WO2020073553A1 - 筛选蛋白酶变体的方法以及获得的蛋白酶变体 - Google Patents

筛选蛋白酶变体的方法以及获得的蛋白酶变体 Download PDF

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WO2020073553A1
WO2020073553A1 PCT/CN2019/071417 CN2019071417W WO2020073553A1 WO 2020073553 A1 WO2020073553 A1 WO 2020073553A1 CN 2019071417 W CN2019071417 W CN 2019071417W WO 2020073553 A1 WO2020073553 A1 WO 2020073553A1
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protease
variant
seq
tev
protease variant
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刘日魁
邹晓龙
万江华
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上饶市康可得生物科技有限公司
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Priority claimed from CN201811540344.6A external-priority patent/CN111019925B/zh
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Priority to EP19870712.7A priority Critical patent/EP3851524A4/en
Priority to JP2021520425A priority patent/JP7386552B2/ja
Priority to US17/277,204 priority patent/US20220025355A1/en
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    • C07K14/665—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans derived from pro-opiomelanocortin, pro-enkephalin or pro-dynorphin
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    • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
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Definitions

  • the present invention relates to the field of proteins, and in particular to methods for screening TEV protease variants and TEV protease variants.
  • TEV Protease is a 27 kDa active domain derived from Tobacco Etch Virus (TEV) Nla protease. Its amino acid sequence is shown in SEQ ID NO.1. TEV protease has strong site specificity and can recognize the EXXYXQ (G / S) seven amino acid sequence, the most commonly used sequence is Glu-Asn-Leu-Tyr-Phe-Gln-Gly (or ENLYFQG), its cleavage site Between glutamine Gln (P1) and glycine Gly (P1 ') (ie between P1 and P1'), the sequence specificity is much higher than proteases such as thrombin, factor Xa, enterokinase and so on.
  • proteases such as thrombin, factor Xa, enterokinase and so on.
  • TEV protease can tolerate a wide range of pH (pH 4-8.5) and temperature (4-34 °C), for some common additives that increase protein solubility or stability (ethylene glycol, EGTA, detergent and reducing agent) There are also different degrees of tolerance. Studies have shown that TEV protease is insensitive to ethylene glycol, EGTA and some detergents (Triton X-100, Tween-20 and NP-40). When 1% CHAPS is present, the activity is reduced, and the denaturant (2M Urea, 1% SDS) and reducing agent (0.7M ⁇ -mercaptoethanol) can still maintain most of the activity (C. Sun et al., 2012).
  • Wild-type TEV enzymes have certain defects in expression and solubility, so there are a lot of reports on mutants improved by genetic engineering technology.
  • the natural TEV protease will undergo self-cleavage. During expression and purification, it will constantly undergo conformational changes due to collisions with other TEV proteases, and self-cleavage will occur at specific sites, thus making the complete protease truncated and active. Greatly reduced.
  • the stability of the S219N mutant found by Lucast et al. Has been greatly improved, but the solubility is not high. About 95% of the protein is in the form of inclusion bodies in the precipitate. Kapust et al.
  • TEV protease Used genetic engineering to mutate the gene sequence of the natural TEV protease to obtain S219V, a mutant with higher stability and slightly improved enzyme activity, whose stability is about 100 times higher than that of S219N.
  • the expression yield of TEV protease is not high, and the solubility is also very low.
  • Approximately 5% of TEV protease is present in the supernatant of cell disruption, and the yield is 12.5 mg / L.
  • Van den Berg and others discovered a mutant TEVSH that can increase the yield to 54mg / L through genetic shuffling and error-prone PCR. Its solubility is greatly improved than that of S219N, and the enzyme The activity does not change much.
  • TEV protease single-point mutants Five mutants were screened out. Their solubility and enzyme activity were improved relative to wild-type TEV protease, and one was also obtained. Compared with single mutant, the solubility and enzyme activity of double mutant are significantly improved. In response to the shortcomings of TEV protease itself, researchers have been looking for improved mutants. For example, the TEV Ser135Gly mutant is more stable than WT and can tolerate higher temperatures (> 40 ° C); there are some mutations (T17S, N68D, N177V) that can significantly increase the solubility of TEV protease.
  • TEV protease mainly focus on the properties of improving TEV protease enzyme activity, solubility and yield.
  • the screening method of TEV protease as in this application has not been reported.
