a recombinant vector suitable for expressing a nucleotide sequence of interest in an organism. "Expression” refers to the production of a functional product.
  • the expression of a nucleotide sequence may refer to the transcription of the nucleotide sequence (such as transcription to generate mRNA or functional RNA) and/or the translation of RNA into a precursor or mature protein.
  • the "expression construct” of the present invention may be a linear nucleic acid fragment, a circular plasmid, a viral vector, or may be an RNA (such as mRNA) that can be translated.
  • the nucleotide sequence of interest is operably linked to a regulatory sequence.
  • regulatory sequence and “regulatory element” are used interchangeably and refer to nucleotide sequences located upstream (5' non-coding sequence), in the middle or downstream (3' non-coding sequence) of a coding sequence and affecting the transcription, RNA processing or stability or translation of the sequence of interest. Regulatory sequences may include, but are not limited to, promoters, translation leader sequences, introns and polyadenylation. N-nucleotide recognition sequence.
  • operably linked refers to a regulatory sequence linked to a target nucleotide sequence such that transcription of the target nucleotide sequence is controlled and regulated by the regulatory sequence.
  • Techniques for operably linking a regulatory sequence to a target nucleotide sequence are known in the art.
  • the sequence of the polynucleotide encoding the fusion protein is operably linked to an expression control sequence to carry out the desired transcription and ultimately produce the fusion protein in a host cell.
  • Suitable expression control sequences include, but are not limited to, promoters, enhancers, ribosome action sites such as ribosome binding sites, polyadenylation sites, transcriptional splicing sequences, transcriptional termination sequences, and sequences that stabilize mRNA, etc.
  • Vectors used in the expression constructs of the present invention include those that replicate autonomously in host cells, such as plasmid vectors; and also include vectors that can be integrated into host cell DNA and replicated with the host cell DNA. Many vectors suitable for the present invention are commercially available.
  • the expression construct of the present invention is derived from pET30a (+) of Novagen.
  • the present invention provides a host cell comprising the polynucleotide of the second aspect of the present invention or transformed with a construct, such as an expression construct, of the fourth aspect of the present invention, wherein the host cell is capable of expressing the fusion protein.
  • Host cells for expressing fusion proteins of the present invention include prokaryotes, yeast and higher eukaryotic cells.
  • Exemplary prokaryotic hosts include the bacterium of Escherichia, Bacillus, Salmonella, Pseudomonas and Streptomyces.
  • the host cell is an Escherichia cell, preferably Escherichia coli.
  • the host cell used is Escherichia coli BL21 (DE3) strain cell (Novagen).
  • a polynucleotide or construct of the invention can be introduced into a host cell by one of a number of well-known techniques, including, but not limited to, heat shock transformation, electroporation, DEAE-dextran transfection, microinjection, liposome-mediated transfection, calcium phosphate precipitation, protoplast fusion, microprojectile bombardment, viral transformation, and the like.
  • the polynucleotide encoding the fusion protein can be integrated into the genome of a host cell, which can express the encoded fusion protein under appropriate conditions or constitutively express the encoded fusion protein.
  • the polynucleotide encoding the fusion protein is present in the host cell in an extrachromosomal form (eg, a plasmid or a construct such as an expression vector).
  • an extrachromosomal form eg, a plasmid or a construct such as an expression vector.
  • the present invention provides a method for producing and purifying a target protein, comprising:
  • the preparation of fusion protein can adopt any suitable technique and means known in the art, for example, using host cells or in vitro cell-free expression system to express fusion protein under suitable expression conditions.
  • Host cells or expression systems expressing fusion protein can be directly used in subsequent steps, or can be separated or purified to remove non-desirable substances to obtain host cells or expression systems expressing fusion protein.
  • step (a) of the method comprises: culturing the host cell of the fifth aspect of the invention under conditions suitable for expression of the fusion protein, and optionally isolating and/or collecting the host cell.
  • step (a) of the method comprises: transforming host cells with the construct, in particular the expression construct, of the fourth aspect of the invention, culturing the transformed host cells under conditions suitable for expression of the fusion protein, and optionally isolating and/or collecting the host cells.
  • the polynucleotide sequence encoding the target protein can be cloned into the multiple cloning site of the polynucleotide of the third aspect of the present invention to obtain a polynucleotide encoding a fusion protein, and then a construct containing the polynucleotide is introduced into a host cell for expression or expressed in an in vitro cell-free system to produce a fusion protein.
  • the host cells include prokaryotes, yeasts and higher eukaryotic cells, such as bacteria including but not limited to Escherichia, Bacillus, Salmonella, Pseudomonas and Streptomyces, preferably Escherichia cells, more preferably Escherichia coli, such as Escherichia coli BL21 (DE3) (Novagen).
  • prokaryotes such as bacteria including but not limited to Escherichia, Bacillus, Salmonella, Pseudomonas and Streptomyces, preferably Escherichia cells, more preferably Escherichia coli, such as Escherichia coli BL21 (DE3) (Novagen).
  • obtaining the insoluble portion containing the fusion protein means that, based on the aggregation properties of the self-aggregating peptide used, appropriate methods or means such as adjusting pH, salt ion concentration, etc. are used for the host cells or expression systems expressing the fusion protein to achieve aggregation of the fusion protein, thereby obtaining active aggregates of the fusion protein.
  • the method step (b) includes: collecting and lysing cells, and/or collecting supernatant or precipitate containing the fusion protein. For example, if the self-aggregating peptide spontaneously or under certain conditions self-aggregates in the host cell, after the fusion protein is expressed, the host cells are collected, the cells are lysed, and the precipitate is collected or the precipitate is collected under the certain conditions (e.g., by centrifugation).
  • Step (b) can be repeated more than once, for example, the obtained precipitate is redissolved by changing the pH or salt concentration, and then precipitated to further reduce the content of substances other than the fusion protein in the obtained precipitate.
  • the conditions for expressing fusion proteins are known in the art, such as temperature, pH, culture medium, etc. Any suitable method for recovering fusion proteins is known in the art, including but not limited to, for example, by chromatography, centrifugation, dialysis, etc.
  • the cleavage site in the fusion protein can be cut using any suitable technique known in the art, so that the target protein is no longer connected to the rest of the fusion protein and becomes a free independent part.
