Protein purification method

Abstract

The present invention relates to a method for the production and purification of a protein of interest, and more particularly, the present invention relates to a fusion protein comprising: a self-aggregating peptide, a linker peptide comprising an amino acid sequence selected from an elastin-like polypeptide (ELP) or a protease propeptide (ProP) and the spacer comprising a cleavage site, and a protein portion of interest, and a method for column-free one-step purification of a target protein by expressing the fusion protein.

Description

Technical Field

[0001] 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. Background Art

[0002] 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. Among the various expression systems currently used, 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. However, when expressing disulfide-bonded or complex-structured proteins such as human insulin and human growth hormone in Escherichia coli, inactive inclusion bodies are often formed due to protein misfolding, which requires a complex process of denaturation / renaturation to restore its activity, resulting in complex production processes, low efficiency and high costs.

[0003] At the same time, the purity of the target protein must be high enough. For example, 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%. However, 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. However, due to the lack of specificity, ion exchange chromatography and size exclusion chromatography are difficult to obtain high-purity target proteins as a single purification method; affinity chromatography requires specific resins and buffers, which makes it more expensive and is not conducive to industrial applications. At present, 4-6 steps of column chromatography are usually required to achieve high purity of the target protein, which greatly increases the time and economic cost of downstream protein purification. It is reported that the cost of the downstream purification process accounts for 50-80% of the overall production cost of recombinant proteins (Markus et al., Trends in Analytical Chemistry, 2021, 20(4): 309-325). Moreover, the complex and specific purification conditions make it difficult to standardize the purification process, which is not conducive to high-throughput protein purification.

[0004] In recent years, researchers have attempted to develop protein purification methods that do not rely on column chromatography, including the use of N proand elastin-like polypeptide (ELP) tags. pro Inducing the production of inactive inclusion bodies, the additional refolding process required downstream will increase time and economic costs. The multiple rounds of phase transitions required for the purification process of the ELP tag (high temperature aggregation-centrifugation-low temperature disaggregation-centrifugation) 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). In addition, the molecular weight of the ELP tag is relatively large (generally more than 30 kDa to ensure the purification effect), resulting in a low recovery rate of the target protein.

[0005] In recent years, the cleavable self-aggregation tag method (cSAT) induces the fusion protein to form active protein aggregates through self-assembling peptides, and then releases the target protein into the supernatant by introducing self-cleavage of the intein, 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). However, 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%.

[0006] Therefore, a low-cost, high-efficiency preparation technology is needed to produce high-purity, high-activity target proteins. Summary of the invention

[0007] 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, especially 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.

[0008] In the first aspect of the present invention, 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.

[0009] In some embodiments, 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 nanoantibody NB21, xylanase, caplacizumab and glutathione sulfhydryltransferase (GST).

[0010] In some embodiments, 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.

[0011] In some embodiments, the self-aggregating peptide comprises a self-assembling peptide, including a surface-active short peptide, such as L6KD (SEQ ID NO: 5), or one or more tandemly repeated amphipathic β-folded short peptides, such as ELK16 (SEQ ID NO: 6). In some embodiments, the self-aggregating peptide comprises a salt concentration-responsive self-aggregating peptide, including α-helical MpA or β-folded short peptides EIK8 (SEQ ID NO: 58) and EFR8 (SEQ ID NO: 59).

[0012] In the second aspect of the present invention, an isolated polynucleotide is provided, which comprises a nucleotide sequence encoding the fusion protein of the first aspect or a complementary sequence thereof.

[0013] In the third aspect of the present invention, an isolated polynucleotide is provided, 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.

[0014] In a fourth aspect of the present invention, a construct, such as an expression construct, is provided, comprising the polynucleotide of the second aspect or the third aspect.

[0015] In the fifth aspect of the present invention, a host cell is provided, 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.

[0016] In some embodiments, 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.

[0017] In a sixth aspect of the present invention, a method for producing and purifying a target protein is provided:

[0018] (a) preparing the fusion protein of the first aspect;

[0019] (b) obtaining an insoluble fraction containing the fusion protein;

[0020] (c) releasing the target protein from the insoluble portion by cleaving a cleavage site in the fusion protein; and

[0021] (d) optionally, recovering and / or purifying the target protein.

[0022] In some embodiments, step (a) of the method comprises: culturing the host cell of the fifth aspect to express the fusion protein. In a further embodiment, 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.

[0023] In some embodiments, a method for high-throughput production and purification of multiple target proteins is provided, the method comprising the following steps:

[0024] (a) preparing the fusion protein of the first aspect in parallel, for example, culturing the host cell of the fifth aspect in a multi-well plate such as a 48, 96, or 384 deep-well plate to express the fusion protein;

[0025] (b) obtaining an insoluble portion containing the fusion protein by aggregation of the self-aggregating peptides, for example, placing it in a multi-well plate such as a 48-, 96-, or 384-well filter plate;

[0026] (c) releasing the target protein from the insoluble portion by cleaving the cleavage site in the fusion protein; and

[0027] (d) optionally, recovering and / or purifying the target protein. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 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 are shown. A: Expression and purification strategy; B: SAP-Linker-Mtu-POI vector structure diagram.

[0029] Figure 2The SDS-PAGE analysis results 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 protein are shown. 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.

[0030] Figure 3 The high-throughput expression and purification strategy of the target protein and the SDS-PAGE analysis results are shown. A: Schematic diagram of the expression and purification strategy; B: SDS-PAGE analysis results of the target protein, Xyl: xylanase, Cap: capecitabine.

[0031] Figure 4 The results of fermentation production, expression and purification of Capacizumab are shown in Figure 1. A: OD and glucose control process of fermentation production process; B: SDS-PAGE analysis results of Capacizumab expression and purification.

[0032] Figure 5 The protein purification method based on salt concentration responsive self-aggregating peptide and linker peptide and the structure diagram of the expression vector used are shown. A: Expression and purification strategy; B: SAP-Linker-Mtu-POI vector structure diagram.

[0033] Figure 6 The figure shows the expression and salt purification of human growth hormone (hGH, SEQ ID NO: 10) fusion protein mediated by 0.7M (NH4)2SO4, as well as the SDS-PAGE result 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.

[0034] Figure 7 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 are shown. A: Expression and purification strategy; B: POI-Mxe-Linker-ELK16 vector structure diagram.

[0035] Figure 8The figure shows the results of SDS-PAGE analysis of the intein Mxe (SEQ ID NO: 4) for the expression and purification of xylanase fusion protein.

[0036] Fig. 9 The SDS-PAGE analysis results of the connecting peptides HD (SEQ ID NO: 44) and HFBII (SEQ ID NO: 45) for fusion protein expression and purification are shown. 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.

[0037] Fig.10 The SDS-PAGE analysis results of the connecting peptides P1-P10 for the expression and purification of the fusion protein are shown. A: SDS-PAGE analysis results of the expression and purification of insulin-like growth factor-1 (IGF-1); B: SDS-PAGE analysis results of the expression and purification of capecitabine.

[0038] Fig.11 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 are shown. 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. DETAILED DESCRIPTION OF THE INVENTION

[0040] 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.

[0041] As used herein, 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. For example, "A and / or B" encompasses "A," "A and B," and "B." For example, "A, B, and / or C" encompasses "A," "B," "C," "A and B," "A and C," "B and C," and "A and B and C."

[0042] Unless otherwise indicated, 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).

[0043] 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.

[0044] 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.

[0045] As used herein, "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.

[0046] In the first aspect, 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.

[0047] As used herein, "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.

[0048] In one embodiment, 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. For example, the targeting molecules can be antibodies that specifically target tumor antigens.

[0049] In some embodiments, 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.

[0050] In some embodiments, 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. In some embodiments, the target protein of the present invention is a nanobody. In some embodiments, the target protein of the present invention is an enzyme. In some embodiments, the length of the target protein can be 20-500 amino acids, for example, about 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500 amino acids.

[0051] In some embodiments, 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).

[0052] In some embodiments, 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.

[0053] As used herein, human growth hormone (hGH) 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 physiologically 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 a variety of diseases, and there are 11 growth hormone indications approved by the US FDA. Currently, the global sales of growth hormone exceed US$3 billion.

[0054] As used herein, insulin-like growth factor (IGF) 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).

[0055] As used herein, nanoantibodies 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 nanoantibodies 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).

[0056] On September 3, 2018, EMA approved Sanofi's nano-antibody drug Cablivi (capacilizumab) for the treatment of acquired thrombotic thrombocytopenic purpura (aTTP) in adults. Acquired or immune-mediated thrombotic thrombocytopenic purpura (TTP) is a rare thrombotic microangiopathy (TMA) characterized by thrombocytopenia and hemolytic anemia. 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.

[0057] As used herein, 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.

[0058] As used herein, glutathione S-transferase (GST) is one of the most important phase II metabolic enzymes for biotransformation in vivo and is the main detoxification system for cell resistance to damage and cancer. Due to its solubilizing effect during fusion expression and its specific affinity for its substrate - glutathione, it is often used as a tag or model protein for protein purification.

[0059] As used herein, "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.

[0060] As used herein, "active aggregate" means that the target protein portion can still be correctly folded and retain activity or that the target protein portion in the aggregate can retain activity after being released from the fusion protein, preferably in a soluble state.

[0061] Self-aggregating peptides (such as amphiphilic self-assembling peptides) have the ability to induce the formation of active aggregates. When these self-aggregating peptides are fused with the target protein and expressed in the host cell, the expressed fusion protein will form insoluble aggregates. The formation of aggregates has many advantages. First, it can prevent the degradation of the fusion protein by intracellular proteases, thereby increasing its stability. Secondly, the formation of aggregates has the potential to reduce the growth burden of the fusion protein on the host cell, thereby increasing the yield of the target protein. After cell lysis, the insoluble aggregates in the cell lysate can be collected by simple centrifugal precipitation or filtration methods, thereby achieving preliminary purification of the fusion protein and removing soluble impurities. Then, by cutting the cleavage site located at the connection site between the self-aggregating peptide and the target protein, the soluble part containing the target protein is released from the insoluble part (precipitate) and dispersed in the supernatant. Finally, simple centrifugal precipitation or filtration methods are used again to remove insoluble impurities to obtain the soluble target protein. This method based on self-aggregating peptides can simplify the protein separation and purification steps, avoid the use of expensive purification columns and other equipment, and thus significantly reduce production costs. This protein production method based on self-aggregating peptides has broad application prospects, especially in the fields of biopharmaceuticals and protein engineering, providing a new approach for efficient and economically feasible protein production.

[0062] Common self-aggregating peptides in this field can be divided into three categories (Yang et al., Biotechnol Lett, 2018, 40:745–753). The first category induces the formation of stable active aggregates; the second category remains soluble in the cell, and under certain external conditions, such as pH and salt concentration, it responds to external conditions to form aggregates; the third category, such as N pro Due to the induction of the formation of inactive aggregates, the subsequent protein purification process requires an additional denaturation process, which is time-consuming and labor-intensive and will not be discussed in detail in the following discussion.

[0063] Some 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 β-folded short peptides, amphiphilic α-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 self-aggregating peptides of the present invention include L6KD, whose amino acid sequence is shown in SEQ ID NO: 5. In addition, surfactant-like peptides with similar structures (such as L6KD, L6K2 (SEQ ID NO: 60), L6D2 (SEQ ID NO: 61), etc.) have similar activities, and can promote the fusion protein to form insoluble active aggregates in cells (Zhou et al., Microbial Cell Factories, 2012, 11, 10). An amphiphilic β-folded short peptide refers to a short peptide consisting of 4-30 amino acid residues, in which hydrophobic amino acids and charged hydrophilic amino acids are arranged alternately to form a β-folded structure. In this structure, the hydrophobic amino acid residues are located on one side, and the alternating positively charged 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. In general, the longer the length of the amphiphilic β-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 β-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.

