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WO2022253266A1 - Procédé de purification de protéine recombinante - Google Patents

Procédé de purification de protéine recombinante Download PDF

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Publication number
WO2022253266A1
WO2022253266A1 PCT/CN2022/096574 CN2022096574W WO2022253266A1 WO 2022253266 A1 WO2022253266 A1 WO 2022253266A1 CN 2022096574 W CN2022096574 W CN 2022096574W WO 2022253266 A1 WO2022253266 A1 WO 2022253266A1
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Prior art keywords
salt
polypeptide
mtu
protein
cpa
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Chinese (zh)
Inventor
林章凛
杨晓锋
曾光
郑寅珍
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South China University of Technology SCUT
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South China University of Technology SCUT
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Priority to CN202280040156.5A priority Critical patent/CN117440963A/zh
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/64—General methods for preparing the vector, for introducing it into the cell or for selecting the vector-containing host

Definitions

  • the invention relates to the field of genetic engineering. More specifically, the present invention relates to a fusion polypeptide comprising a salt concentration-responsive self-aggregating peptide portion and a polypeptide portion of interest, and methods for producing and purifying the polypeptide of interest by expressing the fusion polypeptide.
  • Recombinant proteins have been widely used in medicine, food, chemical industry, energy, textile, environmental protection and other fields.
  • the production of recombinant protein is very important no matter in commercial scale or laboratory scale, and the cost of separation and purification of recombinant protein accounts for about 30%-80% of its total cost, which is the bottleneck technology of recombinant protein preparation (Fields C. et al. , Biotechnology and Bioengineering, 2016, 113(1):11-25).
  • the purification can generally be divided into three steps: sample capture, intermediate purification and fine purification.
  • moderate purification can achieve moderate sample purity, and the processed protein samples can be used for various experimental analysis, such as N-terminal sequence analysis, antigen-antibody reaction, etc.
  • methods that can achieve moderate protein purification include traditional ion-exchange chromatography, hydrophobic interaction chromatography, and affinity chromatography, as well as recent research hotspots such as fusion tag expression of self-cleavage and self-aggregation functions.
  • Ion exchange chromatography and hydrophobic interaction chromatography have certain requirements on the initial conditions of the sample, and their versatility and efficiency are not as good as those of affinity chromatography.
  • Affinity purification often achieves high yields, making it one of the most commonly used methods for intermediate purification of recombinant proteins.
  • the commonly used affinity purification technology in the laboratory includes the fusion expression of polyhistidine tag (his-tag) or glutathione transferase tag (GST-tag) and the target protein, and then through the chelation of metal ions ( Generally nickel ions) or a chromatographic column immobilized with glutathione resin for specific binding and elution for purification, which provides a general purification method for the production of different target proteins (Arnau J. et al., Protein Expression and Purification , 2006, 48(1):1-13).
  • N pro from the N-terminus of typical swine fever virus (CSFV) has protease activity and has a strong tendency to form insoluble aggregates, which has the characteristics of aggregation purification and protein shearing.
  • CSFV typical swine fever virus
  • using the N pro method requires renaturation and further fine purification of the target protein to remove the N pro fusion fragment in the product (Achmuller C. et al., Nature Methods, 2007, 4:1037-1043). It has also been reported that the self-aggregation tendency of amphipathic ⁇ -sheets composed of multiple repeating units is enhanced as the number of repeating units increases (Zhang S.
  • ELP E lastin-Like Polypeptide
  • the protein fused with this tag can be purified by controlling the temperature or ion concentration to continuously switch the fusion protein between the liquid phase (soluble) and the solid phase (precipitation) (Meyer DE et al., Nature Biotechnology, 1999.17 (11):1112-1115; Banki MR et al., Nature Methods, 2005, 2(9):659-661).
  • temperature or ion concentration to continuously switch the fusion protein between the liquid phase (soluble) and the solid phase (precipitation)
  • the protein purity and yield are usually low, and it is difficult to meet the standards for industrial applications, especially It is the higher temperature that may affect the activity of the purified protein, and the multi-step operation is not conducive to simplifying the process flow.
  • the long length of ELP itself at least 300 amino acid residues, it has adverse effects on the expression and purification of the fusion protein.
  • a fusion polypeptide which comprises a target polypeptide part and a salt concentration-responsive self-aggregation peptide part, wherein the target polypeptide part is linked to the salt concentration-responsive self-aggregation peptide part through a spacer , and wherein the spacer comprises a cleavage site,
  • the salt concentration-responsive self-aggregation peptide is a CpA variant, wherein the CpA has an amino acid sequence as shown in SEQ ID NO: 1, and the CpA variant corresponds to the first position of SEQ ID NO: 1 and The position at position 17 contains amino acid substitutions of C1M and C17M.
  • an isolated polynucleotide comprising a nucleotide sequence encoding a fusion polypeptide of the present invention or its complementary sequence.
  • an isolated polynucleotide comprising a nucleotide sequence encoding a CpA variant or its complementary sequence, wherein the CpA has an amino acid sequence as shown in SEQ ID NO: 1,
  • the CpA variant comprises amino acid substitutions of C1M and C17M at positions corresponding to positions 1 and 17 of SEQ ID NO:1.
  • an expression construct comprising a polynucleotide of the present invention.
  • a host cell comprising the polynucleotide of the present invention or transformed by the expression construct of the present invention, wherein said host cell is capable of expressing said fusion polypeptide.
  • a method for producing and purifying a polypeptide of interest comprising the following steps:
  • step (e) releasing soluble polypeptide of interest from the insoluble fraction collected from step (d) by cleaving said cleavage site;
  • step (f) removing the insoluble portion in step (e), and recovering the soluble portion containing the target polypeptide.
  • Fig. 1 shows a protein purification method based on salt concentration-responsive self-aggregating peptide induction and a structural diagram of the expression vector used.
  • A Purification strategy (taking salt concentration-responsive self-aggregating peptide MpA/CpA and intein Mtu ⁇ I-CM as examples);
  • B pET30-MpA-Mtu-hGH, pET30-MpA-Mtu-RFP, pET30-MpA-Mtu - Vector structure map of GST, pET30-MpA-Mtu-LCB3, pET30-MpA-Mtu- ⁇ NSpyCatcher-ELP- ⁇ NSpyCatcher, pET30-CpA-Mtu-hGH, pET30-CpA-Mtu-RFP and pET30a-Xylanase-Mxe-MpA .
  • Fig. 2 shows the expression results of human growth hormone hGH fusion protein and red fluorescent protein RFP fusion protein.
  • A SDS-PAGE analysis results of MpA-Mtu-hGH and MpA-Mtu-RFP expressions
  • B SDS-PAGE analysis results of CpA-Mtu-hGH and CpA-Mtu-RFP expressions.
  • Figure 3 shows the results of SDS-PAGE analysis of human growth hormone hGH and red fluorescent protein RFP purified by 3M NaCl-induced aggregation of the aggregation peptide MpA.
  • A SDS-PAGE analysis results of human growth hormone hGH expression and purification;
  • B red fluorescent protein RFP expression and purification SDS-PAGE analysis results.
  • Fig. 4 shows the results of SDS-PAGE analysis of human growth hormone hGH and red fluorescent protein RFP purified by 0.7M Na 2 SO 4 to induce aggregation of the aggregation peptide MpA.
  • A SDS-PAGE analysis results of human growth hormone hGH expression and purification;
  • B red fluorescent protein RFP expression and purification SDS-PAGE analysis results.
  • Fig. 5 shows the results of SDS-PAGE analysis of human growth hormone hGH and red fluorescent protein RFP purified by 0.7M (NH 4 ) 2 SO 4 to induce aggregation of the aggregation peptide MpA.
  • A SDS-PAGE analysis results of human growth hormone hGH expression and purification;
  • B red fluorescent protein RFP expression and purification SDS-PAGE analysis results.
  • Figure 6 shows the results of SDS-PAGE analysis of purified human growth hormone hGH and red fluorescent protein RFP by 3M NaCl-induced aggregation of the aggregation peptide CpA.
  • A SDS-PAGE analysis results of human growth hormone hGH expression and purification;
  • B red fluorescent protein RFP expression and purification SDS-PAGE analysis results.
  • Fig. 7 shows the results of SDS-PAGE analysis of human growth hormone hGH and red fluorescent protein RFP purified by 0.7M Na 2 SO 4- induced aggregation of the aggregation peptide CpA.
  • A SDS-PAGE analysis results of human growth hormone hGH expression and purification;
  • B red fluorescent protein RFP expression and purification SDS-PAGE analysis results.
  • Fig. 8 shows the results of SDS-PAGE analysis of human growth hormone hGH and red fluorescent protein RFP purified by 0.7M (NH 4 ) 2 SO 4 to induce aggregation of the aggregation peptide CpA.
  • A SDS-PAGE analysis results of human growth hormone hGH expression and purification;
  • B red fluorescent protein RFP expression and purification SDS-PAGE analysis results.
  • Fig. 9 is a diagram showing the activity characterization results of identifying aggregate MpA/CpA-Mtu-RFP and cleaved supernatant RFP by RFP showing red under natural light and RFP showing red fluorescence under 365nm ultraviolet light.
  • A The physical picture of the aggregates formed after adding three kinds of salts (3M NaCl, 0.7M Na 2 SO 4 , 0.7M (NH 4 ) 2 SO 4 ) to the lysed supernatant of MpA-Mtu-RFP under natural light;
  • B Under natural light The physical picture of the cleaved supernatant of MpA-Mtu-RFP under the conditions of three salts (3M NaCl, 0.7M Na 2 SO 4 , 0.7M (NH 4 ) 2 SO 4 );
  • C MpA-Mtu under 365nm ultraviolet light - Fluorescence images of the cleaved supernatant of RFP under three salt conditions (3M NaCl, 0.7M Na 2 SO 4 , 0.7M (NH 4 ) 2 SO 4 );
  • D CpA-Mtu-RFP cleaved supernatant under natural light Physical pictures of aggregates formed after adding three salts (3M NaCl, 0.7M Na 2 SO 4 ,
  • Figure 10 shows the purification of glutathione sulfhydryl transferase GST , new crown polypeptide LCB3 and multivalent backbone protein ⁇ NSpyCatcher- SDS-PAGE analysis results of ELP- ⁇ NSpyCatcher;
  • A SDS-PAGE analysis results of 3M NaCl-mediated GST purification
  • B SDS-PAGE analysis results of 3M NaCl-mediated LCB3 purification
  • C 3M NaCl-mediated ⁇ NSpyCatcher-ELP - SDS-PAGE analysis results of ⁇ NSpyCatcher purification
  • D SDS-PAGE analysis results of 0.7M Na 2 SO 4 mediated GST purification
  • E SDS-PAGE analysis results of 0.7M Na 2 SO 4 mediated LCB3 purification
  • F 0.7M Na 2 SO 4 mediated SDS-PAGE analysis results of ⁇ NSpyCatcher-ELP- ⁇ NSpyCatcher purification
  • G 0.7M (NH 4 ) 2 SO 4 mediated
  • Figure 11 shows the SDS-PAGE analysis results of purifying xylanase xylanase by 3M NaCl, 0.7M Na 2 SO 4 , 0.7M (NH 4 ) 2 SO 4 inducing aggregation of the fusion protein Xylanase-Mxe-MpA;
  • A 3M NaCl mediated xylanase expression and purification SDS-PAGE analysis results;
  • B 0.7M Na 2 SO 4 mediated xylanase expression and purification SDS-PAGE analysis results;
  • C 0.7M (NH 4 ) 2 SO 4 mediated xylanase expression and Purified SDS-PAGE analysis results.