  • the obtained insoluble part containing the fusion protein is placed in a suitable solution, and the pH and other conditions are adjusted so that the cleavage site described herein can be cut, thereby releasing the target protein.
  • cleavage of the fusion protein can be performed by any suitable means known in the art, such as including a spacer sequence between the short peptide tag and the target protein, wherein the spacer sequence comprises a cleavage site that can be cleaved (such as the cleavage site described herein), thereby removing the short peptide tag by cleaving the fusion protein, thereby releasing the target protein.
  • a spacer sequence between the short peptide tag and the target protein wherein the spacer sequence comprises a cleavage site that can be cleaved (such as the cleavage site described herein), thereby removing the short peptide tag by cleaving the fusion protein, thereby releasing the target protein.
  • the target protein released can be recovered by any suitable method or means.
  • the released target protein if it is soluble under the cutting conditions, it can be dissolved in the solution, and the remaining part of the fusion protein cut also forms insoluble aggregates due to the aggregation of the self-aggregating peptide, thereby collecting the supernatant, and optionally removing the precipitate by centrifugation, etc.; if the released target protein is insoluble under the cutting conditions, the conditions of the solution such as pH, salt ion concentration, etc.
  • the fusion protein cut can be changed to make it soluble and the remaining part of the fusion protein cut also forms insoluble aggregates due to the aggregation of the self-aggregating peptide, and then collecting the supernatant, optionally removing the precipitate by centrifugation, etc.
  • the method comprises the following steps: (a) culturing the host cell of the present invention to express the fusion protein of the present invention; (b) lysing the host cell, then removing the soluble part of the cell lysate, recovering the insoluble part (the fusion protein is insoluble in the cytoplasm), or removing the insoluble part of the cell lysate, recovering the soluble part (the fusion protein is soluble in the cytoplasm, such as using a self-aggregating peptide that induces aggregation with salt (which does not aggregate in the cytoplasm)), adding salt to induce the fusion protein to form an insoluble part at a certain salt concentration; (c) releasing a soluble target protein from the fusion protein of the insoluble part by cutting the cleavage site; and (d) recovering the soluble part containing the target protein, and optionally separating or purifying it.
  • a schematic diagram of the method of the present invention can be seen in Figure 1A.
  • the method for lysing the host cells is selected from the commonly used treatment methods in the art, such as ultrasound, homogenization, high pressure (such as in a French press), hypotonicity (osmolysis), detergents, lysing enzymes, organic solvents or combinations thereof, and the lysis is performed under weakly alkaline pH conditions (such as pH 7.5-8.5), thereby lysing the cell membrane of the host cells and releasing the active aggregates from the cells, but still maintaining an insoluble state.
  • the fusion protein is recovered in a soluble form, and the fusion protein in a precipitated state is obtained by changing the ion concentration.
  • the present invention provides a method for high-throughput production and purification of multiple target proteins, comprising:
  • the method of the present invention can be used to prepare multiple, for example, at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300 or more fusion proteins of target proteins in parallel.
  • Multiple target proteins can be made into the fusion proteins of the present invention respectively, for example, the host cells expressing these fusion proteins are placed in an array on a multi-well culture plate such as a 96-well plate, and then the method of producing and purifying the target protein of the present invention is carried out, thereby producing and purifying multiple target proteins simultaneously.
  • purity refers to the purity of the target protein, that is, the proportion of the target protein, such as human growth hormone, to the total protein in the purified solution. Since the target protein is expressed by cells, there are a large number of other proteins in the cells (such as Escherichia coli, which has thousands of proteins). Purifying the target protein from such a large variety of protein mixtures has always been a key technical problem. Through the steps of cell crushing, centrifugation, and separation after cutting, there are basically only proteins and inorganic salts in the purified solution. Therefore, the higher the proportion of the target protein in the purified solution, the higher the purity of the production.
  • the fusion method of the present invention based on self-aggregating peptides and connecting peptides can successfully produce active target proteins in large quantities.
  • the self-aggregating peptides used in the present invention can induce the fusion protein to form a large number of active protein aggregates, which can avoid the degradation of the target protein in the host and help it to fold correctly in prokaryotic cells.
  • the inventors were surprised to find that this method only requires simple centrifugation operations to obtain a target protein with a considerable yield and a purity of >98%.
  • the production and purification method of the present invention has low equipment requirements, does not require column purification, has low production costs, is easy to operate, and has extremely high purity.
  • step means that the step exists or does not exist.
  • the term "about” refers to a numerical range that includes a specific value that a person skilled in the art would reasonably consider to be similar to the specific value. In some embodiments, the term “about” refers to within the standard error of measurement using commonly accepted measurements in the art. In some embodiments, about refers to ⁇ 10%, ⁇ 5%, ⁇ 4%, ⁇ 3%, ⁇ 2%, ⁇ 1% or even ⁇ 0.5% of a specific value.
  • Example 1 Construction of L6KD-Mtu-POI, L6KD-ELP-Mtu-POI, L6KD-PEP-Mtu-POI fusion protein expression vectors
  • POI represents the target protein.
  • POI refers to insulin-like growth factor IGF-1, human growth hormone hGH (SEQ ID NO: 10), anti-new crown nanoantibody NB21 (SEQ ID NO: 40), xylanase, capecitabine or glutathione sulfhydryl transferase GST.
  • the primers required for constructing the plasmid were designed by oligo 7 and synthesized by Shanghai Bioengineering as shown in Table 1.
  • ELP SEQ ID NO: 1
  • PEP SEQ ID NO: 2
  • NCBI No.: WP_014683986 NCBI No.: WP_014683986
  • the PCR reaction used NEB's Q5 polymerase (New England Biolab (NEB)), and the PCR conditions were: 98°C 30sec, 98°C 10sec, 67°C 30sec, 72°C 30sec, a total of 30 cycles; finally 72°C 2min.
  • NEB NEB's Q5 polymerase
  • the PCR amplification product was subjected to 1% agarose gelation. Glue separation and recovery.
  • pET32-L6KD-Mtu ⁇ I-CM-hGH (Lin et al., Chemical Engineering Science, 2022, 262: 118052) as a template, where the L6KD amino acid sequence is shown in SEQ ID NO: 5, the Mtu nucleotide sequence is shown in SEQ ID NO: 36, and the hGH nucleotide sequence is shown in SEQ ID NO: 39, the Mtu-hGH fragment and the Backbone-L6KD fragment were amplified by PCR reaction using M-F, ORI-R and ORI-F, M-R as primers, respectively.