[0064] MpA is an amphipathic α-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 hydrophobic interactions induced by salt ions 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 EW et al., Proceedings of the National Academy of Sciences, 2005, 102: 12656-12661; Lin Zhanglin et al., WO2022253266A1).

[0065] In one embodiment, the salt is selected from monovalent metal salts such as potassium salts or sodium salts, divalent metal salts such as magnesium salts, calcium salts, manganese salts or copper salts, or ammonium salts, preferably ammonium salts, potassium salts or sodium salts. In one embodiment, the anion of the salt is selected from sulfate, hydrogen phosphate, acetate, halide ions such as fluoride, chloride, bromide or iodide, nitrate, perchlorate, or thiocyanate ion, preferably sulfate, hydrogen phosphate, chloride or acetate. In one embodiment, 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.

[0066] As used herein, "spacer" refers to a polypeptide composed of an amino acid sequence of a certain length, which includes a sequence for achieving cutting and connects the various parts of the fusion protein without affecting the structure and activity of each part. These cutting sequences include enzymatic cleavage sequences that can be used for protease recognition, or intein sequences for self-cleavage, etc. In certain applications, such as in the production of polypeptide drugs, 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. For this reason, in some embodiments, the spacer in the fusion protein of the present invention also includes a cleavage site. By cutting the 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.

[0067] In some embodiments, 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, and 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. In some embodiments, the cleavage site is a pH-dependent cleavage site. In some embodiments, 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.

[0068] In some embodiments, 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. In some embodiments, the spacer consists of a cleavage site described herein, such as a self-cleavage site.

[0069] In the fusion polypeptide of the present invention, the spacer is directly connected to the target polypeptide portion and / or the salt concentration responsive self-aggregating peptide portion. In other embodiments, the spacer further comprises an amino acid linker at its N-terminus and / or C-terminus. In other embodiments, the spacer is connected to the target polypeptide portion and / or the salt concentration responsive self-aggregating peptide portion through an amino acid linker. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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.

[0070] In some specific embodiments, the spacer comprises an intein, which comprises a self-cleavage site. As used herein, 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 the chemical method, 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.

[0071] In some specific embodiments of the present invention, the intein is selected from Mxe GyrA, Ssp DnaB or MtuΔI-CM. The "MtuΔI-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 the N-terminal 110 amino acids and the C-terminal 58 amino acids, obtaining a very small intein, and then introducing four mutations C1A, V67L, D24G, D422G (Wood et al., Nature Biotechnology, 1999, 17: 889-892). In some embodiments, the MtuΔI-CM comprises the amino acid sequence shown in SEQ ID NO: 3. In some embodiments, the nucleotide sequence encoding the MtuΔI-CM has the nucleotide sequence shown in SEQ ID NO: 36. In some embodiments, the MtuΔI-CM is connected to the C-terminus of the target polypeptide portion. In some embodiments, the intein MtuΔI-CM can be induced to self-cleave at its carboxyl terminus by a buffer system at pH 5.5-6.8, such as pH 6.2. In some embodiments, the spacer is MtuΔI-CM or a mutant thereof.

[0072] In some embodiments, the intein is Mxe GyrA, which has a sequence shown in SEQ ID NO: 4. In some embodiments, Mxe GyrA is connected to the C-terminus of the target protein. In some embodiments, by adding an appropriate amount of dithiothreitol (DTT) in the buffer system, Mxe GyrA can be induced to self-cleave at its amino terminus. As needed, the technician can determine the concentration and reaction time of DTT. In addition, DTT can also be removed in subsequent operations. In some embodiments, the spacer is Mxe GyrA or a mutant thereof.

[0073] In previous work, it was found that such protein purification methods based on cleavable self-aggregating peptides generally have the phenomenon of partial disaggregation of self-aggregating peptides after cleavage, which reduces the purity of the target protein; and some proteins have low cleavage efficiency and are easy to precipitate after cleavage, resulting in low yield of the target protein. To solve these problems, the inventors introduced a connecting peptide.

[0074] As used herein, "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.

[0075] In some embodiments, 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).

[0076] As used herein, the term "identity" is used to refer to the matching 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. For example, 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)).

[0077] As used herein, elastin-like polypeptide (ELP) is a polypeptide polymer derived from natural elastin and can be synthesized artificially. ELP is composed of multiple VPGXG repeating units (X = A, G, V or E), and its aggregation characteristics are related to the number of repeating units (Banki et al., Nature Methods, 2005, 2 (9): 659-661). ELP can undergo a reversible phase transition process, called inverse temperature transition. When the temperature is lower than 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, and ELP fusion protein also has this characteristic. It should be pointed out that when the phase transition occurs, only the ELP is agglutinated, and the fused exogenous protein does not denature or precipitate. By utilizing this characteristic of ELP, the ELP tag can be used to purify ELP fusion proteins conveniently and quickly, and the purification process does not require chromatographic separation, thus avoiding expensive affinity resins and purification equipment. At the same time, the concentration and replacement of buffers of ELP fusion proteins also become very simple. The aggregation of ELP fusion proteins is triggered by heating or increasing the salt ion concentration, and the ELP fusion proteins are separated from the expression host proteins by centrifugation. The precipitated ELP fusion proteins are re-dissolved with a low-salt and low-temperature solution, and then centrifuged to remove insoluble proteins to obtain ELP fusion proteins. 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). In order to ensure that it has good solubility under experimental conditions, the ELP used in this article contains repeating pentapeptides (VPGXG) 15 (SEQ ID NO: 62), wherein X can independently be Ala, Gly, Val or Glu in a ratio of 4:1, and its phase transition temperature is 45-60°C (Jiang et al., Science Advances, 2020, 6(41):eabc4824-4833).

[0078] 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.

[0079] As used herein, 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). The propeptide is located at the N-terminus of the polypeptide chain and is essential in the folding process of subtilisin E. Experiments have shown that after being treated with a denaturant, subtilisin E becomes an unstructured polypeptide chain. When 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. However, 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. et al., FEBS Letters, 2010, 584(23): 4789-4796) When studying the in vivo folding of nattokinase (NK), it was found that the mature peptide co-expressed with IMC can fold correctly, while in the absence of IMC, the mature peptide becomes an inclusion body, and even in vitro renaturation cannot be successful, indicating that IMC is also indispensable for the folding of the mature peptide in vivo.

[0080] In vitro studies have also found that the propeptide of 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). In the study of some propeptide mutants, it was found that some mutants lost the function of assisting protease folding, but their role as inhibitors was more significant, and unlike the original propeptide, they could spontaneously form secondary structures without the presence of protease (Cynthia et al., Journal of Biological Chemistry, 2001, 305, 1: 151-165), and the chaperone activity and protease inhibitor activity of the propeptide may be separated.

[0081] Sequence analysis of protease propeptides revealed that propeptides are almost always less conserved than their homologous catalytic domains and contain a large number of charged amino acids. The propeptide has two conserved domains N1 and N2 (motifs N1 and N2), including the hydrophobic amino acid residue 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 conserved domain is flanked by charged residues, which are speculated to initiate the folding of the protease through "hydrophobic collapse". Recently, Lin Min et al. found that the propeptide PEP (Peptide for Expression-Promoting) from the S8 protein family protease of Deinococcus gobiensis (CN112481286B, 2022) can enable the soluble expression of four milk proteins that were not originally expressed, including αS2-casein, κ-casein, α-lactalbumin and bovine serum albumin in Escherichia coli.

[0082] In some embodiments, 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. In one embodiment, 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.

[0083] In some embodiments, 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.

[0084] In some embodiments, 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. In particular, the connecting peptide is from Deinococcus gobiensis, preferably the propeptide of a protease of the S8 protein family.

[0085] In some embodiments, 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.

[0086] In some embodiments, 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.

[0087] Those skilled in the art will appreciate that in order to reduce the mutual interference between different parts in the fusion protein of the present invention, different parts of the fusion protein can be connected by an amino acid linker. As used herein, "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). In some embodiments, the amino acid linker is selected from a GS type amino acid linker and a PT type amino acid linker. In one embodiment, the length of the amino acid linker can 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. In some embodiments, the amino acid sequence of the GS type amino acid linker used in the present invention is shown in SEQ ID NO: 8. In other embodiments, the amino acid sequence of the PT type amino acid linker used in the present invention is shown in SEQ ID NO: 9.

[0088] The inventors were surprised to find that 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.

[0089] High-throughput protein purification is of great significance in drug screening and other fields. For example, in the screening process of antibody drugs, the preferred screening method is surface display technology, such as phage surface display, which can quickly screen 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). However, the protein sequences obtained by surface display are likely to be false positives or have 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 techniques require the purity of the test object to be >90%. Therefore, after dozens to hundreds of candidate sequences are screened by surface display technology, the proteins containing these sequences need to be expressed and purified one by one, and affinity tests are performed. There are already some commercial His-Tag-based high-throughput purification technologies, such as small gravity columns, but this process is time-consuming, labor-intensive and costly, limiting its large-scale application.

[0090] 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. For example, 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.

[0091] In a second aspect, the present invention provides an isolated polynucleotide comprising a nucleotide sequence encoding the fusion protein of the first aspect or a complementary sequence thereof.

[0092] In the third aspect, 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.

[0093] As used herein, the term "expression" generally refers to the process of producing polypeptides by transcription and translation of polynucleotides. In this article, the term "expression" can be understood as "heterologous expression", that is, it refers to expressing in a host cell or expressing in vitro a polypeptide encoded by a heterologous nucleic acid.

[0094] In a fourth aspect, the present invention provides a construct, such as an expression construct, comprising the polynucleotide of the second aspect or the third aspect.

[0095] As used herein, "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. For example, 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. Typically, in an expression construct, the nucleotide sequence of interest is operably linked to a regulatory sequence.

[0096] As used herein, "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 recognition sequences.

[0097] As used herein, the term "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.

[0098] In the expression construct of the present invention, 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.

[0099] 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. In a specific embodiment, the expression construct of the present invention is derived from pET30a (+) of Novagen.

[0100] In a fifth aspect, the present invention provides a host cell comprising the polynucleotide of the second aspect of the present invention or transformed with the construct of the fourth aspect of the present invention, such as an expression construct, wherein the host cell is capable of expressing the fusion protein.

[0101] 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. In a preferred embodiment, the host cell is an Escherichia cell, preferably Escherichia coli. In a specific embodiment of the present invention, the host cell used is Escherichia coli BL21 (DE3) strain cell (Novagen).

[0102] A polynucleotide or construct of the invention, e.g., an expression construct, 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.

[0103] In one embodiment, 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.

[0104] In one embodiment, 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).

[0105] In a sixth aspect, the present invention provides a method for producing and purifying a target protein, comprising:

[0106] (a) preparing the target protein into the fusion protein described in the first aspect;

[0107] (b) generating active aggregates by aggregation of self-aggregating peptides to obtain an insoluble fraction containing the fusion protein;

[0108] (c) releasing the target protein from the insoluble portion by cleaving a cleavage site in the fusion protein; and

[0109] (d) optionally, recovering and / or purifying the target protein.

[0110] Herein, 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.

[0111] In one embodiment, 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.

[0112] In one embodiment, 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.

