WO2022253266A1 - Recombinant protein purification method - Google Patents
Recombinant protein purification method Download PDFInfo
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- 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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Images
Classifications
-
- 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
Provided are a fusion polypeptide comprising a portion of a salt concentration-responsive self-aggregating peptide and a portion of a polypeptide of interest, and a method for producing and purifying a polypeptide of interest by expressing the fusion polypeptide.
Description
本发明涉及基因工程领域。更具体地，本发明涉及包含盐浓度响应性自聚集肽部分和目的多肽部分的融合多肽，以及通过表达所述融合多肽来生产和纯化目的多肽的方法。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.
发明背景Background of the invention
重组蛋白已经广泛应用于医药、食品、化工、能源、纺织、环保等领域。重组蛋白质的生产，无论在商业规模还是实验室规模都至关重要，而重组蛋白的分离与纯化成本约占其全部成本的30％-80％，是重组蛋白质制备的瓶颈技术(Fields C.等，Biotechnology and Bioengineering,2016,113(1):11-25)。重组蛋白在制备出来后，纯化一般可分为样品捕获、中度纯化和精细纯化等三步。其中，中度纯化可以达到适中的样品纯度，其处理后的蛋白质样品可以被用于多种实验分析，如N端序列分析、抗原抗体反应等。目前可以实现蛋白质中度纯化的方法包括传统的离子交换层析、疏水性相互作用层析、亲和层析，以及近期的研究热点如自切割自聚集功能的融合标签表达等。离子交换层析和疏水性相互作用层析由于对样品起始条件有一定的要求，通用性和效率不及亲和层析。亲和纯化通常可以获得高收率，使其成为目前最常用的重组蛋白质中度纯化方法之一。实验室中常用的亲和纯化技术包括多组氨酸标签(his-tag)或谷胱甘肽转移酶标签(GST-tag)与目的蛋白的融合表达，再通过装有螯合了金属离子(一般为镍离子)或固定了谷胱甘肽的树脂的层析柱进行特异性结合和洗脱进行纯化，为不同目的蛋白的生产提供了通用的纯化手段(Arnau J.等，Protein Expression and Purification,2006,48(1):1-13)。然而这种方法，1)需要消耗大量价格昂贵的填粒，2)需要专业且贵重的设备来实现纯化过程，3)需要加入蛋白酶进行融合表达标签的移除而使成本升高、步骤增多、回收率降低，这三个主要缺点使亲和纯化成本较高，不利于工业领域的应用。而且，由于树脂价格高、消耗大，其成本占整个蛋白质纯化市场的约1/4。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). After the recombinant protein is prepared, the purification can generally be divided into three steps: sample capture, intermediate purification and fine purification. Among them, 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. Currently, 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). However, this method, 1) needs to consume a lot of expensive pellets, 2) needs professional and expensive equipment to realize the purification process, 3) needs to add protease to remove the fusion expression tag, which increases the cost and steps, The recovery rate is reduced, and these three main disadvantages make the cost of affinity purification higher, which is not conducive to the application in the industrial field. Moreover, due to the high price and large consumption of resin, its cost accounts for about 1/4 of the entire protein purification market.
近年来，许多工作开始设计具有自聚集自切割功能的融合标签以克服上述缺点。来自典型性猪瘟病毒(CSFV)N-端的具有蛋白酶活性且有强烈的形成不可溶聚集体趋势的N
pro，就具备了将聚集纯化和蛋白质剪切集于一身的特点。但是利用N
pro方法需要对目的蛋白进行复性和进一步的精细纯化来去除产物中的N
pro融合片段(Achmuller C.等，Nature Methods,2007,4:1037-1043)。也有报道显示由多个重复单元组成的两亲性β折叠的自聚集倾向随着重复单元数目增加而增强(Zhang S.等，EMBO Journal,1992,11:3787-3796)。本领域还有通过多个重复单元串联重复形成具有自聚集特性的多肽的报道，如类弹性蛋白(E lastin-Like Polypeptide,ELP)，其由数十至上百个VPGXG(其中，X 表示任意氨基酸)重复单元组成，其聚集特性与重复单元数目相关，一般采用60-110个重复(即有300个氨基酸以上的)ELP。将具有诱导聚集功能的ELP与具有自切割功能的内含肽(Intein)结合，构建出的新融合标签可以产生与N
pro相似的功效。与这一标签融合表达的蛋白可以通过控制温度或离子浓度，使融合蛋白在液相(可溶)和固相(沉淀)之间不断转换，从而达到纯化目的(Meyer D.E.等，Nature Biotechnology,1999.17(11):1112-1115；Banki M.R.等，Nature Methods,2005,2(9):659-661)。然而，在实际操作中往往是需要温度和离子浓度同时调节，而且需要采用两轮的盐结合温度的相变来反复诱导聚集，蛋白纯度以及收率通常较低，难以达到工业应用的标准，特别是较高的温度有可能影响所纯化蛋白质的活性，并且多步操作不利于简化工艺流程。另外，由于ELP本身长度较长(至少300个氨基酸残基)，对于融合蛋白的表达以及纯化均有不利的影响。
In recent years, many works have begun to design fusion tags with self-aggregation and self-cleavage functions to overcome the above shortcomings. 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. However, 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 β-sheets composed of multiple repeating units is enhanced as the number of repeating units increases (Zhang S. et al., EMBO Journal, 1992, 11:3787-3796). There are also reports in the art that a polypeptide with self-aggregation properties is formed through multiple repeating units in tandem repetition, such as elastin-like (E lastin-Like Polypeptide, ELP), which consists of tens to hundreds of VPGXG (wherein, X represents any amino acid ) repeating units, and its aggregation characteristics are related to the number of repeating units. Generally, ELPs with 60-110 repeats (that is, more than 300 amino acids) are used. Combining ELP with the function of inducing aggregation and intein with self-cleavage function, the new fusion tag constructed can produce similar efficacy to N pro . 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). However, in actual operation, it is often necessary to adjust the temperature and ion concentration at the same time, and it is necessary to use two rounds of salt-binding temperature phase transitions to repeatedly induce aggregation. 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. In addition, due to 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.
因此，本领域仍然需要低成本、简便、高效的蛋白质纯化方法。Therefore, there is still a need in the art for low-cost, simple and efficient protein purification methods.
发明简述Brief description of the invention
在本发明的第一方面，提供一种融合多肽，其包含目的多肽部分和盐浓度响应性自聚集肽部分，其中所述目的多肽部分通过间隔物连接于所述盐浓度响应性自聚集肽部分，并且其中所述间隔物包含切割位点，In the first aspect of the present invention, a fusion polypeptide is provided, 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,
其中所述盐浓度响应性自聚集肽为CpA变体，其中所述CpA具有如SEQ ID NO:1所示的氨基酸序列，所述CpA变体在对应于SEQ ID NO:1的第1位和第17位的位置包含C1M以及C17M的氨基酸取代。Wherein 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.
在本发明的第二方面，提供一种分离的多核苷酸，其包含编码本发明的融合多肽的核苷酸序列或其互补序列。In a second aspect of the present invention, there is provided an isolated polynucleotide comprising a nucleotide sequence encoding a fusion polypeptide of the present invention or its complementary sequence.
在本发明的第三方面，提供一种分离的多核苷酸，其包含编码CpA变体的核苷酸序列或其互补序列，其中所述CpA具有如SEQ ID NO:1所示的氨基酸序列，所述CpA变体在对应于SEQ ID NO:1的第1位和第17位的位置包含C1M以及C17M的氨基酸取代。In a third aspect of the present invention, there is provided 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.
在本发明的第四方面，提供表达构建体，其包含本发明的多核苷酸。In a fourth aspect of the present invention there is provided an expression construct comprising a polynucleotide of the present invention.
在本发明的第五方面，提供宿主细胞，其包含本发明的多核苷酸或由本发明的表达构建体转化，其中所述宿主细胞能够表达所述融合多肽。In a fifth aspect of the present invention, there is provided 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.
在本发明的第六方面，提供生产和纯化目的多肽的方法，所述方法包括以下步骤：In a sixth aspect of the present invention, there is provided a method for producing and purifying a polypeptide of interest, said method comprising the following steps:
(a)培养本发明的宿主细胞，从而表达融合多肽；(a) cultivating the host cell of the present invention so as to express the fusion polypeptide;
(b)在第一盐条件下，裂解所述宿主细胞，然后去除细胞裂解物的不溶部分，回收可溶部分；(b) under a first salt condition, lysing the host cell, then removing the insoluble portion of the cell lysate, and recovering the soluble portion;
(c)在第二盐条件下，所述融合蛋白形成不溶部分；(c) under a second salt condition, the fusion protein forms an insoluble fraction;
(d)回收步骤(c)中形成的不溶部分；(d) recovering the insoluble fraction formed in step (c);
(e)通过切割所述切割位点从收集自步骤(d)的不溶部分释放可溶的目的多肽；和(e) releasing soluble polypeptide of interest from the insoluble fraction collected from step (d) by cleaving said cleavage site; and
(f)去除步骤(e)中的不溶部分，回收含有所述目的多肽的可溶部分。(f) removing the insoluble portion in step (e), and recovering the soluble portion containing the target polypeptide.
附图简述Brief description of the drawings
图1示出基于盐浓度响应性自聚集肽诱导的蛋白质纯化方法以及所采用的表达载体结构图。A：纯化策略(以盐浓度响应性自聚集肽MpA/CpA和内含肽MtuΔI-CM为例)；B：pET30-MpA-Mtu-hGH、pET30-MpA-Mtu-RFP、pET30-MpA-Mtu-GST、pET30-MpA-Mtu-LCB3、pET30-MpA-Mtu-ΔNSpyCatcher-ELP-ΔNSpyCatcher、pET30-CpA-Mtu-hGH、pET30-CpA-Mtu-RFP和pET30a-Xylanase-Mxe-MpA的载体结构图。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 MtuΔI-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-ΔNSpyCatcher-ELP-ΔNSpyCatcher, pET30-CpA-Mtu-hGH, pET30-CpA-Mtu-RFP and pET30a-Xylanase-Mxe-MpA .
图2示出人生长激素hGH融合蛋白和红色荧光蛋白RFP融合蛋白表达结果图。A：MpA-Mtu-hGH和MpA-Mtu-RFP表达SDS-PAGE分析结果；B：CpA-Mtu-hGH和CpA-Mtu-RFP表达SDS-PAGE分析结果。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.
图3示出通过3M NaCl诱导聚集肽MpA聚集来纯化人生长激素hGH、红色荧光蛋白RFP的SDS-PAGE分析结果图。A：人生长激素hGH表达与纯化SDS-PAGE分析结果；B：红色荧光蛋白RFP表达与纯化SDS-PAGE分析结果。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.
图4示出通过0.7M Na
2SO
4诱导聚集肽MpA聚集来纯化人生长激素hGH和红色荧光蛋白RFP的SDS-PAGE分析结果图。A：人生长激素hGH表达与纯化SDS-PAGE分析结果；B：红色荧光蛋白RFP表达与纯化SDS-PAGE分析结果。
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.
图5示出通过0.7M(NH
4)
2SO
4诱导聚集肽MpA聚集来纯化人生长激素hGH和红色荧光蛋白RFP的SDS-PAGE分析结果图。A：人生长激素hGH表达与纯化SDS-PAGE分析结果；B：红色荧光蛋白RFP表达与纯化SDS-PAGE分析结果。
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.
图6示出通过3M NaCl诱导聚集肽CpA聚集来纯化人生长激素hGH和红色荧光蛋白RFP的SDS-PAGE分析结果图。A：人生长激素hGH表达与纯化SDS-PAGE分析结果；B：红色荧光蛋白RFP表达与纯化SDS-PAGE分析结果。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.
图7示出通过0.7M Na
2SO
4诱导聚集肽CpA聚集来纯化人生长激素hGH和红色荧光蛋白RFP的SDS-PAGE分析结果图。A：人生长激素hGH表达与纯化SDS-PAGE分析结果；B：红色荧光蛋白RFP表达与纯化SDS-PAGE分析结果。
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.
图8示出通过0.7M(NH
4)
2SO
4诱导聚集肽CpA聚集来纯化人生长激素hGH和红色荧光蛋白RFP的SDS-PAGE分析结果图。A：人生长激素hGH表达与纯化SDS-PAGE分析结果；B：红色荧光蛋白RFP表达与纯化SDS-PAGE分析结果。
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.
图9示出通过自然光下RFP呈现红色和365nm紫外光下RFP显红色荧光来鉴定聚集体MpA/CpA-Mtu-RFP和切割上清RFP的活性表征结果图。A：自然光下MpA-Mtu-RFP裂解上清分别加三种盐(3M NaCl、0.7M Na
2SO
4、0.7M(NH
4)
2SO
4)后形成的聚集体实物图；B：自然光下MpA-Mtu-RFP分别在三种盐(3M NaCl、0.7M Na
2SO
4、0.7M(NH
4)
2SO
4)条件下得到的切割上清实物图；C：365nm紫外光下MpA-Mtu-RFP分别在三种盐(3M NaCl、0.7M Na
2SO
4、0.7M(NH
4)
2SO
4)条件下得到的切割上清荧光图；D： 自然光下CpA-Mtu-RFP裂解上清分别加三种盐(3M NaCl、0.7M Na
2SO
4、0.7M(NH
4)
2SO
4)后形成的聚集体实物图；E：自然光下CpA-Mtu-RFP分别在三种盐(3M NaCl、0.7M Na
2SO
4、0.7M(NH
4)
2SO
4)条件下得到的切割上清实物图；F：365nm紫外光下CpA-Mtu-RFP分别在三种盐(3M NaCl、0.7M Na
2SO
4、0.7M(NH
4)
2SO
4)条件下得到的切割上清荧光图。
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 , 0.7M (NH 4 ) 2 SO 4 ); E: CpA-Mtu-RFP in three salts (3M Physical picture of the cut supernatant obtained under the conditions of NaCl, 0.7M Na 2 SO 4 , 0.7M (NH 4 ) 2 SO 4 ); F: CpA-Mtu-RFP in three salts (3M NaCl, 0.7 M Na 2 SO 4 , 0.7M (NH 4 ) 2 SO 4 ) conditions of the cut supernatant fluorescence map.
图10示出通过3M NaCl、0.7M Na
2SO
4、0.7M(NH
4)
2SO
4诱导聚集肽MpA聚集来纯化谷胱甘肽巯基转移酶GST、新冠多肽LCB3和多价骨架蛋白ΔNSpyCatcher-ELP-ΔNSpyCatcher的SDS-PAGE分析结果图；A：3M NaCl介导GST纯化的SDS-PAGE分析结果，B：3M NaCl介导LCB3纯化的SDS-PAGE分析结果，C：3M NaCl介导ΔNSpyCatcher-ELP-ΔNSpyCatcher纯化的SDS-PAGE分析结果，D：0.7M Na
2SO
4介导GST纯化的SDS-PAGE分析结果，E：0.7M Na
2SO
4介导LCB3纯化的SDS-PAGE分析结果，F：0.7M Na
2SO
4介导ΔNSpyCatcher-ELP-ΔNSpyCatcher纯化的SDS-PAGE分析结果，G：0.7M(NH
4)
2SO
4介导GST纯化的SDS-PAGE分析结果，H：0.7M(NH
4)
2SO
4介导LCB3纯化的SDS-PAGE分析结果，I：0.7M(NH
4)
2SO
4介导ΔNSpyCatcher-ELP-ΔNSpyCatcher纯化的SDS-PAGE分析结果。
Figure 10 shows the purification of glutathione sulfhydryl transferase GST , new crown polypeptide LCB3 and multivalent backbone protein ΔNSpyCatcher- SDS-PAGE analysis results of ELP-ΔNSpyCatcher; 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 ΔNSpyCatcher-ELP - SDS-PAGE analysis results of ΔNSpyCatcher 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 ΔNSpyCatcher-ELP-ΔNSpyCatcher purification, G: 0.7M (NH 4 ) 2 SO 4 mediated SDS-PAGE analysis results of GST purification, H: 0.7M (NH 4 ) 2 SO 4 mediated SDS-PAGE analysis results of LCB3 purification, I: 0.7M (NH 4 ) 2 SO 4 mediated SDS-PAGE analysis results of ΔNSpyCatcher-ELP-ΔNSpyCatcher purification.
图11示出通过3M NaCl、0.7M Na
2SO
4、0.7M(NH
4)
2SO
4诱导融合蛋白Xylanase-Mxe-MpA聚集来纯化木聚糖酶xylanase的SDS-PAGE分析结果图；A：3M NaCl介导xylanase表达与纯化SDS-PAGE分析结果；B：0.7M Na
2SO
4介导xylanase表达与纯化SDS-PAGE分析结果；C：0.7M(NH
4)
2SO
4介导xylanase表达与纯化SDS-PAGE分析结果。
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.
图12示出通过生物膜层光学干涉(BLI)技术检测纯化后人生长激素hGH与人生长激素受体蛋白Growth hormone receptor(Abcam,ab180053)的亲和力表征结果图。A：通过3M NaCl介导纯化的人生长激素hGH的亲和力测试；B：通过0.7M Na
2SO
4介导纯化的人生长激素hGH的亲和力测试；C：通过0.7M(NH
4)
2SO
4介导纯化的人生长激素hGH的亲和力测试；D：商业化的人生长激素hGH的亲和力测试。
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. A: 3M NaCl-mediated affinity test of purified human growth hormone hGH; B: 0.7M Na 2 SO 4 -mediated affinity test of purified human growth hormone hGH; C: 0.7M (NH 4 ) 2 SO 4 Mediates the affinity test of purified human growth hormone hGH; D: the affinity test of commercialized human growth hormone hGH.
图13示出通过生物膜层光学干涉(BLI)技术检测纯化后新冠多肽LCB3与其受体蛋白SARS-CoV-2 Spike protein(GenScript,Z03483)的亲和力表征结果图。A：通过3M NaCl介导纯化的新冠多肽LCB3的亲和力测试；B：通过0.7M Na
2SO
4介导纯化的新冠多肽LCB3的亲和力测试；C：通过0.7M(NH
4)
2SO
4介导纯化的新冠多肽LCB3的亲和力测试。
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. A: Affinity test of novel coronavirus peptide LCB3 mediated by 3M NaCl; B: Affinity test of novel coronavirus peptide LCB3 purified by 0.7M Na 2 SO 4 ; C: Mediated by 0.7M (NH 4 ) 2 SO 4 Affinity test of purified novel coronavirus peptide LCB3.
图14示出通过SDS-PAGE鉴定多价骨架蛋白ΔNSpyCatcher-ELP-ΔNSpyCatcher与LCB3-SpyTag的共价结合，根据其共价结合产物的形成来表征多价骨架蛋白的活性。Figure 14 shows the identification of the covalent binding of the multivalent scaffold protein ΔNSpyCatcher-ELP-ΔNSpyCatcher 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.
发明详述Detailed description of the invention
一、定义1. Definition
在本发明中，除非另有说明，否则本文中使用的科学和技术名词具有本领域技术人 员所通常理解的含义。并且，本文中所用的蛋白质和核酸化学、分子生物学、细胞和组织培养、微生物学、免疫学相关术语和实验室操作步骤均为相应领域内广泛使用的术语和常规步骤。同时，为了更好地理解本发明，下面提供相关术语的定义和解释。In the present invention, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Moreover, the terms related to protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, immunology and laboratory operation steps used herein are all terms and routine procedures widely used in the corresponding fields. Meanwhile, in order to better understand the present invention, definitions and explanations of relevant terms are provided below.
如本文所用，术语“和/或”涵盖由该术语连接的项目的所有组合，应视作各个组合已经单独地在本文列出。例如，“A和/或B”涵盖了“A”、“A和B”以及“B”。例如，“A、B和/或C”涵盖“A”、“B”、“C”、“A和B”、“A和C”、“B和C”以及“A和B和C”。As used herein, 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. For example, "A and/or B" includes "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."