  • the PCR reaction used Q5 polymerase from NEB, and the PCR conditions were: 98°C30sec, 98°C10sec, 64°C30sec, 72°C120sec, for a total of 30 cycles; and finally 72°C2min. After the reaction, the PCR amplification products were separated and recovered by 1% agarose gel.
  • the ELP, Mtu-hGH fragments and Backbone-L6KD fragment, as well as the PEP, Mtu-hGH fragments and Backbone-L6KD fragments were assembled by Gibson assembly (for example, see https://www.neb.cn/applications/cloning-and-synthetic-biology/dna-assembly-and-cloning/gibson-assembly), and the homology arm sequences were CCGACGCCGACCCCAGAATTC (SEQ ID NO: 65), GCGCTGGCTGAAGGCACGC (SEQ ID NO: 66) and TGTGACCGTCTCCGGGAGCTG (SEQ ID NO: 16) to obtain pET32-L6KD-ELP-Mtu-hGH and pET32-L6KD-PEP-Mtu-hGH.
  • Gibson assembly for example, see https://www.neb.cn/applications/cloning-and-synthetic-bio
  • the assembled products were transformed into Escherichia coli DH5 ⁇ competent cells, and the transformed cells were spread on LB plates supplemented with 100 ⁇ g/mL carbenicillin to screen positive clones.
  • the plasmids were extracted and sequenced. The sequencing results showed that the cloned plasmid sequences were correct.
  • the amino acid sequence of the NB21 gene (SEQ ID NO: 40) was obtained from the literature (Schoof et al., Science, 2020, 370(6523): 1473-1479), and the gene fragment was obtained by Shanghai Bioengineering after gene codon optimization and synthesis.
  • the synthesized gene was used as a template and POI-F and END-R were used as primers to amplify the NB21 fragment by PCR reaction.
  • the Backbone-KanaR fragment was amplified by PCR reaction using pET30-L6KD-Mtu-IGF (Lin et al., Chemical Engineering Science, 2022, 262: 118052) as a template and END-F and ORI-R as primers.
  • the Backbone-L6KD-Mtu fragment was amplified by PCR reaction using the above-mentioned pET32-L6KD-Mtu ⁇ I-CM-hGH as a template and ORI-F and POI-R as primers. After the PCR reaction, the PCR amplification products were separated and recovered by 1% agarose gel. The NB21, Backbone-KanaR fragments and Backbone-L6KD-Mtu fragments were assembled by Gibson Assembly to obtain the pET30-L6KD-Mtu-NB21 plasmid.
  • the construction process of pET30-L6KD-ELP-Mtu-NB21 and pET30-L6KD-PEP-Mtu-NB21 plasmids was similar, except that the PCR template of the Backbone-L6KD-Mtu fragment was replaced by pET32-L6KD-ELP-Mtu-hGH and pET32-L6KD-PEP-Mtu-hGH, respectively.
  • the Gibson Assembly product was transformed into Escherichia coli DH5 ⁇ competent cells, and the transformed cells were spread on LB plates supplemented with 50 ⁇ g/mL kanamycin to screen positive clones, and the plasmids were extracted and sequenced.
  • IGF-1 SEQ ID NO: 38
  • xylanase SEQ ID NO: 41
  • caplacizumab SEQ ID NO: 42
  • GST SEQ ID NO: 43
  • genes are from pET30-L6KD-Mtu-IGF (Lin et al., Chemical Engineering Science, 2022, 262, 118052), pET30-CpA-Mtu-Xylanase (Lin Zhanglin et al., WO2022253266A1), pET32-L6KD-Mtu-Caplacizumab (Lin et al., Chemical Engineering Science, 2022, 262: 118052) and pET30-CpA-Mtu-GST (Lin Zhanglin et al., WO2022253266A1), respectively.
  • pET30-CpA-Mtu-GST was used as a template, POI-F and ORI-R were used as primers to amplify the GST-Backbone fragment through PCR reaction.
  • pET30-L6KD-Mtu-NB21 was used as a template, ORI-F and POI-R were used as primers to amplify the Backbone-L6KD-Mtu fragment through PCR reaction.
  • the PCR amplification product was separated and recovered by 1% agarose gel. The above two fragments were assembled by Gibson Assembly to obtain the pET30-L6KD-Mtu-GST plasmid.
  • the construction process of pET30-L6KD-ELP-Mtu-GST and pET30-L6KD-PEP-Mtu-GST plasmids was similar, except that the PCR template of the expression vector was replaced with pET30-L6KD-ELP-Mtu-NB21 and pET30-L6KD-PEP-Mtu-NB21, respectively.
  • the Gibson Assembly product was transformed into E. coli DH5 ⁇ competent cells, and the transformed cells were spread on LB plates supplemented with 50 ⁇ g/mL kanamycin to screen positive clones, and the plasmids were extracted and sequenced.
  • Example 2 Expression and purification of IGF-1/hGH/Nb21/xylanase/capacilizumab/GST
  • target proteins include human insulin-like growth factor IGF-1, human growth hormone hGH, new coronavirus nanoantibody Nb21, xylanase, capecitabine and glutathione S-transferase GST.
  • the plasmids constructed in Example 1 (containing plasmids pET32-L6KD-Mtu-hGH, pET32-L6KD-ELP-Mtu-hGH, pET32-L6KD-PEP-Mtu-hGH, pET30-L6KD-Mtu-IGF-1/Nb21/Xylanase/Caplacizumab/GST, pET30-L6KD-ELP-Mtu-IGF-1/Nb21/Xylanase/Caplacizumab/GST and pET30-L6KD-PEP-Mtu-IGF-1/Nb21/Xylanase/Caplacizumab/GST) were transformed into Escherichia coli BL21 (DE3) respectively.
  • 1 OD the cell amount with an OD 600 of 1 in 1 mL is referred to as 1 OD).
  • the cells were resuspended to 20 OD/mL with lysis buffer B1 (2.4 g Tris, 29.22 g NaCl, 0.37 g Na 2 EDTA ⁇ 2H 2 O dissolved in 800 mL water, pH adjusted to 8.5, and water added to 1 L), and ultrasonically disrupted (disruption conditions: power 200 W, ultrasonic time 3 sec, interval time 3 sec, ultrasonic number 99 times). Centrifuged at 4°C, 15000g for 20 min, and the supernatant and precipitate were collected respectively.