  • Fig. 12 shows the results of affinity characterization between purified human growth hormone hGH and human growth hormone receptor protein Growth hormone receptor (Abcam, ab180053) detected by biofilm optical interferometry (BLI) technology.
  • Figure 13 shows the affinity characterization results of the purified novel coronavirus polypeptide LCB3 and its receptor protein SARS-CoV-2 Spike protein (GenScript, Z03483) detected by biofilm layer optical interference (BLI) technology.
  • Figure 14 shows the identification of the covalent binding of the multivalent scaffold protein ⁇ NSpyCatcher-ELP- ⁇ NSpyCatcher to LCB3-SpyTag by SDS-PAGE, and the activity of the multivalent scaffold protein is characterized according to the formation of its covalently bound product.
  • the term “and/or” covers all combinations of the items connected by the term, and each combination should be deemed to have been individually listed herein.
  • “A and/or B” includes “A,” “A and B,” and “B.”
  • “A, B, and/or C” encompasses “A,” “B,” “C,” “A and B,” “A and C,” “B and C,” and “A and B and C.”
  • Polypeptide “peptide”, and “protein” are used interchangeably herein to refer to a polymer of amino acid residues.
  • the term applies to amino acid polymers in which one or more amino acid residues are an artificial chemical analog of the corresponding natural amino acid, as well as to polymers of natural amino acids.
  • the terms “polypeptide”, “peptide”, “amino acid sequence” and “protein” may also include modified forms including, but not limited to, glycosylation, lipid linkage, sulfation, gamma carboxylation of glutamic acid residues, hydroxylation ylation and ADP-ribosylation.
  • variable refers to a polypeptide or polynucleotide comprising one or more amino acid or nucleotide mutations compared to its parent.
  • variant and mutant are used interchangeably.
  • the term "corresponds to” means that those skilled in the art use known sequence alignment methods to align two or more related polypeptide or nucleic acid sequences (including sequences of molecules, regions of molecules) to maximize matching. and/or theoretical sequences) to obtain the highest level of matching, the parts, positions or regions that align with each other. In other words, when two or more polypeptide or nucleic acid sequences are optimally aligned, two similar positions (or portions or regions) align. When aligning two or more sequences, similar parts/positions/regions are identified based on their position along a linear nucleic acid or amino acid sequence.
  • polynucleotide refers to a macromolecule composed of multiple nucleotides linked by 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), so as to improve the expression of the polypeptide. Methods for performing codon optimization are known in the art.
  • hybridizes under stringent conditions refers to the annealing of a polynucleotide molecule to a target nucleic acid molecule by complementary base pairing.
  • Those skilled in the art are familiar with parameters that affect specific hybridization, such as the length and composition of a particular molecule. Parameters particularly relevant to hybridization also include, for example, annealing and washing temperatures, buffer composition and salt concentration.
  • hybridization under stringent conditions refers to hybridization under highly stringent conditions, ie 0.1 ⁇ SSPE, 0.1% SDS, 65°C.
  • hybridization under stringent conditions refers to hybridization under moderately stringent conditions, ie 0.2 ⁇ SSPE, 0.1% SDS, 50°C.
  • hybridization under stringent conditions refers to hybridization under low stringency conditions, ie 0.2 ⁇ SSPE, 0.1% SDS, 40°C.
  • Equivalent stringent conditions are known in the art. Those skilled in the art can adjust the parameters affecting hybridization to achieve hybridization of polynucleotide molecules to target nucleic acid molecules under conditions of low, medium or high stringency.
  • the protein or nucleic acid may consist of the sequence, or may have additional Amino acids or nucleotides, but still have the activity described in the present invention.
  • those skilled in the art know that the methionine encoded by the initiation codon at the N-terminal of the polypeptide may be retained in some practical cases (eg, when expressed in a specific expression system), but it does not substantially affect the function of the polypeptide.
  • expression generally refers to the process by which a polypeptide is produced by transcription and translation of a polynucleotide.
  • expression can be understood as “heterologous expression”, which refers to the expression in a host cell or in vitro expression of a polypeptide encoded by a heterologous nucleic acid.
  • 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.
  • expression of a nucleotide sequence can refer to transcription of the nucleotide sequence (eg, transcription to produce mRNA or functional RNA) and/or translation of the 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 capable of translation (such as mRNA).
  • a nucleotide sequence of interest is operably linked to regulatory sequences in an expression construct.
  • regulatory sequence and “regulatory element” are used interchangeably to refer to a sequence located upstream (5' non-coding sequence), midway or downstream (3' non-coding sequence) of a coding sequence and which affects the transcription, RNA, Processing or stability or translation of nucleotide sequences. Regulatory sequences may include, but are not limited to, promoters, translation leader sequences, introns, and polyadenylation recognition sequences.
  • operably linked means that a regulatory sequence is linked to a nucleotide sequence of interest, such that the transcription of the nucleotide sequence of interest is controlled and regulated by the regulatory sequence.
  • Techniques for operably linking regulatory sequences to a nucleotide sequence of interest are known in the art.
  • self-aggregation refers to a property of a polypeptide, that is, monomers of the polypeptide are assembled into polymers under certain physical and/or chemical conditions.
  • purity refers to the purity of the target protein, that is, the ratio of the target protein 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 cell (such as Escherichia coli, there are thousands of proteins), it has always been a key to purify the target protein from such a large variety of protein mixtures. technical challenge. Through steps such as 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.
  • ionic strength is a measure of the concentration of ions in a solution. Ionic strength is measured in molarity (mol/L) and is calculated by multiplying the mass molarity (mi) of each ion i in solution by the valence of that ion The sum of the items obtained by the square of (zi) is half.
  • CpA or “CpA short peptide” refers to an amphipathic polypeptide known in the art, which has a hydrophilic region and a hydrophobic region separated from each other, wherein the alpha helical state of CpA is limited by the salt concentration
  • a specific self-aggregation structure can be spontaneously formed, and within a certain range as the concentration of salt ions increases, the strength of the aggregate increases and the volume increases. Increase (Daniel E.W. et al., Proceedings of the National Academy of Sciences, 2005, 102:12656–12661).
  • the CpA peptide has the amino acid sequence shown in SEQ ID NO: 1.
  • the CpA peptide has the nucleotide sequence shown in SEQ ID NO:8.
  • the present invention constructs the variant MpA of the short peptide CpA by introducing amino acid mutations, which has better salt concentration-responsive self-aggregation characteristics than CpA, especially higher aggregation efficiency, higher recovery efficiency and higher purity of the target protein.
  • the salt concentration-responsive self-aggregation property refers to the property of being soluble under the first salt condition and capable of self-aggregation under the second salt condition.
  • said first salt condition comprises a first salt concentration and said second salt condition comprises a second salt concentration.
  • said first salt concentration is different than said second salt concentration.
  • said first salt concentration is higher than said second salt concentration.
  • said first salt concentration is lower than said second salt concentration.
  • said first salt condition comprises a first salt species and said second salt condition comprises a second salt species.
  • said first salt species is the same as said second salt species.
  • said first salt species is different from said second salt species.
  • the aggregation efficiency of the CpA variant of the invention is increased by 5% to 300% compared to the aggregation efficiency of CpA.
  • the present invention relates to an isolated polypeptide, which is a variant of a short peptide of CpA, wherein said CpA has an amino acid sequence as shown in SEQ ID NO:1.
  • the CpA variant has mutations, such as deletions, insertions and/or substitutions, at the amino acid residues at positions corresponding to positions 1 and 17 of SEQ ID NO:1.
  • the CpA variant has amino acid substitutions at the amino acid residues at positions corresponding to positions 1 and 17 of SEQ ID NO:1.
  • the amino acid residues of the CpA variant at positions corresponding to positions 1 and 17 of SEQ ID NO: 1 are substituted with methionine (M).
  • the cysteine (C) at the positions corresponding to positions 1 and 17 of SEQ ID NO: 1 is substituted with methionine (M) in the CpA variant.
  • the CpA variant comprises amino acid substitutions of C1M and C17M at positions corresponding to positions 1 and 17 of SEQ ID NO: 1.
  • the amino acid sequence of the CpA variant is shown in SEQ ID NO:2.
  • a peptide with salt concentration-responsive self-aggregation properties and two structurally and functionally unrelated target polypeptides are used to form a fusion polypeptide, and the short peptide with salt-concentration-responsive self-aggregation will make the fusion protein triplet change
  • the fusion protein triplet is induced to aggregate into a precipitate, and the fusion polypeptide triplet can be separated from the impurity components in the bacterial protein extract by simple centrifugation or filtration to obtain a high-purity fusion protein triplet.
  • Such a protein purification process simplifies the steps of protein separation and purification, avoids repeated purifications to ensure yields, avoids the use of expensive purification columns, significantly reduces production costs, and aggregates at low or normal temperatures to avoid the purpose Degradation of polypeptides, and the final purified protein has high purity, high recovery rate and maintains the corresponding protein activity.
  • the inventors unexpectedly found that by using the CpA variant MpA as a salt concentration-responsive self-aggregating peptide to form a fusion polypeptide with the target polypeptide, only one adjustment of the salt concentration is required for protein precipitation, and the target protein with a purity of usually more than 85% can be obtained , its purification efficiency is equivalent to that of a purification column and the steps are simple. On the one hand, it can be used for high-throughput protein purification on a laboratory scale. On the other hand, due to its high economy, it overcomes the bottleneck of industrial applications.