“多肽”、“肽”、和“蛋白质”在本发明中可互换使用，指氨基酸残基的聚合物。该术语适用于其中一个或多个氨基酸残基是相应的天然氨基酸的人工化学类似物的氨基酸聚合物，以及适用于天然氨基酸的聚合物。术语“多肽”、“肽”、“氨基酸序列”和“蛋白质”还可包括修饰形式，包括但不限于糖基化、脂质连接、硫酸盐化、谷氨酸残基的γ羧化、羟化和ADP-核糖基化。"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.
在本文中，术语“变体”是指与其亲本相比包含一个或多个氨基酸或核苷酸突变的多肽或多核苷酸。在本文中，术语“变体”与“突变体”可互换使用。As used herein, the term "variant" refers to a polypeptide or polynucleotide comprising one or more amino acid or nucleotide mutations compared to its parent. Herein, the terms "variant" and "mutant" are used interchangeably.
在本文中，术语“对应于”是指本领域技术人员利用已知的序列比对方法以最大化匹配来比对两个或更多个相关多肽或核酸序列(包括分子的序列、分子的区域和/或理论序列)从而获得最高等级匹配时，互相对齐的部分、位置或区域。换句话说，在两个或更多个多肽或核酸序列最适比对时，两个类似位置(或部分或区域)对齐。当比对两个或更多个序列时，基于沿线性核酸或氨基酸序列的位置鉴定类似部分/位置/区域。As used herein, 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.
如本文所用，“多核苷酸”是指多个核苷酸通过磷酸二酯键连接而成的大分子，其中所述核苷酸包括核糖核苷酸和脱氧核糖核苷酸。本发明的多核苷酸的序列可以针对不同的宿主细胞(如大肠杆菌)进行密码子优化，从而改善多肽的表达。进行密码子优化的方法是本领域已知的。As used herein, "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.
在本文中，术语“在严格条件下杂交”是指多核苷酸分子与靶核酸分子通过互补的碱基配对退火。本领域技术人员熟悉影响特异性杂交的参数，例如特定分子的长度和组成。与杂交特别相关的参数还包括例如退火和洗涤温度、缓冲液组成和盐浓度。在一个实施方式中，在严格条件下杂交是指在高度严格条件下杂交，即0.1×SSPE，0.1％SDS，65℃。在一个实施方式中，在严格条件下杂交是指在中度严格条件下杂交，即0.2×SSPE，0.1％SDS，50℃。在一个实施方式中，在严格条件下杂交是指在低度严格条件下杂交，即0.2×SSPE，0.1％SDS，40℃。等效的严格条件是本领域已知的。本领域技术人员能够调整影响杂交的参数，以在低、中或高度严格条件下实现多核苷酸分子与靶核酸分子的杂交。Herein, the term "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. In one embodiment, hybridization under stringent conditions refers to hybridization under highly stringent conditions, ie 0.1×SSPE, 0.1% SDS, 65°C. In one embodiment, hybridization under stringent conditions refers to hybridization under moderately stringent conditions, ie 0.2×SSPE, 0.1% SDS, 50°C. In one embodiment, 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.
“包含”或“包括”一词在本文中用于描述蛋白质或核酸的序列时，所述蛋白质或核酸可以是由所述序列组成，或者在所述蛋白质或核酸的一端或两端可以具有额外的氨基酸或核苷酸，但仍然具有本发明所述的活性。此外，本领域技术人员清楚多肽N端由起始密码子编码的甲硫氨酸在某些实际情况下(例如在特定表达系统表达时)会被保留， 但不实质影响多肽的功能。因此，本申请说明书和权利要求书中在描述具体的多肽氨基酸序列时，尽管其可能不包含N端由起始密码子编码的甲硫氨酸，然而此时也涵盖包含该甲硫氨酸的序列。相应地，其编码核苷酸序列也可以包含起始密码子。When the word "comprising" or "comprises" is used herein to describe a protein or nucleic acid sequence, 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. In addition, 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. Therefore, when describing a specific amino acid sequence of a polypeptide in the specification and claims of this application, although it may not contain a methionine encoded by a start codon at the N-terminus, it also covers a methionine containing the methionine at this time. sequence. Correspondingly, its coding nucleotide sequence may also contain an initiation codon.
术语“表达”通常是指通过多核苷酸的转录和翻译产生多肽的过程。在本文中，术语“表达”可理解为“异源表达”，即是指在宿主细胞中表达或体外表达由异源核酸编码的多肽。The term "expression" generally refers to the process by which a polypeptide is produced by transcription and translation of a polynucleotide. In this context, the term "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.
如本发明所用，“表达构建体”是指适于感兴趣的核苷酸序列在生物体中表达的载体如重组载体。“表达”指功能产物的产生。例如，核苷酸序列的表达可指核苷酸序列的转录(如转录生成mRNA或功能RNA)和/或RNA翻译成前体或成熟蛋白质。本发明的“表达构建体”可以是线性的核酸片段、环状质粒、病毒载体，或者，可以是能够翻译的RNA(如mRNA)。通常，在表达构建体中，感兴趣的核苷酸序列与调控序列可操作地连接。As used in the present invention, "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, 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). Typically, a nucleotide sequence of interest is operably linked to regulatory sequences in an expression construct.
“调控序列”和“调控元件”可互换使用，指位于编码序列的上游(5'非编码序列)、中间或下游(3'非编码序列)，并且影响相关感兴趣的序列的转录、RNA加工或稳定性或者翻译的核苷酸序列。调控序列可包括但不限于启动子、翻译前导序列、内含子和多腺苷酸化识别序列。"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.
如本文中所用，术语“可操作地连接”指调控序列与目的核苷酸序列连接，使得目的核苷酸序列的转录被所述调控序列控制和调节。用于将调控序列可操作地连接于目的核苷酸序列的技术为本领域已知的。As used herein, the term "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.
如本文所用，“自聚集”是指多肽的一种特性，即多肽单体在一定物理和/或化学条件诱导下组装成多聚体。As used herein, "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.
如本文所用，“纯度”是指目的蛋白的纯度，即在纯化液中，目的蛋白占总蛋白的比例。由于通过细胞表达目的蛋白，在胞内有大量的其他蛋白(如大肠杆菌就有几千种蛋白)，要把目的蛋白从如此多种类且量较大的蛋白混合物中纯化出来一直是一个关键的技术难题。通过细胞的破碎、离心、切割后的分离等步骤，纯化液中基本只有蛋白质和无机盐，因此纯化液中目的蛋白的比例越高，则生产的纯度越高。As used herein, "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.
术语“离子强度”是溶液中离子浓度的度量，离子强度的单位为摩尔浓度(mol/L)，其计算方式为溶液中每种离子i的质量摩尔浓度(mi)乘以该离子的价数(zi)的平方所得诸项之和得一半。The term "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.
二、CpA变体及其融合多肽2. CpA variants and their fusion polypeptides
“CpA”或“CpA短肽”是指本领域中已知的一种两亲性(amphipathic)多肽，其具有彼此分隔的亲水性区域和疏水性区域，其中CpA的α螺旋状态受盐浓度调控，在含盐的溶液中盐离子诱导的疏水相互作用以及其它推动力作用下能自发地形成特定的自聚集结构，并且在一定的范围内随着盐离子浓度提高，聚集体强度增强且体积增大(Daniel E.W.等，Proceedings of the National Academy of Sciences,2005, 102:12656–12661)。在一个实施方案中，CpA肽具有SEQ ID NO:1所示的氨基酸序列。在一个实施方案中，CpA肽具有SEQ ID NO:8所示的核苷酸序列。"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 Under the action of hydrophobic interaction induced by salt ions and other driving forces in salt-containing solution, 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). In one embodiment, the CpA peptide has the amino acid sequence shown in SEQ ID NO: 1. In one embodiment, the CpA peptide has the nucleotide sequence shown in SEQ ID NO:8.
本发明通过引入氨基酸突变构建了短肽CpA的变体MpA，其具有比CpA更优异的盐浓度响应性自聚集特性，尤其是聚集效率更高、回收效率更高且目的蛋白的纯度更高。其中，所述盐浓度响应性自聚集特性是指在第一盐条件下可溶且在第二盐条件下能够自聚集的性质。在一个实施方案中，所述第一盐条件包含第一盐浓度，所述第二盐条件包含第二盐浓度。在一个实施方案中，所述第一盐浓度与所述第二盐浓度不同。在一个实施方案中，所述第一盐浓度高于所述第二盐浓度。在一个实施方案中，所述第一盐浓度低于所述第二盐浓度。在一个实施方案中，所述第一盐条件包含第一盐种类，所述第二盐条件包含第二盐种类。在一个实施方案中，所述第一盐种类与所述第二盐种类相同。在一个实施方案中，所述第一盐种类与所述第二盐种类不同。在一个实施方案中，与CpA的聚集效率相比，本发明的CpA变体的聚集效率升高5％至300％。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. Wherein, 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. In one embodiment, 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 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. In one embodiment, the aggregation efficiency of the CpA variant of the invention is increased by 5% to 300% compared to the aggregation efficiency of CpA.
因此，在第一方面，本发明涉及一种分离的多肽，其为CpA短肽的变体，其中所述CpA具有如SEQ ID NO:1所示的氨基酸序列。在一个实施方案中，所述CpA变体在对应于SEQ ID NO:1的第1位和第17位的位置处的氨基酸残基具有突变，例如缺失、插入和/或取代。在一个实施方案中，所述CpA变体在对应于SEQ ID NO:1的第1位和第17位的位置处的氨基酸残基具有氨基酸取代。在一个实施方案中，所述CpA变体在对应于SEQ ID NO:1的第1位和第17位的位置处的氨基酸残基被甲硫氨酸(M)取代。在一个实施方案中，所述CpA变体在对应于SEQ ID NO:1的第1位和第17位的位置处的半胱氨酸(C)被甲硫氨酸(M)取代。在一个实施方案中，所述CpA变体在对应于SEQ ID NO:1的第1位和第17位的位置包含C1M以及C17M的氨基酸取代。在一个实施方案中，所述CpA变体的氨基酸序列如SEQ ID NO:2所示。Therefore, in a first aspect, 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. In one embodiment, 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. In one embodiment, 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. In one embodiment, 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). In one embodiment, 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. In one embodiment, the CpA variant comprises amino acid substitutions of C1M and C17M at positions corresponding to positions 1 and 17 of SEQ ID NO: 1. In one embodiment, the amino acid sequence of the CpA variant is shown in SEQ ID NO:2.
在多肽中引入氨基酸突变的方法为本领域技术人员所熟知。例如参见Ausubel,Current Protocols in Molecular Biology,John Wiley and Sons,Inc.(1994)；T.Maniatis等,Molecular Cloning:A Laboratory Manual,Cold Spring Harbor laboratory,Cold Spring Harbor,N.Y.(1989)。例如，可使用市售试剂盒，例如QuikChange
TM定点诱变试剂盒Stratagene，或者直接通过化学法合成具有突变的多肽。
Methods for introducing amino acid mutations into polypeptides are well known to those skilled in the art. See, eg, Ausubel, Current Protocols in Molecular Biology, John Wiley and Sons, Inc. (1994); T. Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor laboratory, Cold Spring Harbor, NY (1989). For example, commercially available kits, such as QuikChange ™ site-directed mutagenesis kit Stratagene, or directly synthesized polypeptides with mutations by chemical methods can be used.
本发明通过将具有盐浓度响应性自聚集特性的肽与两种结构上和功能上均不相关的目的多肽构成融合多肽，盐浓度响应性自聚集功能的短肽会使融合蛋白三联体在改变缓冲液盐浓度后将融合蛋白三联体诱导聚集成沉淀，可以利用简单的离心或过滤手段使融合多肽三联体与细菌蛋白提取液中的杂质成分分离，获得高纯度的融合蛋白三联体。这样的蛋白纯化过程简化了蛋白分离纯化的步骤、避免为了保证收率而反复多次进行纯化、避免使用昂贵的纯化柱、显著地降低生产成本、并且在低温或常温下进行聚集从而避免了目的多肽的降解，并最终所得纯化的蛋白纯度高、回收率高且保持相应的蛋白活性。本发明人意外地发现，利用CpA变体MpA作为盐浓度响应性自聚集肽与目的多肽构成融合多肽，仅需要一次盐浓度的调整进行蛋白沉淀，即可获得纯度通常达到85％以 上的目的蛋白，其纯化效率与纯化柱相当且步骤简便，一方面可用于实验室规模的高通量蛋白纯化，另一方面由于其较高的经济性，克服了工业领域应用的瓶颈。In the present invention, 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 After the salt concentration of the buffer solution, 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.
因此，本发明涉及一种融合多肽，其包含目的多肽部分和盐浓度响应性自聚集肽部分，其中所述目的多肽部分通过间隔物连接于所述盐浓度响应性自聚集肽部分，并且其中所述间隔物包含切割位点。在一个实施方案中，所述盐浓度响应性自聚集肽部分包含盐浓度响应性自聚集肽。在一个实施方案中，所述盐浓度响应性自聚集肽为在第一盐条件下可溶且在第二盐条件下能够自聚集的肽。在一个实施方案中，所述盐浓度响应性自聚集肽为CpA变体，其中所述CpA具有如SEQ ID NO:1所示的氨基酸序列。在一个实施方案中，所述CpA变体在对应于SEQ ID NO:1的第1位和第17位的位置处的氨基酸残基具有突变，例如缺失、插入和/或取代。在一个实施方案中，所述CpA变体在对应于SEQ ID NO:1的第1位和第17位的位置处的氨基酸残基具有氨基酸取代。在一个实施方案中，所述CpA变体在对应于SEQ ID NO:1的第1位和第17位的位置处的氨基酸残基被甲硫氨酸(M)取代。在一个实施方案中，所述CpA变体在对应于SEQ ID NO:1的第1位和第17位的位置处的半胱氨酸(C)被甲硫氨酸(M)取代。在一个实施方案中，所述CpA变体在对应于SEQ ID NO:1的第1位和第17位的位置包含C1M以及C17M的氨基酸取代。在一个实施方案中，所述盐浓度响应性自聚集肽的氨基酸序列如SEQ ID NO:2所示。Therefore, 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. In one embodiment, the salt concentration-responsive self-aggregating peptide portion comprises a salt-responsive self-aggregating peptide. In one embodiment, 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. In one embodiment, 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. In one embodiment, 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. In one embodiment, 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. In one embodiment, 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). In one embodiment, 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. In one embodiment, the CpA variant comprises amino acid substitutions of C1M and C17M at positions corresponding to positions 1 and 17 of SEQ ID NO: 1. In one embodiment, the amino acid sequence of the salt concentration-responsive self-aggregating peptide is shown in SEQ ID NO:2.
在一个实施方案中，本发明的盐浓度响应性自聚集肽部分可以包括一或多个串联连接的所述盐浓度响应性自聚集肽。本发明的盐浓度响应性自聚集肽部分可以包含1至150、1至130、1至110、1至90、1至70、1至50、1至30、1至10、1至5个所述盐浓度响应性自聚集肽，例如1、2、3、4、5个所述盐浓度响应性自聚集肽。所述盐浓度响应性自聚集肽部分中的两或多个盐浓度响应性自聚集肽可以形成串联重复。为了便于重组操作以及考虑到生产成本问题，期望使用较少的重复。因此，在一些实施方案中，所述盐浓度响应性自聚集肽部分仅包含一个所述盐浓度响应性自聚集肽。In one embodiment, 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. Thus, in some embodiments, the salt concentration-responsive self-aggregating peptide portion comprises only one of the salt-responsive self-aggregating peptides.
如本文所用，“间隔物”是指具有一定长度的氨基酸组成的多肽，其包括实现切割所需的序列，如用于酶切割的蛋白酶识别序列、用于自切割的内含肽序列等，以连接融合蛋白的各部分并不影响各部分的结构和活性。因此，本发明的间隔物包含“切割位点”。在本发明的融合多肽中，间隔物与目的多肽部分和/或盐浓度响应性自聚集肽部分直接连接。在另一些实施方案中，所述间隔物在其N端和/或C端进一步包含接头。在另一些实施方案中，所述间隔物通过接头与目的多肽部分和/或盐浓度响应性自聚集肽部分连接。在一些实施方案中，所述切割位点位于所述间隔物的C端，且所述切割位点紧邻所述目的多肽部分的N端。在一些实施方案中，所述切割位点位于所述间隔物的N端，且所述切割位点紧邻所述目的多肽部分的C端。在一些实施方案中，所述间隔物通过所述切割位点连接于所述目的多肽部分。在一些实施方案中，所述间隔物通过所述切割位点直接连接于所述目的多肽部分的N端或C端。As used herein, "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. Thus, the spacers of the invention comprise a "cleavage site". In the fusion polypeptide of the present invention, the spacer is directly linked to the polypeptide part of interest and/or the salt concentration-responsive self-aggregating peptide part. In other embodiments, the spacer further comprises a linker at its N-terminus and/or C-terminus. In other embodiments, the spacer is linked to the polypeptide portion of interest and/or the salt concentration-responsive self-aggregating peptide portion via a linker. In some embodiments, 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. In some embodiments, the cleavage site is located N-terminal to the spacer, and the cleavage site is immediately C-terminal to the polypeptide portion of interest. In some embodiments, the spacer is linked to the polypeptide portion of interest through the cleavage site. In some embodiments, the spacer is linked directly to the N- or C-terminus of the polypeptide portion of interest via the cleavage site.
本发明的用于将可溶的目的多肽部分从不可溶部分(沉淀)释放出来的切割位点包括 可以选自温度依赖性切割位点、pH依赖性切割位点、离子依赖性切割位点、酶切割位点或自切割位点，或本领域技术人员已知的其它任何切割位点。在一些具体实施方案中，所述切割位点为自切割位点。在一些实施方案中，所述切割位点是pH依赖性切割位点。在一些实施方案中，所述间隔物连接于所述目的多肽部分的N端或C端。应当理解，本领域技术人员可以根据需要选择合适的间隔物，并选择间隔物合适的连接位置。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. In some embodiments, the cleavage site is a self-cleavage site. In some embodiments, the cleavage site is a pH-dependent cleavage site. In some embodiments, 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.
在一些具体实施方案中，所述间隔物包含内含肽，所述内含肽包含自切割位点。内含肽是一段具有蛋白酶活性的特殊序列多肽，在诱导其蛋白酶活性产生后可以在设计位点的特定氨基酸残基切割，从而目的多肽从融合多肽三联体中分离并释放到可溶的溶液中，即可获得高纯度的目的多肽。因此，基于内含肽的切割方法不需要外加酶或使用如化学法中所用的溴化氢等有害物质，而仅仅需要改变聚集体所处的缓冲环境就能简单地诱导切割(Wu et al.,1998；TELENTI et al.,1997)。本领域已知多种自切割内含肽，例如NEB公司的一系列具有不同自切割特性的内含肽。In some embodiments, 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.
在本发明的一些具体实施方案中，所述内含肽选自Mxe GyrA、Ssp DnaB或MtuΔI-CM。如本文所用，“MtuΔI-CM”衍生自Mtu recA野生型内含肽，其通过缺失Mtu recA特大型内含肽的核酸内切酶结构域，保留N端110个氨基酸和C端58个氨基酸，得到极小型内含肽，再引入四个突变：C1A、V67L、D24G、D422G而得到(Wood等，1999)。在一些可选的实施方案中，所述MtuΔI-CM包含SEQ ID NO:3所示的序列。在一些可选的实施方案中，所述MtuΔI-CM具有SEQ ID NO:10的核苷酸序列。在一些可选的实施方案中，所述MtuΔI-CM连接于所述目的多肽部分的C端。在一个具体的实施方案中，所述内含肽MtuΔI-CM通过在pH 5.5-6.8的缓冲体系可诱导该内含肽在其羧基端的自切割。在一些可选的实施方案中，所述间隔物为MtuΔI-CM的突变体。In some specific embodiments of the present invention, the intein is selected from Mxe GyrA, Ssp DnaB or MtuΔI-CM. As used herein, "MtuΔI-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). In some optional embodiments, the MtuΔI-CM comprises the sequence shown in SEQ ID NO:3. In some optional embodiments, the MtuΔI-CM has the nucleotide sequence of SEQ ID NO:10. In some alternative embodiments, the MtuΔI-CM is linked to the C-terminus of the polypeptide portion of interest. In a specific embodiment, the intein MtuΔI-CM can induce self-cleavage of the intein at its carboxyl terminus by a buffer system at pH 5.5-6.8. In some alternative embodiments, the spacer is a mutant of MtuΔI-CM.