  • the present invention is based at least in part on the inventor's discovery in the production process of polypeptide drugs: the current conventional TEV protease will cleave a large amount of fusion protein (self-digestion) during the expression process, resulting in the early release of the polypeptide and easy hydrolysis by intracellular proteases. Conducive to purification, not suitable for industrial production; conventional TEV protease can still maintain most of its activity in low-density denaturants (2M urea, 1% SDS) (C. Sun et al., 2012), but there are many proteins in 2M urea It is often impossible to completely dissolve under denaturing conditions at low concentration, which limits the use of TEV protease. In this regard, the inventors have provided the following new screening methods to find TEV protease variants suitable for solving this problem.
  • the present invention provides a method for screening protease variants, which includes the following steps:
  • the protease cleavage activity is weak under the host or similar conditions and has protease cleavage activity under in vitro denaturation conditions Protease variants;
  • protease is preferably TEV protease or enterokinase.
  • the method further includes generating the mutation library using a random mutation kit.
  • the library is constructed using a suitable protease template, such as a TEV protease template, for example, S219V TEV protease with the amino acid sequence shown in SEQ ID NO. 10 as a template to construct the library .
  • a suitable protease template such as a TEV protease template, for example, S219V TEV protease with the amino acid sequence shown in SEQ ID NO. 10 as a template to construct the library .
  • the protease variant has lower cleavage activity in the host or similar conditions compared to the original protease and retains the cleavage activity of the original protease under denaturing conditions in vitro.
  • the TEV protease variant has a lower enzyme cleavage activity in the host or similar conditions than the S219V protease having the amino acid sequence shown in SEQ ID NO. 10 and is retained under denaturing conditions in vitro The digestion activity of the S219V protease with the amino acid sequence shown in SEQ ID NO.10.
  • the protease variant is displayed as a fusion protein comprising a protease-protease cleavage sequence-Y1, such as TEVp-sTEV-Y1, preferably a TEV protease variant, preferably a protease variant on the surface of a phage, preferably a TEV protease variant
  • TEVp is a TEV protease
  • sTEV is a TEV protease cleavage sequence
  • Y1 is a protein of interest; preferably wherein the TEV protease cleavage sequence is EXXYXQG / S / H, where X is any amino acid residue, preferably the The TEV protease cleavage sequence is selected from SEQ ID NO: 7 and 8.
  • the method comprises adding part A to the N-terminus of the protease variant, preferably the TEV protease variant, directly or indirectly through a linker in the host.
  • Part A is one of the following: affinity tag, biotin, antibody, antigen, hapten, streptavidin, ligand, receptor, nucleic acid, enzyme, substrate, and aptamer.
  • the method includes incubating a phage library with A added with a solid support immobilized with Part B specific for Part A under in vitro conditions suitable for the protease, and isolating the support from The solution containing the bacteriophage of interest is optionally repeatedly screened one or more times.
  • part B is one of the following: biotin, antibody, antigen, hapten, polystreptavidin, ligand, receptor, nucleic acid, enzyme, substrate, and aptamer, provided that part B Specific binding to part A.
  • the protease preferably TEV protease
  • the protease is Avi-TEVp-sTEV-Y1-PIII in the Avi-protease-protease cleavage site-Y1-PIII, preferably via the pHEN1-Avi-TEVp-sTEV-Y1 construct.
  • the Avi tag is a short peptide tag consisting of 15 amino acid residues GLNDIFEAQKIEWHE; preferably wherein the TEV protease cleavage sequence is EXXYXQG / S / H, where X is any amino acid residue, preferably The TEV protease cleavage sequence is selected from SEQ ID NO: 7 and 8.
  • the cleavage activity is for a fusion protein comprising the protease and its cleavage sequence, such as protease-protease cleavage sequence-Y1, preferably the TEV protease variant and its cleavage sequence, preferably TEVp-sTEV- Measured by Y1.
  • protease-protease cleavage sequence-Y1 preferably the TEV protease variant and its cleavage sequence, preferably TEVp-sTEV- Measured by Y1.
  • the screening comprises biotinylating the phage library of random mutations of the protease, preferably TEV protease, in the host.
  • the screening includes treating the biotinylated phage library with streptavidin magnetic beads, placing the phage captured by the magnetic beads in medium to high concentration urea for a period of time, and separating the magnetic beads from the phage containing the target Solution, optionally repeatedly screening the target bacteriophage one or more times,
  • the library is verified by colony PCR.
  • the primers used in the colony PCR are: upstream primer: 5'-CCACCATGGCCGGTCTGAATGATATTTTTGAAGC-3 '
  • Downstream primer 5'-TTGTTCTGCGGCCGCAAATTCCAGC-3 '.
  • the library capacity of 1x10 9 or more, preferably 2x10 9; overall mutation frequency is preferably in a low level of 1.5-7 / kb.