  • the present invention relates to a fusion polypeptide comprising a target polypeptide part and a salt concentration-responsive self-aggregating peptide part, wherein the target polypeptide part is linked to the salt-concentration-responsive self-aggregating peptide part through a spacer, and wherein the The spacer comprises a cleavage site.
  • the salt concentration-responsive self-aggregating peptide portion comprises a salt-responsive self-aggregating peptide.
  • the salt concentration-responsive self-aggregating peptide is a peptide that is soluble under a first salt condition and capable of self-aggregating under a second salt condition.
  • the salt concentration-responsive self-aggregating peptide is a CpA variant, wherein the CpA has an amino acid sequence as shown in SEQ ID NO:1.
  • the CpA variant has mutations, such as deletions, insertions and/or substitutions, at the amino acid residues at positions corresponding to positions 1 and 17 of SEQ ID NO:1.
  • the CpA variant has amino acid substitutions at the amino acid residues at positions corresponding to positions 1 and 17 of SEQ ID NO:1.
  • the amino acid residues of the CpA variant at positions corresponding to positions 1 and 17 of SEQ ID NO: 1 are substituted with methionine (M).
  • the cysteine (C) at the positions corresponding to positions 1 and 17 of SEQ ID NO: 1 is substituted with methionine (M) in the CpA variant.
  • the CpA variant comprises amino acid substitutions of C1M and C17M at positions corresponding to positions 1 and 17 of SEQ ID NO: 1.
  • the amino acid sequence of the salt concentration-responsive self-aggregating peptide is shown in SEQ ID NO:2.
  • the salt concentration-responsive self-aggregating peptide moiety of the present invention may comprise one or more of said salt-responsive self-aggregating peptides linked in series.
  • the salt concentration-responsive self-aggregating peptide moiety of the present invention may comprise 1 to 150, 1 to 130, 1 to 110, 1 to 90, 1 to 70, 1 to 50, 1 to 30, 1 to 10, 1 to 5
  • the salt concentration-responsive self-aggregation peptide for example, 1, 2, 3, 4, 5 salt concentration-responsive self-aggregation peptides.
  • Two or more salt concentration-responsive self-aggregating peptides in the salt-responsive self-aggregating peptide portion may form tandem repeats. For ease of recombination operations and for production cost considerations, it is desirable to use fewer repetitions.
  • the salt concentration-responsive self-aggregating peptide portion comprises only one of the salt-responsive self-aggregating peptides.
  • spacer refers to a polypeptide having a certain length of amino acid composition, which includes sequences required to achieve cleavage, such as protease recognition sequences for enzymatic cleavage, intein sequences for self-cleavage, etc., and Linking the parts of the fusion protein does not affect the structure and activity of the parts.
  • the spacers of the invention comprise a "cleavage site".
  • the spacer is directly linked to the polypeptide part of interest and/or the salt concentration-responsive self-aggregating peptide part.
  • the spacer further comprises a linker at its N-terminus and/or C-terminus.
  • the spacer is linked to the polypeptide portion of interest and/or the salt concentration-responsive self-aggregating peptide portion via a linker.
  • the cleavage site is located at the C-terminus of the spacer, and the cleavage site is immediately N-terminal to the polypeptide portion of interest.
  • the cleavage site is located N-terminal to the spacer, and the cleavage site is immediately C-terminal to the polypeptide portion of interest.
  • the spacer is linked to the polypeptide portion of interest through the cleavage site.
  • the spacer is linked directly to the N- or C-terminus of the polypeptide portion of interest via the cleavage site.
  • the cleavage site used for releasing the soluble portion of the polypeptide of interest from the insoluble portion (precipitation) of the present invention includes a temperature-dependent cleavage site, a pH-dependent cleavage site, an ion-dependent cleavage site, An enzymatic cleavage site or a self-cleavage site, or any other cleavage site known to those skilled in the art.
  • the cleavage site is a self-cleavage site.
  • the cleavage site is a pH-dependent cleavage site.
  • the spacer is attached to the N- or C-terminus of the polypeptide portion of interest. It should be understood that those skilled in the art can select a suitable spacer as needed, and select a suitable connection position of the spacer.
  • the spacer comprises an intein comprising a self-cleavage site.
  • Intein is a special sequence polypeptide with protease activity, which can be cleaved at specific amino acid residues at the designed site after inducing its protease activity, so that the target polypeptide is separated from the fusion polypeptide triplet and released into a soluble solution , the target polypeptide can be obtained with high purity. Therefore, the intein-based cleavage method does not require the addition of enzymes or the use of harmful substances such as hydrogen bromide used in chemical methods, but can simply induce cleavage by changing the buffer environment in which the aggregates are located (Wu et al. , 1998; TELENTI et al., 1997).
  • Various self-cleaving inteins are known in the art, for example, a series of inteins with different self-cleaving properties from NEB Company.
  • the intein is selected from Mxe GyrA, Ssp DnaB or Mtu ⁇ I-CM.
  • Mtu ⁇ I-CM is derived from the Mtu recA wild-type intein by deleting the endonuclease domain of the Mtu recA extra large intein, retaining the N-terminal 110 amino acids and the C-terminal 58 amino acids, A very small intein was obtained, and then four mutations were introduced: C1A, V67L, D24G, D422G (Wood et al., 1999).
  • the Mtu ⁇ I-CM comprises the sequence shown in SEQ ID NO:3.
  • the Mtu ⁇ I-CM has the nucleotide sequence of SEQ ID NO:10.
  • the Mtu ⁇ I-CM is linked to the C-terminus of the polypeptide portion of interest.
  • the intein Mtu ⁇ I-CM can induce self-cleavage of the intein at its carboxyl terminus by a buffer system at pH 5.5-6.8.
  • the spacer is a mutant of Mtu ⁇ I-CM.
  • linker refers to a polypeptide with a certain length consisting of amino acids with low hydrophobicity and low charge effect. When it is used in a fusion protein, it can fully unfold the connected parts and fully fold into respective native conformations.
  • Linkers commonly used in the art include, for example, flexible GS-type linkers rich in glycine (G) and serine (S); rigid PT-type linkers rich in proline (P) and threonine (T).
  • the linker is selected from a GS-type linker and a PT-type linker.
  • the amino acid sequence of the GS-type linker used in the present invention is shown in SEQ ID NO:6.
  • the amino acid sequence of the PT-type linker used in the present invention is shown in SEQ ID NO:7.
  • the nucleotide sequence of the GS-type adapter used in the present invention is shown in SEQ ID NO: 13.
  • the nucleotide sequence of the PT-type linker used in the present invention is shown in SEQ ID NO:14.
  • the polypeptide of interest is 20, 50, 70, 100, 150, 200, 250, 300, 350, 400, 450, or 500 amino acid residues in length, or between any two of the above-mentioned lengths. Any length.
  • the polypeptide portion of interest is selected from a therapeutic molecule, a detectable molecule, or a targeting molecule.
  • the therapeutic molecules include, but are not limited to, nucleic acid drugs, protein drugs (including therapeutic polypeptides, therapeutic antibodies, etc.) and the like.
  • Exemplary therapeutic molecules include, but are not limited to, toxins, immunomodulators, antagonists, apoptosis inducers, hormones, radiopharmaceuticals, anti-angiogenic agents, gene therapy cytokines, chemokines, prodrugs, chemotherapeutics, etc. , such as human growth hormone (hGH), new crown polypeptide LCB3, etc.
  • the detectable molecules include but are not limited to fluorescent proteins, enzymes, labels, etc., such as red fluorescent protein (RFP), glutathione thiol transferase GST, Xylanase, etc.
  • fluorescent proteins such as red fluorescent protein (RFP), glutathione thiol transferase GST, Xylanase, etc.
  • the targeting molecules include but are not limited to targeting antibodies, specific receptor ligands and the like.
  • the targeting molecule can be an antibody that specifically targets a tumor antigen.
  • the target polypeptide is selected from human growth hormone (hGH), red fluorescent protein (RFP), glutathione thiol transferase GST, new crown polypeptide LCB3, multivalent backbone protein ⁇ NSpyCatcher-ELP- ⁇ NSpyCatcher or Xylanase .
  • the polypeptide portion of interest comprises a polypeptide selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55 or SEQ ID NO: 56 The amino acid sequence shown.
  • the polypeptide of interest is selected from the group consisting of SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59 or SEQ ID NO: 60 the nucleotide sequence.
  • the portion of the polypeptide of interest is located at the N- or C-terminus of the fusion polypeptide.
  • the spacer is attached to the N- or C-terminus of the polypeptide portion of interest.
  • the polypeptide portion of interest is located at the C-terminus of the fusion polypeptide, and the spacer is attached to the N-terminus of the polypeptide portion of interest.
  • the polypeptide portion of interest is located at the N-terminus of the fusion polypeptide, and the spacer is attached to the C-terminus of the polypeptide portion of interest.
  • the fusion polypeptide has the following structure from N-terminus to C-terminus: salt concentration-responsive self-aggregation peptide such as MpA-spacer-purpose polypeptide, or target polypeptide-spacer-salt concentration-responsive self-aggregation peptide For example MpA.
  • the fusion polypeptide has the following structure from N-terminus to C-terminus: salt concentration-responsive self-aggregation peptide such as MpA-linker-spacer-target polypeptide, or target polypeptide-spacer-linker-salt concentration-responsive Self-aggregating peptides such as MpA.
  • the fusion polypeptide has the following structure from N-terminus to C-terminus: MpA-linker-Mtu ⁇ I-CM-polypeptide of interest such as human growth hormone or RFP, or polypeptide of interest such as human growth hormone or RFP-Mxe GyrA- Linker - MpA.
  • the invention provides an isolated polynucleotide comprising a nucleotide sequence encoding a fusion polypeptide of the invention, or the complement thereof.
  • the present invention provides an isolated polynucleotide comprising a nucleotide sequence encoding a CpA variant or a complementary sequence thereof, wherein the CpA has an amino acid sequence as shown in SEQ ID NO: 1, wherein The CpA variants comprise amino acid substitutions of C1M and C17M at positions corresponding to positions 1 and 17 of SEQ ID NO:1.
  • the polynucleotide of the present invention comprises the nucleotide sequence of SEQ ID NO:9 or SEQ ID NO:61.
  • the isolated polynucleotide of the present invention comprises a polynucleotide sequence that hybridizes to the nucleotide sequence shown in SEQ ID NO:9 or SEQ ID NO:61 under stringent conditions.
  • the polypeptide encoded by the polynucleotide of the present invention still maintains an aggregation efficiency comparable to that of MpA.
  • the invention provides an expression construct comprising a polynucleotide of the invention operably linked to an expression control sequence.