[0113] In one embodiment, step (a) of the method comprises: constructing an expression construct (e.g., a plasmid or a viral vector) comprising a nucleotide sequence encoding a fusion protein formed by fusion of the peptide described in the first aspect of the invention and a target protein, transforming host cells with the expression construct, culturing the transformed host cells under conditions suitable for expression of the fusion protein, and optionally isolating and / or collecting the host cells.

[0114] In one embodiment, 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 can be introduced into a host cell for expression or expressed in an in vitro cell-free system to produce a fusion protein.

[0115] 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).

[0116] Herein, 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.

[0117] In one embodiment, the method step (b) includes: collecting and lysing cells, and / or collecting supernatant or precipitate containing fusion protein. For example, if the self-aggregating peptide spontaneously or under certain conditions in the host cell self-aggregates, 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). For example, when using a self-aggregating peptide that induces aggregation with salt (especially when the fusion protein can be soluble after expression), after the fusion protein is expressed, the aggregation of the self-aggregating peptide is induced by increasing the salt concentration (e.g., after the host cell is expressed, the cells are broken to obtain the supernatant, and then salt is added), resulting in the aggregation of the fusion protein, and the active aggregates of the fusion protein are obtained. 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.

[0118] 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.

[0119] Herein, 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. For example, 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.

[0120] As described herein, 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.

[0121] Herein, the target protein released can be recovered by any suitable method or means. For example, if the released target protein 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. 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.

[0122] In one embodiment, the method comprises the following steps: (a) culturing the host cells of the present invention to express the fusion protein of the present invention; (b) lysing the host cells, then removing the soluble portion of the cell lysate and recovering the insoluble portion (the fusion protein is insoluble in the cytoplasm), or removing the insoluble portion of the cell lysate and recovering the soluble portion (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 portion at a certain salt concentration; (c) releasing a soluble target protein from the fusion protein of the insoluble portion by cutting the cleavage site; and (d) recovering the soluble portion containing the target protein, and optionally separating or purifying it. A schematic diagram of the method of the present invention can be found in Figure 1 A.

[0123] In the present invention, 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 carried out under weakly alkaline pH conditions (such as pH 7.5-8.5), thereby lysing the cell membrane of the host cells, so that the active aggregates are released from the cell, but still remain in an insoluble state. In other embodiments, the fusion protein is recovered in a soluble form, and the fusion protein in a precipitated state is obtained by changing the ion concentration.

[0124] In a further embodiment, the present invention provides a method for high-throughput production and purification of multiple target proteins, comprising:

[0125] (a) preparing the fusion proteins of the first aspect in parallel with each other respectively, for example, expressing a polynucleotide encoding the fusion protein in a host cell or an in vitro cell-free system, the polynucleotide being obtained, for example, by cloning a nucleotide sequence encoding the target protein into a multiple cloning site of the polynucleotide of claim 7, for example, culturing the host cell of the fifth aspect in a 96-deep-well plate to express the fusion protein;

[0126] (b) generating active aggregates by aggregation of self-aggregating peptides to obtain an insoluble fraction containing the fusion protein, for example, in a 96-well filter plate;

[0127] (c) releasing the target protein from the insoluble portion by cleaving the cleavage site in the fusion protein; and

[0128] (d) optionally, recovering and / or purifying the target protein.

[0129] 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.

[0130] As used herein, "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.

[0131] Different from the existing production and purification technologies, the fusion method of the present invention based on self-aggregating peptides and connecting peptides can successfully produce a large amount of active target protein. The self-aggregating peptide 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. In particular, the inventors were surprised to find that this method only requires a simple centrifugation operation 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.

[0132] As used herein, the word "or" is intended to include "and" unless the context indicates otherwise.

[0133] As used herein, "optional" or "optionally" means that the subsequently described event or circumstance occurs or does not occur, and the description includes instances where the event or circumstance occurs and instances where it does not occur. For example, an optionally included step means that the step exists or does not exist.

[0134] As used herein, 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.

[0135] As used herein, when a specific value or ratio is listed for a feature in the specification, a range consisting of any two of the values ​​or ratios is also included. For example, when the values ​​1, 2, 3, and 4 are listed, 1-2, 1-3, 1-4, 2-3, 2-4, and 3-4 are also included.

[0136] Although various embodiments and various aspects of the present invention are shown and described herein, it is apparent to those skilled in the art that these embodiments and various aspects are only examples of the present invention. Those skilled in the art can make multiple changes, modifications and substitutions without departing from the scope of the spirit of the present invention. It should be understood that various alternatives of the embodiments of the present invention described herein can be used in the implementation of the present invention. Example

[0137] To make the technical solutions and advantages of the present invention clearer, the embodiments of the present invention will be further described in detail by examples. It should be understood that the examples should not be construed as limiting, and those skilled in the art can make further adjustments to the embodiments based on the principles of the present invention.

[0138] Unless otherwise specified, the methods used in the following examples are conventional methods. For specific steps, see, for example, Molecular Cloning: A Laboratory Manual (Sambrook, J., Russell, David W., Molecular Cloning: A Laboratory Manual, 3rd edition, 2001, NY, Cold Spring Harbor). All primers used were synthesized by Shanghai Bio-Technology Co., Ltd.

[0139] Example 1: Construction of L6KD-Mtu-POI, L6KD-ELP-Mtu-POI, L6KD-PEP-Mtu-POI fusion protein expression vectors

[0140] POI represents the target protein. In the examples of the present application, 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.

[0141] Table 1: Oligonucleotide primers used in this example

[0142] Primers Nucleotide sequence SEQ ID NO ELP-F2 5′-CCGACGCCGACCCCAGAAT-3′ 12 ELP-R2 5′-GCGTGCCTTCAGCCAGC-3′ 13 MF 5′-GCGCTGGCTGAAGGCACG-3′ 14 MR 5′-GAATTCTGGGGTCGGCGTCG-3′ 15 ORI-F 5′-TGTGACCGTCTCCGGGAGCTG-3′ 16 ORI-R 5′-CAGCTCCCGGAGACGGTCACA-3′ 17 END-F 5′-TGAGATCCGGCTGCTAACAA-3′ 18 END-R 5′-GCTTTGTTAGCAGCCGGATC-3′ 19 POI-F 5′-GTTGCGGAAGGCGTTGTGGTTC-3′ 20 POI-R 5′-GAACCACAACGCCTTCCGCAAC-3′ 21

[0143] First, the ELP (SEQ ID NO: 1) polynucleotide sequence was designed (Banki MR et al., Nature Methods, 2005, 2 (9): 659-661), and the PEP (SEQ ID NO: 2) (NCBI No.: WP_014683986) polynucleotide sequence was obtained from NCBI, and the gene fragment was obtained by gene synthesis by Beijing Ruiboxingke. Using the synthesized gene as a template, ELP-F2 and ELP-R2 as primers, PCR reaction amplification was performed to obtain ELP and PEP polynucleotide fragments respectively. 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. After the reaction was completed, the PCR amplification product was separated and recovered by 1% agarose gel.

[0144] Using pET32-L6KD-MtuΔI-CM-hGH (Lin et al., Chemical Engineering Science, 2022, 262: 118052) as a template, wherein 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, MF, ORI-R and ORI-F, MR were used as primers to amplify the Mtu-hGH fragment and the Backbone-L6KD fragment by PCR reaction. The PCR reaction used Q5 polymerase from NEB, and the PCR conditions were: 98°C 30sec, 98°C 10sec, 64°C 30sec, 72°C 120sec, for a total of 30 cycles; finally 72°C 2min. After the reaction, the PCR amplification product was separated and recovered by 1% agarose gel. The ELP, Mtu-hGH fragment and Backbone-L6KD fragment, as well as the PEP, Mtu-hGH fragment and Backbone-L6KD fragment were respectively subjected to Gibson assembly (for example, see https: / / www.neb.cn / applications / cloning-and-synthetic-biology / dna-assembly-and-cloning / gib son-assembly), and the homology arm sequences were CCGACGCCGACCCCAGAATTC (SEQID 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. The assembled product was transformed into Escherichia coli DH5α competent cells, and the transformed cells were spread on LB plates supplemented with 100 μg / mL carbenicillin to screen positive clones. The plasmids were extracted and sequenced. The sequencing results showed that the cloned plasmid sequences were correct.

[0145] 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, END-F and ORI-R as primers, and the Backbone-L6KD-Mtu fragment was amplified by PCR reaction using the above-mentioned pET32-L6KD-MtuΔI-CM-hGH as a template, and ORI-F and POI-R as primers. After the PCR reaction was completed, the PCR amplification product was 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 templates of the Backbone-L6KD-Mtu fragment were replaced with pET32-L6KD-ELP-Mtu-hGH and pET32-L6KD-PEP-Mtu-hGH, respectively. The Gibson Assembly product was transformed into Escherichia coli DH5α competent cells, and the transformed cells were spread on LB plates supplemented with 50 μg / mL kanamycin to screen positive clones, and the plasmids were extracted and sequenced.

[0146] The construction process of other plasmids is similar. IGF-1 (SEQ ID NO: 38), xylanase (SEQ ID NO: 41), caplacizumab (SEQ ID NO: 42) and GST (SEQ ID NO: 43) genes are respectively 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). Take GST as an example: pET30-CpA-Mtu-GST was used as a template, and POI-F and ORI-R were used as primers to amplify the GST-Backbone fragment by PCR reaction. Next, pET30-L6KD-Mtu-NB21 was used as a template, and ORI-F and POI-R were used as primers to amplify the Backbone-L6KD-Mtu fragment by PCR reaction. After the PCR reaction was completed, 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 is similar, except that the PCR templates of the expression vectors are replaced with pET30-L6KD-ELP-Mtu-NB21 and pET30-L6KD-PEP-Mtu-NB21, respectively. The Gibson Assembly product was transformed into E. coli DH5α competent cells, and the transformed cells were plated on LB plates supplemented with 50 μg / mL kanamycin to screen positive clones, and the plasmids were extracted and sequenced. The structures of the constructed pET30-L6KD-Mtu-POI, pET30-L6KD-ELP-Mtu-POI and pET30-L6KD-PEP-Mtu-POI plasmids are shown in Figure 1 As shown in B.

[0147] Example 2: Expression and purification of IGF-1 / hGH / Nb21 / xylanase / capacilizumab / GST

[0148] Six different types of target proteins were purified, including human insulin-like growth factor IGF-1, human growth hormone hGH, new coronavirus nanoantibody Nb21, xylanase, capecitabine and glutathione S-transferase GST.

[0149] 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. The above expression strains were inoculated into LB liquid medium containing 50 μg / mL kanamycin or 100 μg / mL carbenicillin, and cultured in a shaking incubator at 37°C until the logarithmic phase (OD 600 =0.4-0.6), add 0.2mM IPTG, induce at 18℃ for 24 hours, measure bacterial concentration OD 600 (The following will be 1mL OD 600 The cell mass of 1 is called 1OD).

[0150] The cells were resuspended to 20 OD / mL with lysis buffer B1 (2.4 g Tris, 29.22 g NaCl, 0.37 g Na2EDTA·2H2O 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 separately. 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 precipitate was centrifuged at 4°C, 15000g for 20 min, and the precipitate was resuspended with an equal volume of lysis buffer. The obtained supernatant and precipitate were subjected to SDS-PAGE detection together with the supernatant and precipitate before cleavage. The results are as follows Figure 2As shown, lanes ES, EP, CP, and CS are the expression and purification samples of the target protein, respectively, ES: cell lysate supernatant; EP: cell lysate precipitate, in which clear aggregates expressed by the fusion protein can be detected; CP: precipitate separated after cleavage; CS: supernatant separated after cleavage, in which 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 5μg, 2.5μg, 1.25μg, 0.625μg, and 0.3125μg, respectively.