本领域技术人员能够理解，为了减少本发明的融合蛋白中不同部分之间的相互干扰，可以通过接头连接融合蛋白的不同部分。如本文所用，“接头”是指具有一定长度的由低疏水性和低电荷效应的氨基酸组成的多肽，其用于融合蛋白时可以使所连接的各部分充分展开，互不干扰地充分折叠成各自的天然构象。Those skilled in the art can understand that, in order to reduce the mutual interference between different parts of the fusion protein of the present invention, different parts of the fusion protein can be connected through a linker. As used herein, "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.
本领域常用的接头包括例如，富含甘氨酸(G)和丝氨酸(S)的柔性的GS型接头；富含脯氨酸(P)和苏氨酸(T)的刚性的PT型接头。在一些实施方案中，所述接头选自GS型接头和PT型接头。在一些实施方案中，本发明所使用的GS型接头的氨基酸序列示于SEQ ID NO:6。在另一些实施方案中，本发明所使用的PT型接头的氨基酸序列示于SEQ ID NO:7。在一些实施方案中，本发明所使用的GS型接头的核苷酸序列示于SEQ ID NO:13。在另一些实施方案中，本发明所使用的PT型接头的核苷酸序列示于SEQ ID NO:14。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). In some embodiments, the linker is selected from a GS-type linker and a PT-type linker. In some embodiments, the amino acid sequence of the GS-type linker used in the present invention is shown in SEQ ID NO:6. In other embodiments, the amino acid sequence of the PT-type linker used in the present invention is shown in SEQ ID NO:7. In some embodiments, the nucleotide sequence of the GS-type adapter used in the present invention is shown in SEQ ID NO: 13. In other embodiments, the nucleotide sequence of the PT-type linker used in the present invention is shown in SEQ ID NO:14.
在一些实施方案中，所述目的多肽长度为20、50、70、100、150、200、250、300、350、400、450或500个氨基酸残基，或为任意两个上述长度之间的任意长度。在一些实施方案中，所述目的多肽部分选自治疗性分子、可检测分子或靶向性分子。In some embodiments, 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. In some embodiments, the polypeptide portion of interest is selected from a therapeutic molecule, a detectable molecule, or a targeting molecule.
所述治疗性分子包括但不限于核酸药物、蛋白药物(包括治疗性多肽、治疗性抗体 等)等。示例性的治疗性分子包括但不限于毒素、免疫调节剂、拮抗剂、细胞凋亡诱导剂、激素、放射性药物、抗血管生成剂、基因药物细胞因子、趋化因子、前药、化疗药物等，例如人生长激素(hGH)、新冠多肽LCB3等。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.
所述可检测分子包括但不限于荧光蛋白、酶、标签等，例如红色荧光蛋白(RFP)、谷胱甘肽巯基转移酶GST、Xylanase等。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.
所述靶向性分子包括但不限于靶向性抗体、特异性受体配体等。例如，所述靶向性分子可以是特异性靶向肿瘤抗原的抗体。The targeting molecules include but are not limited to targeting antibodies, specific receptor ligands and the like. For example, the targeting molecule can be an antibody that specifically targets a tumor antigen.
在一个实施方案中，所述目的多肽选自人生长激素(hGH)、红色荧光蛋白(RFP)、谷胱甘肽巯基转移酶GST、新冠多肽LCB3、多价骨架蛋白ΔNSpyCatcher-ELP-ΔNSpyCatcher或Xylanase。在一个实施方案中，所述目的多肽部分包含选自如SEQ ID NO:4、SEQ ID NO:5、SEQ ID NO:53、SEQ ID NO:54、SEQ ID NO:55或SEQ ID NO:56所示的氨基酸序列。在一个实施方案中，所述目的多肽具有选自如SEQ ID NO:11、SEQ ID NO:12、SEQ ID NO:57、SEQ ID NO:58、SEQ ID NO:59或SEQ ID NO:60所示的核苷酸序列。In one embodiment, 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 ΔNSpyCatcher-ELP-ΔNSpyCatcher or Xylanase . In one embodiment, 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. In one embodiment, 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.
在一些实施方案中，所述目的多肽部分位于所述融合多肽的N端或C端。在一些实施方案中，所述间隔物连接于所述目的多肽部分的N端或C端。在一些实施方案中，所述目的多肽部分位于所述融合多肽的C端，且所述间隔物连接于所述目的多肽部分的N端。在一些实施方案中，所述目的多肽部分位于所述融合多肽的N端，且所述间隔物连接于所述目的多肽部分的C端。在一些实施方案中，所述融合多肽从N端到C端具有以下结构：盐浓度响应性自聚集肽例如MpA－间隔物－目的多肽，或目的多肽－间隔物－盐浓度响应性自聚集肽例如MpA。在一些实施方案中，所述融合多肽从N端到C端具有以下结构：盐浓度响应性自聚集肽例如MpA－接头－间隔物－目的多肽，或目的多肽－间隔物－接头－盐浓度响应性自聚集肽例如MpA。在一些实施方案中，所述融合多肽从N端到C端具有以下结构：MpA－接头－MtuΔI-CM－目的多肽例如人生长激素或RFP，或目的多肽例如人生长激素或RFP－Mxe GyrA－接头－MpA。In some embodiments, the portion of the polypeptide of interest is located at the N- or C-terminus of the fusion polypeptide. In some embodiments, the spacer is attached to the N- or C-terminus of the polypeptide portion of interest. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, the fusion polypeptide has the following structure from N-terminus to C-terminus: MpA-linker-MtuΔI-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.
三、多核苷酸、表达构建体、宿主细胞和融合多肽制备方法3. Preparation method of polynucleotide, expression construct, host cell and fusion polypeptide
在另一方面，本发明提供一种分离的多核苷酸，其包含编码本发明的融合多肽的核苷酸序列或其互补序列。In another aspect, the invention provides an isolated polynucleotide comprising a nucleotide sequence encoding a fusion polypeptide of the invention, or the complement thereof.
在另一方面，本发明提供一种分离的多核苷酸，其包含编码CpA变体的核苷酸序列或其互补序列，其中所述CpA具有如SEQ ID NO:1所示的氨基酸序列，所述CpA变体在对应于SEQ ID NO:1的第1位和第17位的位置包含C1M以及C17M的氨基酸取代。In another aspect, 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.
在一些实施方式中，本发明的多核苷酸包含SEQ ID NO:9或SEQ ID NO:61的核苷酸序列。在一个实施方案中，本发明的分离的多核苷酸包括在严格条件下与SEQ ID NO:9或SEQ ID NO:61所示的核苷酸序列杂交的多核苷酸序列。在上述实施方案中，本发明的多核苷酸编码的多肽仍保持与MpA相当的聚集效率。In some embodiments, the polynucleotide of the present invention comprises the nucleotide sequence of SEQ ID NO:9 or SEQ ID NO:61. In one embodiment, 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. In the above embodiments, the polypeptide encoded by the polynucleotide of the present invention still maintains an aggregation efficiency comparable to that of MpA.
在另一方面，本发明提供了一种表达构建体，其包含与表达调控序列可操作地连接的本发明的多核苷酸。在另一个实施方案中，本发明的表达构建体包含与表达调控序列可操作地连接的本发明的多核苷酸。In another aspect, the invention provides an expression construct comprising a polynucleotide of the invention operably linked to an expression control sequence. In another embodiment, an expression construct of the invention comprises a polynucleotide of the invention operably linked to an expression control sequence.
用于本发明的表达构建体的载体包括那些在宿主细胞中自主复制的载体，如质粒载体；还包括能够整合到宿主细胞DNA中并和宿主细胞DNA一起复制的载体。可商购获得许多适于本发明的载体。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.
在另一方面，本发明提供一种宿主细胞，其含有本发明的多核苷酸或以本发明的表达构建体转化，其中所述宿主细胞能够表达本发明的融合多肽或能够表达本发明的CpA变体。In another aspect, 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.
可用于表达本发明的融合多肽或本发明的CpA变体的宿主细胞包括原核生物、酵母和高等真核细胞。示例性的原核宿主包括埃希氏菌属(Escherichia)、芽孢杆菌属(Bacillus)、沙门氏菌属(Salmonella)以及假单胞菌属(Pseudomonas)和链霉菌属(Streptomyces)的细菌。在优选的实施方案中，宿主细胞是埃希氏菌属细胞，优选是大肠杆菌。在本发明的一个具体实施方案中，所使用的宿主细胞为大肠杆菌BL21(DE3)菌株细胞。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. In a preferred embodiment, the host cell is an Escherichia cell, preferably E. coli. In a specific embodiment of the present invention, the host cells used are Escherichia coli BL21 (DE3) strain cells.
可以通过许多已熟知的技术之一将本发明的重组表达构建体导入宿主细胞，这样的技术包括但不限于：热激转化，电穿孔，DEAE-葡聚糖转染，显微注射，脂质体接介导的转染，磷酸钙沉淀，原生质融合，微粒轰击，病毒转化及类似技术。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.
在另一方面，本发明提供了一种产生本发明的融合多肽的方法，包括：In another aspect, the invention provides a method of producing a fusion polypeptide of the invention, comprising:
a)在允许融合多肽表达的条件下培养本发明的宿主细胞；a) cultivating the host cell of the present invention under conditions that allow the expression of the fusion polypeptide;
b)从得自步骤a)的培养物获得由所述宿主细胞表达的融合多肽。b) obtaining the fusion polypeptide expressed by said host cell from the culture obtained from step a).
四、生产和纯化目的多肽的方法4. Methods for producing and purifying target polypeptides
本发明还涉及生产和纯化目的多肽的方法，所述方法包括以下步骤：(a)培养本发明的宿主细胞，从而表达融合多肽；(b)在第一盐条件下，裂解所述宿主细胞，然后去除细胞裂解物的不溶部分，回收可溶部分；(c)在第二盐条件下，所述融合蛋白形成不溶部分；(d)回收步骤(c)中形成的不溶部分；(e)通过切割所述切割位点从收集自步骤(d)的不溶部分释放可溶的目的多肽；和(f)去除步骤(e)中的不溶部分，回收含有所述目的多肽的可溶部分。本发明的方法的示意图可参见图1A。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.
在本发明中，使宿主细胞裂解的方法选自本领域常用的处理方式，例如超声、匀浆、高压(例如在弗氏压碎器中)、低渗(osmolysis)、去垢剂、裂解酶、有机溶剂或其组合。In the present invention, 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.
在一个实施方案中，所述第一盐条件包含第一盐浓度，所述第二盐条件包含第二盐浓度。在一个实施方案中，所述第一盐浓度与所述第二盐浓度不同。在一个实施方案中，所述第一盐浓度高于所述第二盐浓度。在一个实施方案中，所述第一盐浓度低于所述第二盐浓度。在一个实施方案中，所述第一盐条件包含第一离子强度，所述第二盐条件包含第二离子强度。在一个实施方案中，所述第一离子强度与所述第二离子强度不同。在 一个实施方案中，所述第一离子强度高于所述第二离子强度。在一个实施方案中，所述第一离子强度低于所述第二离子强度。在一个实施方案中，所述第一盐条件包含第一盐种类，所述第二盐条件包含第二盐种类。在一个实施方案中，所述第一盐种类与所述第二盐种类相同。在一个实施方案中，所述第一盐种类与所述第二盐种类不同。In one embodiment, 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.
在一个实施方案中，所述第一盐条件下和/或第二盐条件下的所述盐(即所述第一盐种类和/或所述第二盐种类)选自一价金属盐如钾盐或钠盐等，二价金属盐如镁盐、钙盐、锰盐或铜盐等，或铵盐，优选为铵盐、钾盐或钠盐。在一个实施方案中，所述第一盐条件下和/或第二盐条件下的所述盐的阴离子选自硫酸根，磷酸氢根，乙酸根，卤离子如氟离子、氯离子、溴离子或碘离子等，硝酸根，高氯酸根，或硫氰酸根离子，优选为硫酸根、磷酸氢根、氯离子或乙酸根。在一个实施方案中，所述第一盐条件下和/或第二盐条件下的所述盐选自氯化钠、硫酸钠、硝酸钠、磷酸氢二钠、磷酸二氢钠、碳酸钠、氯化钾、硫酸钾、硝酸钾、磷酸氢二钾、磷酸二氢钾、碳酸钾、硝酸铵、硫酸铵或氯化铵，优选为氯化钠、硫酸钠或硫酸铵。In one embodiment, said salt under said first salt condition and/or under said second salt condition (i.e. said first salt species and/or said second salt species) 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. In one embodiment, 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. In one embodiment, 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.
在一个实施方案中，所述第一离子强度为0-0.2mol/L。在一个实施方案中，所述第一离子强度为0-0.1mol/L或0.1-0.2mol/L。在一个实施方案中，所述第一离子强度为约0mol/L、约0.1mol/L或约0.2mol/L。在一个实施方案中，所述第二离子强度为0.5-5.0mol/L。在一个实施方案中，所述第二离子强度为1.0-4.5mol/L、1.5-4.0mol/L、1.5-2.5mol/L、2.0-3.5mol/L或2.5-3.0mol/L。在一个实施方案中，所述第二离子强度为约0.5mol/L、约0.8mol/L、约1.0mol/L、约1.2mol/L、约1.5mol/L、约1.8mol/L、约2.0mol/L、约2.1mol/L、约2.2mol/L、约2.5mol/L、约2.8mol/L、约3.0mol/L、约3.2mol/L、约3.5mol/L、约3.8mol/L、约4.0mol/L、约4.2mol/L、约4.5mol/L、约4.8mol/L、约5.0mol/L，或为任意两个上述离子强度之间的任意离子强度。In one embodiment, 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. In one embodiment, 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.
在一个实施方案中，所述第二盐条件选自0.5～4M NaCl，优选为3M NaCl；0.2～1.5M Na
2SO
4，优选为0.7M Na
2SO
4；或0.2～1.5M(NH
4)
2SO
4，优选为0.7M(NH
4)
2SO
4。在一个实施方案中，所述第一盐条件为约0M。在一个实施方案中，步骤(e)在第二盐条件下进行。在一个实施方案中，所述第二盐种类为NaCl，且所述第二离子强度为2.5-3.0mol/L，优选为约3mo/L。在一个实施方案中，所述第二盐种类为Na
2SO
4，且所述第二离子强度为1.5-2.5mol/L，优选为约2.1mo/L。在一个实施方案中，所述第二盐种类为(NH
4)
2SO
4，且所述第二离子强度为1.5-2.5mol/L，优选为约2.1mo/L。在一个实施方案中，所述第二盐种类为K
2SO
4，且所述第二离子强度为1.5-2.5mol/L，优选为约2.1mo/L。在一个实施方案中，所述第二盐种类为Na
2HPO
4，且所述第二离子强度为1.5-2.5mol/L，优选为约2.1mo/L。
In one embodiment, 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 . In one embodiment, the first salt condition is about OM. In one embodiment, step (e) is performed under second salt conditions. In one embodiment, the second salt species is NaCl, and the second ionic strength is 2.5-3.0 mol/L, preferably about 3 mo/L. In one embodiment, 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. In one embodiment, 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.
在一个具体的实施方案中，在生理条件(如：正常温度18-37℃、中性pH值7.4-7.8)下培养宿主细胞以表达本发明的融合蛋白。因此，由于表达是在正常生理条件下培养的宿主细胞内进行，既避免了宿主细胞培养周期的延长，同时因培养条件适宜可提高融合蛋白的产量和产率。In a specific embodiment, 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.
在一个实施方案中，所述步骤(c)在4℃-25℃的温度下进行。在一个实施方案中，所述步骤(c)在4℃-20℃、4℃-15℃或4℃-10℃的温度下进行。在一个实施方案中，所述步骤(c)在4℃、10℃、15℃、20℃或25℃的温度下进行，优选在4℃下进行。在一个实施方案中，所述步骤(e)在4℃-25℃的温度下进行。在一个实施方案中，所述步骤(e)在4℃-20℃、4℃-15℃或4℃-10℃的温度下进行。在一个实施方案中，所述步骤(e)在4℃、10℃、15℃、20℃或25℃的温度下进行，优选在25℃下进行。在一个实施方案中，所述步骤(c)以及所述步骤(e)均在4℃-25℃的温度下进行。在一个实施方案中，所述步骤(c)以及所述步骤(e)均不在高于25℃的温度下进行。在一个实施方案中，所述步骤(b)至所述步骤(f)均在4℃-25℃的温度下进行。在一个实施方案中，所述步骤(b)至所述步骤(f)均不在高于25℃的温度下进行。因此，本发明省略了通过反复改变温度条件以获得沉淀状态的融合蛋白的步骤，也避免了过高的温度对蛋白质稳定性及活性的影响。In one embodiment, 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. In one embodiment, 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.
在一个实施方案中，所述步骤(c)包括调节含有收集自步骤(b)的可溶部分的溶液的盐浓度。在一个实施方案中，所述步骤(c)包括降低含有收集自步骤(b)的可溶部分的溶液的盐浓度。在一个实施方案中，所述步骤(c)包括升高含有收集自步骤(b)的可溶部分的溶液的盐浓度。In one embodiment, 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).
在一个实施方案中，所述步骤(c)和步骤(d)进行1、2或3次。在一个实施方案中，所述步骤(e)和步骤(f)进行1、2或3次。在一个实施方案中，所述步骤(c)和所述步骤(e)仅进行一次。在一个实施方案中，所述步骤(c)至所述步骤(f)仅进行一次。In one embodiment, 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.
在一个实施方案中，所述步骤(b)在中性至弱碱性的pH条件下进行。在一个特定实施方案中，所述中性至弱碱性的pH条件为pH 7.2-8.5。在一个优选的实施方案中，所述中性至弱碱性的pH条件是pH 7.4-8.3。在一个更优选的实施方案中，所述中性至弱碱性的pH条件是pH 7.6-8.2。在一个最优选的实施方案中，所述中性至弱碱性的pH条件是pH 8.0。在一个实施方案中，所述步骤(c)在中性至弱碱性的pH条件下进行。在一个特定实施方案中，所述中性至弱碱性的pH条件为pH 7.2-8.5。在一个优选的实施方案中，所述中性至弱碱性的pH条件是pH 7.4-8.3。在一个更优选的实施方案中，所述中性至弱碱性的pH条件是pH 7.6-8.2。在一个最优选的实施方案中，所述中性至弱碱性的pH条件是pH 8.0。在一个实施方案中，所述步骤(e)在弱酸性的pH条件下进行。在一个特定实施方案中，所述弱酸性的pH条件为pH 5.5-6.8，且优选5.5-6.5。在一个最优选的实施方案中，所述弱酸性的pH条件是pH 6.2。In one embodiment, the step (b) is carried out under neutral to slightly alkaline pH conditions. In a particular embodiment, the neutral to slightly alkaline pH condition is pH 7.2-8.5. In a preferred embodiment, the neutral to slightly alkaline pH condition is pH 7.4-8.3. In a more preferred embodiment, 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, the step (c) is carried out under neutral to slightly alkaline pH conditions. In a particular embodiment, the neutral to slightly alkaline pH condition is pH 7.2-8.5. In a preferred embodiment, the neutral to slightly alkaline pH condition is pH 7.4-8.3. In a more preferred embodiment, 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.