  • an expression construct of the invention comprises a polynucleotide of the invention operably linked to an expression control sequence.
  • 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 are capable of integrating into and replicating with host cell DNA. Many vectors suitable for the present invention are commercially available.
  • the present invention provides a host cell containing the polynucleotide of the present invention or transformed with the expression construct of the present invention, wherein the host cell is capable of expressing the fusion polypeptide of the present invention or capable of expressing the CpA of the present invention Variants.
  • Host cells useful for expressing fusion polypeptides of the invention or CpA variants of the invention include prokaryotes, yeast, and higher eukaryotic cells.
  • Exemplary prokaryotic hosts include bacteria of the genera Escherichia, Bacillus, Salmonella, and the genera Pseudomonas and Streptomyces.
  • the host cell is an Escherichia cell, preferably E. coli.
  • the host cells used are Escherichia coli BL21 (DE3) strain cells.
  • the recombinant expression constructs of the present invention can be introduced into host cells by one of many well-known techniques including, but not limited to: heat shock transformation, electroporation, DEAE-dextran transfection, microinjection, lipid Infection-mediated transfection, calcium phosphate precipitation, protoplast fusion, particle bombardment, viral transformation and similar techniques.
  • the invention provides a method of producing a fusion polypeptide of the invention, comprising:
  • step b) obtaining the fusion polypeptide expressed by said host cell from the culture obtained from step a).
  • the present invention also relates to a method for producing and purifying a target polypeptide, the method comprising the following steps: (a) culturing the host cell of the present invention to express the fusion polypeptide; (b) lysing the host cell under the first salt condition, The insoluble fraction of the cell lysate is then removed, and the soluble fraction is recovered; (c) under a second salt condition, the fusion protein forms an insoluble fraction; (d) recovering the insoluble fraction formed in step (c); (e) by cleaving said cleavage site to release soluble polypeptide of interest from the insoluble fraction collected from step (d); and (f) removing the insoluble fraction from step (e), recovering a soluble fraction containing said polypeptide of interest.
  • a schematic diagram of the method of the present invention can be seen in Figure 1A.
  • the method for lysing the host cells is selected from the usual treatment methods in the art, such as ultrasound, homogenization, high pressure (such as in a French press), osmolysis, detergent, lyase , organic solvents or combinations thereof.
  • said first salt condition comprises a first salt concentration and said second salt condition comprises a second salt concentration. In one embodiment, said first salt concentration is different than said second salt concentration. In one embodiment, said first salt concentration is higher than said second salt concentration. In one embodiment, said first salt concentration is lower than said second salt concentration. In one embodiment, said first salt condition comprises a first ionic strength and said second salt condition comprises a second ionic strength. In one embodiment, said first ionic strength is different from said second ionic strength. In one embodiment, said first ionic strength is higher than said second ionic strength. In one embodiment, said first ionic strength is lower than said second ionic strength. In one embodiment, said first salt condition comprises a first salt species and said second salt condition comprises a second salt species. In one embodiment, said first salt species is the same as said second salt species. In one embodiment, said first salt species is different from said second salt species.
  • said salt under said first salt condition and/or under said second salt condition is selected from monovalent metal salts such as Potassium salt or sodium salt, etc., divalent metal salts such as magnesium salt, calcium salt, manganese salt or copper salt, etc., or ammonium salt, preferably ammonium salt, potassium salt or sodium salt.
  • monovalent metal salts such as Potassium salt or sodium salt, etc.
  • divalent metal salts such as magnesium salt, calcium salt, manganese salt or copper salt, etc.
  • ammonium salt preferably ammonium salt, potassium salt or sodium salt.
  • the anion of said salt under said first salt condition and/or under said second salt condition is selected from sulfate, hydrogenphosphate, acetate, halides such as fluoride, chloride, bromide Or iodide ion etc., nitrate, perchlorate, or thiocyanate ion, preferably sulfate, hydrogenphosphate, chloride or acetate.
  • said salt under said first salt condition and/or under said second salt condition is selected from sodium chloride, sodium sulfate, sodium nitrate, disodium hydrogen phosphate, sodium dihydrogen phosphate, sodium carbonate, Potassium chloride, potassium sulfate, potassium nitrate, dipotassium hydrogenphosphate, potassium dihydrogenphosphate, potassium carbonate, ammonium nitrate, ammonium sulfate or ammonium chloride, preferably sodium chloride, sodium sulfate or ammonium sulfate.
  • the first ionic strength is 0-0.2 mol/L. In one embodiment, the first ionic strength is 0-0.1 mol/L or 0.1-0.2 mol/L. In one embodiment, the first ionic strength is about 0 mol/L, about 0.1 mol/L, or about 0.2 mol/L. In one embodiment, the second ionic strength is 0.5-5.0 mol/L. In one embodiment, the second ionic strength is 1.0-4.5 mol/L, 1.5-4.0 mol/L, 1.5-2.5 mol/L, 2.0-3.5 mol/L or 2.5-3.0 mol/L.
  • the second ionic strength is about 0.5 mol/L, about 0.8 mol/L, about 1.0 mol/L, about 1.2 mol/L, about 1.5 mol/L, about 1.8 mol/L, about 2.0mol/L, about 2.1mol/L, about 2.2mol/L, about 2.5mol/L, about 2.8mol/L, about 3.0mol/L, about 3.2mol/L, about 3.5mol/L, about 3.8mol /L, about 4.0mol/L, about 4.2mol/L, about 4.5mol/L, about 4.8mol/L, about 5.0mol/L, or any ionic strength between any two of the aforementioned ionic strengths.
  • the second salt condition is selected from 0.5-4M NaCl, preferably 3M NaCl; 0.2-1.5M Na 2 SO 4 , preferably 0.7M Na 2 SO 4 ; or 0.2-1.5M (NH 4 ) 2 SO 4 , preferably 0.7M (NH 4 ) 2 SO 4 .
  • the first salt condition is about OM.
  • step (e) is performed under second salt conditions.
  • the second salt species is NaCl, and the second ionic strength is 2.5-3.0 mol/L, preferably about 3 mo/L.
  • the second salt species is Na 2 SO 4 , and the second ionic strength is 1.5-2.5 mol/L, preferably about 2.1 mo/L.
  • the second salt species is (NH 4 ) 2 SO 4 , and the second ionic strength is 1.5-2.5 mol/L, preferably about 2.1 mol/L. In one embodiment, the second salt species is K 2 SO 4 , and the second ionic strength is 1.5-2.5 mol/L, preferably about 2.1 mo/L. In one embodiment, the second salt species is Na 2 HPO 4 , and the second ionic strength is 1.5-2.5 mol/L, preferably about 2.1 mo/L.
  • host cells are cultured under physiological conditions (eg normal temperature 18-37° C., neutral pH 7.4-7.8) to express the fusion protein of the present invention. Therefore, since the expression is carried out in the host cells cultured under normal physiological conditions, the prolongation of the culture period of the host cells is avoided, and at the same time, the yield and yield of the fusion protein can be increased due to the appropriate culture conditions.
  • physiological conditions eg normal temperature 18-37° C., neutral pH 7.4-7.8
  • said step (c) is performed at a temperature between 4°C and 25°C. In one embodiment, said step (c) is performed at a temperature of 4°C-20°C, 4°C-15°C or 4°C-10°C. In one embodiment, said step (c) is performed at a temperature of 4°C, 10°C, 15°C, 20°C or 25°C, preferably at 4°C. In one embodiment, said step (e) is performed at a temperature between 4°C and 25°C. In one embodiment, said step (e) is performed at a temperature of 4°C-20°C, 4°C-15°C or 4°C-10°C.
  • said step (e) is carried out at a temperature of 4°C, 10°C, 15°C, 20°C or 25°C, preferably at 25°C. In one embodiment, both step (c) and step (e) are performed at a temperature of 4°C to 25°C. In one embodiment, neither said step (c) nor said step (e) is performed at a temperature higher than 25°C. In one embodiment, said step (b) to said step (f) are all carried out at a temperature of 4°C-25°C. In one embodiment, none of said step (b) to said step (f) is performed at a temperature higher than 25°C. Therefore, the present invention omits the step of repeatedly changing the temperature condition to obtain the fusion protein in a precipitated state, and also avoids the influence of excessive temperature on protein stability and activity.
  • said step (c) comprises adjusting the salt concentration of the solution comprising the soluble fraction collected from step (b). In one embodiment, said step (c) comprises reducing the salt concentration of the solution containing the soluble fraction collected from step (b). In one embodiment, said step (c) comprises increasing the salt concentration of the solution containing the soluble fraction collected from step (b).
  • said step (c) and step (d) are performed 1, 2 or 3 times. In one embodiment, said step (e) and step (f) are performed 1, 2 or 3 times. In one embodiment, said step (c) and said step (e) are performed only once. In one embodiment, said step (c) to said step (f) are performed only once.
  • the step (b) is carried out under neutral to slightly alkaline pH conditions.
  • the neutral to slightly alkaline pH condition is pH 7.2-8.5.
  • the neutral to slightly alkaline pH condition is pH 7.4-8.3.
  • the neutral to slightly alkaline pH condition is pH 7.6-8.2.
  • the neutral to slightly alkaline pH condition is pH 8.0.
  • the step (c) is carried out under neutral to slightly alkaline pH conditions.
  • the neutral to slightly alkaline pH condition is pH 7.2-8.5.
  • the neutral to slightly alkaline pH condition is pH 7.4-8.3.
  • the neutral to slightly alkaline pH condition is pH 7.6-8.2. In a most preferred embodiment, the neutral to slightly alkaline pH condition is pH 8.0. In one embodiment, step (e) is carried out under slightly acidic pH conditions. In a specific embodiment, the slightly acidic pH condition is pH 5.5-6.8, and preferably 5.5-6.5. In a most preferred embodiment, the slightly acidic pH condition is pH 6.2.
  • Embodiment 1 Construct MpA-Mtu-POI, CpA-Mtu-POI, POI-Mxe-MpA fusion protein expression vector
  • POI refers to the target protein.
  • POI refers to human growth hormone hGH, red fluorescent protein RFP, glutathione sulfhydryl transferase GST, new crown polypeptide LCB3, multivalent backbone protein ⁇ NSpyCatcher-ELP- ⁇ NSpyCatcher or xylan Enzyme Xylanase.