  • lysis buffer B1 2.4 g Tris, 29.22 g NaCl, 0.37 g Na 2 EDTA ⁇ 2H 2 O dissolved in 800 mL water, pH adjusted to 8.5, and water added to 1 L
  • Lanes ES, EP, CP, and CS are the expression and purification samples of the target protein, which are ES: cell lysate supernatant; EP: cell lysate precipitate, where clear aggregates expressed by the fusion protein can be detected; CP: precipitate separated after cutting; CS: supernatant separated after cutting, where clear target protein bands can be detected; Lane IV is a protein quantitative standard containing bovine serum albumin BSA or aprotinin APR, and the loading amounts are 5 ⁇ g, 2.5 ⁇ g, 1.25 ⁇ g, 0.625 ⁇ g, and 0.3125 ⁇ g, respectively.
  • the optical density analysis of the target band was performed using ImageJ gel quantitative analysis software, and the aggregate yield formed by the fusion protein, the target protein yield released into the supernatant after intein-mediated self-cleavage, the Mtu ⁇ I-CM cleavage efficiency, the recovery rate and its purity in the supernatant were calculated.
  • the results are shown in Table 2.
  • the 18 fusion proteins (L6KD-Mtu-IGF-1/hGH/Nb21/Xylanase/Caplacizumab/GST, L6KD-ELP-Mtu-IGF-1/hGH/Nb21/Xylanase/Caplacizumab/GST and L6KD-PEP-Mtu-IGF-1/hGH/Nb21/Xylanase/Caplacizumab/GST) used were all present in the form of precipitation, among which the aggregate expression of the L6KD-Mtu system was 205-545 mg/L LB culture medium.
  • the target protein was separated from L6KD-Mtu by self-cleavage of the intein Mtu ⁇ I-CM, and the cleavage efficiency was 38-70%.
  • the yield of the target protein released into the supernatant after the L6KD-Mtu tag was 2.2-57 mg/L LB culture medium, and the purity was 27-93%.
  • the aggregate expression level of the L6KD-ELP-Mtu system was 19-419 mg/L LB culture medium, the cleavage efficiency was 82-99%, the yield of the target protein released into the supernatant after cleavage was 7.3-42 mg/L LB culture medium, and the purity was 17-74%.
  • the aggregate expression level of the L6KD-PEP-Mtu system was 365-557 mg/L LB culture medium, the cleavage efficiency was 50-91%, and the yield of the target protein released into the supernatant after tag cleavage was 6.7-78 mg/L LB culture medium, and the purity was >98%.
  • FIG. 3A The high-throughput protein expression and purification process is shown in Figure 3A.
  • the strain obtained in Example 2 (containing plasmid pET30-L6KD-PEP-Mtu-Nb21/Xylanase/Caplacizumab/GST or pET32-L6KD-PEP-Mtu-hGH) was streaked and revived.
  • Escherichia coli BL21 (DE3) cells carrying the target plasmid were inoculated into a 96-well culture plate, each well containing 200 ⁇ L LB medium supplemented with 50 ⁇ g/mL kanamycin or 100 ⁇ g/mL carbenicillin, and cultured overnight at 37°C, 800 rpm shaking.
  • the overnight culture was transferred to a 96 deep-well plate at a ratio of 1:50, each well containing 800 ⁇ L fresh LB medium supplemented with the corresponding antibiotics, and then cultured at 37°C for 2 hours until OD 600 reached 0.4-0.8.
  • the culture temperature was lowered to 18°C, and 200 ⁇ L of LB medium containing 1 mM IPTG was added to start protein expression. Then the culture was continued at 18°C for 24 hours. After the culture was completed, the cells were harvested by centrifugation at 4,000g, 4°C for 30 minutes. 200 ⁇ L of BugBuster reagent (Millipore) was added to each well to lyse E. coli.
  • Target protein yield after intein-mediated self-cleavage (volume calculated per liter of LB medium).
  • capecitabine was selected as an example for further scale-up production.
  • the fermentation process was consistent with our previous work (Lin et al., Chemical Engineering Science, 2022, 262: 118052), and the scale-up fermentation was carried out in a 5-L fermenter using a DO-stat fed-batch feeding strategy.
  • Example 2 The strain obtained in Example 2 (containing plasmid pET30-L6KD-PEP-Mtu-Caplacizumab) was streaked and revived. 140mL LB medium was inoculated as seed liquid one day in advance.
  • the initial fermentation liquid volume was 2L
  • the seed culture liquid was inoculated at a ratio of 7%
  • the initial fermentation temperature was 37°C
  • the rotation speed was 200rpm
  • the entire fermentation process was ventilated at 3L/min
  • the dissolved oxygen was controlled at 20%. When the dissolved oxygen was lower than 20%, the dissolved oxygen was maintained by increasing the rotation speed, and the maximum speed was 1,000rpm.
  • the initial glucose concentration was 10g/L, which was consumed about 5h after inoculation.
  • the dissolved oxygen and pH value of the culture medium rose, and feeding began.
  • the concentration of glucose in the fermentation medium was controlled at 0.1-1.5g/L.
  • samples were taken every 2 hours to measure the OD 600 of the bacteria and the glucose concentration in the culture solution.
  • the temperature was lowered to 18 ° C, and then IPTG with a final concentration of 0.2 mM was added for induction. After 28 hours of induction, the OD 600 of the bacteria was 160.
  • Lanes I-VI are protein quantitative standards containing bovine serum albumin BSA, and the sample loading is 5 ⁇ g, 2.5 ⁇ g, 1.25 ⁇ g, 0.625 ⁇ g, 0.3125 ⁇ g, and 0.15625 ⁇ g, respectively.
  • the relative gray analysis statistics of the caprazumab released into the supernatant after cutting and the detailed statistical results of the purification process are shown in Table 4.
  • the yield of fusion protein aggregates was 12.1 g/L of culture medium, the yield of capecitabine released into the supernatant after cleavage was 1.1 g/L of culture medium, and the purity was 98%; at 28 hours of induction, the yield of fusion protein aggregates was 13.5 g/L of culture medium, the yield of capecitabine released into the supernatant after cleavage was 1.4 g/L of culture medium, and the purity was 99%, which was about 33 times that of shake flask horizontal fermentation.