[0151] According to the protein quantitative standard, 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 MtuΔI-CM cleavage efficiency, the recovery rate and its purity in the supernatant were calculated. The results are shown in Table 2.

[0152] Table 2: Expression and purification of IGF-1 / hGH / Nb21 / xylanase / capacilizumab / GST

[0153]

[0154] a Protein aggregate yield (volume per liter of LB medium), b The yield of the target protein after intein-mediated self-cleavage (volume per liter of LB medium), c Intein-mediated cleavage efficiency = 100% × (aggregate expression before cleavage - aggregate remaining after cleavage) / aggregate yield before cleavage, d Recovery rate = 100% × actual yield of target protein / theoretical yield of target protein that can be produced when protein aggregates are completely cleaved.

[0155] 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 are all present in the form of precipitation, wherein the aggregate expression amount of the L6KD-Mtu system is 205-545 mg / L LB culture fluid. The target protein is separated from L6KD-Mtu by self-cleavage of the intein MtuΔI-CM, the cleavage efficiency is 38-70%, and the yield of the target protein released into the supernatant after the L6KD-Mtu tag is 2.2-57 mg / L LB culture fluid, and the purity is 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%.

[0156] Example 3: High-throughput expression and purification of target protein

[0157] High-throughput protein expression and purification processes such as Figure 3 As shown in A. 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 μL LB medium supplemented with 50 μg / mL kanamycin or 100 μg / mL carbenicillin, and cultured overnight at 37°C with shaking at 800 rpm. The overnight culture was transferred to a 96 deep-well plate at a ratio of 1:50, each well containing 800 μL fresh LB medium supplemented with the corresponding antibiotics, and then cultured at 37°C for 2 hours until OD 600Reach 0.4-0.8. The culture temperature was lowered to 18°C, and 200 μL of LB medium containing 1 mM IPTG was added to start protein expression. Then continue to culture at 18°C ​​for 24 hours. After the culture is completed, the cells are harvested by centrifugation at 4,000g, 4°C for 30 minutes. 200 μL of BugBuster reagent (Millipore) was added to each well to lyse E. coli. The cell lysate was then transferred to a 96-well filter plate, centrifuged at 2,500g, 4°C for 30 minutes, the filtrate was discarded, and 200 μL of buffer B1 was added to wash the protein aggregates on the filter membrane twice. Then 100 μL of cutting buffer was added, incubated at 25°C for 24 hours, and the filtrate was collected by centrifugation for SDS-PAGE analysis. The purification results are as shown below. Figure 3 As shown, lanes I-IV are protein quantitative standards containing bovine serum albumin BSA, and the loading amounts are 5 μg, 2.5 μg, 1.25 μg, 0.625 μg, 0.3125 μg, and 0.15625 μg, respectively. The statistical results of the relative grayscale analysis of the purified target protein are shown in Table 3.

[0158] Table 3: Fermentation production and protein expression and purification

[0159] Target protein NB21 Xyl Cap GST hGH <![CDATA[Production a (mg / L)]]> 26 16 10 10 17 Final protein concentration (mg / L) 208 128 80 80 136 purity(%) 96 98 90 94 98

[0160] a Target protein yield after intein-mediated self-cleavage (volume per liter of LB medium).

[0161] The results showed that after high-throughput expression and purification, our method can still obtain the above target protein with a yield of 10-26 mg / L culture fluid and a purity of 90-98%. The final protein concentration is 80-208 mg / L and the purity is >90%, which can meet the needs of most downstream detection or screening.

[0162] Example 4: Fermentation production and expression and purification of capecitabine

[0163] In order to explore the commercial application potential of the present invention, 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 fermentor using a DO-stat fed-batch feeding strategy.

[0164] The strain obtained in Example 2 (containing the plasmid pET30-L6KD-PEP-Mtu-Caplacizumab) was streaked and revived. 140 mL of LB medium was inoculated as seed liquid one day in advance. For fermentation in a 5-L fermenter, the initial fermentation broth volume was 2L, the seed culture broth was inoculated at a ratio of 7%, the initial fermentation temperature was 37°C, the rotation speed was 200 rpm, the entire fermentation process was ventilated at 3L / min, and 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 1,000 rpm. During the fermentation and culture process, the initial glucose concentration was 10 g / L, which was consumed about 5 hours after inoculation. At this time, the dissolved oxygen and pH value of the culture medium rose, and feeding began. During the feeding process, in order to prevent the accumulation of acetate in the culture medium during the fermentation process, the concentration of glucose in the fermentation medium was controlled at 0.1-1.5 g / L. Samples were taken every 2 hours after inoculation to measure the bacterial OD 600 and the glucose concentration in the culture medium, when the bacterial OD 600 When the temperature reaches 30-40℃, the temperature is lowered to 18℃, and IPTG with a final concentration of 0.2mM is added for induction. After 28h of induction, the OD of the bacteria is 600 During the fermentation process, samples were taken at 24h and 28h of induction, and the cells were collected and purified according to the method shown in Example 2. The expression and purification results are shown in FIG. Figure 4 As shown, lanes I-VI are protein quantitative standards containing bovine serum albumin BSA, and the loading amounts are 5 μg, 2.5 μg, 1.25 μg, 0.625 μg, 0.3125 μg, and 0.15625 μg, respectively. The relative gray analysis statistics of capecitabine released into the supernatant after cleavage and the detailed statistical results of the purification process are shown in Table 4.

[0165] Table 4: Fermentation production and protein expression and purification of capecitabine

[0166]

[0167] a Protein aggregate yield (volume per liter of LB medium), b The yield of the target protein after intein-mediated self-cleavage (volume per liter of LB medium), c Intein-mediated cleavage efficiency = 100% × (aggregate expression before cleavage - aggregate remaining after cleavage) / aggregate yield before cleavage, d Recovery rate = 100% × actual yield of target protein / theoretical yield of target protein that can be produced when protein aggregates are completely cleaved.

[0168] The results showed that at 24 hours of induction, the output of fusion protein aggregates was 12.1 g / L of culture medium, the output 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 output of fusion protein aggregates was 13.5 g / L of culture medium, the output 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.

[0169] Example 5: Construction of SAP-Mtu-hGH, SAP-PEP-Mtu-hGH fusion protein expression vector

[0170] SAP represents aggregation peptide. In the present embodiment, 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.

[0171] Table 5: Oligonucleotide primers used in this example

[0172]

[0173] Using the pET30-L6KD-PEP-Mtu-IGF obtained in Example 1 as a template, PEP-F and POI-R as primers were used to amplify the PEP-Mtu fragment by PCR reaction, and using pET30-MpA-Mtu-hGH (Lin Zhanglin et al., WO2022253266A1) as a template, POI-F and 11-R as primers were used to amplify the hGH-Backbone-MpA fragment by PCR reaction. 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. After the reaction was completed, the PCR amplification product was separated and recovered by 1% agarose gel. The PEP-Mtu fragment and the hGH-Backbone-MpA fragment were assembled by Gibson Assembly, and the assembled products were transformed into Escherichia coli DH5α competent cells. The transformed cells were spread on LB plates supplemented with 50 μg / mL kanamycin to screen positive clones, and the plasmids were extracted and sequenced. The sequencing results showed that the cloned plasmid sequences were correct.

[0174] 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. Taking 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°C for 30 seconds, 98°C for 10 seconds, 64°C for 30 seconds, 72°C for 120 seconds, for a total of 30 cycles; and finally 72°C for 2 minutes. After the reaction was completed, 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, respectively. The construction process of EFR8 was similar, except that the corresponding primers were replaced with EFR8-F and EFR8-R, respectively. The assembly product was transformed into Escherichia coli DH5α competent cells, and the transformed cells were spread on LB plates supplemented with 50 μg / 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 Figure 5 As shown in B.

[0175] Example 6: hGH protein purification mediated by MpA, EIK8 and EFR8 phase transition and Mtu-mediated cleavage

[0176] The plasmids 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) were transformed into Escherichia coli BL21 (DE3) competent cells to obtain 6 hGH fusion protein expression strains. The 6 expression strains were inoculated into LB liquid culture medium containing 50 μg / mL kanamycin and cultured in a 37°C shaker until the logarithmic phase (OD 600 =0.4-0.6), add 0.2mM IPTG, induce at 18℃ for 24 hours, measure bacterial concentration OD 600 , centrifuge at 4000 rpm for 25 min at 4°C to harvest the cells, remove the supernatant medium and freeze the cells at -80°C. 600 The cell mass of 1 is called 1OD).

[0177] 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.

[0178] Na2SO4 was added to the lysate supernatant to 0.7M and placed at 4°C overnight for 12h to allow the self-aggregating peptide to fully aggregate. The suspension was then centrifuged at 4°C, 15,000g for 30min, and the precipitate was washed with buffer B4 containing 0.7M Na2SO4 (99.43g Na2SO4, 2.4g Tris, 0.37g EDTA·2N aDissolve in 800 mL of water, adjust pH to 8.0, and add water to 1 L) and wash once, then centrifuge under the same conditions to separate the supernatant and precipitate. Use cleavage buffer B5 containing 0.7 M Na2SO4 (PBS without NaCl, supplemented with 0.7 M Na2SO4, supplemented with 40 mM Bis-Tris, pH 6.2, 2 mM EDTA) to fully resuspend the precipitate and place it at 25°C for 24 hours to allow the intein to fully self-cleave. Incubate at 4°C for 3 hours to allow the aggregated peptide to fully aggregate, and then centrifuge the suspension at 4°C, 16,000g for 30 minutes to separate. The supernatant after cell lysis, the supernatant and precipitate after salt aggregation, and the supernatant and precipitate after cleavage were subjected to SDS-PAGE detection together. The results are as follows Figure 6 As shown, lanes ES, EP, ESS, ESP, CP, and CS are the expression and purification samples of the target protein, respectively, ES: cell lysate supernatant, in which a clear fusion protein band can be detected; EP: cell lysate precipitate, which almost does not contain fusion protein; ESS: supernatant separated after salt aggregation of lysate supernatant; ESP: precipitate separated after salt aggregation of lysate supernatant, in which a clear fusion protein band can be detected; CP: precipitate after cutting of aggregates obtained by adding salt; CS: supernatant after cutting of aggregates obtained by adding salt, in which a clear target protein band can be detected; lane IV is a protein quantitative standard containing bovine serum albumin BSA, and the loading amounts are 5μg, 2.5μg, 1.25μg, 0.625μg, and 0.3125μg, respectively.

[0179] According to the protein quantitative standard, the optical density analysis of the target band was performed using ImageJ (National Institutes of Health) gel quantitative analysis software, and the target protein yield released into the supernatant after the intein-mediated self-cleavage, the aggregation efficiency after salt addition, the MtuΔI-CM cleavage efficiency and the human growth hormone hGH recovery rate and its purity in the supernatant were calculated. The results are shown in Table 6.

[0180] Table 6: hGH purification by 0.7M Na2SO4-mediated MpA / EIK8 / EFR8 phase transition and Mtu-mediated cleavage

[0181]

[0182] a The expression level of the fusion protein in the lysis supernatant (volume per liter of LB medium), b The yield of the target protein after intein-mediated self-cleavage (volume per liter of LB medium), c Aggregation efficiency = 100% × the amount of fusion protein in the precipitate after salt addition / (the amount of fusion protein in the precipitate after salt addition + the amount of fusion protein in the supernatant after salt addition), dIntein-mediated self-cleavage efficiency = 100% × (the amount of fusion protein in the precipitate after salt addition - the amount of fusion protein in the precipitate after cleavage) / the amount of fusion protein in the precipitate after salt addition, e Recovery rate = 100% × actual yield of target protein / theoretical yield of target protein that can be produced by expression supernatant under complete cleavage.