  • the expression vectors used include pET30-MpA-Mtu-hGH, pET30-MpA-Mtu-RFP, pET30-CpA-Mtu-hGH, pET30-CpA-Mtu-RFP, pET30a-MpA-Mtu-GST, pET30a-MpA- Mtu-LCB3, pET30a-MpA- ⁇ NSpyCatcher-ELP- ⁇ NSpyCatcher and pET30a-MpA-Mtu-Xylanase, the primers required for constructing the plasmid, were designed by oligo 6 and synthesized by Shanghai Sangong as shown in Table 1. primers.
  • the underlined parts of the primers are the recognition sites of restriction endonucleases NdeI and XhoI, respectively.
  • the nucleotide sequences of the MpA and PT linkers were designed with the online tool DNAworks (https://hpcwebapps.cit.nih.gov/dnaworks/).
  • Design and synthesize 4 oligonucleotide primers (SEQ ID No: 15-18) as shown in Table 1 MpA by DNAWorks, mix the 4 oligonucleotide primers of 10 ⁇ M in equal volume and take 2 ⁇ L, Then add 1 ⁇ L of 10 ⁇ M dNTP, 4 ⁇ L of 5 ⁇ Q5 reaction buffer, 12.8 ⁇ L of ddH 2 O, and 0.2 ⁇ L of Q5 DNA polymerase.
  • the PCR conditions are: 98°C for 30 sec, 98°C for 10 sec, 60°C for 20 sec, and 72°C for 15 sec, a total of 14 cycles. Finally, 72°C for 2min.
  • the PCR conditions are: 98°C for 30sec, 98°C for 10sec, 68°C for 20sec, and 72°C 15sec, a total of 29 cycles, the last 2min at 72°C.
  • the PCR amplification product was separated and recovered on 1% agarose gel to obtain the NdeI-MpA-PT polynucleotide fragment.
  • Mtu-hGH-XhoI was obtained by PCR amplification using primers Mtu-F and Mtu-hGH-3-R polynucleotide fragments.
  • NdeI-MpA-PT and Mtu-hGH-XhoI as templates, using primers MpA-F and Mtu-hGH-3-R, NdeI-MpA-PT-Mtu was obtained by overlapping PCR (overlapping PCR) method - hGH-XhoI polynucleotide sequence.
  • the polynucleotide fragments purified by overlapping PCR and the pET30a plasmid (Novagen) were double-digested with restriction endonucleases Nde I and Xho I, respectively, and then the corresponding fragments were recovered for purification. After purification, they were ligated with T4 DNA ligase.
  • the ligation product was transformed into Escherichia coli DH5 ⁇ competent cells, and the transformed cells were spread on LB plates supplemented with 50 ⁇ g/mL kanamycin to screen positive clones, and the plasmids were extracted with a plasmid extraction kit and sequenced.
  • Plasmids pET30a-MpA-Mtu-GST and pET30a-MpA-Mtu- ⁇ NSpyCatcher-ELP- ⁇ NSpyCatcher are constructed in a similar way, using pET30-MpA-Mtu-hGH and the target gene (GST or ⁇ NSpyCatcher-ELP- ⁇ NSpyCatcher) as templates respectively, by The method of Gibson assembly is obtained.
  • pET30a-MpA-Mtu-GST using the plasmid pET30-MpA-Mtu-hGH as a template, using primers GST-Backbone-F and Mtu-R to amplify the Backbone-MpA-Mtu polynucleotide fragment, using GST base Because the template was amplified with primers GST-F and GST-R to obtain the GST fragment, the purified Backbone-MpA-Mtu and GST polynucleotide fragments were assembled by Gibson assembly, and the assembled product was transformed into Escherichia coli DH5 ⁇ competent cells.
  • Transformed cells were plated on LB plates supplemented with 50 ⁇ g/mL kanamycin to screen for positive clones, and plasmids were extracted with a plasmid extraction kit and sequenced.
  • the templates used for the construction of pET30-MpA-Mtu- ⁇ NSpyCatcher-ELP- ⁇ NSpyCatcher are pET30-MpA-Mtu-hGH plasmid and ⁇ NSpyCatcher-ELP- ⁇ NSpyCatcher gene, the primers and operation procedures required for cloning are the same as pET30a-MpA-Mtu-GST similar.
  • the plasmid pET30a-MpA-Mtu-LCB3 first use the LCB3 gene as a template to amplify the LCB3 polynucleotide fragment using primers LCB3-F and LCB3-R, and then use pET32-L6KD-Mtu ⁇ I-CM-hGH (Lin Zhanglin etc., PCT/CN2020/125054, 2020) as a template, using primers Mtu-F and Mtu-R to amplify the Mtu polynucleotide fragment, and then using the polynucleotide fragment Mtu and LCB3 as a template, using primers Mtu-F and LCB3 -R, the Mtu-LCB3 polynucleotide fragment was obtained by overlapping PCR (overlapping PCR), and then using pET30a-MpA-Mtu-hGH as a template, using primers LCB3-Backbone
  • pET30-CpA-Mtu-hGH using the plasmid pET30-MpA-Mtu-hGH as a template, using primers Backbone-2-F and CpA-R to amplify the Backbone-CpA polynucleotide fragment, using primer CpA -F and Backbone-2-R amplified to obtain CpA-Mtu-hGH-Backbone polynucleotide fragments, the purified Backbone-CpA and CpA-Mtu-hGH-Backbone polynucleotide fragments were assembled by Gibson assembly, and the assembled products were transformed To Escherichia coli DH5 ⁇ competent cells, the transformed cells were spread on LB plates supplemented with 50 ⁇ g/mL kanamycin to screen positive clones, and the plasmids were extracted with a plasmid extraction kit and sequenced.
  • the template used in the construction of pET30-CpA-Mtu-RFP is pET30-MpA-Mtu-RFP, and the primers and operating procedures required for cloning are similar to those of pET30-CpA-Mtu-hGH.
  • the structures of the constructed plasmids pET30-MpA-Mtu-hGH, pET30-MpA-Mtu-RFP, pET30-CpA-Mtu-hGH and pET30-CpA-Mtu-RFP are shown in Figure 1B.
  • plasmid pET30a-Xylanase-Mxe-MpA first use the method similar to the synthesis of NdeI-MpA-PT polynucleotide fragments to design primers GS-MpA-1, GS-MpA-2, GS-MpA-3 and GS through DNAWorks -MpA-4 was amplified by PCR, and then using the DNAWorks product as a template, using primers GS-MpA-F and MpA-R to amplify the GS-MpA polynucleotide fragment; then use pET30a-hGH-Mxe GyrA-L6KD as Template, use primers Xylanase-Mxe-F and Mxe-R to amplify to obtain Mxe polynucleotide fragments; then use Xylanase gene as template, use primers Xylanase-F and Xylanas
  • Embodiment 2 Expression of MpA-Mtu-POI and CpA-Mtu-POI fusion protein
  • the plasmid constructed in Example 1 (containing plasmids pET30-MpA-Mtu-hGH, pET30-MpA-Mtu-RFP, pET30-CpA-Mtu-hGH and pET30-CpA-Mtu-RFP) was transformed into Escherichia coli BL21 ( DE3) Four MpA/CpA-Mtu-POI fusion protein expression strains were obtained from the competent cells.
  • the cells were resuspended to 100OD/mL with lysis buffer B1 (dissolve 2.4g of Tris and 0.37g of EDTA ⁇ 2Na in 800mL of water, adjust the pH to 8.0, add water to 1L), and perform ultrasonic disruption (the breaking condition is : Horn ⁇ 2, 20% power, ultrasonic time 2sec, interval 2sec, running 25min ⁇ 30min). Centrifuge at 4°C and 15,000g for 30 min, collect the supernatant and precipitate respectively for sample preparation, and detect the expression of the fusion protein in the lysed supernatant and the lysed precipitate by SDS-PAGE.
  • lysis buffer B1 dissolve 2.4g of Tris and 0.37g of EDTA ⁇ 2Na in 800mL of water, adjust the pH to 8.0, add water to 1L
  • ultrasonic disruption the breaking condition is : Horn ⁇ 2, 20% power, ultrasonic time 2sec, interval 2sec, running 25min ⁇ 30min.
  • lanes a-d are the lysed supernatant and lysed pellet of MpA-Mtu-hGH and MpA-Mtu-RFP, respectively, a: MpA-Mtu-hGH cell lysed supernatant, and obvious fusion protein bands can be detected; b : MpA-Mtu-hGH cell lysate pellet, a faint fusion protein band can be detected; c: MpA-Mtu-RFP cell lysate supernatant, an obvious fusion protein band can be detected; d: MpA-Mtu-RFP Cells were lysed and precipitated, and fusion protein bands were hardly observed.
  • swimming lanes 1-5 are protein quantification standards containing bovine serum albumin BSA, and the loading amounts are 0.5 ⁇ g, 1.0 ⁇ g, 2.0 ⁇ g, 4.0 ⁇ g, and 8.0 ⁇ g.
  • lanes a-d are the lysed supernatant and lysed pellet of CpA-Mtu-hGH and CpA-Mtu-RFP respectively, a: CpA-Mtu-hGH cell lysed supernatant, and obvious fusion protein bands can be detected; b : CpA-Mtu-hGH cell lysate pellet, a faint fusion protein band can be detected; c: CpA-Mtu-RFP cell lysate supernatant, an obvious fusion protein band can be detected; d: CpA-Mtu-RFP Cells were lysed and precipitated, and fusion protein bands were hardly observed.
  • swimming lanes 1-5 are protein quantification standards containing bovine serum albumin BSA, and
  • fusion protein Amount of soluble expressiona (mg/L) Soluble ratiob (%) MpA-Mtu-hGH 614 ⁇ 9 93 MpA-Mtu-RFP 401 ⁇ 15 99 CpA-Mtu-hGH 446 ⁇ 40 98 CpA-Mtu-RFP 244 ⁇ 34 99
  • the four fusion proteins (MpA-Mtu-hGH, MpA-Mtu-RFP, CpA-Mtu-hGH and CpA-Mtu-RFP) were expressed in soluble form, and the amount of soluble expression was 614 ⁇ 9mg/ L, 401 ⁇ 15mg/L, 446 ⁇ 40mg/L and 244 ⁇ 34mg/L, the soluble proportions were 93%, 99%, 98% and 99%, respectively.
  • Example 3 3M NaCl-mediated MpA-Mtu-hGH/RFP phase transition and Mtu-mediated cleavage protein purification
  • Example 2 NaCl was added to the lysed supernatant obtained in Example 2 to 3 M and left overnight at 4° C. for 12 hours, so that the self-aggregating peptides were fully aggregated.