  • SAP represents aggregation peptide.
  • SAP refers to MpA (SEQ ID NO: 7), EIK8 (SEQ ID NO: 58) and EFR8 (SEQ ID NO: 59).
  • the expression vectors used include pET30-MpA/EIK8/EFR8-Mtu-hGH and pET30-MpA/EIK8/EFR8-PEP-Mtu-hGH.
  • the primers required for constructing the plasmid were designed by oligo 7 and synthesized by Shanghai Biotechnology Co., Ltd. as shown in Table 5.
  • the PEP-Mtu fragment was amplified by PCR reaction, and the hGH-Backbone-MpA fragment was amplified by PCR reaction using pET30-MpA-Mtu-hGH (Lin Zhanglin et al., WO2022253266A1) as a template and POI-F and 11-R as primers.
  • the PCR reaction used NEB's Q5 polymerase, and the PCR conditions were: 98°C 30sec, 98°C 10sec, 64°C 30sec, 72°C 120sec, a total of 30 cycles; finally 72°C 2min.
  • the PCR amplification product was separated and recovered by 1% agarose gel.
  • the PEP-Mtu fragment and hGH-Backbone-MpA fragment were assembled by Gibson Assembly, and the assembled products were transformed into Escherichia coli DH5 ⁇ competent cells.
  • the transformed cells were spread on LB plates supplemented with 50 ⁇ g/mL kanamycin to screen positive clones, and the plasmids were extracted and sequenced. The sequencing results showed that the cloned plasmid sequences were correct.
  • the plasmid construction process of pET30-EIK8/EFR8-Mtu-hGH and pET30-EIK8/EFR8-PEP-Mtu-hGH is similar.
  • the nucleotide sequence of EIK8/EFR8 is shorter and is directly introduced by primers.
  • EIK8 as an example, the Mtu-hGH-Backbone fragment was amplified by PCR reaction using pET30-MpA-Mtu-hGH as a template and EIK8-F and ORI-R as primers.
  • the Backbone-EIK8 fragment was amplified by PCR reaction using pET30-MpA-Mtu-hGH as a template and ORI-F and EIK8-R as primers.
  • the PEP-Mtu-hGH-Backbone fragment was amplified by PCR reaction using pET30-MpA-PEP-Mtu-hGH as a template and EIK8-F and ORI-R as primers.
  • the PCR reaction used NEB's Q5 polymerase, and the PCR conditions were: 98°C30sec, 98°C10sec, 64°C30sec, 72°C120sec, 30 cycles in total; finally 72°C2min.
  • the PCR amplification products were separated and recovered by 1% agarose gel.
  • the Mtu-hGH-Backbone, Backbone-EIK8 fragments and the PEP-Mtu-hGH-Backbone, Backbone-EIK8 fragments were assembled by Gibson Assembly.
  • the construction process of EFR8 was similar, except that the corresponding primers were replaced with EFR8-F and EFR8-R, respectively.
  • the assembly products were transformed into Escherichia coli DH5 ⁇ competent cells, and the transformed cells were spread on LB plates supplemented with 50 ⁇ g/mL kanamycin to screen positive clones, extract plasmids, and sequence them. The sequencing results showed that the cloned plasmid sequences were correct.
  • the structures of the constructed SAP-Mtu-hGH and SAP-PEP-Mtu-hGH plasmids are shown in FIG5B .
  • Example 6 hGH protein purification mediated by MpA, EIK8 and EFR8 phase transition and Mtu-mediated cleavage
  • the plasmid constructed in Example 5 (containing plasmids pET30-MpA-Mtu-hGH, pET30-MpA-PEP-Mtu-hGH, pET30-EIK8-Mtu-hGH, pET30-EIK8-PEP-Mtu-hGH, pET30-EFR8-Mtu-hGH and pET30-EFR8-PEP-Mtu-hGH) was transformed into Escherichia coli BL21 (DE3) competent cells to obtain 6 hGH fusion protein expression strains.
  • the cells were resuspended to 100 OD/mL with lysis buffer B3 (2.4 g Tris, 0.37 g EDTA ⁇ 2Na dissolved in 800 mL water, pH adjusted to 8.0, and water added to 1 L), and ultrasonically disrupted (the disruption conditions were: 2 horn, 20% power, ultrasonic time 2 sec, interval time 2 sec, running 25 min to 30 min). Centrifuged at 4°C, 15,000 g for 30 min, and the supernatant and precipitate were collected for sample preparation. The expression of the fusion protein in the lysis supernatant and lysis precipitate was detected by SDS-PAGE.
  • Na 2 SO 4 was added to the lysate supernatant to 0.7 M, and the mixture was placed at 4°C overnight for 12 h to allow the self-aggregating peptide to fully aggregate.
  • the suspension was then centrifuged at 4°C, 15,000 g for 30 min, and the precipitate was washed once with buffer B4 containing 0.7 M Na 2 SO 4 (99.43 g of Na 2 SO 4 , 2.4 g of Tris, and 0.37 g of EDTA ⁇ 2N a dissolved in 800 mL of water, adjusted to pH 8.0, and fixed to 1 L with water), and then centrifuged under the same conditions to separate the supernatant and precipitate.
  • the precipitate was fully resuspended in cleavage buffer B5 containing 0.7 M Na 2 SO 4 (PBS without NaCl, supplemented with 0.7 M Na 2 SO 4 , supplemented with 40 mM Bis-Tris, pH 6.2, 2 mM EDTA), and placed at 25°C for 24 h to allow the intein to fully self-cleave.
  • the suspension was incubated at 4°C for 3 h to allow the aggregated peptides to aggregate fully, and then the suspension was centrifuged at 4°C and 16,000 g for 30 min to separate.
  • the optical density analysis of the target band was performed using ImageJ (National Institutes of Health) gel quantitative analysis software.
  • the target protein yield released into the supernatant after intein-mediated self-cleavage, the aggregation efficiency after salt addition, the Mtu ⁇ I-CM cleavage efficiency and the human growth hormone hGH recovery rate and its purity in the supernatant could be calculated.
  • the results are shown in Table 6.