[0183] 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 were all expressed in soluble form, and the amount of soluble expression was 353-537 mg / L. Most of the fusion proteins in the lysate supernatant changed from soluble to precipitate under the condition of 0.7M Na2SO4, and the aggregation efficiency was 92-99%. After the self-cleavage of the intein MtuΔI-CM, the target protein was separated from MpA / EIK8 / EFR8-Mtu, and the cleavage efficiency was 53-93%. The yield of hGH released into the supernatant after cleavage was 51-95 mg / L, and the purity was 80-99%. Among them, the hGH purity of the SAP-PEP-Mtu-hGH fusion protein was the highest, which was 99%.

[0184] Example 7: Construction of a fusion protein expression construct containing the intein Mxe GyrA (SEQ ID NO: 4)

[0185] The required primers for the expression vectors pET30-Xylanase-Mxe-ELK16 and pET30-Xylanase-Mxe-PEP-ELK16 used in the examples of the present application were designed by oligo 7 and synthesized by Shanghai Biotechnology Co., Ltd. as shown in Table 7.

[0186] Table 7: Oligonucleotide primers used in this example

[0187]

[0188]

[0189] Using 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°C 30sec, 98°C 10sec, 64°C 30sec, 72°C 120sec, a total of 30 cycles; finally 72°C 2min. After the reaction, the PCR amplification product was separated and recovered by 1% agarose gel. The Mxe-ELK16-Backbone fragment and the Backbone-Xylanase fragment were assembled by Gibson Assembly.

[0190] Using the pET30-L6KD-PEP-Mtu-IGF obtained in Example 1 as a template, PG-F and PM-R as primers, a PEP fragment was amplified by PCR reaction to obtain a PEP fragment, using pET30-LipA-Mxe-ELK16 as a template, KL-F and ORI-R as primers, and ELK16-Backbone fragment was amplified by PCR reaction, and pET30-Xylanase-Mxe-MpA was used as a template, and ORI-F and Mxe-RP were used as primers to amplify the Backbone-Xylanase-Mxe fragment by PCR reaction. The PEP fragment, ELK16-Backbone fragment, and Backbone-Xylanase-Mxe fragment were assembled by Gibson Assembly. The assembly product was transformed into Escherichia coli DH5α competent cells, and the transformed cells were coated on LB plates supplemented with 50 μg / mL kanamycin to screen positive clones, extract plasmids, and sequence them. The sequencing results showed that the cloned plasmid sequences were all correct. The structures of the constructed pET30-Xylanase-Mxe-ELK16 and pET30-Xylanase-Mxe-PEP-ELK16 plasmids are as follows: Figure 7 As shown in B.

[0191] Example 8: Expression and purification of xylanase

[0192] The strain constructed in Example 7 (containing plasmids pET30-Xylanase-Mxe-ELK16 and pET30-Xylanase-Mxe-PEP-ELK16) was inoculated into LB liquid medium containing 50 μg / mL kanamycin and cultured in a shaking incubator at 37°C until the logarithmic phase (OD 600 =0.4-0.6), 0.2 mM IPTG was added, cells were harvested at 18°C ​​for 24 hours, and the bacterial concentration OD was measured. 600 (The following will be 1mL OD 60 The number of cells equal to 0 is called 1OD)

[0193] The cells were resuspended to 20 OD / mL with lysis buffer B1 (2.4 g Tris, 29.22 g NaCl, 0.37 g Na2EDTA·2H2O 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). Centrifuge at 4 ° C, 12000 rpm for 20 min, and collect the supernatant and precipitate respectively. After washing the precipitate twice with lysis buffer, it was fully resuspended with cleavage buffer (20 mM Tris-HCl, 500 mM NaCl, 40 mM dithiothreitol, 1 mM EDTA, pH 8.5), and placed at 25 ° C overnight for 24 hours to allow the intein to fully self-cleave. The suspension was then centrifuged and the resulting supernatant and precipitate were subjected to SDS-PAGE detection together with the precipitate before cutting (the precipitate was resuspended in the same volume of lysis buffer as in the previous resuspension step). Figure 8 As shown, lanes ES, EP, CP, and CS are the expression and purification samples of the target protein, respectively, ES: cell lysate supernatant; EP: cell lysate precipitate, in which clear aggregates expressed by the fusion protein can be detected; CP: precipitate separated after cleavage; CS: supernatant separated after cleavage, in which clear target protein bands can be detected; lanes I-VI are protein quantitative standards containing bovine serum albumin BSA, and the loading amounts are 5μg, 2.5μg, 1.25μg, 0.625μg, 0.3125μg, and 0.15625μg, respectively.

[0194] According to the protein quantitative standard, 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 xylanase yield released into the supernatant after intein-mediated self-cleavage, the Mxe GyrA cleavage efficiency, the xylanase recovery rate and its purity in the supernatant were calculated. The results are shown in Table 8.

[0195] Table 8: Expression and purification of xylanase

[0196]

[0197] a Protein aggregate yield (volume per liter of LB medium), b The yield of the target protein after intein-mediated self-cleavage (volume per liter of LB medium), c Intein-mediated cleavage efficiency = 100% × (aggregate expression before cleavage - aggregate remaining after cleavage) / aggregate yield before cleavage, d Recovery rate = 100% × actual yield of target protein / theoretical yield of target protein that can be produced when protein aggregates are completely cleaved.

[0198] The two fusion proteins (Xylanase-Mxe-ELK16 and Xylanase-Mxe-PEP-ELK16) used are both present in the form of precipitation, and the aggregate expression amount is 278-282 mg / L LB culture fluid. The two different fusion proteins are self-cleaved by the intein MxeGyrA, and the xylanase is separated from Mxe-ELK16, the cleavage efficiency is 51-70%, the yield of the xylanase released into the supernatant after cleavage is 78-107 mg / L LB culture fluid, and the purity of the xylanase recovered after cleavage is 92-98%. Among them, the xylanase purity of the Xylanase-Mxe-PEP-ELK16 fusion protein is the highest, that is, the xylanase yield of 107 mg / L LB culture fluid and the purity of 98% can be purified in one step through the purification technology based on the self-aggregating peptide and the self-cleavage tag.

[0199] Example 9: Construction of L6KD-HD / HFBII-Mtu-POI, L6KD-ProP-Mtu-POI, L6KD-N1 / N2 fusion protein expression vectors

[0200] POI represents the target protein. In the present application example, HD (SEQ ID NO: 44) refers to the hydrophilic domain, HFBII (SEQ ID NO: 45) refers to hydrophobic protein, ProP refers to protease propeptide, including P1-P10, N1 (SEQ ID NO: 56) and N2 (SEQ ID NO: 57) are truncated PEP, 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 Biotech as shown in Table 9.

[0201] Table 9: Oligonucleotide primers used in this example

[0202] Primers Nucleotide sequence SEQ ID NO ELP-F2 5′-CCGACGCCGACCCCAGAAT-3′ 12 ELP-R2 5′-GCGTGCCTTCAGCCAGC-3′ 13 MF 5′-GCGCTGGCTGAAGGCACG-3′ 14 MR 5′-GAATTCTGGGGTCGGCGTCG-3′ 15 ORI-F 5′-TGTGACCGTCTCCGGGAGCTG-3′ 16 ORI-R 5′-CAGCTCCCGGAGACGGTCACA-3′ 17

[0203] First, the amino acid sequences of the hydrophilic domain HD (Liu et al., Microbial Biotechnology, 2019, 12: 752-762) and the hydrophobic protein HFBII (Uniprot ID: P79073 · HYP2_HYPJE) were searched, and the gene fragments were obtained by gene synthesis by Shanghai Shenggong according to their amino terminal sequences. Various protease propeptide (ProP) sequences P1-P10 homologous to PEP were obtained by BLAST, among which P1 (SEQ ID NO: 46) was derived from yeast protease propeptide, P2 (SEQ ID NO: 47) was derived from cucumber protease propeptide, P3 (SEQ ID NO: 48) was derived from cyanobacteria protease propeptide, P4 (SEQ ID NO: 49) was derived from subtilisin propeptide, which was also the earliest discovered and most studied propeptide, P5 (SEQ ID NO: 50) was derived from human PCSK9 protease propeptide, and the remaining P6-P10 (SEQ ID NO: 51-55) were derived from bacterial protease propeptide. The sequence identity of the 10 propeptides with PEP is 38-95%. Based on its amino terminal sequence, gene fragments were obtained by gene synthesis by Shanghai Biotech. N1 and N2 of truncated PEP were also obtained by gene synthesis by Shanghai Biotech based on its amino terminal sequence. The synthesized gene was used as a template, and ELP-F2 and ELP-R2 were used as primers to amplify the polynucleotide fragments of each connecting peptide by PCR reaction. The PCR reaction used Q5 polymerase from NEB (New England Biolab (NEB)), and the PCR conditions were: 98℃30sec, 98℃10sec, 67℃30sec, 72℃30sec, a total of 30 cycles; finally 72℃2min. After the reaction, the PCR amplification product was separated and recovered by 1% agarose gel.

[0204] The above-mentioned pET32-L6KD-MtuΔI-CM-IGF was used as a template, and the Mtu-IGF fragment and the Backbone-L6KD fragment were amplified by PCR reaction using MF, ORI-R and ORI-F, MR as primers. The PCR reaction used Q5 polymerase from NEB, and the PCR conditions were: 98℃30sec, 98℃10sec, 64℃30sec, 72℃120sec, for a total of 30 cycles; finally 72℃2min. 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 DH5α competent cells, and the transformed cells were spread on LB plates supplemented with 50 μg / mL kanamycin to screen positive clones. The plasmids were extracted and sequenced. The sequencing results showed that the cloned plasmid sequences were correct.

[0205] Example 10: Hydrophilic domain HD and hydrophobic protein HFBII as connecting peptides for expression and purification of target protein

[0206] 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 four fusion protein expression strains containing different connecting peptides. Two expression strains were inoculated into LB liquid culture medium containing 50 μg / mL kanamycin, and cultured in a 37°C shaker until the logarithmic phase (OD 600 =0.4-0.6), add 0.2mM IPTG, induce at 18℃ for 24 hours, measure bacterial concentration OD 600 (The following will be 1mL OD 600 The cell mass of 1 is called 1OD).

[0207] The cells were resuspended to 20 OD / mL with lysis buffer B1 (2.4 g Tris, 29.22 g NaCl, 0.37 g Na2EDTA·2H2O 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 separately. 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 precipitate was centrifuged at 4°C, 15000g for 20 min, and the precipitate was resuspended with an equal volume of lysis buffer. The obtained supernatant and precipitate were subjected to SDS-PAGE detection together with the supernatant and precipitate before cleavage. The results are as follows Fig. 9 As shown, lanes ES, EP, CP, and CS are the expression and purification samples of the target protein, respectively, ES: cell lysate supernatant; EP: cell lysate precipitate, in which clear aggregates expressed by the fusion protein can be detected; CP: precipitate separated after cleavage; CS: supernatant separated after cleavage, 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 5μg, 2.5μg, 1.25μg, 0.625μg, 0.3125μg, and 0.15625μg, respectively.

[0208] According to the protein quantitative standard, 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 MtuΔI-CM cleavage efficiency, the target protein recovery rate and its purity in the supernatant were calculated. The results are shown in Table 10.