  • the suspension was centrifuged at 4°C and 15,000 g for 30 min, and the centrifuged pellet was washed with an equal volume of 3M NaCl-containing buffer B2 (175.32 g of NaCl, 2.4 g of Tris, 0.37 g of EDTA ⁇ 2N a Dissolve in 800mL water, adjust pH to 8.0, add water to volume to 1L) wash once, centrifuge and separate supernatant and precipitate under other conditions, use cleavage buffer B3 containing 3M NaCl (PBS supplemented with NaCl to 3M, make up Add 40mM Bis-Tris, pH 6.2, 2mM EDTA) to fully resuspend the pellet, and place it at 25°C for 24h, so that the intein is fully self-
  • lanes ad are human growth hormone hGH expression and purification samples, respectively: a: cell lysate supernatant, a clear fusion protein band can be detected; b: lysate supernatant separated after salt aggregation ; c: The precipitate separated from the supernatant of the lysate after adding salt and aggregation, and a clear fusion protein band can be detected; d: The precipitate obtained after cleavage of the aggregate obtained by adding salt; e: The upper part of the aggregate obtained after adding salt Clear, a clear band of human growth hormone hGH can be detected.
  • swimming lanes 1-6 are protein quantitative standards containing bovine serum albumin BSA, and the loading amounts are 0.125 ⁇ g, 0.25 ⁇ g, 0.5 ⁇ g, 1.0 ⁇ g, 2.0 ⁇ g, and 4.0 ⁇ g.
  • lanes ad are red fluorescent protein RFP expression and purification samples, respectively a: supernatant of cell lysate, clear fusion protein bands can be detected; b: supernatant of lysate supernatant after adding salt and aggregation ; c: The precipitate separated from the supernatant of the lysate after adding salt and aggregation, and a clear fusion protein band can be detected; d: The precipitate obtained after cleavage of the aggregate obtained by adding salt; e: The upper part of the aggregate obtained after adding salt Clearly, a clear red fluorescent protein RFP band can be detected.
  • swimming lanes 1-6 are protein quantitative standards containing bovine serum albumin BSA, and the loading amounts are 0.125 ⁇ g, 0.25 ⁇
  • a intein-mediated self-cleavage target protein yield (volume is calculated per liter of LB medium)
  • b aggregation efficiency 100% ⁇ the amount of fusion protein in the precipitate after adding salt/(in the precipitate after adding salt The amount of fusion protein+the amount of fusion protein in the supernatant after adding salt)
  • c -intein-mediated self-cleavage efficiency 100% ⁇ (the amount of fusion protein in the precipitate after adding salt-the amount of fusion protein in the precipitate after cleavage )/the amount of the fusion protein in the precipitate after adding salt
  • d recovery rate 100% ⁇ actual yield of the target protein/theoretical yield of the target protein that the expression supernatant can produce under the condition of complete cutting.
  • Example 4 0.7M Na 2 SO 4 mediated MpA-Mtu-hGH/RFP phase transition and Mtu-mediated cleavage protein purification
  • lanes ad are human growth hormone hGH expression and purification samples, respectively a: cell lysate supernatant, a clear fusion protein band can be detected; b: lysate supernatant separated after salt aggregation ; c: The precipitate separated from the supernatant of the lysate after adding salt and aggregation, and a clear fusion protein band can be detected; d: The precipitate obtained after cleavage of the aggregate obtained by adding salt; e: The upper part of the aggregate obtained after adding salt Clear, a clear band of human growth hormone hGH can be detected.
  • swimming lanes 1-6 are protein quantitative standards containing bovine serum albumin BSA, and the loading amounts are 0.125 ⁇ g, 0.25 ⁇ g, 0.5 ⁇ g, 1.0 ⁇ g, 2.0 ⁇ g, and 4.0 ⁇ g.
  • lanes ad are red fluorescent protein RFP expression and purification samples, respectively a: cell lysate supernatant, a clear fusion protein band can be detected; b: lysate supernatant separated after salt aggregation ; c: The precipitate separated from the supernatant of the lysate after adding salt and aggregation, and a clear fusion protein band can be detected; d: The precipitate obtained after cleavage of the aggregate obtained by adding salt; e: The upper part of the aggregate obtained after adding salt Clearly, a clear red fluorescent protein RFP band can be detected.
  • swimming lanes 1-6 are protein quantitative standards containing bovine serum albumin BSA, and the loading amounts are 0.125 ⁇ g, 0.25 ⁇ g, 0.5
  • a intein-mediated self-cleavage target protein yield (volume is calculated per liter of LB medium)
  • b aggregation efficiency 100% ⁇ the amount of fusion protein in the precipitate after adding salt/(in the precipitate after adding salt The amount of fusion protein+the amount of fusion protein in the supernatant after adding salt)
  • c -intein-mediated self-cleavage efficiency 100% ⁇ (the amount of fusion protein in the precipitate after adding salt-the amount of fusion protein in the precipitate after cleavage )/the amount of the fusion protein in the precipitate after adding salt
  • d recovery rate 100% ⁇ actual yield of the target protein/theoretical yield of the target protein that the expression supernatant can produce under the condition of complete cutting.
  • the lysed supernatants of the two fusion proteins were mostly changed from soluble to precipitated under the condition of 0.7M Na 2 SO 4 (96% of MpA- Mtu-hGH is converted into precipitate, 62% of MpA-Mtu-RFP is converted into precipitate), the intein Mtu ⁇ I-CM is self-cleaved, and the target protein is separated from MpA-Mtu with a cleavage efficiency of 61-95%, which is released on the
  • the yields of hGH and RFP in serum were 91 mg/L and 164 mg/L, respectively, and the purity of recovered hGH and RFP after cleavage were 99% and 87%, respectively.
  • Example 5 0.7M (NH 4 ) 2 SO 4 mediated MpA-Mtu-hGH/RFP phase transition and Mtu-mediated cleavage protein purification
  • lanes ad are human growth hormone hGH expression and purification samples, respectively a: cell lysate supernatant, a clear band of fusion protein can be detected; b: lysate supernatant separated after salt aggregation ; c: The precipitate separated from the supernatant of the lysate after adding salt and aggregation, and a clear fusion protein band can be detected; d: The precipitate obtained after cleavage of the aggregate obtained by adding salt; e: The upper part of the aggregate obtained after adding salt Clear, a clear band of human growth hormone hGH can be detected.
  • swimming lanes 1-6 are protein quantitative standards containing bovine serum albumin BSA, and the loading amounts are 0.125 ⁇ g, 0.25 ⁇ g, 0.5 ⁇ g, 1.0 ⁇ g, 2.0 ⁇ g, and 4.0 ⁇ g.
  • lanes ad are red fluorescent protein RFP expression and purification samples, respectively a: cell lysate supernatant, a clear fusion protein band can be detected; b: lysate supernatant separated after salt aggregation ; c: The precipitate separated from the supernatant of the lysate after adding salt and aggregation, and a clear fusion protein band can be detected; d: The precipitate obtained after cleavage of the aggregate obtained by adding salt; e: The upper part of the aggregate obtained after adding salt Clearly, a clear red fluorescent protein RFP band can be detected.
  • swimming lanes 1-6 are protein quantitative standards containing bovine serum albumin BSA, and the loading amounts are 0.125 ⁇ g, 0.25 ⁇ g, 0.5
  • a intein-mediated self-cleavage target protein yield (volume is calculated per liter of LB medium)
  • b aggregation efficiency 100% ⁇ the amount of fusion protein in the precipitate after adding salt/(in the precipitate after adding salt The amount of fusion protein+the amount of fusion protein in the supernatant after adding salt)
  • c -intein-mediated self-cleavage efficiency 100% ⁇ (the amount of fusion protein in the precipitate after adding salt-the amount of fusion protein in the precipitate after cleavage )/the amount of the fusion protein in the precipitate after adding salt
  • d recovery rate 100% ⁇ actual yield of the target protein/theoretical yield of the target protein that the expression supernatant can produce under the condition of complete cutting.
  • the lysed supernatant of the two fusion proteins (MpA-Mtu-hGH, MpA - Mtu -RFP) used was changed from soluble to precipitated (93 % of MpA -Mtu-hGH is converted into a precipitate, 50% of MpA-Mtu-RFP is converted into a precipitate), the intein Mtu ⁇ I-CM is self-cleaved, and the target protein is separated from MpA-Mtu with a cleavage efficiency of 72-98%, which is released into
  • the yields of hGH and RFP in the supernatant were 115 mg/L and 87 mg/L, respectively, and the purity of hGH and RFP recovered after cleavage were 93% and 94%, respectively.
  • Example 6 3M NaCl-mediated CpA-Mtu-hGH/RFP phase transition and protein purification of Mtu-mediated cleavage
  • Example 2 NaCl was added to the lysed supernatant obtained in Example 2 to 3 M and left overnight at 4° C. for 12 hours, so that the self-aggregating peptides were fully aggregated.
  • the suspension was centrifuged at 4°C and 15,000 g for 30 min, and the centrifuged pellet was washed with an equal volume of 3M NaCl-containing buffer B2 (175.32 g of NaCl, 2.4 g of Tris, 0.37 g of EDTA ⁇ 2N a Dissolve in 800mL water, adjust pH to 8.0, add water to volume to 1L) wash once, centrifuge and separate supernatant and precipitate under other conditions, use cleavage buffer B3 containing 3M NaCl (PBS supplemented with NaCl to 3M, make up Add 40mM Bis-Tris, pH 6.2, 2mM EDTA) to fully resuspend the pellet, and place it at 25°C for 24h, so that the intein is fully self-
  • lanes ad are human growth hormone hGH expression and purification samples, respectively: a: cell lysate supernatant, a clear fusion protein band can be detected; b: lysate supernatant separated after adding salt and aggregation ; c: The precipitate separated from the supernatant of the lysate after adding salt and aggregation, and a clear fusion protein band can be detected; d: The precipitate obtained after cleavage of the aggregate obtained by adding salt; e: The upper part of the aggregate obtained after adding salt Clear, a clear band of human growth hormone hGH can be detected.
  • swimming lanes 1-5 are protein quantitative standards containing bovine serum albumin BSA, and the loading amounts are 0.25 ⁇ g, 0.5 ⁇ g, 1.0 ⁇ g, 2.0 ⁇ g, and 4.0 ⁇ g.
  • lanes ad are red fluorescent protein RFP expression and purification samples, respectively a: cell lysate supernatant, a clear fusion protein band can be detected; b: lysate supernatant separated after salt aggregation ; c: The precipitate separated from the supernatant of the lysate after adding salt and aggregation, and a clear fusion protein band can be detected; d: The precipitate obtained after cleavage of the aggregate obtained by adding salt; e: The upper part of the aggregate obtained after adding salt Clearly, a clear red fluorescent protein RFP band can be detected.