  • the six fusion proteins (MpA-Mtu-hGH, MpA-PEP-Mtu-hGH, EIK8-Mtu-hGH, EIK8-PEP-Mtu-hGH, EFR8-Mtu-hGH and EFR8-PEP-Mtu-hGH) used are all expressed in soluble form, and the amount of soluble expression is 353-537 mg/L. In the case of 0.7M Na2SO4 , most of the fusion proteins in the lysis supernatant are changed from soluble to precipitate, and the aggregation efficiency is 92-99%.
  • the target protein After the self-cleavage of the intein Mtu ⁇ I-CM, the target protein is separated from MpA/EIK8/EFR8-Mtu, and the cleavage efficiency is 53-93%. After cleavage, the yield of hGH released into the supernatant is 51-95 mg/L, and the purity is 80-99%. Among them, the hGH purity of the SAP-PEP-Mtu-hGH fusion protein is the highest, which is 99%.
  • Example 7 Construction of a fusion protein expression construct containing the intein Mxe GyrA (SEQ ID NO: 4)
  • the required primers for the expression vectors pET30-Xylanase-Mxe-ELK16 and pET30-Xylanase-Mxe-PEP-ELK16 used in the examples of this application were designed using oligo 7 and synthesized by Shanghai Biotechnology Co., Ltd. as shown in Table 7.
  • pET30-LipA-Mxe-ELK16 Zhou et al., Microbial Cell Factories, 2012, 11, 10) as a template, Mxe-F and ORI-R as primers, the Mxe-ELK16-Backbone fragment was amplified by PCR reaction, and using pET30-Xylanase-Mxe-MpA (Lin Zhanglin et al., WO2022253266A1) as a template, ORI-F and Mxe-R1 as primers, the Backbone-Xylanase fragment was amplified by PCR reaction.
  • the PCR reaction used Q5 polymerase from NEB, and the PCR conditions were: 98°C30sec, 98°C10sec, 64°C30sec, 72°C120sec, a total of 30 cycles; finally 72°C2min. After the reaction, the PCR amplification products were separated and recovered by 1% agarose gel. The Mxe-ELK16-Backbone fragment and the Backbone-Xylanase fragment were assembled by Gibson Assembly.
  • a PEP fragment was amplified by PCR reaction to obtain the PEP fragment, using pET30-LipA-Mxe-ELK16 as a template, KL-F and ORI-R as primers, a PCR reaction was amplified to obtain the ELK16-Backbone fragment, and using pET30-Xylanase-Mxe-MpA as a template, ORI-F and Mxe-RP as primers, a PCR reaction was amplified to obtain the Backbone-Xylanase-Mxe fragment.
  • the PEP fragment, ELK16-Backbone fragment, and Backbone-Xylanase-Mxe fragment were assembled by Gibson Assembly.
  • the assembled product was transformed into E. coli DH5 ⁇ competent cells, and the transformed cells were spread on LB plates supplemented with 50 ⁇ g/mL kanamycin to screen positive clones, and the plasmids were extracted and sequenced.
  • the sequencing results showed that the cloned plasmid sequences were correct.
  • the structures of the constructed pET30-Xylanase-Mxe-ELK16 and pET30-Xylanase-Mxe-PEP-ELK16 plasmids are shown in Figure 7B.
  • the optical density analysis of the target band was performed using ImageJ gel quantitative analysis software.
  • the aggregate yield formed by the fusion protein, the xylanase yield released into the supernatant after self-cleavage mediated by the intein, the Mxe GyrA cleavage efficiency, the xylanase recovery rate and its purity in the supernatant could be calculated.
  • the results are shown in Table 8.
  • the two fusion proteins (Xylanase-Mxe-ELK16 and Xylanase-Mxe-PEP-ELK16) used were both present in the form of precipitation, and the aggregate expression was 278-282 mg/L LB culture medium.
  • the two different fusion proteins were self-cleaved by the intein Mxe GyrA, and the xylanase was separated from Mxe-ELK16.
  • the cleavage efficiency was 51-70%, and the yield of xylanase released into the supernatant after cleavage was 78-107 mg/L LB culture medium.
  • the purity of the xylanase recovered after cleavage was 92-98%.
  • the xylanase of the Xylanase-Mxe-PEP-ELK16 fusion protein had the highest xylanase purity, that is, the xylanase yield of 107 mg/L LB culture medium and the purity of 98% could be purified in one step through the purification technology based on self-aggregating peptides and self-cleavage tags.
  • Example 9 Construction of L6KD-HD/HFBII-Mtu-POI, L6KD-ProP-Mtu-POI, L6KD-N1/N2 fusion protein expression vectors
  • POI represents the target protein.
  • HD SEQ ID NO: 44
  • HFBII SEQ ID NO: 45
  • ProP refers to the protease propeptide, including P1-P10
  • N1 SEQ ID NO: 56
  • N2 SEQ ID NO: 57
  • POI refers to insulin-like growth factor, capecitabine.
  • the expression vector used includes a combination of pET30-L6KD-ProP-Mtu and two POIs.
  • the primers required for constructing the plasmid were designed by oligo 7 and synthesized by Shanghai Biotechnology as shown in Table 9.
  • ProP protease propeiptide sequences P1-P10 homologous to PEP, among which P1 (SEQ ID NO: 46) was derived from yeast protease propeiptide, P2 (SEQ ID NO: 47) was derived from cucumber protease propeiptide, P3 (SEQ ID NO: 48) was derived from cyanobacteria protease propeiptide, P4 (SEQ ID NO: 49) was derived from subtilisin propeiptide, which is also the earliest discovered and most studied propeiptide, P5 (SEQ ID NO: 50) was derived from human PCSK9 protease propeiptide, and the remaining P6-P10 (SEQ ID NO: 51-55) were derived from bacterial protease propeiptide.
  • P1 SEQ ID NO: 46
  • P2 SEQ ID NO: 47
  • the sequence identity of the 10 propeiptides with PEP was 38-95%. According to its amino terminal sequence, the gene fragment was obtained by gene synthesis by Shanghai Biotech. The truncated PEP N1 and N2 were also synthesized by Shanghai Biotech according to their amino terminal sequences to obtain gene fragments. The synthesized genes were used as templates, and ELP-F2 and ELP-R2 were used as primers to amplify the polynucleotide fragments of each connecting peptide through PCR reaction.