[0209] Table 10: HD and hydrophobic protein HFBII as linker peptides for expression and purification of target proteins

[0210]

[0211]

[0212] a Protein aggregate yield (volume per liter of LB medium), b The yield of the target protein after intein-mediated self-cleavage (volume per liter of LB medium), c Intein-mediated cleavage efficiency = 100% × (aggregate expression before cleavage - aggregate remaining after cleavage) / aggregate yield before cleavage,d Recovery rate = 100% × actual yield of target protein / theoretical yield of target protein that can be produced when protein aggregates are completely cleaved.

[0213] 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 amount is 25-94 mg / L LB culture medium. The four different fusion proteins are self-cleaved by the intein MtuΔI-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.

[0214] Example 11: Various ProPs used as connecting peptides for expression and purification of target proteins

[0215] The plasmids constructed in Example 9 (containing plasmids pET30-L6KD-ProP-Mtu-IGF and pET30-L6KD-ProP-Mtu-Caplacizumab) were transformed into Escherichia coli BL21 (DE3) competent cells to obtain 20 strains expressing fusion proteins containing different ProPs. The two expression strains were inoculated into LB liquid culture medium containing 50 μg / mL kanamycin and cultured in a shaking incubator at 37°C until the logarithmic phase (OD 600 =0.4-0.6), add 0.2mM IPTG, induce at 18℃ for 24 hours, measure bacterial concentration OD 600 (The following will be 1mL OD 600 The cell mass of 1 is called 1OD).

[0216] The cells were resuspended to 20 OD / mL with lysis buffer B1 (2.4 g Tris, 29.22 g NaCl, 0.37 g Na2EDTA·2H2O 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 separately. 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 precipitate was centrifuged at 4°C, 15000g for 20 min, and the precipitate was resuspended with an equal volume of lysis buffer. The obtained supernatant and precipitate were subjected to SDS-PAGE detection together with the supernatant and precipitate before cleavage. The results are as follows Fig.10 As shown, lanes ES, EP, CP, and CS are the expression and purification samples of the target protein, respectively, ES: cell lysate supernatant; EP: cell lysate precipitate, in which clear aggregates expressed by the fusion protein can be detected; CP: precipitate separated after cleavage; CS: supernatant separated after cleavage, 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 5μg, 2.5μg, 1.25μg, 0.625μg, 0.3125μg, and 0.15625μg, respectively.

[0217] According to the protein quantitative standard, 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 MtuΔI-CM cleavage efficiency, the target protein recovery rate and its purity in the supernatant were calculated. The results are shown in Table 11.

[0218] Table 11: Various ProPs used as linker peptides for expression and purification of target proteins

[0219]

[0220]

[0221] a Protein aggregate yield (volume per liter of LB medium), b The yield of the target protein after intein-mediated self-cleavage (volume per liter of LB medium), c Intein-mediated cleavage efficiency = 100% × (aggregate expression before cleavage - aggregate remaining after cleavage) / aggregate yield before cleavage,d Recovery rate = 100% × actual yield of target protein / theoretical yield of target protein that can be produced by complete cleavage of protein aggregates. e Sequence similarity between ProP and PEP, algorithm from (Chen et al., Molecular Plant, 2020, 13(8): 1194-1202).

[0222] The 20 fusion proteins (L6KD-ProP-Mtu-IGF and L6KD-ProP-Mtu-Caplacizumab) used all exist in the form of precipitation, and the aggregate expression amount is 52-433 mg / L LB culture fluid. The 20 different fusion proteins are self-cleaved by the intein MtuΔI-CM, and the target protein is separated from L6KD-Mtu-ProP. The cleavage efficiency is 17-91%, and the yield of the target protein released into the supernatant after cleavage is 0.9-26 mg / L LB culture fluid. The purity of the target protein recovered after cleavage is 19-99%. Among them, the purity of the purified protein of the propeptide P7-P10 with a sequence similarity of more than 60% with PEP is all more than 96%, which proves that a variety of protease propeptides with high sequence similarity with PEP have the potential to be applied to the method of the present invention to obtain high-purity proteins.

[0223] Example 12: PEP truncated form: N1 and N2 used as connecting peptides for expression and purification of target protein

[0224] The plasmids 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) were transformed into Escherichia coli BL21 (DE3) competent cells to obtain 6 fusion protein expression strains containing different truncated PEPs. The two expression strains were inoculated into LB liquid culture medium containing 50 μg / mL kanamycin, and cultured in a 37°C shaker until the logarithmic phase (OD 600 =0.4-0.6), add 0.2mM IPTG, induce at 18℃ for 24 hours, measure bacterial concentration OD 600 (The following will be 1mL OD 600 The cell mass of 1 is called 1OD).

[0225] The cells were resuspended to 20 OD / mL with lysis buffer B1 (2.4 g Tris, 29.22 g NaCl, 0.37 g Na2EDTA·2H2O 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 separately. 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 precipitate was centrifuged at 4°C, 15000g for 20 min, and the precipitate was resuspended with an equal volume of lysis buffer. The obtained supernatant and precipitate were subjected to SDS-PAGE detection together with the supernatant and precipitate before cleavage. The results are as follows Fig.11 As shown, lanes ES, EP, CP, and CS are the expression and purification samples of the target protein, respectively, ES: cell lysate supernatant; EP: cell lysate precipitate, in which clear aggregates expressed by the fusion protein can be detected; CP: precipitate separated after cleavage; CS: supernatant separated after cleavage, 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 5μg, 2.5μg, 1.25μg, 0.625μg, 0.3125μg, and 0.15625μg, respectively.

[0226] According to the protein quantitative standard, 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 MtuΔI-CM cleavage efficiency, the target protein recovery rate and its purity in the supernatant were calculated. The results are shown in Table 12.

[0227] Table 12: PEP truncated N1 and N2 used as connecting peptides for expression and purification of target proteins

[0228]

[0229] a Protein aggregate yield (volume per liter of LB medium), b The yield of the target protein after intein-mediated self-cleavage (volume per liter of LB medium), c Intein-mediated cleavage efficiency = 100% × (aggregate expression before cleavage - aggregate remaining after cleavage) / aggregate yield before cleavage, dRecovery rate = 100% × actual yield of target protein / theoretical yield of target protein that can be produced when protein aggregates are completely cleaved.

[0230] The six fusion proteins used all exist in the form of precipitation, and the aggregate expression amount is 216-335 mg / L LB culture medium. The six different fusion proteins are self-cleaved by the intein MtuΔI-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.

[0231] Sequence Listing

[0232] SEQ ID NO:1 ELP amino acid sequence, length 78aa

[0233] GVGVPGVGVPGVGVPGEGVPGVGVPGVGVPGVGVPGVGVPGEGVPGVGVPGVGVPGVGVPGVGVPGEGVPGVGVPGVG

[0234] SEQ ID NO:2 PEP amino acid sequence, length 76aa

[0235] QYIVVLKEGTQSALSAQSAGGLIGSGLDPQGITVLSVYGQAIEGFAAKLSAQNLEKVRANANVAYVEQDGMMYAS

[0236] SEQ ID NO:3 Mtu amino acid sequence, length 168aa

[0237] ALAEGTRIFDPVTGTTHRIEDVVDGRKPIHVVAAAKDGTLHARPVVSWFDQGTRDVIGLRIAGGAILWATPDHKVLTEYGWRAAGELRKGDRVAQPRRFDGFGDSAPIPARVQALADALDDKFLHDMLAEELRYSVIREVLPTRRARTFGLEVEELHTLVAEGVVVHN

[0238] SEQ ID NO:4 Mxe amino acid sequence, length 198aa

[0239] CITGDALVALPEGESVRIADIVPGARPNSDNAIDLKVLDRHGNPVLADRLFHSGEHPVYTVRTVEGLRVTGTANHPLLCLVDVAGVPTLLWKLIDEIKPGDYAVIQRSAFSVDCAGFARGKPEFAPTTYTVGVPGLVRFLEAHHRDPDAQAIADELTDGRFYYAKVASVTDAGVQPVYSLRVDTADHAFITNGFVSHA

[0240] SEQ ID NO:5 L6KD amino acid sequence, 8aa in length

[0241] LLLLLLKD

[0242] SEQ ID NO:6 ELK16 amino acid sequence, length 16aa

[0243] LELELKLKLELELKLK

[0244] SEQ ID NO:7 MpA amino acid sequence, 30aa in length

[0245] MKQLEDKIEELLSKAAMKQLEDKIEELLSK

[0246] SEQ ID NO:8 GS amino acid linker amino acid sequence, length 15aa

[0247] GGGGSGGGGSGGGGS

[0248] SEQ ID NO:9 PT amino acid linker amino acid sequence, length 17aa

[0249] PTPPTTPTPPTTPTPTP

[0250] SEQ ID NO: 10 hGH amino acid sequence, length 191aa

[0251] FPTIPLSRLFDNAMLRAHRLHQLAFDTYQEFEEAYIPKEQKYSFLQNPQTSLCFSESIPTPSNREETQQKSNLELLRISLLLIQSWLEPVQFLRSVFANSLVYGASDSNVYDLLKDLEEGIQTLMGRLEDGSPRTGQIFKQTYSKFDTNSHNDDALLKNYGLLYCFRKDMDKVETFLRIVQCRSVEGSCGF

[0252] SEQ ID NO:11 RFP amino acid sequence, length 235aa

[0253] VSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGVVTVTQDSSL QDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKGEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAEGRHSTGGMDELYK

[0254] SEQ ID NO: 12 Primer ELP-F2, length 19 nt

[0255] CCGACGCCGACCCCAGAAT

[0256] SEQ ID NO: 13 Primer ELP-R2, length 17 nt

[0257] GCGTGCCTTCAGCCAGC

[0258] SEQ ID NO:14 Primer MF, length 22 nt

[0259] GCGCTGGCTGAAGGCACG

[0260] SEQ ID NO:15 Primer MR, length 20 nt

[0261] GAATTCTGGGGTCGGCGTCG

[0262] SEQ ID NO: 16 Primer ORI-F, length 21 nt

[0263] TGTGACCGTCTCCGGGAGCTG

[0264] SEQ ID NO: 17 Primer ORI-R, length 21 nt

[0265] CAGCTCCCGGAGACGGTCACA

[0266] SEQ ID NO:18 Primer END-F, length 20 nt

[0267] TGAGATCCGGCTGCTAACAA

[0268] SEQ ID NO: 19 Primer END-R, length 20 nt

[0269] GCTTTGTTAGCAGCCGGATC

[0270] SEQ ID NO:20 Primer POI-F, length 22 nt

[0271] GTTGCGGAAGGCGTTGTGGTTC

[0272] SEQ ID NO:21 Primer POI-R, length 22 nt

[0273] GAACCACAACGCCTTCCGCAAC

[0274] SEQ ID NO:22 Primer PEP-F, length 24 nt

[0275] GAATTCCAGTACATCGTGGTCCTC

[0276] SEQ ID NO:23 Primer 11-R, length 33 nt

[0277] AGGACCACGATGTACTGGAATTCCGGCGTCGGG

[0278] SEQ ID NO:24 Primer EIK8-F, length 45 nt

[0279] ATTGAAATTAAAATTGAGATTAAACCGACCCCACCGACCACGCCA

[0280] SEQ ID NO:25 Primer EIK8-R, length 45 nt

[0281] TTTAATCTCAATTTTAATTTCAATCATATGTATATCTCCTTCTTA

[0282] SEQ ID NO:26 Primer EFR8-F, length 45 nt

[0283] TTCGAGTTCCGCTTCGAATTCCGTCCGACCCCACCGACCACGCCA

[0284] SEQ ID NO:27 Primer EFR8-R, length 45 nt

[0285] ACGGAATTCGAAGCGGAACTCGAACATATGTATATCTCCTTCTTA

[0286] SEQ ID NO:28 Primer Mxe-F, length 23 nt

[0287] CATCACGGGAGATGCACTAGTTG

[0288] SEQ ID NO:29 Primer Mxe-R1, length 23 nt

[0289] CAACTAGTGCATCTCCCGTGATG

[0290] SEQ ID NO:30 Primer PG-F, length 59 nt

[0291] TGGTTCTGGCGGTGGCGGTTCCGGTGGCGGCGGTAGCCAGTACATCGTGGTCCTCAAGG

[0292] SEQ ID NO:31 Primer PM-R, length 40 nt

[0293] GGTCGGTGGGGTCGGAAGCTTGGAGGCGTACATCATGCCG

[0294] SEQ ID NO:32 Primer KL-F, length 18 nt

[0295] AAGCTTCCGACCCCACCG

[0296] SEQ ID NO:33 Primer Mxe-RP, length 53 nt

[0297] GAACCGCCACCGCCAGAACCACCGCCGCCAAGCTTAGCGTGGCTGACGAACCC

[0298] SEQ ID NO:34 ELP nucleotide sequence, 234 bp in length

[0299] GGTGTTGGTGTGCCGGGTGTGGGTGTTCCGGGCGTTGGCGTGCCGGGCGAAGGCGTGCCGGGTGTGGGTGTTCCGGGTGTGGGCGTGCCGGGCGTTGGCGTTCCGGGCGTGGGTGTTCCGGGCGAAGGCGTGCCGGGTGTGGGTGTGCCGGGTGTTGGTGTGCCGGGCGTTGGTGTTCCGGGCGTTGGTGTTCCGGGCGAAGGCGTTCCGGGCGTTGGCGTGCCGGGTGTGGGC

[0300] SEQ ID NO:35 PEP nucleotide sequence, length 228bp

[0301] CAGTACATCGTGGTCCTCAAGGAAGGCACCCAGAGCGCCCTGAGCGCCCAGAGCGCCGGCGGCCTGATCGGCAGCCTGGGTCTGGACCCGCAGGGCATCACGGTCCTGAGCGTGTACGGTCAGGCCATTGAGGGCTTCGCCGCCAAGCTCAGCGCCCAGAACCTGGAGAAGGTGCGCGCCAACGCGAACGTCGCCTACGTCGAGCAGGACGGCATGATGTACGCCTCC

[0302] SEQ ID NO:36 Mtu nucleotide sequence, length 504bp

[0303] GCGCTGGCTGAAGGCACGCGCATTTTTGATCCGGTCACGGGCACGACGCACCGCATTGAAGATGTTGTTGATGGCCGCAAGCCGATTCATGTGGTTGCGGCCGCAAAAGATGGCACCCTGCACGCCCGTCCGGTCGTGAGTTGGTTTGATCAGGGTACGCGTGACGTCATTGGTCTGCGTATCGCGGGCGGTGCAATTCTGTGGGCAACCCCGGATCATAAAGTGCTGACGGAATATGGCTGGCGTGCTGCGGGTGAACTGCGTAAGGGTGACCGTGTTGCACAGCCGCGTCGCTTTGATGGCTTCGGTGACAGCGCACCGATTCCGGCTCGCGTTCAAGCCCTGGCAGATGCTCTGGATGACAAGTTCCTGCACGACATGCTGGCGGAAGAACTGCGTTACTCTGTTATCCGCGAAGTCCTGCCGACCCGTCGCGCCCGCACGTTTGGTCTGGAAGTGGAAGAACTGCATACCCTGGTTGCGGAAGGCGTTGTGGTTCATAAC

[0304] SEQ ID NO:37 Mxe nucleotide sequence, 594 bp in length

[0305] TGCATCACGGGAGATGCACTAGTTGCCCTACCCGAGGGCGAGTCGGTACGCATCGCCGACATCGTGCCGGGTGCGCGGCCCAACAGTGACAACGCCATCGACCTGAAAGTCCTTGACCGGCATGGCAATCCCGTGCTCGCCGACCGGCTGTTCCACTCCGGCGAGCATCCGGTGTACACGGTGCGTACGGTCGAAGGTCTGCGTGTGACGGGCACCGCGAACCACCCGTTGTTGTGTTTGGTCGACGTCGCCGGGGTGCCGACCCTGCTGTGGAAGCTGATCGACGAAATCAAGCCGGGCGATTACGCGGTGATTCAACGCAGCGCATTCAGCGTCGACTGTGCAGGTTTTGCCCGCGGGAAACCCGAATTTGCGCCCACAACCTACACAGTCGGCGTCCCTGGACTGGTGCGTTTCTTGGAAGCACACCACCGAGACCCGGACGCCCAAGCTATCGCCGACGAGCTGACCGACGGGCGGTTCTACTACGCGAAAGTCGCCAGTGTCACCGACGCCGGCGTGCAGCCGGTGTATAGCCTTCGTGTCGACACGGCAGACCACGCGTTTATCACGAACGGGTTCGTCAGCCACGCT

[0306] SEQ ID NO:38 IGF-1 nucleotide sequence, with a length of 210 bp

[0307] GGTCCGGAAACCCTGTGCGGTGCTGAACTGGTTGACGCTCTGCAGTTCGTTTGCGGTGACCGTGGTTTCTACTTCAACAAACCGACCGGTTACGGTTCTTCTTCTCGTCGTGCTCCGCAGACCGGTATCGTTGACGAATGCTGCTTCCGTTCTTGCGACCTGCGTCGTCTGGAAATGTACTGCGCTCCGCTGAAACCGGCTAAATCTGCT

[0308] SEQ ID NO:39 hGH nucleotide sequence, with a length of 573 bp

[0309] TTCCCGACCATCCCGCTGTCTCGTCTGTTCGACAACGCTATGCTGCGTGCTCACCGTCTGCACCAGCTGGCTTTCGACACCTACCAGGAATTCGAAGAAGCTTACATCCCGAAAGAACAGAAATACTCTTTCCTGCAGAACCCGCAGACCTCTCTGTGCTTCTCTGAATCTATCCCGACCCCGTCTAACCGTGAAGAAACCCAGCAGAAATCTAACCTGGAACTGCTGCGTATCTCTCTGCTGCTGATCCAGTCTTGGCTGGAACCGGTTCAGTTCCTGCGTTCTGTTTTCGCTAACTCTCTGGTTTACGGTGCTTCTGACTCTAACGTTTACGACCTGCTGAAAGACCTGGAAGAAGGTATCCAGACCCTGATGGGTCGTCTGGAAGACGGTTCTCCGCGTACCGGTCAGATCTTCAAACAGACCTACTCTAAATTCGACACCAACTCTCACAACGACGACGCTCTGCTGAAAAACTACGGTCTGCTGTACTGCTTCCGTAAAGACATGGACAAAGTTGAAACCTTCCTGCGTATCGTTCAGTGCCGTTCTGTTGAAGGTTCTTGCGGTTTC

[0310] SEQ ID NO: 40 NB21 nucleotide sequence, 351 bp in length

[0311] CAGGTTCAGTTAGTGGAAAGTGGTGGTGGCTTAGTTCAGGCCGGCGGTAGCCTGCGTCTGTCATGCGCTGTGAGCGGCTTAGGCGCACATCGTGTTGGCTGGTTTCGTCGCGCTCCGGGCAAAGAACGCGAATTTGTTGCAGCCATCGGTGCTAATGGCGGTAATACCAATTATTTAGATTCTGTTAAAGGTCGCTTTACAATCTCTCGCGATAATGCTAAAAATACGATCTATCTCCAAATGAATAGTCTGAAACCTCAGGATACAGCAGTGTATTATTGTGCCGCACGTGACATTGAAACGGCCGAATATACATATTGGGGCCAGGGAACACAGGTGACCGTGTCAAGC

[0312] SEQ ID NO:41 Nucleotide sequence of xylanase, length 1218bp

[0313]

[0314] SEQ ID NO: 42 Nucleotide sequence of caplacizumab, with a length of 777 bp

[0315] GAAGTTCAGCTGGTTGAATCTGGTGGTGGTCTGGTTCAGCCGGGTGGTAGCCTGCGTCTGTCTTGCGCTGCGAGCGGCCGTACCTTCAGCTACAACCCGATGGGCTGGTTCCGTCAGGCGCCGGGTAAAGGTCGTGAACTGGTGGCGGCGATCAGCCGTACCGGCGGTAGCACCTATTACCCGGATTCCGTTGAAGGCCGTTTCACCATCTCTCGTGATAACGCGAAACGTATGGTTTACCTGCAAATGAACTCTCTGCGTGCGGAAGATACTGCGGTTTACTACTGCGCGGCGGCTGGCGTTCGCGCGGAAGATGGCCGTGTTCGCACCCTGCCGAGCGAATACACCTTCTGGGGCCAGGGTACCCAGGTGACCGTTAGCAGCGCTGCGGCGGAAGTTCAGCTGGTCGAATCGGGTGGCGGCCTGGTTCAGCCGGGTGGTTCTCTGCGTCTGTCTTGTGCGGCGTCTGGCCGTACCTTCAGCTACAACCCGATGGGTTGGTTCCGTCAGGCGCCGGGTAAAGGCCGTGAACTGGTTGCGGCAATTAGCCGTACCGGTGGCAGCACCTACTACCCGGATTCTGTTGAAGGCCGCTTCACCATTAGCCGTGATAACGCTAAACGTATGGTGTACTTGCAGATGAACAGCCTGCGTGCAGAAGATACCGCAGTTTACTATTGCGCGGCGGCGGGCGTGCGTGCGGAAGATGGTCGTGTTCGCACCCTGCCGAGCGAATACACCTTCTGGGGCCAGGGCACCCAGGTTACCGTTTCTAGC

[0316] SEQ ID NO: 43 GST nucleotide sequence, with a length of 654 bp

[0317] ATGTCCCCTATACTAGGTTATTGGAAAATTAAGGGCCTTGTGCAACCCACTCGACTTCTTTTGGAATATCTTGAAGAAAAATATGAAGAGCATTTGTATGAGCGCGATGAAGGTGATAAATGGCGAAACAAAAAGTTTGAATTGGGTTTGGAGTTTCCCAATCTTCCTTATTATATTGATGGTGATGTTAAATTAACACAGTCTATGGCCATCATACGTTATATAGCTGACAAGCACAACATGTTGGGTGGTTGTCCAAAAGAGCGTGCAGAGATTTCAATGCTTGAAGGAGCGGTTTTGGATATTAGATACGGTGTTTCGAGAATTGCATATAGTAAAGACTTTGAAACTCTCAAAGTTGATTTTCTTAGCAAGCTACCTGAAATGCTGAAAATGTTCGAAGATCGTTTATGTCATAAAACATATTTAAATGGTGATCATGTAACCCATCCTGACTTCATGTTGTATGACGCTCTTGATGTTGTTTTATACATGGACCCAATGTGCCTGGATGCGTTCCCAAAATTAGTTTGTTTTAAAAAACGTATTGAAGCTATCCCACAAATTGATAAGTACTTGAAATCCAGCAAGTATATAGCATGGCCTTTGCAGGGCTGGCAAGCCACGTTTGGTGGTGGCGACCATCCTCCAAAA