  • swimming lanes 1-5 are protein quantitative standards containing bovine serum albumin BSA, and the loading amounts are 0.25 ⁇ g, 0.5 ⁇ g, 1.0 ⁇ g, 2.0 ⁇
  • a intein-mediated self-cleavage target protein yield (volume is calculated per liter of LB medium)
  • b aggregation efficiency 100% ⁇ the amount of fusion protein in the precipitate after adding salt/(in the precipitate after adding salt The amount of fusion protein+the amount of fusion protein in the supernatant after adding salt)
  • c -intein-mediated self-cleavage efficiency 100% ⁇ (the amount of fusion protein in the precipitate after adding salt-the amount of fusion protein in the precipitate after cleavage )/the amount of the fusion protein in the precipitate after adding salt
  • d recovery rate 100% ⁇ actual yield of the target protein/theoretical yield of the target protein that the expression supernatant can produce under the condition of complete cutting.
  • the lysed supernatant of the two fusion proteins (CpA-Mtu-hGH, CpA-Mtu-RFP) used was changed from soluble to precipitated (89% of CpA-Mtu-hGH converted to precipitated) under the condition of 3M NaCl , 25% of CpA-Mtu-RFP is converted into precipitate), the intein Mtu ⁇ I-CM is self-cleaved, the target protein is separated from CpA-Mtu, the cleavage efficiency is 52-80%, and the hGH and RFP are released into the supernatant after cleavage
  • the yields were 60 mg/L and 21 mg/L, respectively, and the purity of hGH and RFP recovered after cleavage were 73% and 74%, respectively.
  • Example 7 0.7M Na 2 SO 4 mediated CpA-Mtu-hGH/RFP phase transition and Mtu-mediated cleavage protein purification
  • lanes ad are human growth hormone hGH expression and purification samples, respectively a: cell lysate supernatant, a clear fusion protein band can be detected; b: lysate supernatant separated after adding salt and aggregation ; c: The precipitate separated from the supernatant of the lysate after adding salt and aggregation, and a clear fusion protein band can be detected; d: The precipitate obtained after cleavage of the aggregate obtained by adding salt; e: The upper part of the aggregate obtained after adding salt Clear, a clear band of human growth hormone hGH can be detected.
  • swimming lanes 1-5 are protein quantitative standards containing bovine serum albumin BSA, and the loading amounts are 0.25 ⁇ g, 0.5 ⁇ g, 1.0 ⁇ g, 2.0 ⁇ g, and 4.0 ⁇ g.
  • lanes ad are red fluorescent protein RFP expression and purification samples, respectively: a: cell lysate supernatant, a clear fusion protein band can be detected; b: lysate supernatant separated after salt aggregation ; c: The precipitate separated from the supernatant of the lysate after adding salt and aggregation, and a clear fusion protein band can be detected; d: The precipitate obtained after cleavage of the aggregate obtained by adding salt; e: The upper part of the aggregate obtained after adding salt Clearly, a clear red fluorescent protein RFP band can be detected.
  • swimming lanes 1-5 are protein quantitative standards containing bovine serum albumin BSA, and the loading amounts are 0.25 ⁇ g, 0.5 ⁇ g, 1.0 ⁇ g, 2.0
  • a intein-mediated self-cleavage target protein yield (volume is calculated per liter of LB medium)
  • b aggregation efficiency 100% ⁇ the amount of fusion protein in the precipitate after adding salt/(in the precipitate after adding salt The amount of fusion protein+the amount of fusion protein in the supernatant after adding salt)
  • c -intein-mediated self-cleavage efficiency 100% ⁇ (the amount of fusion protein in the precipitate after adding salt-the amount of fusion protein in the precipitate after cleavage )/the amount of the fusion protein in the precipitate after adding salt
  • d recovery rate 100% ⁇ actual yield of the target protein/theoretical yield of the target protein that the expression supernatant can produce under the condition of complete cutting.
  • the lysed supernatant of the two fusion proteins (CpA-Mtu-hGH, CpA-Mtu-RFP) used was changed from soluble to precipitated in 0.7M Na 2 SO 4 (87% of CpA-Mtu- hGH is converted into a precipitate, 26% of CpA-Mtu-RFP is converted into a precipitate), the intein Mtu ⁇ I-CM is self-cleaved, and the target protein is separated from CpA-Mtu with a cleavage efficiency of 60-97%, which is released into the supernatant after cleavage
  • the yields of hGH and RFP were 75 mg/L and 36 mg/L, respectively, and the purity of hGH and RFP recovered after cleavage were 79% and 96%, respectively.
  • Example 8 0.7M (NH 4 ) 2 SO 4 mediated CpA-Mtu-hGH/RFP phase transition and protein purification of Mtu-mediated cleavage
  • lanes ad are human growth hormone hGH expression and purification samples, respectively a: cell lysate supernatant, a clear fusion protein band can be detected; b: lysate supernatant separated after adding salt and aggregation ; c: The precipitate separated from the supernatant of the lysate after adding salt and aggregation, and a clear fusion protein band can be detected; d: The precipitate obtained after cleavage of the aggregate obtained by adding salt; e: The upper part of the aggregate obtained after adding salt Clear, a clear band of human growth hormone hGH can be detected.
  • swimming lanes 1-5 are protein quantitative standards containing bovine serum albumin BSA, and the loading amounts are 0.25 ⁇ g, 0.5 ⁇ g, 1.0 ⁇ g, 2.0 ⁇ g, and 4.0 ⁇ g.
  • lanes ad are red fluorescent protein RFP expression and purification samples, respectively: a: cell lysate supernatant, a clear fusion protein band can be detected; b: lysate supernatant separated after adding salt ; c: The precipitate separated from the supernatant of the lysate after adding salt and aggregation, and a clear fusion protein band can be detected; d: The precipitate obtained after cleavage of the aggregate obtained by adding salt; e: The upper part of the aggregate obtained after adding salt Clearly, a clear red fluorescent protein RFP band can be detected.
  • swimming lanes 1-5 are protein quantitative standards containing bovine serum albumin BSA, and the loading amounts are 0.25 ⁇ g, 0.5 ⁇ g, 1.0 ⁇ g, 2.0 ⁇ g,
  • a intein-mediated self-cleavage target protein yield (volume is calculated per liter of LB medium)
  • b aggregation efficiency 100% ⁇ the amount of fusion protein in the precipitate after adding salt/(in the precipitate after adding salt The amount of fusion protein+the amount of fusion protein in the supernatant after adding salt)
  • c -intein-mediated self-cleavage efficiency 100% ⁇ (the amount of fusion protein in the precipitate after adding salt-the amount of fusion protein in the precipitate after cleavage )/the amount of the fusion protein in the precipitate after adding salt
  • d recovery rate 100% ⁇ actual yield of the target protein/theoretical yield of the target protein that the expression supernatant can produce under the condition of complete cutting.
  • the lysed supernatant of the two fusion proteins (CpA-Mtu- hGH , CpA-Mtu-RFP) used was changed from soluble to precipitated (74% CpA -Mtu-hGH is converted into precipitation, 17% of CpA-Mtu-RFP is converted into precipitation), the intein Mtu ⁇ I-CM is self-cleaved, and the target protein is separated from CpA-Mtu with a cleavage efficiency of 76-99%.
  • the cleavage supernatant of MpA/CpA-Mtu-RFP in Examples 3 to 8 was incubated with salt for 12 hours overnight to form aggregates and the cutting supernatant of MpA/CpA-Mtu-RFP were photographed, and RFP appeared red and red under natural light. RFP showed red fluorescence under 365nm ultraviolet light to identify the activity of the aggregate MpA/CpA-Mtu-RFP and the cleaved supernatant RFP, and the results are shown in FIG. 9 .
  • Example 10 3M NaCl, 0.7M Na 2 SO 4 and 0.7M (NH 4 ) 2 SO 4 mediated MpA-Mtu-GST/LCB3/ ⁇ NSpyCatcher-ELP- ⁇ NSpyCatcher phase transition and protein purification of Mtu-mediated cleavage
  • centrifuge the suspension at 4°C and 15,000g for 30 minutes, wash the centrifuged precipitate once with an equal volume of buffer solution B2, B4 or B6 containing the same salt, and then centrifuge the supernatant and precipitate under the same conditions. Divide in half the cleavage buffer B3, B5 or B7 containing the same salt to fully resuspend the pellet, and place it at 25°C for 24 hours to make the intein fully self-cleaved. Then the suspension was centrifuged at 16,000 g for 30 min at 4°C.
  • lanes af are the expression and purification samples of the three target proteins, respectively a: cell lysate supernatant, where a clear band of fusion protein can be detected; b: lysate supernatant separated by salt aggregation Supernatant; c: The precipitate separated after adding salt to the lysate supernatant, and a clear band of fusion protein can be detected; d: The precipitate obtained after cleavage of the aggregate obtained by adding salt; e and f: The cleavage of the aggregate obtained by adding salt After the supernatant, a clear target protein band can be detected, in which the e lane is 2 times the loading amount of the ad lane, and the f lane is 10 times the loading amount of
  • swimming lanes 1-5 are protein quantification standards containing bovine serum albumin BSA, and the loading amounts are 0.125 ⁇ g, 0.25 ⁇ g, 0.5 ⁇ g, 1.0 ⁇ g, and 2.0 ⁇ g.
  • 5 is a protein quantification standard containing bovine serum albumin BSA and isopeptidase APR, and the respective loading amounts of BSA and APR in the swimming lanes are 0.125 ⁇ g, 0.25 ⁇ g, 0.5 ⁇ g, 1.0 ⁇ g, and 2.0 ⁇ g.
  • Lanes M1 and M2 are protein molecular weight standards.
  • a intein-mediated self-cleavage target protein yield (volume is calculated per liter of LB medium)
  • b aggregation efficiency 100% ⁇ the amount of fusion protein in the precipitate after adding salt/(in the precipitate after adding salt The amount of fusion protein+the amount of fusion protein in the supernatant after adding salt)
  • c -intein-mediated self-cleavage efficiency 100% ⁇ (the amount of fusion protein in the precipitate after adding salt-the amount of fusion protein in the precipitate after cleavage )/the amount of the fusion protein in the precipitate after adding salt
  • d recovery rate 100% ⁇ actual yield of the target protein/theoretical yield of the target protein that the expression supernatant can produce under the condition of complete cutting.