为使本发明的技术方案和优点更加清楚，下面将通过实施例对本发明实施方式作进一步地详细描述。应当理解实施例不应理解为限制性的，本领域技术人员能够基于本发明的原理对实施方式做进一步的调整。In order to make the technical solutions and advantages of the present invention clearer, the implementation of the present invention will be further described in detail through examples below. 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 implementation based on the principles of the present invention.
以下实施例中所用方法如无特别说明均为常规方法，具体步骤可参见，例如，Molecular Cloning:A Laboratory Manual(Sambrook J.等，Molecular Cloning:A Laboratory Manual，3rd edition，2001，NY，Cold Spring Harbor)。所用引物均由上海生工生物合成。The methods used in the following examples are conventional methods unless otherwise specified, and the specific steps can be referred to, for example, Molecular Cloning: A Laboratory Manual (Sambrook J. etc., Molecular Cloning: A Laboratory Manual, 3rd edition, 2001, NY, Cold Spring Harbor). The primers used were synthesized by Shanghai Sangon Biotech.
实施例1：构建MpA-Mtu-POI，CpA-Mtu-POI，POI-Mxe-MpA融合蛋白表达载体Embodiment 1: Construct MpA-Mtu-POI, CpA-Mtu-POI, POI-Mxe-MpA fusion protein expression vector
POI表示目的蛋白，在本申请实施例中，POI指人生长激素hGH，红色荧光蛋白RFP，谷胱甘肽巯基转移酶GST，新冠多肽LCB3，多价骨架蛋白ΔNSpyCatcher-ELP-ΔNSpyCatcher或木聚糖酶Xylanase。所使用的表达载体包括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-ΔNSpyCatcher-ELP-ΔNSpyCatcher和pET30a-MpA-Mtu-Xylanase，构建质粒所需要的引物，通过oligo 6设计并由上海生工合成如表1所示的寡聚核苷酸引物。POI refers to the target protein. In the examples of this application, POI refers to human growth hormone hGH, red fluorescent protein RFP, glutathione sulfhydryl transferase GST, new crown polypeptide LCB3, multivalent backbone protein ΔNSpyCatcher-ELP-ΔNSpyCatcher 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-ΔNSpyCatcher-ELP-ΔNSpyCatcher 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.
表1本实施例所用寡聚核苷酸引物Table 1 Oligonucleotide primers used in this embodiment
a引物下划线部分分别为限制性内切酶NdeI和Xho I的识别位点。a The underlined parts of the primers are the recognition sites of restriction endonucleases NdeI and XhoI, respectively.
首先用在线工具DNAworks(https://hpcwebapps.cit.nih.gov/dnaworks/)设计MpA和PT型接头的核苷酸序列。通过DNAWorks设计并合成如表1中MpA所示的4条寡聚核苷酸引物(SEQ ID No:15～18)，将这4条10μM的寡聚核苷酸引物等体积混合后取2μL，再加入1μL 10μM的dNTP、4μL 5×Q5reaction buffer、ddH
2O 12.8μL、Q5DNA聚合酶0.2μL，PCR条件为：98℃30sec，98℃10sec，60℃20sec，72℃15sec，共14个循环，最后72℃2min。反应结束后，以DNAworks的产物作为模板，使用寡聚核苷酸引物MpA-F和PT-Mtu-R进行PCR扩增，PCR条件为：98℃30sec，98℃10sec，68℃20sec，72℃15sec，共29个循环，最后72℃2min。对PCR扩增产物进行1％琼脂糖凝胶分离回收，获得NdeI-MpA-PT多核苷酸片段。以pET32-L6KD-MtuΔI-CM-hGH(林章凛等，PCT/CN2020/125054，2020)为模板，使用引物Mtu-F和Mtu-hGH-3-R通过PCR扩增获得Mtu-hGH-XhoI多核苷酸片段。进而以多核苷酸片段NdeI-MpA-PT和Mtu-hGH-XhoI为模板，使用引物MpA-F和Mtu-hGH-3-R，通过重叠PCR(overlapping PCR)法得到NdeI-MpA-PT-Mtu-hGH-XhoI多核苷酸序列。重叠PCR纯化后的多核苷酸片段和pET30a质粒(Novagen)分别用限制性内切酶Nde I和Xho I进行双酶切，然后回收相应的片段进行纯化，纯化后用T4 DNA连接酶连接，将连接产物转化到大肠杆菌DH5α感受态细胞，将转化细胞涂布于添加有50μg/mL卡纳霉素的LB平板上筛选阳性克隆，用质粒提取试剂盒提取质粒，对其进行测序。
Firstly, 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 μM in equal volume and take 2 μL, Then add 1 μL of 10 μM dNTP, 4 μL of 5×Q5 reaction buffer, 12.8 μL of ddH 2 O, and 0.2 μL 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. After the reaction, use the DNAworks product as a template, and use the oligonucleotide primers MpA-F and PT-Mtu-R for PCR amplification. 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. Using pET32-L6KD-MtuΔI-CM-hGH (Lin Zhanglin et al., PCT/CN2020/125054, 2020) as a template, Mtu-hGH-XhoI was obtained by PCR amplification using primers Mtu-F and Mtu-hGH-3-R polynucleotide fragments. Further, using 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 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 with a plasmid extraction kit and sequenced.
然后以构建的质粒pET30a-MpA-Mtu-hGH为模板，使用引物Backbone-F和Mtu-R通过PCR扩增获得Backbone-MpA-PT-Mtu多核苷酸片段，以pET30a-SpyTag-GSlinker-RFP(Lin Z.等，Biotechnology and Bioengineering,2020,117:2923-2932)为模板使用引物RFP-F和Backbone-R通过PCR扩增获得RFP-Backbone多核苷酸片段，将纯化后的Backbone-MpA-PT-Mtu和RFP-Backbone多核苷酸片段进行Gibson assembly组装，组装产物转化到大肠杆菌DH5α感受态细胞，将转化细胞涂布于添加有50μg/mL卡纳霉素的LB平板上筛选阳性克隆，用质粒提取试剂盒提取质粒，对其进行测序。Then take the constructed plasmid pET30a-MpA-Mtu-hGH as a template, use primers Backbone-F and Mtu-R to obtain the Backbone-MpA-PT-Mtu polynucleotide fragment by PCR amplification, and use pET30a-SpyTag-GSlinker-RFP( Lin Z. et al., Biotechnology and Bioengineering, 2020, 117:2923-2932) used primers RFP-F and Backbone-R as templates to obtain RFP-Backbone polynucleotide fragments by PCR amplification, and the purified Backbone-MpA-PT -Mtu and RFP-Backbone polynucleotide fragments were assembled by Gibson assembly, and the assembled product was transformed into Escherichia coli DH5α competent cells, and the transformed cells were spread on LB plates added with 50 μg/mL kanamycin to screen positive clones, and Plasmid extraction kits were used to extract plasmids and sequence them.
质粒pET30a-MpA-Mtu-GST和pET30a-MpA-Mtu-ΔNSpyCatcher-ELP-ΔNSpyCatcher的构建方法类似，分别以 pET30-MpA-Mtu-hGH和目的基因(GST或ΔNSpyCatcher-ELP-ΔNSpyCatcher)为模板，通过Gibson assembly的方法获得。以pET30a-MpA-Mtu-GST为例，以质粒pET30-MpA-Mtu-hGH为模板，使用引物GST-Backbone-F和Mtu-R扩增获得Backbone-MpA-Mtu多核苷酸片段，以GST基因为模板，使用引物GST-F和GST-R扩增获得GST片段，将纯化后的Backbone-MpA-Mtu和GST多核苷酸片段进行Gibson assembly组装，组装产物转化到大肠杆菌DH5α感受态细胞，将转化细胞涂布于添加有50μg/mL卡纳霉素的LB平板上筛选阳性克隆，用质粒提取试剂盒提取质粒，对其进行测序。pET30-MpA-Mtu-ΔNSpyCatcher-ELP-ΔNSpyCatcher的构建所使用的模板为pET30-MpA-Mtu-hGH质粒和ΔNSpyCatcher-ELP-ΔNSpyCatcher基因，克隆所需的引物和操作流程与pET30a-MpA-Mtu-GST类似。所构建的pET30a-MpA-Mtu-GST和pET30a-MpA-Mtu-ΔNSpyCatcher-ELP-ΔNSpyCatcher质粒的结构如图1B所示。Plasmids pET30a-MpA-Mtu-GST and pET30a-MpA-Mtu-ΔNSpyCatcher-ELP-ΔNSpyCatcher are constructed in a similar way, using pET30-MpA-Mtu-hGH and the target gene (GST or ΔNSpyCatcher-ELP-ΔNSpyCatcher) as templates respectively, by The method of Gibson assembly is obtained. Taking pET30a-MpA-Mtu-GST as an example, 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 DH5α competent cells. Transformed cells were plated on LB plates supplemented with 50 μg/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-ΔNSpyCatcher-ELP-ΔNSpyCatcher are pET30-MpA-Mtu-hGH plasmid and ΔNSpyCatcher-ELP-ΔNSpyCatcher gene, the primers and operation procedures required for cloning are the same as pET30a-MpA-Mtu-GST similar. The structures of the constructed pET30a-MpA-Mtu-GST and pET30a-MpA-Mtu-ΔNSpyCatcher-ELP-ΔNSpyCatcher plasmids are shown in Figure 1B.
质粒pET30a-MpA-Mtu-LCB3的构建，首先以LCB3基因为模板使用引物LCB3-F和LCB3-R扩增获得LCB3多核苷酸片段，然后以pET32-L6KD-MtuΔI-CM-hGH(林章凛等，PCT/CN2020/125054，2020)为模板，使用引物Mtu-F和Mtu-R扩增获得Mtu多核苷酸片段，进而以多核苷酸片段Mtu和LCB3为模板，使用引物Mtu-F和LCB3-R，通过重叠PCR(overlapping PCR)法得到Mtu-LCB3多核苷酸片段，接着以pET30a-MpA-Mtu-hGH为模板，使用引物LCB3-Backbone-F和LCB3-Backbone-R扩增获得Backbone-MpA多核苷酸片段，最后将纯化后的Mtu-LCB3多核苷酸片段和Backbone-MpA多核苷酸片段进行Gibson assembly组装，组装产物转化到大肠杆菌DHα感受态细胞，将转化细胞涂布于添加有50μg/mL卡纳霉素的LB平板上筛选阳性克隆，用质粒提取试剂盒提取质粒，对其进行测序。To construct 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-MtuΔI-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-F and LCB3-Backbone-R to amplify to obtain Backbone- MpA polynucleotide fragments, finally the purified Mtu-LCB3 polynucleotide fragments and Backbone-MpA polynucleotide fragments were assembled by Gibson assembly, the assembled products were transformed into Escherichia coli DHα competent cells, and the transformed cells were coated with Positive clones were screened on LB plates with 50 μg/mL kanamycin, and the plasmids were extracted with a plasmid extraction kit and sequenced.
质粒pET30-CpA-Mtu-hGH和pET30-CpA-Mtu-RFP的构建方法类似，分别以pET30-MpA-Mtu-hGH和pET30-MpA-Mtu-RFP为模板，通过Gibson assembly的方法获得。以pET30-CpA-Mtu-hGH的构建为例，以质粒pET30-MpA-Mtu-hGH为模板，使用引物Backbone-2-F和CpA-R扩增获得Backbone-CpA多核苷酸片段，使用引物CpA-F和Backbone-2-R扩增获得CpA-Mtu-hGH-Backbone多核苷酸片段，将纯化后的Backbone-CpA和CpA-Mtu-hGH-Backbone多核苷酸片段进行Gibson assembly组装，组装产物转化到大肠杆菌DH5α感受态细胞，将转化细胞涂布于添加有50μg/mL卡纳霉素的LB平板上筛选阳性克隆，用质粒提取试剂盒提取质粒，对其进行测序。pET30-CpA-Mtu-RFP的构建所使用的模板为pET30-MpA-Mtu-RFP，克隆所需的引物和操作流程与pET30-CpA-Mtu-hGH类似。所构建的pET30-MpA-Mtu-hGH、pET30-MpA-Mtu-RFP、pET30-CpA-Mtu-hGH和pET30-CpA-Mtu-RFP质粒的结构如图1B所示。The construction methods of plasmids pET30-CpA-Mtu-hGH and pET30-CpA-Mtu-RFP are similar, using pET30-MpA-Mtu-hGH and pET30-MpA-Mtu-RFP as templates respectively, and obtaining them by Gibson assembly method. Taking the construction of pET30-CpA-Mtu-hGH as an example, 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 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 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.
质粒pET30a-Xylanase-Mxe-MpA的构建，首先采用与合成NdeI-MpA-PT多核苷酸片段相似的方法通过DNAWorks设计引物GS-MpA-1、GS-MpA-2、GS-MpA-3和GS-MpA-4进行PCR扩增，然后以DNAWorks的产物为模板，使用引物GS-MpA-F和 MpA-R扩增获得GS-MpA多核苷酸片段；然后以pET30a-hGH-Mxe GyrA-L6KD为模板，使用引物Xylanase-Mxe-F和Mxe-R扩增获得Mxe多核苷酸片段；接着以Xylanase基因为模板，使用引物Xylanase-F和Xylanase-R扩增获得Xylanase多核苷酸片段；再然后以质粒pET30a(Novagen)为模板，使用引物30a-F和30a-R扩增获得30a-Backbone多核苷酸片段；最后将纯化后的30a-Backbone、Xylanase、Mxe和GS-MpA多核苷酸片段进行Gibson assembly组装，组装产物转化到大肠杆菌DHα感受态细胞，将转化细胞涂布于添加有50μg/mL卡纳霉素的LB平板上筛选阳性克隆，用质粒提取试剂盒提取质粒，对其进行测序。pET30a-Xylanase-Mxe-MpA的质粒的结构如图1C所示。For the construction of 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 Xylanase-R to amplify to obtain Xylanase polynucleotide fragments; then use Plasmid pET30a (Novagen) was used as a template, and primers 30a-F and 30a-R were used to amplify the 30a-Backbone polynucleotide fragment; finally, the purified 30a-Backbone, Xylanase, Mxe and GS-MpA polynucleotide fragments were subjected to Gibson Assembly, the assembly product was transformed into E. coli DHα competent cells, the transformed cells were spread on the LB plate supplemented with 50 μg/mL kanamycin to screen positive clones, the plasmid was extracted with a plasmid extraction kit, and sequenced. The structure of the plasmid pET30a-Xylanase-Mxe-MpA is shown in Figure 1C.
实施例2：MpA-Mtu-POI和CpA-Mtu-POI融合蛋白的表达Embodiment 2: Expression of MpA-Mtu-POI and CpA-Mtu-POI fusion protein
将实施例1中构建好的质粒(含有质粒pET30-MpA-Mtu-hGH、pET30-MpA-Mtu-RFP、pET30-CpA-Mtu-hGH和pET30-CpA-Mtu-RFP)转化到大肠杆菌BL21(DE3)感受态细胞获得四种MpA/CpA-Mtu-POI融合蛋白表达菌株。分别将四种表达菌株接种到含50μg/mL卡那霉素的LB液体培养基中，并在37℃摇床中培养至对数期(OD
600＝0.4-0.6)，加入0.2mM IPTG，在18℃诱导24小时，测量菌浓度OD
600，4℃4,000rpm离心25min收获细胞，除去上清的培养基将菌体冻存-80℃。(以下将1mL的OD
600为1的细胞量称为1OD)
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 four expression strains were inoculated into LB liquid medium containing 50 μg/mL kanamycin, and cultured in a shaker at 37°C to logarithmic phase (OD 600 =0.4-0.6), adding 0.2mM IPTG, in After induction at 18°C for 24 hours, the OD 600 of the bacterial concentration was measured, and the cells were harvested by centrifugation at 4,000 rpm for 25 minutes at 4°C, and the culture medium from which the supernatant was removed was frozen and stored at -80°C. (Hereafter, 1 mL of cells whose OD600 is 1 is called 1OD)
将菌体用裂解缓冲液B1(2.4g的Tris、0.37g的EDTA·2Na溶解于800mL水中，调pH至8.0，加水定容至1L)重悬至100OD/mL，进行超声破碎(破碎条件为：变幅杆Φ2，20％功率，超声时间2sec，间隔时间2sec，运行25min～30min)。在4℃，15,000g的条件下离心30min，分别收集上清和沉淀部分进行制样，SDS-PAGE检测裂解上清和裂解沉淀中融合蛋白的表达情况。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 Φ2, 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.
结果如图2所示。图2A中，泳道a-d为分别是MpA-Mtu-hGH和MpA-Mtu-RFP的裂解上清和裂解沉淀，a：MpA-Mtu-hGH细胞裂解上清，可检测到明显的融合蛋白条带；b：MpA-Mtu-hGH细胞裂解沉淀，可检测到较淡的融合蛋白条带；c：MpA-Mtu-RFP细胞裂解上清，可检测到明显的融合蛋白条带；d：MpA-Mtu-RFP细胞裂解沉淀，几乎观察不到融合蛋白条带。泳道1-5为含有牛血清蛋白BSA的蛋白定量标准品，上样量依次为0.5μg、1.0μg、2.0μg、4.0μg、8.0μg。图2B中，泳道a-d为分别是CpA-Mtu-hGH和CpA-Mtu-RFP的裂解上清和裂解沉淀，a：CpA-Mtu-hGH细胞裂解上清，可检测到明显的融合蛋白条带；b：CpA-Mtu-hGH细胞裂解沉淀，可检测到较淡的融合蛋白条带；c：CpA-Mtu-RFP细胞裂解上清，可检测到明显的融合蛋白条带；d：CpA-Mtu-RFP细胞裂解沉淀，几乎观察不到融合蛋白条带。泳道1-5为含有牛血清蛋白BSA的蛋白定量标准品，上样量依次为0.25μg、0.5μg、1.0μg、2.0μg、4.0μg、8.0μg。The result is shown in Figure 2. In Figure 2A, 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 μg, 1.0 μg, 2.0 μg, 4.0 μg, and 8.0 μg. In Figure 2B, 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 the loading amounts are 0.25 μg, 0.5 μg, 1.0 μg, 2.0 μg, 4.0 μg, and 8.0 μg.
依照蛋白定量标准品，应用ImageJ(National Institutes of Health)凝胶定量分析软件对目的条带进行光密度分析，可计算得出融合蛋白上清和沉淀表达的产量，结果如表2所示。According to the protein quantification standard, apply ImageJ (National Institutes of Health) gel quantitative analysis software to analyze the optical density of the target band, and calculate the yield of fusion protein supernatant and precipitate expression. The results are shown in Table 2.
表2MpA-Mtu-POI和CpA-Mtu-POI融合蛋白的表达The expression of table 2MpA-Mtu-POI and CpA-Mtu-POI fusion protein
| 融合蛋白fusion protein | 可溶表达的量 a(mg/L) Amount of soluble expressiona (mg/L) | 可溶占比 b(％) Soluble ratiob (%) |
| MpA-Mtu-hGHMpA-Mtu-hGH | 614±9614±9 | 9393 |
| MpA-Mtu-RFPMpA-Mtu-RFP | 401±15401±15 | 9999 |
| CpA-Mtu-hGHCpA-Mtu-hGH | 446±40446±40 | 9898 |
| CpA-Mtu-RFPCpA-Mtu-RFP | 244±34244±34 | 9999 |
a裂解上清中融合蛋白的表达量(体积以每升LB培养基来计算)，
b可溶占比＝100％×裂解上清中融合蛋白的表达量/(裂解上清中融合蛋白的表达量+裂解沉淀中融合蛋白的表达量)。
a The expression of the fusion protein in the lysed supernatant (volume is calculated per liter of LB medium), b The soluble ratio=100%×The expression of the fusion protein in the lysed supernatant/(The expression of the fusion protein in the lysed supernatant amount+the expression amount of the fusion protein in the lysed pellet).