  • the PCR reaction used Q5 polymerase from NEB (New England Biolab (NEB)), and the PCR conditions were: 98°C30sec, 98°C10sec, 67°C30sec, 72°C30sec, a total of 30 cycles; and finally 72°C2min. After the reaction, the PCR amplification products were separated and recovered by 1% agarose gel.
  • the above-mentioned pET32-L6KD-Mtu ⁇ I-CM-IGF was used as a template, and M-F, ORI-R and ORI-F, M-R were used as primers to amplify the Mtu-IGF fragment and the Backbone-L6KD fragment by PCR reaction.
  • the PCR reaction used Q5 polymerase from NEB, and the PCR conditions were: 98°C30sec, 98°C10sec, 64°C30sec, 72°C120sec, for a total of 30 cycles; finally 72°C2min. After the reaction, the PCR amplification product was separated and recovered by 1% agarose gel.
  • the connecting peptide, Mtu-IGF fragment and Backbone-L6KD fragment were assembled by Gibson Assembly.
  • the construction process of the pET30-L6KD-connecting peptide-Mtu-Caplacizumab plasmid was similar, except that the PCR template of the expression vector was replaced with pET30-L6KD-Mtu-Caplacizumab.
  • the assembled products were transformed into Escherichia coli DH5 ⁇ competent cells, and the transformed cells were spread on LB plates supplemented with 50 ⁇ g/mL kanamycin to screen positive clones.
  • the plasmids were extracted and sequenced. The sequencing results showed that the cloned plasmid sequences were correct.
  • Example 10 Hydrophilic domain HD and hydrophobic protein HFBII as connecting peptides for expression and purification of target protein
  • the plasmids constructed in Example 9 (containing plasmids pET30-L6KD-HD-Mtu-IGF, pET30-L6KD-HD-Mtu-Caplacizumab, pET30-L6KD-HFBII-Mtu-IGF and pET30-L6KD-HFBII-Mtu-Caplacizumab) were transformed into Escherichia coli BL21 (DE3) competent cells to obtain 4 fusion protein expression strains containing different connecting peptides.
  • the cells were resuspended to 20 OD/mL with lysis buffer B1 (2.4 g Tris, 29.22 g NaCl, 0.37 g Na 2 EDTA ⁇ 2H 2 O dissolved in 800 mL water, pH adjusted to 8.5, and water added to 1 L), and ultrasonically disrupted (disruption conditions: power 200 W, ultrasonic time 3 sec, interval time 3 sec, ultrasonic number 99 times). Centrifuged at 4°C, 15000g for 20 min, and the supernatant and precipitate were collected respectively.
  • lysis buffer B1 2.4 g Tris, 29.22 g NaCl, 0.37 g Na 2 EDTA ⁇ 2H 2 O dissolved in 800 mL water, pH adjusted to 8.5, and water added to 1 L
  • the precipitate was washed twice with an equal volume of lysis buffer, and then fully resuspended with an equal volume of cleavage buffer (PBS supplemented with 40 mM Bis-Tris, pH 6.2, 2 mM EDTA), and placed at 25°C for 24 h to allow the intein to fully self-cleave. After that, the mixture was centrifuged at 4°C and 15,000 g for 20 min, and the precipitate was resuspended with an equal volume of lysis buffer, and the obtained supernatant and precipitate were subjected to SDS-PAGE detection together with the supernatant and precipitate before cleavage.
  • cleavage buffer PBS supplemented with 40 mM Bis-Tris, pH 6.2, 2 mM EDTA
  • the optical density analysis of the target band was performed using ImageJ gel quantitative analysis software, and the aggregate yield formed by the fusion protein, the target protein yield released into the supernatant after the intein-mediated self-cleavage, the Mtu ⁇ I-CM cleavage efficiency, the target protein recovery rate and its purity in the supernatant were calculated.
  • the results are shown in Table 10.
  • Table 10 HD and hydrophobic protein HFBII as linker peptides for expression and purification of target proteins
  • the four fusion proteins (L6KD-ProP-Mtu-IGF and L6KD-ProP-Mtu-Caplacizumab) used all exist in the form of precipitation, and the aggregate expression level is 25-94 mg/L LB culture medium.
  • the four different fusion proteins are self-cleaved by the intein Mtu ⁇ I-CM, and the target protein is separated from L6KD-Mtu-HD/HFBII.
  • the cleavage efficiency is 31-97%, and the yield of the target protein released into the supernatant after cleavage is 0.2-5.8 mg/L LB culture medium.
  • the purity of the target protein recovered after cleavage is 4-70%. Simple hydrophilicity and hydrophobicity cannot be used as a basis for judging whether the connecting peptide has the ability to be applied to the method of the present invention to obtain high-purity proteins.
  • Example 11 Various ProPs used as connecting peptides for expression and purification of target proteins
  • the plasmids constructed in Example 9 were transformed into competent cells of Escherichia coli BL21 (DE3) to obtain 20 strains expressing fusion proteins containing different ProPs.
  • 1OD the cell amount with an OD 600 of 1 in 1 mL
  • the cells were resuspended to 20 OD/mL with lysis buffer B1 (2.4 g Tris, 29.22 g NaCl, 0.37 g Na 2 EDTA ⁇ 2H 2 O dissolved in 800 mL water, pH adjusted to 8.5, and water added to 1 L), and ultrasonically disrupted (disruption conditions: power 200 W, ultrasonic time 3 sec, interval time 3 sec, ultrasonic number 99 times). Centrifuged at 4°C, 15000g for 20 min, and the supernatant and precipitate were collected respectively.
  • lysis buffer B1 2.4 g Tris, 29.22 g NaCl, 0.37 g Na 2 EDTA ⁇ 2H 2 O dissolved in 800 mL water, pH adjusted to 8.5, and water added to 1 L
  • the precipitate was washed twice with an equal volume of lysis buffer, and then fully resuspended with an equal volume of cleavage buffer (PBS supplemented with 40 mM Bis-Tris, pH 6.2, 2 mM EDTA), and placed at 25°C for 24 h to allow the intein to fully self-cleave. After that, centrifuge at 4°C and 15000g for 20min, resuspend the precipitate with an equal volume of lysis buffer, and perform SDS-PAGE detection on the obtained supernatant and precipitate together with the supernatant and precipitate before cutting. The results are shown in Figure 10.