  • Y1 is human or porcine ACTH or GLP-1.
  • the host is E. coli.
  • the in vitro denaturation conditions are medium to high denaturation conditions, preferably 3M-5M urea, preferably 3.5M-4.5M urea, more preferably 4M urea or 1M-2M guanidine hydrochloride, preferably 1.5M guanidine hydrochloride.
  • the invention provides protease variants obtained by the method of the invention.
  • the protease variant has low cleavage activity during expression in the host, and it is preferred that the TEV protease variant is more compared to the S219V variant having the amino acid sequence shown in SEQ ID NO.
  • the cleavage sequence of the TEV protease variant is selected from EXXYXQG / S / H, where X is any amino acid residue, preferably the TEV protease cleavage sequence is selected from SEQ ID NO: 7 and 8; preferably
  • the protease variants or TEV protease variants under moderate to high denaturation conditions are preferably 3M-5M urea, preferably 3.5M-4.5M urea, more preferably 4M urea or 1M-2M guanidine hydrochloride, preferably 1.5M guanidine hydrochloride
  • the initial protease such as the in vitro cleavage activity of the S219V protease, is retained in an in vitro environment; preferably, the cleavage activity is to include the protease variant and its cleavage sequence, such as protease-protease cleavage sequence-Y1, preferably including the TEV
  • the TEV protease variant comprises one or more mutations selected from the group:
  • the TEV protease variant comprises a combination of mutations selected from:
  • the mutation from leucine (L) to phenylalanine (F) at the position corresponding to the 111th position of the sequence shown in SEQ ID NO: 1 corresponds to the 138th position of the sequence shown in SEQ ID NO: 1 Mutation at the position from isoleucine (I) to lysine (K);
  • the TEV protease variant comprises a mutation from serine (S) to valine (V) at a position corresponding to position 219 of the sequence shown in SEQ ID NO: 1; preferably, the TEV protease further comprises One or more mutations other than the above mutations, provided that the TEV protease variant has low enzymatic activity during expression in the host, preferably with the S219V variant having the amino acid sequence shown in SEQ ID NO.
  • guanidine hydrochloride has high digestion activity in an in vitro environment, and preferably retains the in vitro digestion activity of the S219V variant.
  • the homologue comprises an amino acid sequence having at least 90%, preferably at least 95%, more preferably at least 98%, most preferably at least 99% sequence identity with SEQ ID NO: 4, 5 or 6; preferably , Wherein the homologues contain at least 1, preferably at least 2, more preferably at least 3, and most preferably at least 4 amino acid positions substituted, deleted or added amino acids with SEQ ID NO: 4, 5 or 6 Sequence; preferably wherein the homologue has low enzymatic activity during expression in the host, preferably lower enzymatic activity and / or lower S219V variant with the amino acid sequence shown in SEQ ID NO.10 Or the TEV protease variants under medium to high denaturation conditions, preferably in 3M-5M urea, preferably 3.5M-4.5M urea, more preferably 4M urea or 1M-2M guanidine hydrochloride, preferably 1.5M guanidine hydrochloride in vitro environment The in vitro digestion activity of the S219V variant is retained.
  • the present invention provides fusion proteins.
  • the fusion protein comprises a protease variant according to the invention; preferably the fusion protein comprises a structural protease-protease cleavage sequence-Y1, preferably TEVp-sTEV-Y1, where Y1 is the polypeptide of interest; TEVp is the TEV protease according to the invention Variant; sTEV is a TEV protease cleavage sequence, which is EXXYXQG / S / H, where X is any amino acid residue, preferably the cleavage sequence is selected from SEQ ID NO: 7 and 8; preferably wherein the polypeptide of interest is selected from ACTH, GLP-1 / GLP-2, IFN- ⁇ , IFN- ⁇ , Histatin, CCL5, SDF-1 ⁇ , IGF-1, Leptin, BNP, Ex-4, preferably ACTH, preferably human ACTH, more preferably SEQ ID NO Human ACTH of the amino acid sequence shown in .2; preferably
  • the present invention provides polynucleotide sequences of protease variants or fusion proteins, preferably selected from SEQ ID NO. 14-16.
  • the present invention provides a polynucleotide construct comprising the polynucleotide sequence of the present invention.
  • the invention provides an expression vector comprising the polynucleotide sequence according to the invention or the polynucleotide construct of the invention.
  • the expression vector is a eukaryotic expression vector or a prokaryotic expression vector; preferably selected from the group consisting of pRS314, pYES2, baculovirus-S2 expression system and pcDNA3.1 or preferably selected from the group consisting of pET series Expression vector, pQE series expression vector and pBAD series expression vector.