所采用的4种融合蛋白(MpA-Mtu-hGH、MpA-Mtu-RFP、CpA-Mtu-hGH和CpA-Mtu-RFP)均以可溶形式表达，可溶表达的量分别为614±9mg/L、401±15mg/L、446±40mg/L和244±34mg/L，可溶占比分别为93％、99％、98％和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.
实施例3：3M NaCl介导MpA-Mtu-hGH/RFP相变和Mtu介导切割的蛋白纯化Example 3: 3M NaCl-mediated MpA-Mtu-hGH/RFP phase transition and Mtu-mediated cleavage protein purification
向实施例2中获得的裂解上清中加入NaCl至3M置于4℃过夜12h，使得自聚集肽充分聚集。之后将悬浊液在4℃，15,000g的条件下离心30min，将离心后的沉淀用等体积含3M NaCl的缓冲液B2(175.32g的NaCl、2.4g的Tris、0.37g的EDTA·2N
a溶解于800mL水中，调pH至8.0，加水定容至1L)洗涤1次后等条件离心分离上清和沉淀，使用体积减半的含3M NaCl的切割缓冲液B3(PBS补加NaCl至3M，补加40mM Bis-Tris，pH 6.2，2mM EDTA)充分重悬沉淀，置于25℃24h，使得内含肽充分自切割。4℃孵育3h使聚集肽充分聚集，之后将悬浊液4℃，16,000g的条件下离心30min分离。裂解细胞后的上清、加盐聚集后的上清和沉淀、切割后的上清和沉淀一起进行SDS-PAGE检测，结果如图3所示。图3A中，泳道a-d为人生长激素hGH表达与纯化样品，分别是a：细胞裂解物上清，可检测到清晰的融合蛋白条带；b：裂解物上清加盐聚集后分离的上清；c：裂解物上清加盐聚集后分离的沉淀，可检测到清晰的融合蛋白条带；d：加盐获得的聚集体切割后的沉淀；e：加盐获得的聚集体切割后的上清，可检测到清晰的人生长激素hGH条带。泳道1-6为含有牛血清蛋白BSA的蛋白定量标准品，上样量依次为0.125μg、0.25μg、0.5μg、1.0μg、2.0μg、4.0μg。图3B中，泳道a-d为红色荧光蛋白RFP表达与纯化样品，分别是a：细胞裂解物上清，可检测到清晰的融合蛋白条带；b：裂解物上清加盐聚集后分离的上清；c：裂解物上清加盐聚集后分离的沉淀，可检测到清晰的融合蛋白条带；d：加盐获得的聚集体切割后的沉淀；e：加盐获得的聚集体切割后的上清，可检测到清晰的红色荧光蛋白RFP条带。泳道1-6为含有牛血清蛋白BSA的蛋白定量标准品，上样量依次为0.125μg、0.25μg、0.5μg、1.0μg、2.0μg、4.0μg。
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. Afterwards, 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-cleaved. Incubate at 4°C for 3h to fully aggregate the aggregated peptide, and then centrifuge the suspension at 4°C and 16,000g for 30min to separate. The supernatant after lysing the cells, the supernatant and the precipitate after adding salt to aggregate, and the supernatant and the precipitate after cleavage were tested by SDS-PAGE together, and the results are shown in Figure 3. In Figure 3A, 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 μg, 0.25 μg, 0.5 μg, 1.0 μg, 2.0 μg, and 4.0 μg. In Figure 3B, 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 μg, 0.25 μg, 0.5 μg, 1.0 μg, 2.0 μg, and 4.0 μg.
依照蛋白定量标准品，应用ImageJ(National Institutes of Health)凝胶定量分析软件对目的条带进行光密度分析，可计算得出在内含肽介导的自切割之后释放到上清中的目 的蛋白产量、加盐后的聚集效率、MtuΔI-CM切割效率、人生长激素hGH和红色荧光蛋白RFP回收率及其在上清中的纯度，结果如表3所示。According to the protein quantification standard, apply ImageJ (National Institutes of Health) gel quantitative analysis software to analyze the optical density of the target band, and calculate the target protein released into the supernatant after intein-mediated self-cleavage The yield, aggregation efficiency after adding salt, cutting efficiency of MtuΔI-CM, recovery rate of human growth hormone hGH and red fluorescent protein RFP and their purity in the supernatant are shown in Table 3.
表3 3M NaCl介导MpA-Mtu-hGH/RFP相变和Mtu介导切割的蛋白纯化情况Table 3 3M NaCl-mediated MpA-Mtu-hGH/RFP phase transition and Mtu-mediated cleavage protein purification
a内含肽介导的自切割后的目的蛋白的产量(体积以每升LB培养基来计算)，
b聚集效率＝100％×加盐后沉淀中融合蛋白的量/(加盐后沉淀中融合蛋白的量+加盐后上清中融合蛋白的量)，
c内含肽介导的自切割效率＝100％×(加盐后沉淀中融合蛋白的量-切割后沉淀中融合蛋白的量)/加盐后沉淀中融合蛋白的量，
d回收率＝100％×目的蛋白实际产量/表达上清在完全切割的情况下可生产目的蛋白的理论产量。
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.
所采用的2种融合蛋白(MpA-Mtu-hGH、MpA-Mtu-RFP)的裂解上清在3M NaCl的情况下融合蛋白由可溶变为沉淀(96％的MpA-Mtu-hGH转化为沉淀、54％的MpA-Mtu-RFP转化为沉淀)，内含肽MtuΔI-CM自切割，目的蛋白同MpA-Mtu分离，切割效率是45～65％，切割后释放到上清中hGH和RFP的产量分别为72mg/L和79mg/L，切割后回收的hGH和RFP纯度分别为99％和86％。The lysed supernatants of the two fusion proteins (MpA-Mtu-hGH, MpA-Mtu-RFP) that were used changed from soluble to precipitated under the condition of 3M NaCl (96% of MpA-Mtu-hGH was converted into precipitated , 54% of MpA-Mtu-RFP is converted into precipitate), the intein MtuΔI-CM is self-cleaved, the target protein is separated from MpA-Mtu, the cleavage efficiency is 45-65%, and the hGH and RFP are released into the supernatant after cleavage The yields were 72 mg/L and 79 mg/L, respectively, and the purity of hGH and RFP recovered after cleavage were 99% and 86%, respectively.
实施例4：0.7M Na
2SO
4介导MpA-Mtu-hGH/RFP相变和Mtu介导切割的蛋白纯化
Example 4: 0.7M Na 2 SO 4 mediated MpA-Mtu-hGH/RFP phase transition and Mtu-mediated cleavage protein purification
向实施例2中获得的裂解上清中加入Na
2SO
4至0.7M，置于4℃过夜12h，使得自聚集肽充分聚集。之后将悬浊液在4℃，15,000g的条件下离心30min，将沉淀用含0.7M Na
2SO
4的缓冲液B4(99.43g的Na
2SO
4、2.4g的Tris、0.37g的EDTA·2N
a溶解于800mL水中，调pH至8.0，加水定容至1L)洗涤1次后等条件离心分离上清和沉淀，使用含0.7M Na
2SO
4的切割缓冲液B5(不含NaCl的PBS，补加0.7M Na
2SO
4，补加40mM Bis-Tris，pH6.2，2mM EDTA)充分重悬，置于25℃24h，使得内含肽充分自切割。4℃孵育3h使聚集肽充分聚集，之后将悬浊液4℃，16,000g的条件下离心30min分离。裂解细胞后的上清、加盐聚集后的上清和沉淀、切割后的上清和沉淀一起进行SDS-PAGE检测，结果如图4所示。图4A中，泳道a-d为人生长激素hGH表达与纯化样品，分别是a：细胞裂解物上清，可检测到清晰的融合蛋白条带；b：裂解物上清加盐聚集后分离的上清；c：裂解物上清加盐聚集后分离的沉淀，可检测到清晰的融合蛋白条带；d：加盐获得的聚集体切割后的沉淀；e：加盐获得的聚集体切割后的上清，可检测到清晰的人生长激素hGH条带。泳道1-6为含有牛血清蛋白BSA的蛋白定量标准品， 上样量依次为0.125μg、0.25μg、0.5μg、1.0μg、2.0μg、4.0μg。图4B中，泳道a-d为红色荧光蛋白RFP表达与纯化样品，分别是a：细胞裂解物上清，可检测到清晰的融合蛋白条带；b：裂解物上清加盐聚集后分离的上清；c：裂解物上清加盐聚集后分离的沉淀，可检测到清晰的融合蛋白条带；d：加盐获得的聚集体切割后的沉淀；e：加盐获得的聚集体切割后的上清，可检测到清晰的红色荧光蛋白RFP条带。泳道1-6为含有牛血清蛋白BSA的蛋白定量标准品，上样量依次为0.125μg、0.25μg、0.5μg、1.0μg、2.0μg、4.0μg。
Add Na 2 SO 4 to 0.7 M to the lysed supernatant obtained in Example 2, and place it at 4° C. overnight for 12 hours to fully aggregate the self-aggregating peptide. Afterwards, the suspension was centrifuged at 15,000 g for 30 min at 4°C, and the precipitate was washed with buffer B4 containing 0.7M Na 2 SO 4 (99.43 g of Na 2 SO 4 , 2.4 g of Tris, 0.37 g of EDTA· Dissolve 2N a in 800mL water, adjust the pH to 8.0, add water to make up to 1L) wash once, centrifuge and separate the supernatant and precipitate under the same conditions, use cleavage buffer B5 containing 0.7M Na 2 SO 4 (PBS without NaCl, Add 0.7M Na 2 SO 4 , add 40mM Bis-Tris, pH 6.2, 2mM EDTA) to fully resuspend, and place at 25°C for 24 hours to fully self-cleavage the intein. Incubate at 4°C for 3h to fully aggregate the aggregated peptide, and then centrifuge the suspension at 4°C and 16,000g for 30min to separate. The supernatant after lysing the cells, the supernatant and the precipitate after adding salt to aggregate, and the supernatant and the precipitate after cleavage were tested by SDS-PAGE together, and the results are shown in Figure 4. In Figure 4A, 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 μg, 0.25 μg, 0.5 μg, 1.0 μg, 2.0 μg, and 4.0 μg. In Figure 4B, 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 μg, 0.25 μg, 0.5 μg, 1.0 μg, 2.0 μg, and 4.0 μg.
依照蛋白定量标准品，应用ImageJ(National Institutes of Health)凝胶定量分析软件对目的条带进行光密度分析，可计算得出在内含肽介导的自切割之后释放到上清中的目的蛋白产量、加盐后的聚集效率、MtuΔI-CM切割效率、人生长激素hGH和红色荧光蛋白RFP回收率及其在上清中的纯度，结果如表4所示。According to the protein quantification standard, apply ImageJ (National Institutes of Health) gel quantitative analysis software to analyze the optical density of the target band, and calculate the target protein released into the supernatant after intein-mediated self-cleavage The results of yield, aggregation efficiency after salt addition, MtuΔI-CM cleavage efficiency, human growth hormone hGH and red fluorescent protein RFP recovery and their purity in the supernatant are shown in Table 4.
表4 0.7M Na
2SO
4介导MpA-Mtu-hGH/RFP相变和Mtu介导切割的蛋白纯化情况
Table 4 0.7M Na 2 SO 4 mediated MpA-Mtu-hGH/RFP phase transition and Mtu-mediated cleavage protein purification
a内含肽介导的自切割后的目的蛋白的产量(体积以每升LB培养基来计算)，
b聚集效率＝100％×加盐后沉淀中融合蛋白的量/(加盐后沉淀中融合蛋白的量+加盐后上清中融合蛋白的量)，
c内含肽介导的自切割效率＝100％×(加盐后沉淀中融合蛋白的量-切割后沉淀中融合蛋白的量)/加盐后沉淀中融合蛋白的量，
d回收率＝100％×目的蛋白实际产量/表达上清在完全切割的情况下可生产目的蛋白的理论产量。
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.
所采用的2种融合蛋白(MpA-Mtu-hGH、MpA-Mtu-RFP)的裂解上清在0.7M Na
2SO
4的情况下融合蛋白大部分由可溶变为沉淀(96％的MpA-Mtu-hGH转化为沉淀、62％的MpA-Mtu-RFP转化为沉淀)，内含肽MtuΔI-CM自切割，目的蛋白同MpA-Mtu分离，切割效率是61～95％，切割后释放到上清中hGH和RFP的产量分别为91mg/L和164mg/L，切割后回收的hGH和RFP纯度分别为99％和87％。
The lysed supernatants of the two fusion proteins (MpA-Mtu-hGH, MpA-Mtu-RFP) 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 MtuΔI-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.
实施例5：0.7M(NH
4)
2SO
4介导MpA-Mtu-hGH/RFP相变和Mtu介导切割的蛋白纯化
Example 5: 0.7M (NH 4 ) 2 SO 4 mediated MpA-Mtu-hGH/RFP phase transition and Mtu-mediated cleavage protein purification
向实施例2中获得的裂解上清中加入(NH
4)
2SO
4至3M置于4℃过夜12h，使得自聚集肽充分聚集。之后将悬浊液在4℃，15,000g的条件下离心30min，将沉淀用等体积含0.7M(NH
4)
2SO
4的缓冲液B6(92.50g的(NH
4)
2SO
4、2.4g的Tris、0.37g的EDTA·2N
a 溶解于800mL水中，调pH至8.0，加水定容至1L)洗涤1次后，使用体积减半的含3M NaCl的切割缓冲液B7(不含NaCl的PBS，补加0.7M(NH
4)
2SO
4，补加40mM Bis-Tris，pH6.2，2mM EDTA)充分重悬，置于25℃24h，使得内含肽充分进行自切割。4℃孵育3h使聚集肽充分聚集，之后将悬浊液4℃，16,000g的条件下离心30min分离。裂解细胞后的上清、加盐聚集后的上清和沉淀、切割后的上清和沉淀一起进行SDS-PAGE检测，结果如图5所示。图5A中，泳道a-d为人生长激素hGH表达与纯化样品，分别是a：细胞裂解物上清，可检测到清晰的融合蛋白条带；b：裂解物上清加盐聚集后分离的上清；c：裂解物上清加盐聚集后分离的沉淀，可检测到清晰的融合蛋白条带；d：加盐获得的聚集体切割后的沉淀；e：加盐获得的聚集体切割后的上清，可检测到清晰的人生长激素hGH条带。泳道1-6为含有牛血清蛋白BSA的蛋白定量标准品，上样量依次为0.125μg、0.25μg、0.5μg、1.0μg、2.0μg、4.0μg。图5B中，泳道a-d为红色荧光蛋白RFP表达与纯化样品，分别是a：细胞裂解物上清，可检测到清晰的融合蛋白条带；b：裂解物上清加盐聚集后分离的上清；c：裂解物上清加盐聚集后分离的沉淀，可检测到清晰的融合蛋白条带；d：加盐获得的聚集体切割后的沉淀；e：加盐获得的聚集体切割后的上清，可检测到清晰的红色荧光蛋白RFP条带。泳道1-6为含有牛血清蛋白BSA的蛋白定量标准品，上样量依次为0.125μg、0.25μg、0.5μg、1.0μg、2.0μg、4.0μg。
Add (NH 4 ) 2 SO 4 to 3 M to the lysed supernatant obtained in Example 2 and place it at 4° C. overnight for 12 hours, so that the self-aggregating peptides are fully aggregated. Afterwards, the suspension was centrifuged at 15,000 g for 30 min at 4°C, and the pellet was washed with an equal volume of buffer B6 containing 0.7M (NH 4 ) 2 SO 4 (92.50 g of (NH 4 ) 2 SO 4 , 2.4 g Dissolve 0.37g of Tris and 0.37g of EDTA·2N a in 800mL of water, adjust the pH to 8.0, add water to make up to 1L) after washing once, use cleavage buffer B7 containing 3M NaCl (NaCl-free PBS , supplemented with 0.7M (NH 4 ) 2 SO 4 , supplemented with 40mM Bis-Tris, pH 6.2, 2mM EDTA) to fully resuspend, and placed at 25°C for 24 hours, so that the intein can be fully self-cleaved. Incubate at 4°C for 3h to fully aggregate the aggregated peptide, and then centrifuge the suspension at 4°C and 16,000g for 30min to separate. The supernatant after lysing the cells, the supernatant and the precipitate after adding salt to aggregate, and the supernatant and the precipitate after cleavage were tested by SDS-PAGE together, and the results are shown in Figure 5. In Figure 5A, 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 μg, 0.25 μg, 0.5 μg, 1.0 μg, 2.0 μg, and 4.0 μg. In Figure 5B, 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 μg, 0.25 μg, 0.5 μg, 1.0 μg, 2.0 μg, and 4.0 μg.
依照蛋白定量标准品，应用ImageJ(National Institutes of Health)凝胶定量分析软件对目的条带进行光密度分析，可计算得出在内含肽介导的自切割之后释放到上清中的目的蛋白产量、加盐后的聚集效率、MtuΔI-CM切割效率、人生长激素hGH和红色荧光蛋白RFP回收率及其在上清中的纯度，结果如表5所示。According to the protein quantification standard, apply ImageJ (National Institutes of Health) gel quantitative analysis software to analyze the optical density of the target band, and calculate the target protein released into the supernatant after intein-mediated self-cleavage The results of yield, aggregation efficiency after salt addition, MtuΔI-CM cleavage efficiency, human growth hormone hGH and red fluorescent protein RFP recovery and their purity in the supernatant are shown in Table 5.
表5 0.7M(NH
4)
2SO
4介导MpA-Mtu-hGH/RFP相变和Mtu介导切割的蛋白纯化
Table 5 0.7M(NH 4 ) 2 SO 4 mediated MpA-Mtu-hGH/RFP phase transition and Mtu-mediated cleavage protein purification
a内含肽介导的自切割后的目的蛋白的产量(体积以每升LB培养基来计算)，
b聚集效率＝100％×加盐后沉淀中融合蛋白的量/(加盐后沉淀中融合蛋白的量+加盐后上清中融合蛋白的量)，
c内含肽介导的自切割效率＝100％×(加盐后沉淀中融合蛋白的量-切割后沉淀中融合蛋白的量)/加盐后沉淀中融合蛋白的量，
d回收率＝100％×目的蛋白实际产量/表达上清在完全切割的情况下可生产目的蛋白的理论产量。
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.
所采用的2种融合蛋白(MpA-Mtu-hGH、MpA-Mtu-RFP)的裂解上清在0.7M(NH
4)
2SO
4的情况下融合蛋白由可溶变为沉淀(93％的MpA-Mtu-hGH转化为沉淀、50％的MpA-Mtu-RFP转化为沉淀)，内含肽MtuΔI-CM自切割，目的蛋白同MpA-Mtu分离， 切割效率是72～98％，切割后释放到上清中hGH和RFP的产量分别为115mg/L和87mg/L，切割后回收的hGH和RFP纯度分别为93％和94％。
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 MtuΔI-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.