  • the lysed supernatants of the three fusion proteins were dissolved in 3M NaCl, 0.7M Na 2 SO 4 and 0.7M (NH 4 ) 2 SO 4 , the fusion protein changed from soluble to precipitated, and the aggregation effect (aggregation efficiency: 65% to 96%) of 3M NaCl and 0.7M Na 2 SO 4 was higher than that of 0.7M ( NH 4 ) 2 SO 4 (aggregation efficiency: 51%-93%) is more prominent, the intein Mtu ⁇ I-CM is self-cleaved, the target protein is separated from MpA-Mtu, and the cleavage efficiency is 68-99%.
  • the yields of the three target proteins were 10-125mg/L, and the purity was 68%-97%; in the case of 0.7M Na2SO4 induction, the yields of the three target proteins were 12-102mg/L, The purity is 72%-97%; when induced by 0.7M (NH 4 ) 2 SO 4 , the yields of the three target proteins are 2-117 mg/L, and the purity is 33%-85%.
  • Example 11 3M NaCl, 0.7M Na 2 SO 4 and 0.7M (NH 4 ) 2 SO 4 mediated Xylanase-Mxe-MpA phase transition and Mxe-mediated cleavage protein purification
  • NaCl to 3M, Na 2 SO 4 to 3M and (NH 4 ) 2 SO 4 to 0.7M were added to the lysed supernatant, and placed at 4°C for 30 minutes to induce aggregation. Afterwards, centrifuge the suspension at 4°C and 15,000g for 30 minutes, wash the centrifuged precipitate once with an equal volume of buffer solution B2, B4 or B6 containing the same salt, and then centrifuge the supernatant and precipitate under the same conditions.
  • lanes af are the expression and purification samples of the three target proteins, respectively a: cell lysate supernatant, a clear band of fusion protein can be detected; b: lysate supernatant separated by adding salt Supernatant; c: The precipitate separated from the supernatant of the lysate after adding salt and aggregation, and a clear fusion protein band can be detected; d: The precipitate obtained after cleavage of the aggregate obtained by adding salt; e and f: The cleavage of the aggregate obtained by adding salt After the supernatant, a clear target protein band can be detected, in which the e lane is 2 times the loading amount of the ad lane, and the f lane is 10 times the loading amount of the ad lane.
  • swimming lanes 1-5 are protein quantitative standards containing bovine serum albumin BSA, and the loading amounts are 0.125 ⁇ g, 0.25 ⁇ g, 0.5 ⁇ g, 1.0 ⁇ g, and 2.0 ⁇ g in sequence.
  • swimming lanes M1 and M2 are protein molecular weight standards.
  • a intein-mediated self-cleavage target protein yield (volume is calculated per liter of TB medium)
  • b aggregation efficiency 100% ⁇ the amount of fusion protein in the precipitate after adding salt/(in the precipitate after adding salt The amount of fusion protein+the amount of fusion protein in the supernatant after adding salt)
  • c -intein-mediated self-cleavage efficiency 100% ⁇ (the amount of fusion protein in the precipitate after adding salt-the amount of fusion protein in the precipitate after cleavage )/the amount of the fusion protein in the precipitate after adding salt
  • d recovery rate 100% ⁇ actual yield of the target protein/theoretical yield of the target protein that the expression supernatant can produce under the condition of complete cutting.
  • the lysed supernatant of the used Mxe intein fusion protein (Xylanase- Mxe -MpA) changed from soluble to Precipitation, the aggregation efficiency of 0.7M Na 2 SO 4 can reach up to 69%, the aggregation efficiency of 3M NaCl and 0.7M Na 2 SO 4 is 41% and 37%, respectively, the intein Mxe GyrA self-cleavage, the target protein is the same as Mxe -MpA separation, the cutting efficiency is 63-85%.
  • target protein Xylanase under the situation of 3M NaCl induction is 35mg/L, and purity is 49%; 4 ) In the case of 2 SO 4 induction, the yield of the target protein Xylanase was 37mg/L, and the purity was 54%.
  • Example 12 Detection of the affinity of purified human growth hormone hGH by biofilm layer optical interference (BLI) technology
  • the method of filtration is to replace the cleavage buffer of the cleavage supernatant with the starting buffer for ion exchange (2.4g Tris, dissolved in 800mL water, adjust the pH to 7.2, add water to 1L), and then use a 0.22 ⁇ m filter Membrane to filter samples, using The KTA TM protein purification chromatography system was used to load the sample, and 20 column volumes of 0-1M NaCl were used for linear gradient elution, and the eluted samples obtained by ion exchange purification were collected. The purity and mass concentration of hGH samples obtained by ion exchange purification were determined by SDS-PAGE and BCA Kit (Thermo Fisher, USA), respectively.
  • the human growth hormone hGH initially purified by this method can bind and dissociate with the human growth hormone receptor protein hGH receptor after ion exchange refining purification, and the effect is similar to that of the commercialized human growth hormone hGH.
  • 3M NaCl, 0.7M Na 2 SO 4 and 0.7M (NH 4 ) 2 SO 4 salt-mediated purification of hGH compared with the positive control (commercialized hGH) the binding and dissociation constant K D values are all less than 10 -12
  • the Kon (10 5 M -1 s -1 ) value of association is between 1.82 and 3.53
  • the Koff (s -1 ) value of dissociation is all less than 10 -7 .
  • Example 13 Detecting the affinity of the purified new crown polypeptide LCB3 by biofilm layer optical interference (BLI) technology
  • LCB3 was refined and purified, and its purity and mass concentration were determined.
  • the entire BLI experiment was detected by the molecular interaction instrument Octet RED96 (ForteBio), and 20 ⁇ g/ml of the receptor protein of LCB3, the new coronavirus spike protein SARS- CoV-2 Spike protein (GenScript, China) was immobilized on the AR2G sensor in a solution of 10 mM sodium acetate (pH 6.0), and then the sensor was blocked with 1 M ethanolamine solution, followed by kinetic buffer (containing 0.1% bovine serum white).
  • the new crown polypeptide LCB3 initially purified by this method can bind and dissociate with the new crown virus spike protein SARS-CoV-2 Spike protein after ion exchange purification, and the binding dissociation constant K D is consistent with the literature (Cao, L. et al. Science , 2020.370(6515): p.426-431.) reported less than 10 -9 is similar.
  • Example 14 Xylanase-Mxe-MpA cleavage supernatant activity verification
  • the enzymatic activity of xylanase xylanase in the Xylanase-Mxe-MpA cut supernatant was determined by DNS method (Miller, GL et al. Analytical Chemistry, 1959.31(3): p.426-428.).
  • Xylan Sigma, USA
  • xylose Alkadin, China
  • the catalyzed reaction of xylanase is carried out in 50mM phosphate buffer (pH 7.0) containing 0.5% (W/V) xylan, the reaction condition is 55°C for 15min, and the required amount of hydrolyzing substrate to generate 1 ⁇ moL reducing sugar per minute is The amount of enzyme is defined as one enzyme activity unit (IU).
  • the cleaved supernatants obtained by the mediated purification of the three salts 3M NaCl, 0.7M Na 2 SO 4 and 0.7M (NH 4 ) 2 SO 4 were diluted to appropriate concentrations, and then the enzyme activity was determined. The results are shown in Table 13 .
  • 3M NaCl, 0.7M Na 2 SO 4 and 0.7M (NH 4 ) 2 SO 4 mediated the activity of purified xylanase xylanase, which was determined by DNS method, to be greater than that of commercial xylanase (Sigma, 253-439-7 ) enzyme activity ( ⁇ 2.5units/mg).
  • Glutathione thiol transferase activity detection kit (Sangon Biotech, D799612) was used to measure the enzyme activity of glutathione thiol transferase GST in the cut supernatant of MpA-Mtu-GST, and the amino acid sequence was selected from the same source GST recombinant protein from Schistosoma japonicum (Shenzhou, 11213-HNAE) was used as a positive control. The results of the enzyme activity determined according to the method of the kit instructions are shown in Table 14.
  • 3M NaCl, 0.7M Na 2 SO 4 and 0.7M (NH 4 ) 2 SO 4 mediated the activity of purified glutathione sulfhydryl transferase GST (5.4 ⁇ 6.1units/mg) and the enzyme activity of GST standard (6.8 units/mg) are similar.
  • Example 16 MpA-Mtu- ⁇ NSpyCatcher-ELP- ⁇ NSpyCatcher cutting supernatant activity verification
  • ⁇ NSpyCatcher (Liu, Z. et al. Sci Rep, 2014.4: p.7266.) can interact with SpyTag Isopeptide bonds were formed spontaneously, and the formation of covalently bound products was identified by SDS-PAGE, thereby verifying the activity of the scaffold protein ⁇ NSpyCatcher-ELP- ⁇ NSpyCatcher in the cleavage supernatant.
  • the three salts 3M NaCl, 0.7M Na 2 SO 4 and 0.7M (NH 4 ) 2 SO 4 mediated purification of MpA-Mtu- ⁇ NSpyCatcher-ELP- ⁇ NSpyCatcher cutting supernatant and purified LCB3-SpyTag were diluted with PBS to 20 ⁇ M and 120 ⁇ M, then the three cut supernatants were mixed with LCB3-SpyTag in equal volumes, incubated at 25°C for 2 hours, and the samples before and after the reaction were identified by SDS-PAGE.
  • the SDS-PAGE results are shown in Figure 14.
  • Lane 1 is the LBS3-SpyTag before the reaction
  • lanes 2, 4 and 6 are the cleaved supernatants corresponding to the three salt-mediated purifications before the reaction
  • lanes 3, 5 and 7 are after the reaction sample.
  • the bivalent binding product ( ⁇ NSpyC-ELP- ⁇ NSpyC:2 LCB3-SpyTag, 44.6kDa) formed by the backbone protein ⁇ NSpyCatcher-ELP- ⁇ NSpyCatcher and LCB3-SpyTag can be clearly observed, and ⁇ NSpyC-ELP- The bands corresponding to ⁇ NSpyC basically disappeared, indicating that the three salt-mediated purification of the backbone protein ⁇ NSpyC-ELP- ⁇ NSpyC almost completely participated in the reaction, that is, the three salts 3M NaCl, 0.7M Na 2 SO 4 and 0.7M (NH 4 ) 2 SO 4 -mediated purification of ⁇ NSpyC-ELP- ⁇

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Abstract

L'invention concerne un polypeptide de fusion comprenant une partie d'un peptide auto-agrégant sensible à la concentration de sel et une partie d'un polypeptide d'intérêt, ainsi qu'un procédé de production et de purification d'un polypeptide d'intérêt par expression du polypeptide de fusion.
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