  • the present invention provides a cell comprising a polynucleotide sequence according to the present invention, a polynucleotide construct according to the present invention or an expression vector according to the present invention; preferably a eukaryotic cell or a prokaryotic cell; preferably the eukaryotic cell is selected From the group below: Saccharomyces cerevisiae, insect cell expression system; preferably the prokaryotic cells are selected from the group consisting of BL21, BL21 (DE3), BL21 (DE3) pLysS, Rosetta2, Rosetta2pLysS, Tuner (DE3), or Origami2.
  • the invention provides a method for preparing a protease variant according to the invention, preferably a TEV protease variant, which comprises:
  • the present invention provides the use of a protease variant according to the invention, preferably a TEV protease variant, for the preparation of a polypeptide of interest, wherein the protease variant, preferably the TEV protease variant and the polypeptide of interest are in the form of a fusion protein Expression, preferably, the fusion protein is the fusion protein of the present invention.
  • the present invention provides a method for preparing a polypeptide of interest, which includes:
  • protease variants preferably TEV protease
  • buffer such as Tris-HCl, preferably 50 mM Tris-HCl
  • the inventors obtained new TEV protease variants 4D, 12D and 32C, indicating that using the method of the present invention can obtain TEV protease cleavage activity in the host is weak and in vitro denaturation conditions TEV protease enzyme Active TEV protease variant.
  • TEV protease recognition sequence EXXYXQH is used for screening, a TEV protease mutant that efficiently cleaves ENLYFQ ⁇ H can also be obtained. It is desirable for many polypeptides whose N-terminal begins with histidine.
  • TEV protease variants of the present invention can be widely used to produce various polypeptides or proteins with a natural N-terminus.
  • TEV protease variants of the present invention are obtained by screening, but the directly obtained mutants cannot meet the requirements or partially meet the requirements. Through a series of mutation point combinations, an improved mutant with unique acquisition performance can be obtained, such as L111F combined with other sites, which is characterized by no or very low activity in vivo, but higher in vitro in moderate denaturation conditions active.
  • the method of the present invention uses DNA recombination technology and a prokaryotic expression system to express the target protein, and has wide applicability.
  • the production cost of the method for preparing human or porcine ACTH by the method of the present invention can be reduced by hundreds times compared with the traditional pig brain extraction production technology, which greatly shortens the production cycle and saves Production time and cost, and no longer rely on a large number of pigs slaughtered to obtain pig pituitary gland, avoiding the risk of immunogenic allergic reactions caused by using pig-derived ACTH, greatly improving drug safety.
  • the ACTH prepared by this method is exactly the same as the ACTH secreted by the human body, with extremely high amino acid sequence accuracy and product purity, excellent biological activity, improved drug efficacy, and reduced the occurrence of adverse reactions.
  • the difficulty and cost of preparing full-length human ACTH by this method are significantly reduced, the operation is simple, no expensive catalyst and high-pressure equipment are required, the yield is high, and it is suitable for large-scale production.
  • the production process of this method is clear, simple, has good repeatability, is easy to achieve large-scale production, and reduces pollution to the environment.
  • Figure 1 Coomassie brilliant blue staining diagram of TEDS mutant 12D digestion efficiency in different concentrations of urea. The arrow indicates the target protein.
  • Figure 2 Coomassie brilliant blue staining diagram of TEDS mutant 4D digestion efficiency in different concentrations of urea. The arrow indicates the target protein.
  • Figure 3 Coomassie brilliant blue staining of SDS-PAGE gel of TEVP mutant 32C in different concentrations of urea. The arrow indicates the target protein.
  • Figure 4A Coomassie brilliant blue staining diagram of TEDS mutant 4D digestion efficiency in different concentrations of urea.
  • Figure 4B Comparison of grayscale values of electrophoretic bands.
  • Figure 5 Comparison of in vivo and in vitro digestion activities of TEVP mutants 4D, 12D and 32C with S219V.
  • the arrow indicates the target protein.
  • Figure 6 Solubility detection of fusion proteins of TEVP mutants 4D, 12D, 32C and S219V in 0.5M urea.
  • FIG. 7 Map of plasmid pET-28b-PD1-Avi.
  • Figure 8 Plasmid pET-28b-ACTH map.
  • Figure 9 Structure diagram of plasmid Avi-TEV-sTEV-ACTH gene.
  • the term "host” may be any eukaryotic or prokaryotic cell suitable for expressing a protein.
  • the host cell is an E. coli cell.