实施例6：3M NaCl介导CpA-Mtu-hGH/RFP相变和Mtu介导切割的蛋白纯化Example 6: 3M NaCl-mediated CpA-Mtu-hGH/RFP phase transition and protein purification of Mtu-mediated cleavage
向实施例2中获得的裂解上清中加入NaCl至3M置于4℃过夜12h，使得自聚集肽充分聚集。之后将悬浊液在4℃，15,000g的条件下离心30min，将离心后的沉淀用等体积含3M NaCl的缓冲液B2(175.32g的NaCl、2.4g的Tris、0.37g的EDTA·2N
a溶解于800mL水中，调pH至8.0，加水定容至1L)洗涤1次后等条件离心分离上清和沉淀，使用体积减半的含3M NaCl的切割缓冲液B3(PBS补加NaCl至3M，补加40mM Bis-Tris，pH 6.2，2mM EDTA)充分重悬沉淀，置于25℃24h，使得内含肽充分自切割。4℃孵育3h使聚集肽充分聚集，之后将悬浊液4℃，16,000g的条件下离心30min分离。裂解细胞后的上清、加盐聚集后的上清和沉淀、切割后的上清和沉淀一起进行SDS-PAGE检测，结果如图6所示。图6A中，泳道a-d为人生长激素hGH表达与纯化样品，分别是a：细胞裂解物上清，可检测到清晰的融合蛋白条带；b：裂解物上清加盐聚集后分离的上清；c：裂解物上清加盐聚集后分离的沉淀，可检测到清晰的融合蛋白条带；d：加盐获得的聚集体切割后的沉淀；e：加盐获得的聚集体切割后的上清，可检测到清晰的人生长激素hGH条带。泳道1-5为含有牛血清蛋白BSA的蛋白定量标准品，上样量依次为0.25μg、0.5μg、1.0μg、2.0μg、4.0μg。图6B中，泳道a-d为红色荧光蛋白RFP表达与纯化样品，分别是a：细胞裂解物上清，可检测到清晰的融合蛋白条带；b：裂解物上清加盐聚集后分离的上清；c：裂解物上清加盐聚集后分离的沉淀，可检测到清晰的融合蛋白条带；d：加盐获得的聚集体切割后的沉淀；e：加盐获得的聚集体切割后的上清，可检测到清晰的红色荧光蛋白RFP条带。泳道1-5为含有牛血清蛋白BSA的蛋白定量标准品，上样量依次为0.25μg、0.5μg、1.0μg、2.0μg、4.0μg。
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. Afterwards, 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-cleaved. Incubate at 4°C for 3h to fully aggregate the aggregated peptide, and then centrifuge the suspension at 4°C and 16,000g for 30min to separate. The supernatant after lysing the cells, the supernatant and the precipitate after adding salt to aggregate, and the supernatant and the precipitate after cleavage were tested by SDS-PAGE together, and the results are shown in Figure 6. In Figure 6A, 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 μg, 0.5 μg, 1.0 μg, 2.0 μg, and 4.0 μg. In Figure 6B, 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 μg, 0.5 μg, 1.0 μg, 2.0 μg, and 4.0 μg.
依照蛋白定量标准品，应用ImageJ(National Institutes of Health)凝胶定量分析软件对目的条带进行光密度分析，可计算得出在内含肽介导的自切割之后释放到上清中的目的蛋白产量、加盐后的聚集效率、MtuΔI-CM切割效率、人生长激素hGH和红色荧光蛋白RFP回收率及其在上清中的纯度，结果如表6所示。According to the protein quantification standard, apply ImageJ (National Institutes of Health) gel quantitative analysis software to analyze the optical density of the target band, and calculate the target protein released into the supernatant after intein-mediated self-cleavage The results of yield, aggregation efficiency after salt addition, MtuΔI-CM cleavage efficiency, human growth hormone hGH and red fluorescent protein RFP recovery and their purity in the supernatant are shown in Table 6.
表6 3M NaCl介导CpA-Mtu-hGH/RFP相变和Mtu介导切割的蛋白纯化情况Table 6 3M NaCl-mediated CpA-Mtu-hGH/RFP phase transition and Mtu-mediated cleavage protein purification
a内含肽介导的自切割后的目的蛋白的产量(体积以每升LB培养基来计算)，
b聚集效率＝100％×加盐后沉淀中融合蛋白的量/(加盐后沉淀中融合蛋白的量+加盐后上清中融合蛋白的量)，
c内含肽介导的自切割效率＝100％×(加盐后沉淀中融合蛋白的量-切割后沉淀中融合蛋白的量)/加盐后沉淀中融合 蛋白的量，
d回收率＝100％×目的蛋白实际产量/表达上清在完全切割的情况下可生产目的蛋白的理论产量。
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.
所采用的2种融合蛋白(CpA-Mtu-hGH、CpA-Mtu-RFP)的裂解上清在3M NaCl的情况下融合蛋白由可溶变为沉淀(89％的CpA-Mtu-hGH转化为沉淀、25％的CpA-Mtu-RFP转化为沉淀)，内含肽MtuΔI-CM自切割，目的蛋白同CpA-Mtu分离，切割效率是52～80％，切割后释放到上清中hGH和RFP的产量分别为60mg/L和21mg/L，切割后回收的hGH和RFP纯度分别为73％和74％。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 MtuΔI-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.
实施例7：0.7M Na
2SO
4介导CpA-Mtu-hGH/RFP相变和Mtu介导切割的蛋白纯化
Example 7: 0.7M Na 2 SO 4 mediated CpA-Mtu-hGH/RFP phase transition and Mtu-mediated cleavage protein purification
向实施例2中获得的裂解上清中加入Na
2SO
4至0.7M，置于4℃过夜12h，使得自聚集肽充分聚集。之后将悬浊液在4℃，15,000g的条件下离心30min，将沉淀用含0.7M Na
2SO
4的缓冲液B4(99.43g的Na
2SO
4、2.4g的Tris、0.37g的EDTA·2N
a溶解于800mL水中，调pH至8.0，加水定容至1L)洗涤1次后等条件离心分离上清和沉淀，使用含0.7M Na
2SO
4的切割缓冲液B5(不含NaCl的PBS，补加0.7M Na
2SO
4，补加40mM Bis-Tris，pH6.2，2mM EDTA)充分重悬，置于25℃24h，使得内含肽充分自切割。4℃孵育3h使聚集肽充分聚集，之后将悬浊液4℃，16,000g的条件下离心30min分离。裂解细胞后的上清、加盐聚集后的上清和沉淀、切割后的上清和沉淀一起进行SDS-PAGE检测，结果如图7所示。图7A中，泳道a-d为人生长激素hGH表达与纯化样品，分别是a：细胞裂解物上清，可检测到清晰的融合蛋白条带；b：裂解物上清加盐聚集后分离的上清；c：裂解物上清加盐聚集后分离的沉淀，可检测到清晰的融合蛋白条带；d：加盐获得的聚集体切割后的沉淀；e：加盐获得的聚集体切割后的上清，可检测到清晰的人生长激素hGH条带。泳道1-5为含有牛血清蛋白BSA的蛋白定量标准品，上样量依次为0.25μg、0.5μg、1.0μg、2.0μg、4.0μg。图7B中，泳道a-d为红色荧光蛋白RFP表达与纯化样品，分别是a：细胞裂解物上清，可检测到清晰的融合蛋白条带；b：裂解物上清加盐聚集后分离的上清；c：裂解物上清加盐聚集后分离的沉淀，可检测到清晰的融合蛋白条带；d：加盐获得的聚集体切割后的沉淀；e：加盐获得的聚集体切割后的上清，可检测到清晰的红色荧光蛋白RFP条带。泳道1-5为含有牛血清蛋白BSA的蛋白定量标准品，上样量依次为0.25μg、0.5μg、1.0μg、2.0μg、4.0μg。
Add Na 2 SO 4 to 0.7 M to the lysed supernatant obtained in Example 2, and place it at 4° C. overnight for 12 hours to fully aggregate the self-aggregating peptide. Afterwards, the suspension was centrifuged at 15,000 g for 30 min at 4°C, and the precipitate was washed with buffer B4 containing 0.7M Na 2 SO 4 (99.43 g of Na 2 SO 4 , 2.4 g of Tris, 0.37 g of EDTA· Dissolve 2N a in 800mL water, adjust the pH to 8.0, add water to make up to 1L) wash once, centrifuge and separate the supernatant and precipitate under the same conditions, use cleavage buffer B5 containing 0.7M Na 2 SO 4 (PBS without NaCl, Add 0.7M Na 2 SO 4 , add 40mM Bis-Tris, pH 6.2, 2mM EDTA) to fully resuspend, and place at 25°C for 24 hours to fully self-cleavage the intein. Incubate at 4°C for 3h to fully aggregate the aggregated peptide, and then centrifuge the suspension at 4°C and 16,000g for 30min to separate. The supernatant after lysing the cells, the supernatant and the precipitate after adding salt to aggregate, and the supernatant and the precipitate after cleavage were tested by SDS-PAGE together, and the results are shown in Figure 7. In Figure 7A, 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 μg, 0.5 μg, 1.0 μg, 2.0 μg, and 4.0 μg. In Figure 7B, 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 μg, 0.5 μg, 1.0 μg, 2.0 μg, and 4.0 μg.
依照蛋白定量标准品，应用ImageJ(National Institutes of Health)凝胶定量分析软件对目的条带进行光密度分析，可计算得出在内含肽介导的自切割之后释放到上清中的目的蛋白产量、加盐后的聚集效率、MtuΔI-CM切割效率、人生长激素hGH和红色荧光蛋白RFP回收率及其在上清中的纯度，结果如表7所示。According to the protein quantification standard, apply ImageJ (National Institutes of Health) gel quantitative analysis software to analyze the optical density of the target band, and calculate the target protein released into the supernatant after intein-mediated self-cleavage The results of yield, aggregation efficiency after salt addition, MtuΔI-CM cleavage efficiency, human growth hormone hGH and red fluorescent protein RFP recovery and their purity in the supernatant are shown in Table 7.
表7 0.7M Na
2SO
4介导CpA-Mtu-hGH/RFP相变和Mtu介导切割的蛋白纯化情况
Table 7 0.7M Na 2 SO 4 mediated CpA-Mtu-hGH/RFP phase transition and Mtu-mediated cleavage protein purification
a内含肽介导的自切割后的目的蛋白的产量(体积以每升LB培养基来计算)，
b聚集效率＝100％×加盐后沉淀中融合蛋白的量/(加盐后沉淀中融合蛋白的量+加盐后上清中融合蛋白的量)，
c内含肽介导的自切割效率＝100％×(加盐后沉淀中融合蛋白的量-切割后沉淀中融合蛋白的量)/加盐后沉淀中融合蛋白的量，
d回收率＝100％×目的蛋白实际产量/表达上清在完全切割的情况下可生产目的蛋白的理论产量。
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.
所采用的2种融合蛋白(CpA-Mtu-hGH、CpA-Mtu-RFP)的裂解上清在0.7M Na
2SO
4的情况下融合蛋白由可溶变为沉淀(87％的CpA-Mtu-hGH转化为沉淀、26％的CpA-Mtu-RFP转化为沉淀)，内含肽MtuΔI-CM自切割，目的蛋白同CpA-Mtu分离，切割效率是60～97％，切割后释放到上清中hGH和RFP的产量分别为75mg/L和36mg/L，切割后回收的hGH和RFP纯度分别为79％和96％。
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 MtuΔI-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.
实施例8：0.7M(NH
4)
2SO
4介导CpA-Mtu-hGH/RFP相变和Mtu介导切割的蛋白纯化
Example 8: 0.7M (NH 4 ) 2 SO 4 mediated CpA-Mtu-hGH/RFP phase transition and protein purification of Mtu-mediated cleavage
向实施例2中获得的裂解上清中加入(NH
4)
2SO
4至3M置于4℃过夜12h，使得自聚集肽充分聚集。之后将悬浊液在4℃，15,000g的条件下离心30min，将沉淀用等体积含0.7M(NH
4)
2SO
4的缓冲液B6(92.50g的(NH
4)
2SO
4、2.4g的Tris、0.37g的EDTA·2N
a溶解于800mL水中，调pH至8.0，加水定容至1L)洗涤1次后，使用体积减半的含3M NaCl的切割缓冲液B7(不含NaCl的PBS，补加0.7M(NH
4)
2SO
4，补加40mM Bis-Tris，pH6.2，2mM EDTA)充分重悬，置于25℃24h，使得内含肽充分进行自切割。4℃孵育3h使聚集肽充分聚集，之后将悬浊液4℃，16,000g的条件下离心30min分离。裂解细胞后的上清、加盐聚集后的上清和沉淀、切割后的上清和沉淀一起进行SDS-PAGE检测，结果如图8所示。图8A中，泳道a-d为人生长激素hGH表达与纯化样品，分别是a：细胞裂解物上清，可检测到清晰的融合蛋白条带；b：裂解物上清加盐聚集后分离的上清；c：裂解物上清加盐聚集后分离的沉淀，可检测到清晰的融合蛋白条带；d：加盐获得的聚集体切割后的沉淀；e：加盐获得的聚集体切割后的上清，可检测到清晰的人生长激素hGH条带。泳道1-5为含有牛血清蛋白BSA的蛋白定量标准品，上样量依次为0.25μg、0.5μg、1.0μg、2.0μg、4.0μg。图8B中，泳道a-d为红色荧光蛋白RFP表达与纯化样品，分别是a：细胞裂解物上清，可检测到清晰的融合蛋白条带；b：裂解物上清加盐聚集后分离的上清；c：裂解物上清加盐聚集后分离的沉淀，可检测到清晰的融合蛋白条带；d：加盐获得的聚集体切割后的沉淀；e：加盐获得的聚集体切割后的上清，可检测到清晰的红色荧光蛋白RFP条带。泳道1-5为含有牛血清蛋白BSA的蛋白定量标准品，上样量依次为0.25μg、0.5μg、1.0μg、2.0μg、4.0μg。
Add (NH 4 ) 2 SO 4 to 3 M to the lysed supernatant obtained in Example 2 and place it at 4° C. overnight for 12 hours, so that the self-aggregating peptides are fully aggregated. Afterwards, the suspension was centrifuged at 15,000 g for 30 min at 4°C, and the pellet was washed with an equal volume of buffer B6 containing 0.7M (NH 4 ) 2 SO 4 (92.50 g of (NH 4 ) 2 SO 4 , 2.4 g Dissolve 0.37g of Tris and 0.37g of EDTA·2N a in 800mL of water, adjust the pH to 8.0, add water to make up to 1L) after washing once, use cleavage buffer B7 containing 3M NaCl (NaCl-free PBS , supplemented with 0.7M (NH 4 ) 2 SO 4 , supplemented with 40mM Bis-Tris, pH 6.2, 2mM EDTA) to fully resuspend, and placed at 25°C for 24 hours, so that the intein can be fully self-cleaved. Incubate at 4°C for 3h to fully aggregate the aggregated peptide, and then centrifuge the suspension at 4°C and 16,000g for 30min to separate. The supernatant after lysing the cells, the supernatant and the precipitate after adding salt to aggregate, and the supernatant and the precipitate after cleavage were tested by SDS-PAGE together, and the results are shown in Figure 8. In Figure 8A, 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 μg, 0.5 μg, 1.0 μg, 2.0 μg, and 4.0 μg. In Figure 8B, 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 μg, 0.5 μg, 1.0 μg, 2.0 μg, and 4.0 μg.
依照蛋白定量标准品，应用ImageJ(National Institutes of Health)凝胶定量分析软件对目的条带进行光密度分析，可计算得出在内含肽介导的自切割之后释放到上清中的目的蛋白产量、加盐后的聚集效率、MtuΔI-CM切割效率、人生长激素hGH和红色荧光 蛋白RFP回收率及其在上清中的纯度，结果如表8所示。According to the protein quantification standard, apply ImageJ (National Institutes of Health) gel quantitative analysis software to analyze the optical density of the target band, and calculate the target protein released into the supernatant after intein-mediated self-cleavage The yield, aggregation efficiency after adding salt, cutting efficiency of MtuΔI-CM, recovery rate of human growth hormone hGH and red fluorescent protein RFP and their purity in the supernatant are shown in Table 8.
表8 0.7M(NH
4)
2SO
4介导CpA-Mtu-hGH/RFP相变和Mtu介导切割的蛋白纯化
Table 8 0.7M(NH 4 ) 2 SO 4 mediated CpA-Mtu-hGH/RFP phase transition and Mtu-mediated cleavage protein purification
a内含肽介导的自切割后的目的蛋白的产量(体积以每升LB培养基来计算)，
b聚集效率＝100％×加盐后沉淀中融合蛋白的量/(加盐后沉淀中融合蛋白的量+加盐后上清中融合蛋白的量)，
c内含肽介导的自切割效率＝100％×(加盐后沉淀中融合蛋白的量-切割后沉淀中融合蛋白的量)/加盐后沉淀中融合蛋白的量，
d回收率＝100％×目的蛋白实际产量/表达上清在完全切割的情况下可生产目的蛋白的理论产量。
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.
所采用的2种融合蛋白(CpA-Mtu-hGH、CpA-Mtu-RFP)的裂解上清在0.7M(NH
4)
2SO
4的情况下融合蛋白由可溶变为沉淀(74％的CpA-Mtu-hGH转化为沉淀、17％的CpA-Mtu-RFP转化为沉淀)，内含肽MtuΔI-CM自切割，目的蛋白同CpA-Mtu分离，切割效率是76～99％，切割后释放到上清中hGH和RFP的产量分别为97mg/L和18mg/L，切割后回收的hGH和RFP纯度分别为57％和95％。
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 MtuΔI-CM is self-cleaved, and the target protein is separated from CpA-Mtu with a cleavage efficiency of 76-99%. After cleavage, it is released into The yields of hGH and RFP in the supernatant were 97 mg/L and 18 mg/L, respectively, and the purity of hGH and RFP recovered after cleavage were 57% and 95%, respectively.
实施例9：MpA/CpA-Mtu-RFP聚集体和切割上清中RFP的活性表征Example 9: Characterization of RFP activity in MpA/CpA-Mtu-RFP aggregates and cleavage supernatants
分别对实施例3～8中MpA/CpA-Mtu-RFP的裂解上清加盐孵育过夜12h后形成的聚集体和MpA/CpA-Mtu-RFP的切割上清进行拍照，自然光下RFP呈现红色和365nm紫外光下RFP显红色荧光来鉴定聚集体MpA/CpA-Mtu-RFP和切割上清RFP的活性，结果如图9所示。图9A中从左到右依次为3M NaCl、0.7M Na
2SO
4和0.7M(NH
4)
2SO
4三种盐加入到MpA-Mtu-RFP裂解上清孵育12h后形成的聚集体，在自然光下三种聚集体都显红色，说明MpA-Mtu-RFP加盐后形成的聚集体都是具有活性的。图9B中从左到右依次为3M NaCl、0.7M Na
2SO
4和0.7M(NH
4)
2SO
4三种盐对应的MpA-Mtu-RFP切割上清，在自然光下三种盐对应的切割上清都显红色，图9C中在365nm紫外光下切割上清都显红色荧光，说明三种盐条件下MpA-Mtu-RFP切割上清中的RFP都是具有活性的。图9D中从左到右依次为3M NaCl、0.7M Na
2SO
4和0.7M(NH
4)
2SO
4三种盐加入到CpA-Mtu-RFP裂解上清孵育12h后形成的聚集体，在自然光下三种聚集体都显红色，说明CpA-Mtu-RFP加盐后形成的聚集体都是具有活性的。图9E中从左到右依次为3M NaCl、0.7M Na
2SO
4和0.7M(NH
4)
2SO
4三种盐对应的CpA-Mtu-RFP切割上清，在自然光下三种盐对应的切割上清都显红色，图9F中在365nm紫外光下切割上清都显红色荧光，说明三种盐条件下CpA-Mtu-RFP切割上清中的RFP都是具有活性的。
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 . In Figure 9A, from left to right, three salts of 3M NaCl, 0.7M Na 2 SO 4 and 0.7M (NH 4 ) 2 SO 4 were added to the MpA-Mtu-RFP lysed supernatant and incubated for 12h to form aggregates. The three aggregates all appeared red under natural light, indicating that the aggregates formed after adding salt to MpA-Mtu-RFP were all active. In Fig. 9B, from left to right, the cut supernatants of MpA-Mtu-RFP corresponding to three salts of 3M NaCl, 0.7M Na 2 SO 4 and 0.7M (NH 4 ) 2 SO 4 , and the supernatants corresponding to the three salts under natural light The cutting supernatants were all red, and in Figure 9C under 365nm ultraviolet light, the cutting supernatants all showed red fluorescence, indicating that the RFP in the cutting supernatants of MpA-Mtu-RFP were all active under the three salt conditions. In Figure 9D, from left to right, three salts of 3M NaCl, 0.7M Na 2 SO 4 and 0.7M (NH 4 ) 2 SO 4 were added to the CpA-Mtu-RFP lysed supernatant and incubated for 12 hours to form aggregates. Under natural light, the three aggregates all appeared red, indicating that the aggregates formed after adding salt to CpA-Mtu-RFP were all active. In Fig. 9E, from left to right, the cut supernatants of CpA-Mtu-RFP corresponding to the three salts of 3M NaCl, 0.7M Na 2 SO 4 and 0.7M (NH 4 ) 2 SO 4 , and the supernatants corresponding to the three salts under natural light The cutting supernatants were all red, and in Figure 9F under 365nm ultraviolet light, the cutting supernatants all showed red fluorescence, indicating that the RFP in the cutting supernatant of CpA-Mtu-RFP was active under the three salt conditions.