  • the optical density analysis of the target band was performed using ImageJ gel quantitative analysis software, and the aggregate yield formed by the fusion protein, the target protein yield released into the supernatant after the intein-mediated self-cleavage, the Mtu ⁇ I-CM cleavage efficiency, the target protein recovery rate and its purity in the supernatant were calculated.
  • the results are shown in Table 11.
  • the 20 fusion proteins (L6KD-ProP-Mtu-IGF and L6KD-ProP-Mtu-Caplacizumab) used were all present in the form of precipitation, and the aggregate expression level was 52-433 mg/L LB culture medium.
  • the 20 different fusion proteins were self-cleaved by the intein Mtu ⁇ I-CM, and the target protein was separated from L6KD-Mtu-ProP.
  • the cleavage efficiency was 17-91%, and the target protein was released into the supernatant after cleavage.
  • the yield of the target protein is 0.9-26 mg/L LB culture medium, and the purity of the target protein recovered after cutting is 19-99%.
  • the purity of the purified protein of the propeptides P7-P10 with a sequence similarity of >60% to PEP is >96%, which proves that a variety of protease propeptides with high sequence similarity to PEP have the potential to be applied to the method of the present invention to obtain high-purity proteins.
  • Example 12 PEP truncated form: N1 and N2 used as connecting peptides for expression and purification of target protein
  • the plasmid constructed in Example 9 (containing plasmids pET30-L6KD-N1-Mtu-IGF, pET30-L6KD-N1-Mtu-Caplacizumab, pET30-L6KD-N1-Mtu-Xylanase, pET30-L6KD-N2-Mtu-IGF, pET30-L6KD-N2-Mtu-Caplacizumab and pET30-L6KD-N2-Mtu-Xylanase) was transformed into Escherichia coli BL21 (DE3) competent cells to obtain 6 fusion protein expression strains containing different truncated PEPs.
  • the cell volume with an OD 600 of 1 in 1 mL is referred to as 1 OD).
  • the cells were resuspended to 20 OD/mL with lysis buffer B1 (2.4 g Tris, 29.22 g NaCl, 0.37 g Na 2 EDTA ⁇ 2H 2 O dissolved in 800 mL water, pH adjusted to 8.5, and water added to 1 L), and ultrasonically disrupted (disruption conditions: power 200 W, ultrasonic time 3 sec, interval time 3 sec, ultrasonic number 99 times). Centrifuged at 4°C, 15000g for 20 min, and the supernatant and precipitate were collected respectively.
  • lysis buffer B1 2.4 g Tris, 29.22 g NaCl, 0.37 g Na 2 EDTA ⁇ 2H 2 O dissolved in 800 mL water, pH adjusted to 8.5, and water added to 1 L
  • the precipitate was washed twice with an equal volume of lysis buffer, and then fully resuspended with an equal volume of cleavage buffer (PBS supplemented with 40 mM Bis-Tris, pH 6.2, 2 mM EDTA), and placed at 25°C for 24 h to allow the intein to fully self-cleave. After that, the mixture was centrifuged at 4°C and 15000g for 20 minutes, and the precipitate was resuspended with an equal volume of lysis buffer, and the obtained supernatant and precipitate were subjected to SDS-PAGE detection together with the supernatant and precipitate before cutting. The results are shown in Figure 11.
  • Lanes ES, EP, CP, and CS are the expression and purification samples of the target protein, which are ES: cell lysate supernatant; EP: cell lysate precipitate, in which clear aggregates expressed by the fusion protein can be detected; CP: precipitate separated after cutting; CS: supernatant separated after cutting, in which clear target protein bands can be detected; Lanes I-VI are protein quantitative standards containing aprotinin APR or bovine serum albumin BSA, and the loading amounts are 5 ⁇ g, 2.5 ⁇ g, 1.25 ⁇ g, 0.625 ⁇ g, 0.3125 ⁇ g, and 0.15625 ⁇ g, respectively.
  • the optical density analysis of the target band was performed using ImageJ gel quantitative analysis software, and the aggregate yield formed by the fusion protein, the target protein yield released into the supernatant after the intein-mediated self-cleavage, the Mtu ⁇ I-CM cleavage efficiency, the target protein recovery rate and its purity in the supernatant were calculated.
  • the results are shown in Table 12.
  • the six fusion proteins used all exist in the form of precipitation, and the aggregate expression level is 216-335 mg/L LB culture medium.
  • the six different fusion proteins are self-cleaved by the intein Mtu ⁇ I-CM, and the target protein is separated from L6KD-Mtu-N1/N2.
  • the cleavage efficiency is 1-85%, and the yield of the target protein released into the supernatant after cleavage is 0.9-78 mg/L LB culture medium.
  • the purity of the target protein recovered after cleavage is 47-99%. It is proved that the truncated PEP cannot guarantee the potential of obtaining high-purity protein by the method of the present invention.
  • SEQ ID NO:1 ELP amino acid sequence, length 78aa
  • SEQ ID NO:2 PEP amino acid sequence, length 76aa
  • SEQ ID NO:40NB21 nucleotide sequence, length 351 bp
  • SEQ ID NO:42 Capacillinammonab nucleotide sequence, 777 bp in length
  • SEQ ID NO:44 HD amino acid sequence, length 67aa
  • VPGXG (VPGXG) 15 , wherein X is independently Ala, Gly, Val or Glu
  • VPGXG where X is Ala, Gly, Val or Glu

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Abstract

L'invention concerne un procédé de production et de purification d'une protéine cible, et plus particulièrement, l'invention concerne une protéine de fusion contenant un peptide d'auto-agrégation, un peptide de liaison, un espaceur et une fraction de protéine cible, le peptide de liaison contenant une séquence d'acides aminés choisie parmi un polypeptide de type élastine (ELP) ou un propeptide de protéase (ProP) et l'espaceur contenant un site de clivage, et un procédé de purification en une étape sans colonne de la protéine cible au moyen de l'expression de la protéine de fusion.
PCT/CN2024/116250 2023-09-15 2024-09-02 Procédé de purification de protéine Pending WO2025055767A1 (fr)

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WO2022253266A1 (fr) * 2021-06-03 2022-12-08 华南理工大学 Procédé de purification de protéine recombinante
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