[0318] SEQ ID NO:44 HD amino acid sequence, with a length of 67 aa

[0319] ADASKAADQAKDKAQDVAQNVKQGAQQAASDAKDKVQDVKADASRAADQAKDKAQDVAQNVKQSAQD

[0320] SEQ ID NO:45 HFBII amino acid sequence, with a length of 71 aa

[0321] AVCPTGLFSNPLCCATNVLDLIGVDCKTPTIAVDTGAIFQAHCASKGSKPLCCVAPVADQALLCQKAIGTF

[0322] SEQ ID NO:46P1 amino acid sequence, length 81aa

[0323] QFIVVFDSSATVDQIASEIQKLDSLVDEDSSNGITSALDLPVYTDGSGFLGFVGKFNSTIVDKLKESSVLTVEPDTIVSLP

[0324] SEQ ID NO:47P2 amino acid sequence, length 77aa

[0325] YIVYMGRKLEDPDSAHLHHRAMLEQVVGSTFAPESVLHTYKRSFNGFAVKLTEEEAEKIASMEGVVSVFLNEMNELH

[0326] SEQ ID NO:48P3 amino acid sequence, length 66aa

[0327] QYIVVLRDGHDPLTVAQQHANVHALSVGFVYKHALKGYAARIPEGRLAALARDPRVLFISEDRPVK

[0328] SEQ ID NO:49P4 amino acid sequence, length 77aa

[0329] AGKSSTEKKYIVGFKQTMSAMSSAKKKDVISEKGGKVQKQFKYVNAAAATLDEKAVKELKKDPSVAYVEEDHIAHEY

[0330] SEQ ID NO:50P5 amino acid sequence, length 73aa

[0331] TYVVVLKEETHLSQSERTARRLQAQAARRGYLTKILHVFHGLLPGFLVKMSGDLLELALKLPHVDYIEEDSSV

[0332] SEQ ID NO:51P6 amino acid sequence, length 70aa

[0333] QYIVVFKKGAAAPVLQQLQSGNVSGLGVSDQDVQLLYVYSTALPGFAAVLSDAALVQLARDPRVAYIEAD

[0334] SEQ ID NO:52P7 amino acid sequence, length 79aa

[0335] QYIVVFRKTATVQGQSISKLSAGQMVQRLGLDRQGAQIQAVYTEVLDGFAAKLSASNIQKLASDPRVAYLQAVQTYYAT

[0336] SEQ ID NO:53P8 amino acid sequence, length 77aa

[0337] EYIVVLNKGAISTAMSAQNVNGLVTQLGLDPQGVSVLHVYTAALNGFAAKLSDANLAKLKSNPAVAYVEQNKVVKAT

[0338] SEQ ID NO:54P9 amino acid sequence, length 76aa

[0339] QYIVVMSAGATASALASQSGGLVQALGLDPQGVSILSVYTQALNGFAVKLNAQNLARLQADPRVKYIQQDSVAHAT

[0340] SEQ ID NO:55P10 amino acid sequence, length 76aa

[0341] QYIVVLKEGTQNNLSAQSAGGLIGSGLDPQGITVLSVYGQAIEGFAAKLSTQNVDKLRANTNVEYVEQDGMMYAN

[0342] SEQ ID NO:56N1 amino acid sequence, length 40aa QYIVVLKEGTQSALSAQSAGGLIGSLGLDPQGITVLSVYG

[0343] SEQ ID NO:57N2 amino acid sequence, length 36aa QAIEGFAAKLSAQNLEKVRANANVAYVEQDGMMYAS

[0344] SEQ ID NO:58 EIK8 amino acid sequence

[0345] IEIKIEIK

[0346] SEQ ID NO:59 EFR8 amino acid sequence

[0347] FEFRFEFR

[0348] SEQ ID NO:60L6K2 amino acid sequence

[0349] LLLLLLKK

[0350] SEQ ID NO:61L6D2 amino acid sequence

[0351] LLLLLLDD

[0352] SEQ ID NO:62

[0353] (VPGXG) 15 , wherein X is independently Ala, Gly, Val or Glu

[0354] SEQ ID NO:63

[0355] VPGXG, where X is Ala, Gly, Val or Glu

[0356] SEQ ID NO:64

[0357] (VPGXG)n wherein X is independently Ala, Gly, Val or Glu, and n=10-100 SEQ ID NO: 65

[0358] CCGACGCCGACCCCAGAATTC

[0359] SEQ ID NO:66

[0360] GCGCTGGCTGAAGGCACGC.

Claims

1. A fusion protein comprising, 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 connecting peptide comprises an elastin-like polypeptide (ELP) or a protease propeptide (ProP), in particular an anisoglobulin protease propeptide PEP or a homologue thereof, preferably, the connecting peptide comprises 30-100, preferably 60-80 amino acid residues; and The spacer comprises a cleavage site, and preferably the spacer has a length of 10-250 amino acid residues; Optionally, an amino acid linker is included between the aggregating peptide and the connecting peptide, the connecting peptide and the spacer, and / or the spacer and the target protein, and preferably the amino acid linker comprises an amino acid sequence selected from any one of SEQ ID NOs: 8-9.

2. The fusion protein of claim 1, wherein the connecting peptide comprises: (a) an amino acid sequence selected from any one of SEQ ID NOs: 1-2 and 46-55, or an amino acid sequence having at least 60% identity thereto, preferably comprising 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 thereto; or (b) comprises the (VPGXG)n motif, wherein X is independently A, G, V or E, preferably V or E, more preferably V:E=4:1; n is an integer, preferably n is 10-20, more preferably 15, for example comprising the amino acid sequence shown in SEQ ID NO:

1.

3. The fusion protein of claim 1 or 2, wherein the self-aggregating peptide is selected from amphipathic self-assembling short peptides, such as amphipathic β-folded short peptides, amphipathic α-helical short peptides, surfactant-like short peptides and salt concentration-responsive self-aggregating peptides, such as selected from SEQ ID NOs: 5-7 and 58-61.

4. The fusion protein of any one of claims 1-3, wherein the cleavage site is 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, preferably comprising an intein such as MxeGyrA, Ssp DnaB or MtuΔI-CM, for example comprising the amino acid sequence shown in any one of SEQ ID NOs: 3-4.

5. The fusion protein according to any one of claims 1 to 4, wherein the target protein: (a) located at the C-terminus of the fusion protein and the spacer is connected to the N-terminus of the target protein, or the target protein is located at the N-terminus of the fusion protein and the spacer is connected to the C-terminus of the target protein; (b) selected from therapeutic molecules, detectable molecules and targeting molecules, such as toxins, immunomodulators, antagonists, apoptosis inducers, hormones, radiopharmaceuticals, anti-angiogenic agents, cytokines, chemokines, prodrugs, chemotherapeutic drugs, fluorescent proteins, enzymes, tags, targeting antibodies or specific receptor ligands, in particular selected from red fluorescent protein (RFP), insulin-like growth factors such as insulin-like growth factor-1 (IGF-1), growth hormones such as human growth hormone (hGH), nanobodies such as anti-new crown nanobody NB21, xylanase, capecitabine and glutathione sulfhydryltransferase (GST), in particular, 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 shown in any one of SEQ ID NOs: 38-43; and / or (c) 20-500 amino acids in length.

6. An isolated polynucleotide comprising a nucleotide sequence encoding the fusion protein of any one of claims 1 to 5 or a complementary sequence thereof.

7. 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 a fusion protein according to any one of claims 1 to 5, wherein the self-aggregating peptide, the connecting peptide and the spacer are as defined in any one of claims 1 to 5.

8. The polynucleotide of claim 7, wherein the target protein has a length of 20-500 amino acids, for example, selected from therapeutic molecules, detectable molecules and targeting molecules, such as toxins, immunomodulators, antagonists, apoptosis inducers, hormones, radiopharmaceuticals, anti-angiogenic agents, cytokines, chemokines, prodrugs, chemotherapeutic drugs, fluorescent proteins, enzymes, tags, targeted antibodies or specific receptor ligands, in particular selected from red fluorescent protein (RFP), insulin-like growth factors such as insulin-like growth factor-1 (IGF-1), growth hormones such as human growth hormone (hGH), nanobodies such as anti-new crown nanoantibody NB21, xylanase, capecitabine and glutathione sulfhydryltransferase (GST), in particular, 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 shown in any one of SEQ ID NOs: 38-43.

9. The polynucleotide of any one of claims 6-8, which encodes from 5' to 3' direction: (a) a self-aggregating peptide, a connecting peptide, a spacer and a target protein; or (b) a target protein, a spacer, a connecting peptide and a self-aggregating peptide, optionally encoding an amino acid linker between an aggregating peptide and a connecting peptide, a connecting peptide and a spacer, and / or a spacer and a target protein.

10. A construct, preferably an expression construct, comprising the polynucleotide of any one of claims 6 to 9.

11. A host cell comprising the polynucleotide of claim 6 or transformed by a construct, preferably an expression construct, comprising the polynucleotide, wherein the host cell is capable of expressing the fusion protein, preferably selected from prokaryotes, yeasts and higher eukaryotic cells, such as bacteria of the genera Escherichia, Bacillus, Salmonella and Pseudomonas and Streptomyces, preferably Escherichia coli.

12. A method for producing and purifying a target protein, preferably a soluble target protein, the method comprising: (a) preparing the target protein into a fusion protein as described in any one of claims 1 to 5, for example, expressing a polynucleotide encoding the fusion protein in a host cell or an in vitro cell-free system, wherein the polynucleotide is obtained, for example, by cloning a nucleotide sequence encoding the target protein into a multiple cloning site of a polynucleotide according to claim 7; (b) generating active aggregates by aggregation of self-aggregating peptides to obtain an insoluble fraction containing the fusion protein; (c) releasing the target protein from the insoluble portion by cleaving the cleavage site in the fusion protein; and (d) optionally, recovering and / or purifying the target protein, Preferably, the host cell is selected from prokaryotes, yeasts and higher eukaryotic cells, such as bacteria of the genera Escherichia, Bacillus, Salmonella, Pseudomonas and Streptomyces, preferably Escherichia coli.

13. A method for high-throughput production and purification of multiple target proteins, preferably soluble target proteins, comprising the following steps: (a) preparing each target protein into a fusion protein according to any one of claims 1 to 5, for example, expressing a polynucleotide encoding the fusion protein in a host cell or an in vitro cell-free system, the polynucleotide being obtained, for example, by cloning a nucleotide sequence encoding the target protein into a multiple cloning site of a polynucleotide according to claim 7, for example, culturing a host cell according to claim 11 in a multi-well plate to express the fusion protein; (b) generating active aggregates by aggregation of self-aggregating peptides to obtain an insoluble fraction comprising the fusion protein, for example in a multi-well plate; (c) releasing the target protein from the insoluble portion by cleaving the cleavage site in the fusion protein; and (d) optionally, recovering and / or purifying the target protein.

14. The method of claim 12 or 13, wherein step (a) comprises: Cultivating the host cell of claim 11 to express the fusion protein, optionally, step (b) comprises: lysing the host cell to obtain an insoluble fraction containing the fusion protein.

15. The method of any one of claims 12-14, wherein the target protein is 20-500 amino acids in length, for example, selected from therapeutic molecules, detectable molecules and targeting molecules, such as toxins, immunomodulators, antagonists, apoptosis inducers, hormones, radiopharmaceuticals, anti-angiogenic agents, cytokines, chemokines, prodrugs, chemotherapeutic drugs, fluorescent proteins, enzymes, tags, targeted antibodies or specific receptor ligands, in particular selected from red fluorescent protein (RFP), insulin-like growth factors such as insulin-like growth factor-1 (IGF-1), growth hormones such as human growth hormone (hGH), nanobodies such as anti-new crown nanoantibody NB21, xylanase, capecitabine and glutathione sulfhydryltransferase (GST), in particular, 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 shown in any one of SEQ ID NOs: 38-43.

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