实施例10：3M NaCl、0.7M Na
2SO
4和0.7M(NH
4)
2SO
4介导 MpA-Mtu-GST/LCB3/ΔNSpyCatcher-ELP-ΔNSpyCatcher相变和Mtu介导切割的蛋白纯化
Example 10: 3M NaCl, 0.7M Na 2 SO 4 and 0.7M (NH 4 ) 2 SO 4 mediated MpA-Mtu-GST/LCB3/ΔNSpyCatcher-ELP-ΔNSpyCatcher phase transition and protein purification of Mtu-mediated cleavage
为了证明MpA-Mtu-POI方法的普适性，我们使用该方法对另外三种不同类型的目的蛋白进行纯化，谷胱甘肽巯基转移酶GST、新冠多肽LCB3(Cao,L.等Science,2020.370(6515):p.426-431.)和多价骨架蛋白ΔNSpyCatcher-ELP-ΔNSpyCatcher。首先将实施1中已介绍的三种表达质粒pET30a-MpA-Mtu-GST、pET30a-MpA-Mtu-LCB3和pET30a-MpA-Mtu-ΔNSpyCatcher-ELP-ΔNSpyCatcher导入到表达菌株E.coli BL21(DE3)中，采用与实施例2相同的方法获得了上述三种融合蛋白对应表达菌株的裂解上清。向裂解上清中分别加入NaCl至3M、Na
2SO
4至3M和(NH
4)
2SO
4至0.7M，置于4℃诱导聚集30分钟。之后将悬浊液在4℃，15,000g的条件下离心30min，将离心后的沉淀用等体积含相同盐的缓冲液B2、B4或B6洗涤1次后等条件离心分离上清和沉淀，使用体积减半含相同盐的切割缓冲液B3、B5或B7充分重悬沉淀，置于25℃24h，使得内含肽充分自切割。然后将悬浊液4℃，16,000g的条件下离心30min分离。裂解细胞后的上清、加盐聚集后的上清和沉淀、切割后的上清和沉淀一起进行SDS-PAGE检测，结果如图10所示。图10中，泳道a-f为三种目的蛋白的表达与纯化样品，分别是a：细胞裂解物上清，可检测到清晰的融合蛋白条带；b：裂解物上清加盐聚集后分离的上清；c：裂解物上清加盐聚集后分离的沉淀，可检测到清晰的融合蛋白条带；d：加盐获得的聚集体切割后的沉淀；e和f：加盐获得的聚集体切割后的上清，可检测到清晰的目的蛋白条带，其中e泳道是a-d泳道上样量的2倍，f泳道是a-d泳道上样量的10倍。泳道1-5为含有牛血清蛋白BSA的蛋白定量标准品，上样量依次为0.125μg、0.25μg、0.5μg、1.0μg、2.0μg，其中图10B、图10E和图10H中，泳道1-5为含有牛血清蛋白BSA和异肽酶APR的蛋白定量标准品，BSA和APR各自在泳道中的上样量依次为0.125μg、0.25μg、0.5μg、1.0μg、2.0μg。泳道M1和M2为蛋白分子量标准品。
In order to prove the universality of the MpA-Mtu-POI method, we used this method to purify three other different types of target proteins, glutathione sulfhydryl transferase GST, new crown peptide LCB3 (Cao, L. et al. Science, 2020.370 (6515): p.426-431.) and the multivalent scaffold protein ΔNSpyCatcher-ELP-ΔNSpyCatcher. First, introduce the three expression plasmids pET30a-MpA-Mtu-GST, pET30a-MpA-Mtu-LCB3 and pET30a-MpA-Mtu-ΔNSpyCatcher-ELP-ΔNSpyCatcher introduced in Implementation 1 into the expression strain E.coli BL21(DE3) In the same method as in Example 2, the lysed supernatants of the expression strains corresponding to the above three fusion proteins were obtained. 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. 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. The supernatant after lysing the cells, the supernatant and the precipitate after adding salt to aggregate, and the supernatant and the precipitate after cleavage were tested by SDS-PAGE together, and the results are shown in Figure 10. In Figure 10, 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 the ad lane. Swimming lanes 1-5 are protein quantification standards containing bovine serum albumin BSA, and the loading amounts are 0.125 μg, 0.25 μg, 0.5 μg, 1.0 μg, and 2.0 μg. 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 μg, 0.25 μg, 0.5 μg, 1.0 μg, and 2.0 μg. Lanes M1 and M2 are protein molecular weight standards.
依照蛋白定量标准品，应用ImageJ(National Institutes of Health)凝胶定量分析软件对目的条带进行光密度分析，可计算得出在内含肽介导的自切割之后释放到上清中的目的蛋白产量、加盐后的聚集效率、MtuΔI-CM切割效率和回收率及其在上清中的纯度，结果如表9所示。According to the protein quantification standard, apply ImageJ (National Institutes of Health) gel quantitative analysis software to analyze the optical density of the target band, and calculate the target protein released into the supernatant after intein-mediated self-cleavage The yield, aggregation efficiency after adding salt, cutting efficiency and recovery of MtuΔI-CM and its purity in the supernatant are shown in Table 9.
表9 3M NaCl、0.7M Na
2SO
4和0.7M(NH
4)
2SO
4介导MpA-Mtu-GST/LCB3/ΔNSpyCatcher-ELP-ΔNSpyCatcher相变和Mtu介导切割的蛋白纯化情况
Table 9 3M NaCl, 0.7M Na 2 SO 4 and 0.7M (NH 4 ) 2 SO 4 mediated MpA-Mtu-GST/LCB3/ΔNSpyCatcher-ELP-ΔNSpyCatcher phase transition and Mtu-mediated cleavage protein purification
a内含肽介导的自切割后的目的蛋白的产量(体积以每升LB培养基来计算)，
b聚集效率＝100％×加盐后沉淀中融合蛋白的量/(加盐后沉淀中融合蛋白的量+加盐后上清中融合蛋白的量)，
c内含肽介导的自切割效率＝100％×(加盐后沉淀中融合蛋白的量-切割后沉淀中融合蛋白的量)/加盐后沉淀中融合蛋白的量，
d回收率＝100％×目的蛋白实际产量/表达上清在完全切割的情况下可生产目的蛋白的理论产量。
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.
所采用的3种融合蛋白(pET30a-MpA-Mtu-GST、pET30a-MpA-Mtu-LCB3和pET30a-MpA-Mtu-ΔNSpyCatcher-ELP-ΔNSpyCatcher)的裂解上清在3M NaCl、0.7M Na
2SO
4和0.7M(NH
4)
2SO
4的情况下融合蛋白由可溶变为沉淀，其中3M NaCl和0.7M Na
2SO
4的诱导聚集效果(聚集效率：65％～96％)比0.7M(NH
4)
2SO
4(聚集效率：51％～93％)的更突出，内含肽MtuΔI-CM自切割，目的蛋白同MpA-Mtu分离，切割效率是68～99％。3M NaCl诱导的情况下3种目的蛋白的产量为10～125mg/L，纯度为68％～97％；0.7M Na
2SO
4诱导的情况下3种目的蛋白的产量为12～102mg/L，纯度为72％～97％；0.7M(NH
4)
2SO
4诱导的情况下3种目的蛋白的产量为2～117mg/L，纯度为33％～85％。
The lysed supernatants of the three fusion proteins (pET30a-MpA-Mtu-GST, pET30a-MpA-Mtu-LCB3 and pET30a-MpA-Mtu-ΔNSpyCatcher-ELP-ΔNSpyCatcher) 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 MtuΔI-CM is self-cleaved, the target protein is separated from MpA-Mtu, and the cleavage efficiency is 68-99%. In the case of 3M NaCl induction, 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%.
实施例11：3M NaCl、0.7M Na
2SO
4和0.7M(NH
4)
2SO
4介导Xylanase-Mxe-MpA相变和Mxe介导切割的蛋白纯化
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
为了测试内含肽Mxe GyrA能否应用于该纯化方法，我们将实施1中已介绍的表达质粒pET30a-Xylanase-Mxe-MpA导入到表达菌株E.coli BL21(DE3)中，采用实施例2中类似的表达方法，将Xylanase-Mxe-MpA表达菌株接种到含50μg/mL卡那霉素的TB液体培养基中，并在37℃摇床中培养至对数期(OD
600＝0.4-0.6)，加入0.2mM IPTG，在18℃诱导24小时，测量菌浓度OD
600，4℃4,000rpm离心25min收获细胞，除去上清的培养基将菌体冻存-80℃，采用与实施例2相同的方法获得了融合蛋白Xylanase-Mxe-MpA对应表达菌株的裂解上清。向裂解上清中分别加入NaCl至3M、Na
2SO
4至3M和(NH
4)
2SO
4至0.7M，置于4℃诱导聚集30分钟。之后将悬浊液在4℃，15,000g的条件下离心30min，将离心后的沉淀用等体积含相同盐的缓冲液B2、B4或B6洗涤1次后等条件离心分离上清和沉淀，使用体积减半含相同盐的切割缓冲液 B8(175.32g的NaCl、2.4g的Tris、0.37g的EDTA·2Na、6.17g二硫苏糖醇DTT溶解于800mL水中，调pH至8.0，加水定容至1L)、B9(99.428g的Na
2SO
4、2.4g的Tris、0.37g的EDTA·2Na、6.17g二硫苏糖醇DTT溶解于800mL水中，调pH至8.0，加水定容至1L)或B10(92.498g的(NH
4)
2SO
4、2.4g的Tris、0.37g的EDTA·2Na、6.17g二硫苏糖醇DTT溶解于800mL水中，调pH至8.0，加水定容至1L)充分重悬沉淀，置于25℃24h，使得内含肽充分自切割。然后将悬浊液4℃，16,000g的条件下离心30min分离。裂解细胞后的上清、加盐聚集后的上清和沉淀、切割后的上清和沉淀一起进行SDS-PAGE检测，结果如图11所示。图11中，泳道a-f为三种目的蛋白的表达与纯化样品，分别是a：细胞裂解物上清，可检测到清晰的融合蛋白条带；b：裂解物上清加盐聚集后分离的上清；c：裂解物上清加盐聚集后分离的沉淀，可检测到清晰的融合蛋白条带；d：加盐获得的聚集体切割后的沉淀；e和f：加盐获得的聚集体切割后的上清，可检测到清晰的目的蛋白条带，其中e泳道是a-d泳道上样量的2倍，f泳道是a-d泳道上样量的10倍。泳道1-5为含有牛血清蛋白BSA的蛋白定量标准品，上样量依次为0.125μg、0.25μg、0.5μg、1.0μg、2.0μg，泳道M1和M2为蛋白分子量标准品。
In order to test whether the intein Mxe GyrA can be applied to this purification method, we introduced the expression plasmid pET30a-Xylanase-Mxe-MpA introduced in Implementation 1 into the expression strain E.coli BL21(DE3), using the method described in Example 2 In a similar expression method, the Xylanase-Mxe-MpA expression strain was inoculated into TB liquid medium containing 50 μg/mL kanamycin, and cultured in a shaker at 37°C to logarithmic phase (OD 600 =0.4-0.6) , add 0.2mM IPTG, induce at 18°C for 24 hours, measure the bacterial concentration OD 600 , centrifuge at 4,000rpm at 4°C for 25min to harvest the cells, remove the supernatant medium and freeze the cells at -80°C, using the same method as in Example 2 Methods The lysed supernatant of the strain expressing the fusion protein Xylanase-Mxe-MpA was obtained. 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. Cut in half the cleavage buffer B8 containing the same salt (175.32g of NaCl, 2.4g of Tris, 0.37g of EDTA 2Na, 6.17g of dithiothreitol DTT dissolved in 800mL of water, adjust the pH to 8.0, add water to 1L), B9 (dissolve 99.428g of Na 2 SO 4 , 2.4g of Tris, 0.37g of EDTA·2Na, 6.17g of dithiothreitol DTT in 800mL of water, adjust the pH to 8.0, add water to make up to 1L) or B10 (dissolve 92.498g of (NH 4 ) 2 SO 4 , 2.4g of Tris, 0.37g of EDTA·2Na, 6.17g of dithiothreitol DTT in 800mL of water, adjust the pH to 8.0, add water to make up to 1L) fully The pellet was resuspended and placed at 25°C for 24 hours to allow the intein to fully self-cleavage. Then the suspension was centrifuged at 16,000 g for 30 min at 4°C. The supernatant after lysing the cells, the supernatant and the precipitate after adding salt to aggregate, and the supernatant and the precipitate after cleavage were tested by SDS-PAGE together, and the results are shown in Figure 11. In Figure 11, 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 μg, 0.25 μg, 0.5 μg, 1.0 μg, and 2.0 μg in sequence. Swimming lanes M1 and M2 are protein molecular weight standards.
依照蛋白定量标准品，应用ImageJ(National Institutes of Health)凝胶定量分析软件对目的条带进行光密度分析，可计算得出在内含肽介导的自切割之后释放到上清中的目的蛋白产量、加盐后的聚集效率、Mxe GyrA切割效率和回收率及其在上清中的纯度，结果如表10所示。According to the protein quantification standard, apply ImageJ (National Institutes of Health) gel quantitative analysis software to analyze the optical density of the target band, and calculate the target protein released into the supernatant after intein-mediated self-cleavage Yield, aggregation efficiency after adding salt, Mxe GyrA cutting efficiency and recovery rate and its purity in the supernatant, the results are shown in Table 10.
表10 3M NaCl、0.7M Na
2SO
4和0.7M(NH
4)
2SO
4介导Xylanase-Mxe-MpA相变和Mxe介导切割的蛋白纯化情况
Table 10 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 protein purification
a内含肽介导的自切割后的目的蛋白的产量(体积以每升TB培养基来计算)，
b聚集效率＝100％×加盐后沉淀中融合蛋白的量/(加盐后沉淀中融合蛋白的量+加盐后上清中融合蛋白的量)，
c内含肽介导的自切割效率＝100％×(加盐后沉淀中融合蛋白的量-切割后沉淀中融合蛋白的量)/加盐后沉淀中融合蛋白的量，
d回收率＝100％×目的蛋白实际产量/表达上清在完全切割的情况下可生产目的蛋白的理论产量。
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.
所采用的Mxe内含肽融合蛋白(Xylanase-Mxe-MpA)的裂解上清在3M NaCl、0.7 M Na
2SO
4和0.7M(NH
4)
2SO
4的情况下融合蛋白由可溶变为沉淀，其中0.7M Na
2SO
4的聚集效率最高可达69％，3M NaCl和0.7M Na
2SO
4的聚集效率分别为41％和37％，内含肽Mxe GyrA自切割，目的蛋白同Mxe-MpA分离，切割效率是63～85％。3M NaCl诱导的情况下目的蛋白Xylanase的产量为35mg/L，纯度为49％；0.7M Na
2SO
4诱导的情况下目的蛋白Xylanase的产量为103mg/L，纯度为84％；0.7M(NH
4)
2SO
4诱导的情况下目的蛋白Xylanase的产量为37mg/L，纯度为54％。
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%. The output of 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%.
实施例12：通过生物膜层光学干涉(BLI)技术检测纯化后人生长激素hGH的亲和力Example 12: Detection of the affinity of purified human growth hormone hGH by biofilm layer optical interference (BLI) technology
首先采用标准的色谱柱纯化法对MpA-Mtu-hGH的切割上清进行进一步纯化，根据hGH的等电点(pI＝5.27)选择阴离子交换柱Capto Q(GE Healthcare,USA),通过透析或超滤的方法将切割上清的切割缓冲液更换为用于离子交换的起始缓冲液(2.4g的Tris，溶解于800mL水中，调pH至7.2，加水定容至1L)，然后使用0.22μm滤膜对样品进行过滤，使用
KTA
TM蛋白纯化层析系统进行上样，采用20个柱体积0～1M NaCl的线性梯度洗脱，收集离子交换纯化获得的洗脱样品。将离子交换纯化获得hGH样品分别通过SDS-PAGE和BCA Kit(Thermo Fisher,USA)进行纯度和质量浓度的测定。使用商品化的人重组生长激素hGH(金赛药业，中国)作为阳性对照，整个BLI实验使用分子相互作用仪Octet RED96(ForteBio)进行检测，按照Amine Reactive 2nd Generation(AR2G)生物传感器(ForteBio,CA)说明书将20μg/ml的人生长激素受体蛋白hGH receptor(Abcam,UK)在10mM醋酸钠(pH 4.0)的溶液中固定到AR2G传感器上，然后使用1M乙醇胺溶液封闭传感器，接着在动力学缓冲液(含0.1％牛血清白蛋白BSA和0.02％吐温-20的PBS,pH 7.4)中平衡已经固定了生长激素受体蛋白的传感器，再然后将平衡后的传感器与待测的hGH样品(浓度梯度为25nM、50nM、100nM、200nM和400nM的hGH)在动力学缓冲液中亲和600秒，然后再将亲和后的传感器在动力学缓冲液中解离600秒，结合和解离的动力学结果如图12所示。按照Octet动力学操作手册进行结合解离常数K
D、结合的动力学常数Kon和解离的动力学常数Koff的计算，结果如表11所示。
First, the cut supernatant of MpA-Mtu-hGH was further purified by standard chromatographic column purification method, and the anion exchange column Capto Q (GE Healthcare, USA) was selected according to the isoelectric point (pI=5.27) of hGH, and was purified by dialysis or ultrafiltration. 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 0.22μm 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. Commercialized human recombinant growth hormone hGH (Kinsey Pharmaceuticals, China) was used as a positive control, and the entire BLI experiment was detected using a molecular interaction instrument Octet RED96 (ForteBio), according to the Amine Reactive 2nd Generation (AR2G) biosensor (ForteBio, CA) Instructions 20 μg/ml human growth hormone receptor protein hGH receptor (Abcam, UK) was immobilized on the AR2G sensor in a solution of 10 mM sodium acetate (pH 4.0), and then the sensor was blocked with 1M ethanolamine solution, followed by kinetic Equilibrate the sensor with immobilized growth hormone receptor protein in the buffer (PBS containing 0.1% bovine serum albumin BSA and 0.02% Tween-20, pH 7.4), and then mix the equilibrated sensor with the hGH sample to be tested (concentration gradient is 25nM, 50nM, 100nM, 200nM and 400nM hGH) affinity in the kinetic buffer for 600 seconds, and then dissociate the sensor after affinity in the kinetic buffer for 600 seconds, the binding and dissociation The kinetic results are shown in Figure 12. The binding and dissociation constant K D , the binding kinetic constant Kon and the dissociation kinetic constant Koff were calculated according to the Octet kinetic operation manual, and the results are shown in Table 11.
表11 3M NaCl、0.7M Na
2SO
4和0.7M(NH
4)
2SO
4介导纯化的人生长激素hGH与人生长激素受体蛋白hGH receptor的亲和力测试结果
Table 11 3M NaCl, 0.7M Na 2 SO 4 and 0.7M (NH 4 ) 2 SO 4 mediated affinity test results of purified human growth hormone hGH and human growth hormone receptor protein hGH receptor
*商品化的冻干粉直接使用
* Commercialized freeze-dried powder is used directly
采用该方法初纯化的人生长激素hGH经过离子交换精纯化后都能与人生长激素受体蛋白hGH receptor发生结合和解离且与商品化的人生长激素hGH效果相近。3M NaCl、0.7M Na
2SO
4和0.7M(NH
4)
2SO
4三种盐介导纯化获得的hGH和阳性对照(商品化的hGH)相比较，结合解离常数K
D值都小于10
-12，结合的动力学常数Kon(10
5M
-1s
-1)值在1.82～3.53之间，解离的动力学常数Koff(s
-1)值都小于10
-7
。
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, and the Koff (s -1 ) value of dissociation is all less than 10 -7 .
实施例13：通过生物膜层光学干涉(BLI)技术检测纯化后新冠多肽LCB3的亲和力Example 13: Detecting the affinity of the purified new crown polypeptide LCB3 by biofilm layer optical interference (BLI) technology
首先采用标准的色谱柱纯化法对MpA-Mtu-LCB3的切割上清进行进一步纯化，根据LCB3的等电点(pI＝4.94)选择阴离子交换柱Capto Q(GE Healthcare,USA),采用实施例12中同样的方法对LCB3进行精纯化、纯度和质量浓度的测定。整个BLI实验使用分子相互作用仪Octet RED96(ForteBio)进行检测，按照Amine Reactive 2nd Generation(AR2G)生物传感器(ForteBio,CA)说明书将20μg/ml的LCB3的受体蛋白即新冠病毒刺突蛋白SARS-CoV-2 Spike protein(金斯瑞，中国)在10mM醋酸钠(pH 6.0)的溶液中固定到AR2G传感器上，然后使用1M乙醇胺溶液封闭传感器，接着在动力学缓冲液(含0.1％牛血清白蛋白BSA和0.02％吐温-20的PBS,pH 7.4)中平衡已经固定了新冠病毒刺突蛋白的传感器，再然后将平衡后的传感器与待测的LCB3样品(浓度梯度为12.5nM、25nM、50nM、100nM和200nM的LCB3)在动力学缓冲液中亲和600秒，然后再将亲和后的传感器在动力学缓冲液中解离600秒，结合和解离的动力学结果如图13所示。按照Octet动力学操作手册进行结合解离常数K
D、结合的动力学常数Kon和解离的动力学常数Koff的计算，结果如表12所示。
First, the cut supernatant of MpA-Mtu-LCB3 was further purified by standard chromatographic column purification method, and an anion exchange column Capto Q (GE Healthcare, USA) was selected according to the isoelectric point (pI=4.94) of LCB3, using Example 12 In the same method, 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 μg/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). protein BSA and 0.02% Tween-20 PBS, pH 7.4) to balance the sensor that has fixed the spike protein of the new coronavirus, and then mix the balanced sensor with the LCB3 sample to be tested (concentration gradient is 12.5nM, 25nM, 50nM, 100nM and 200nM LCB3) were affinity in the kinetic buffer for 600 seconds, and then dissociated the affinity sensor in the kinetic buffer for 600 seconds, the kinetic results of binding and dissociation are shown in Figure 13 . The binding and dissociation constant K D , the binding kinetic constant Kon and the dissociation kinetic constant Koff were calculated according to the Octet Kinetics Manual, and the results are shown in Table 12.
表12 3M NaCl、0.7M Na
2SO
4和0.7M(NH
4)
2SO
4介导纯化的新冠多肽LCB3与新冠病毒刺突蛋白SARS-CoV-2 Spike protein的亲和力测试结果
Table 12 Affinity test results of 3M NaCl, 0.7M Na 2 SO 4 and 0.7M (NH 4 ) 2 SO 4 mediated purification of novel coronavirus polypeptide LCB3 and SARS-CoV-2 Spike protein
采用该方法初纯化的新冠多肽LCB3经过离子交换精纯化后都能与新冠病毒刺突蛋白SARS-CoV-2 Spike protein发生结合和解离，结合解离常数K
D与文献(Cao,L.等Science,2020.370(6515):p.426-431.)报道的小于10
-9相近。
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.
实施例14：Xylanase-Mxe-MpA切割上清活性验证Example 14: Xylanase-Mxe-MpA cleavage supernatant activity verification
采用DNS法(Miller,G.L.等Analytical Chemistry,1959.31(3):p.426-428.)对Xylanase-Mxe-MpA切割上清中的木聚糖酶xylanase进行酶活测定。选择木聚糖(Sigma， USA)作为底物，木糖(阿拉丁，中国)作为标准品用于制作还原糖含量的标准曲线。木聚糖酶的催化反应在含0.5％(W/V)木聚糖的50mM磷酸缓冲液(pH 7.0)中进行，反应条件55℃15min，以每分钟水解底物生成1μmoL还原糖所需的酶量定义为一个酶活性单位(IU)。将三种盐3M NaCl、0.7M Na
2SO
4和0.7M(NH
4)
2SO
4介导纯化得到的切割上清分别稀释到适当的浓度，然后进行酶活测定，结果如表13所示。
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) was selected as a substrate, and xylose (Aladdin, China) was used as a standard to prepare a standard curve for reducing sugar content. 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 μmoL 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 .
| 待测样品Sample to be tested | 初纯化所用的盐Salt used for initial purification | 酶活(units/mg)Enzyme activity (units/mg) |
| Purified xylanasePurified xylanase |
3M NaCl |
3030 |
| Purified xylanasePurified xylanase | 0.7M Na 2SO 4 0.7M Na2SO4 | 1616 |
| Purified xylanasePurified xylanase | 0.7M(NH 4) 2SO 4 0.7M(NH 4 ) 2 SO 4 | 1212 |
3M NaCl、0.7M Na
2SO
4和0.7M(NH
4)
2SO
4介导纯化的木聚糖酶xylanase通过DNS法测定的酶活大于商业的木聚糖酶(Sigma,253-439-7)的酶活(≥2.5units/mg)。
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).
实施例15：MpA-Mtu-GST切割上清活性验证Example 15: MpA-Mtu-GST cutting supernatant activity verification
采用谷胱甘肽巯基转移酶活性检测试剂盒(生工生物，D799612)的方法对MpA-Mtu-GST切割上清中的谷胱甘肽巯基转移酶GST进行酶活测定，选择氨基酸序列同样来源于日本血吸虫的GST重组蛋白(义翘神州，11213-HNAE)作为阳性对照。按照试剂盒说明书的方法测定的酶活结果如表14所示。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.
| 待测样品Sample to be tested | 初纯化所用的盐Salt used for initial purification | 酶活(units/mg)Enzyme activity (units/mg) |
|
Purified GST |
3M NaCl3M NaCl | 6.16.1 |
| Purified GSTPurified GST | 0.7M Na 2SO 4 0.7M Na2SO4 | 5.45.4 |
| Purified GSTPurified GST | 0.7M(NH 4) 2SO 4 0.7M(NH 4 ) 2 SO 4 | 5.75.7 |
| GST standardGST standard | -- | 6.86.8 |
3M NaCl、0.7M Na
2SO
4和0.7M(NH
4)
2SO
4介导纯化的谷胱甘肽巯基转移酶GST酶活(5.4～6.1units/mg)与GST标准品的酶活(6.8units/mg)相近。
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.
实施例16：MpA-Mtu-ΔNSpyCatcher-ELP-ΔNSpyCatcher切割上清活性验证Example 16: MpA-Mtu-ΔNSpyCatcher-ELP-ΔNSpyCatcher cutting supernatant activity verification
基于Spy化学(Zakeri,B.等Proceedings of the National Academy of Sciences,2012.109(12):p.E690-E697.)，ΔNSpyCatcher(Liu,Z.等Sci Rep,2014.4:p.7266.)能够与SpyTag自发形成异肽键，采用SDS-PAGE法鉴定共价结合产物的形成，从而验证切割上清中骨架蛋白ΔNSpyCatcher-ELP-ΔNSpyCatcher的活性。将三种盐3M NaCl、0.7M Na
2SO
4和0.7M(NH
4)
2SO
4介导纯化的MpA-Mtu-ΔNSpyCatcher-ELP-ΔNSpyCatcher切割上清和已纯化的LCB3-SpyTag分别用PBS稀释到20μM和120μM，然后将三种切割上清分别等体积与LCB3-SpyTag混合，25℃孵育2小时，将反应前后的样品通过SDS-PAGE鉴定。SDS-PAGE结果如图14所示，泳道1为反应前的LBS3-SpyTag，泳道2、4和6为反应前三种盐介导纯化对应的切割上清，泳道3、5和7为反应后的样品。在泳道3、5和7中能够明显观察到骨架蛋白ΔNSpyCatcher-ELP-ΔNSpyCatcher与LCB3-SpyTag形成的二价结合产物(ΔNSpyC-ELP-ΔNSpyC:2 LCB3-SpyTag,44.6kDa)， 且ΔNSpyC-ELP-ΔNSpyC对应条带都基本消失，说明三种盐介导纯化的骨架蛋白ΔNSpyC-ELP-ΔNSpyC都几乎完全参与反应，即三种盐3M NaCl、0.7M Na
2SO
4和0.7M(NH
4)
2SO
4介导纯化的ΔNSpyC-ELP-ΔNSpyC均具有活性。
Based on Spy chemistry (Zakeri, B. et al. Proceedings of the National Academy of Sciences, 2012.109 (12): p.E690-E697.), ΔNSpyCatcher (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 ΔNSpyCatcher-ELP-ΔNSpyCatcher 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-ΔNSpyCatcher-ELP-ΔNSpyCatcher cutting supernatant and purified LCB3-SpyTag were diluted with PBS to 20 μM and 120 μM, 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, and lanes 3, 5 and 7 are after the reaction sample. In lanes 3, 5 and 7, the bivalent binding product (ΔNSpyC-ELP-ΔNSpyC:2 LCB3-SpyTag, 44.6kDa) formed by the backbone protein ΔNSpyCatcher-ELP-ΔNSpyCatcher and LCB3-SpyTag can be clearly observed, and ΔNSpyC-ELP- The bands corresponding to ΔNSpyC basically disappeared, indicating that the three salt-mediated purification of the backbone protein ΔNSpyC-ELP-ΔNSpyC 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 ΔNSpyC-ELP-ΔNSpyC were all active.
序列表sequence listing
参考文献references
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Claims (29)
- 一种融合多肽，其包含目的多肽部分和盐浓度响应性自聚集肽部分，其中所述目的多肽部分通过间隔物连接于所述盐浓度响应性自聚集肽部分，并且其中所述间隔物包含切割位点，A fusion polypeptide comprising a polypeptide part of interest and a salt concentration-responsive self-aggregation peptide part, wherein the polypeptide part of interest is linked to the salt concentration-responsive self-aggregation peptide part through a spacer, and wherein the spacer comprises a cleavage site,其中所述盐浓度响应性自聚集肽为CpA变体，其中所述CpA具有如SEQ ID NO:1所示的氨基酸序列，所述CpA变体在对应于SEQ ID NO:1的第1位和第17位的位置包含C1M以及C17M的氨基酸取代。Wherein 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.
- 权利要求1的融合多肽，其中所述CpA变体的氨基酸序列如SEQ ID NO:2所示。The fusion polypeptide of claim 1, wherein the amino acid sequence of the CpA variant is as shown in SEQ ID NO:2.
- 权利要求1的融合多肽，其中所述盐浓度响应性自聚集肽为在第一盐条件下可溶且在第二盐条件下能够自聚集的肽。The fusion polypeptide of claim 1, wherein 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.
- 权利要求3的融合多肽，其中所述第一盐条件包含第一盐浓度，所述第二盐条件包含第二盐浓度，所述第一盐浓度低于所述第二盐浓度。The fusion polypeptide of claim 3, wherein said first salt condition comprises a first salt concentration, said second salt condition comprises a second salt concentration, said first salt concentration being lower than said second salt concentration.
- 权利要求1的融合多肽，其中所述间隔物与所述目的多肽部分和/或所述盐浓度响应性自聚集肽部分直接连接。The fusion polypeptide of claim 1, wherein said spacer is directly linked to said polypeptide portion of interest and/or said salt concentration responsive self-aggregating peptide portion.
- 权利要求1的融合多肽，其中所述间隔物在其N端和/或C端进一步包含接头。The fusion polypeptide of claim 1, wherein the spacer further comprises a linker at its N-terminus and/or C-terminus.
- 权利要求6的融合多肽，其中所述接头选自GS型接头和PT型接头。The fusion polypeptide of claim 6, wherein said linker is selected from a GS-type linker and a PT-type linker.
- 权利要求1的融合多肽，其中所述目的多肽部分位于所述融合多肽的C端，且所述间隔物连接于所述目的多肽部分的N端。The fusion polypeptide of claim 1, wherein the polypeptide portion of interest is located at the C-terminus of the fusion polypeptide, and the spacer is connected to the N-terminus of the polypeptide portion of interest.
- 权利要求8的融合多肽，其中所述间隔物通过所述切割位点连接于所述目的多肽部分。8. The fusion polypeptide of claim 8, wherein said spacer is attached to said polypeptide portion of interest through said cleavage site.
- 权利要求1的融合多肽，其中所述切割位点选自温度依赖性切割位点、pH依赖性切割位点、离子依赖性切割位点、酶切割位点或自切割位点。The fusion polypeptide of claim 1, 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.
- 权利要求1的融合多肽，其中所述间隔物包含内含肽，所述内含肽包含自切割位点。The fusion polypeptide of claim 1, wherein said spacer comprises an intein comprising a self-cleavage site.
- 权利要求11的融合多肽，其中所述内含肽选自Mxe GyrA、Ssp DnaB或MtuΔI-CM。The fusion polypeptide of claim 11, wherein said intein is selected from Mxe GyrA, Ssp DnaB or MtuΔI-CM.
- 权利要求11的融合多肽，其中所述内含肽为MtuΔI-CM，其包含SEQ ID NO:3所示的序列。The fusion polypeptide of claim 11, wherein said intein is MtuΔI-CM, which comprises the sequence shown in SEQ ID NO:3.
- 权利要求1的融合多肽，其中所述目的多肽长度为20、50、70、100、150、200、250、300、350、400、450或500个氨基酸残基，或为任意两个上述长度之间的任意长度。The fusion polypeptide of claim 1, wherein the length of the target polypeptide is 20, 50, 70, 100, 150, 200, 250, 300, 350, 400, 450 or 500 amino acid residues, or any two of the above-mentioned lengths any length in between.
- 一种分离的多核苷酸，其包含编码权利要求1-14中任一项的融合多肽的核苷酸序列或其互补序列。An isolated polynucleotide comprising a nucleotide sequence encoding the fusion polypeptide of any one of claims 1-14, or its complement.
- 一种分离的多核苷酸，其包含编码CpA变体的核苷酸序列或其互补序列，其中所述CpA具有如SEQ ID NO:1所示的氨基酸序列，所述CpA变体在对应于SEQ ID NO:1 的第1位和第17位的位置处包含C1M和C17M的氨基酸取代。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, and the CpA variant is corresponding to SEQ ID NO: 1 The positions of positions 1 and 17 of ID NO: 1 comprise amino acid substitutions of C1M and C17M.
- 表达构建体，其包含权利要求15的多核苷酸。An expression construct comprising the polynucleotide of claim 15.
- 宿主细胞，其包含权利要求15的多核苷酸或由权利要求17的表达构建体转化，其中所述宿主细胞能够表达所述融合多肽。A host cell comprising the polynucleotide of claim 15 or transformed by the expression construct of claim 17, wherein said host cell is capable of expressing said fusion polypeptide.
- 权利要求18的宿主细胞，其选自原核生物、酵母和高等真核细胞。The host cell of claim 18 selected from the group consisting of prokaryotes, yeast and higher eukaryotic cells.
- 权利要求19的宿主细胞，其中所述原核生物包括埃希氏菌属(Escherichia)、芽孢杆菌属(Bacillus)、沙门氏菌属(Salmonella)以及假单胞菌属(Pseudomonas)和链霉菌属(Streptomyces)的细菌。The host cell of claim 19, wherein said prokaryotes include Escherichia, Bacillus, Salmonella, and Pseudomonas and Streptomyces bacteria.
- 权利要求19的宿主细胞，其中所述原核生物是埃希氏菌属，优选大肠杆菌(E.coli)。19. The host cell of claim 19, wherein said prokaryote is Escherichia, preferably Escherichia coli (E. coli).
- 生产和纯化目的多肽的方法，所述方法包括以下步骤：A method for producing and purifying a polypeptide of interest, said method comprising the steps of:(a)培养权利要求18-21中任一项的宿主细胞，从而表达融合多肽；(a) cultivating the host cell of any one of claims 18-21, thereby expressing the fusion polypeptide;(b)在第一盐条件下，裂解所述宿主细胞，然后去除细胞裂解物的不溶部分，回收可溶部分；(b) under a first salt condition, lysing the host cell, then removing the insoluble portion of the cell lysate, and recovering the soluble portion;(c)在第二盐条件下，所述融合蛋白形成不溶部分；(c) under a second salt condition, the fusion protein forms an insoluble fraction;(d)回收步骤(c)中形成的不溶部分；(d) recovering the insoluble fraction formed in step (c);(e)通过切割所述切割位点从收集自步骤(d)的不溶部分释放可溶的目的多肽；和(e) releasing soluble polypeptide of interest from the insoluble fraction collected from step (d) by cleaving said cleavage site; and(f)去除步骤(e)中的不溶部分，回收含有所述目的多肽的可溶部分。(f) removing the insoluble portion in step (e), and recovering the soluble portion containing the target polypeptide.
- 权利要求22的方法，其中所述第一盐条件包含第一离子强度，所述第二盐条件包含第二离子强度，所述第一离子强度低于所述第二离子强度。22. The method of claim 22, wherein said first salt condition comprises a first ionic strength and said second salt condition comprises a second ionic strength, said first ionic strength being lower than said second ionic strength.
- 权利要求22的方法，其中所述步骤(c)包括调节含有收集自步骤(b)的可溶部分的溶液的盐浓度。22. The method of claim 22, wherein said step (c) comprises adjusting the salt concentration of the solution containing the soluble fraction collected from step (b).
- 权利要求22的方法，其中所述步骤(c)包括升高含有收集自步骤(b)的可溶部分的溶液的盐浓度。22. The method of claim 22, wherein said step (c) comprises increasing the salt concentration of the solution containing the soluble fraction collected from step (b).
- 权利要求22的方法，其中步骤(e)在第二盐条件下进行。The method of claim 22, wherein step (e) is performed under second salt conditions.
- 权利要求22的方法，其中所述第一盐条件下和/或第二盐条件下的所述盐选自氯化钠、硫酸钠、硝酸钠、磷酸氢二钠、磷酸二氢钠、碳酸钠、氯化钾、硫酸钾、硝酸钾、磷酸氢二钾、磷酸二氢钾、碳酸钾、硝酸铵、硫酸铵或氯化铵，优选为氯化钠、硫酸钠或硫酸铵。The method of claim 22, wherein said salt under said first salt condition and/or said salt under 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 hydrogen phosphate, potassium dihydrogen phosphate, potassium carbonate, ammonium nitrate, ammonium sulfate or ammonium chloride, preferably sodium chloride, sodium sulfate or ammonium sulfate.
- 权利要求22的方法，其中所述第一离子强度为0-0.2mol/L。The method of claim 22, wherein said first ionic strength is 0-0.2 mol/L.
- 权利要求22的方法，其中所述第二离子强度为0.5-5mol/L。The method of claim 22, wherein said second ionic strength is 0.5-5 mol/L.
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