  • in vitro denaturation conditions refers to conditions that dissolve fusion proteins containing TEV protease to have enzymatic cleavage activity, such as in 3M-5M urea, preferably 3.5M-4.5M urea, more preferably 4M urea or 1M- Conditions for 2M guanidine hydrochloride, preferably 1.5M guanidine hydrochloride.
  • TEV protease is an active domain of 27 kDa in Nla protease derived from tobacco etch virus (TEV).
  • TEV protease S219V TEV protease with the amino acid sequence shown in SEQ ID NO. 10 is used as the starting TEV protease.
  • S219V TEV protease with the amino acid sequence shown in SEQ ID NO. 10 is used as the starting TEV protease.
  • various other TEV proteases can be used as the starting TEV protease in the method of the present invention.
  • TEV protease has strong site specificity and can recognize the EXXYXQ (G / S) seven amino acid sequence, the most commonly used sequence is Glu-Asn-Leu-Tyr-Phe-Gln-Gly (or ENLYFQG), its cleavage site Between glutamine Gln (P1) and glycine Gly (P1 ') (that is, between P1 and P1'), the sequence specificity is much higher than that of proteases such as thrombin, factor Xa, enterokinase and so on.
  • proteases such as thrombin, factor Xa, enterokinase and so on.
  • TEV protease variant refers to a TEV protease that has differences in amino acid residues compared to the starting TEV protease used for screening, where the variant does not imply that the TEV protease has a specific sequence and / or structure.
  • TEV protease cleavage activity refers to the cleavage activity of the TEV protease itself to cleave the fusion protein containing the TEV protease and its cleavage site.
  • the TEV protease cleavage activity of the present invention is a cleavage activity for a fusion protein containing EXXYXQ (G / S / H).
  • Phage display technology is used herein to display fusion proteins. Phage display technology is to insert the DNA sequence of the foreign protein or peptide into the appropriate position of the phage coat protein structural gene, so that the foreign gene is expressed with the expression of the coat protein, and at the same time, the foreign protein is displayed to the bacteriophage with the reassembly of the phage Surface biotechnology. Those skilled in the art will understand that the selection of random mutation libraries using other TEV proteases will allow those skilled in the art to perform the methods of the present invention.
  • the phage display system may be a single-stranded filamentous phage display system, a lambda phage display system, and a T4 phage display system.
  • a single-chain filamentous phage display system it may be a PIII display system and a PVIII display system.
  • the lambda phage display system it may be a PV display system and a D protein display system.
  • the phage display system is a PIII display system.
  • Part A and Part B refer to a pair of binding partners, for example selected from biotin, antibody, antigen, hapten, streptavidin, ligand, receptor, nucleic acid, enzyme , Substrate and aptamer, provided that "Part A” and "Part B” specifically bind to each other.
  • a solid support containing Part B is used to capture the fusion protein containing Part A.
  • the solid support may be streptavidin-coated beads or resin, such as Dynabeads M-280 streptavidin, Dynabeads MyOne streptavidin , Dynabeads M-270 streptavidin (Invitrogen), streptavidin agarose resin (Pierce), streptavidin super linked resin, MagnaBind streptavidin beads (ThermoFisher Scientific), BioMag Streptomyces Avidin, ProMag streptavidin, silica streptavidin (Bangs Laboratories), efficient streptavidin agarose (Streptavidin Sepharose High Performance) (GE Healthcare), streptavidin polyphenylene Ethylene microspheres (Microspheres-Nanospheres), streptavidin-coated polystyrene particles (Spherotech), or any other streptavidin
  • the TEV protease can be displayed at the end (N-terminal) of the bacteriophage PIII protein in the manner of Avi-TEVp-sTEV-Y1-PIII, where the Avi tag is a short peptide tag composed of 15 amino acid residues GLNDIFEAQKIEWHE.
  • Avi-TEVp-sTEV-Y1-PIII is biotinylated by the Avi tag during host expression.
  • enterokinase is a heterodimeric serine protease present in the duodenum of mammals.
  • the cleavage site of enterokinase is well-known such as DDDDK, and the nucleotide sequence and amino acid sequence of enterokinase are also available to those skilled in the art, such as GenBank: AAC50138.1, AAB40026.1, and so on.
  • TEV protease is often used to efficiently cleave fusion proteins, it is limited to cleavage when both are soluble.
  • most polypeptide drugs are poorly soluble and are usually expressed in the form of inclusion bodies, which does not match the digestion conditions of TEV protease. Inclusion bodies need to be dissolved with high concentration of urea or guanidine hydrochloride, but digestion under these conditions is extremely challenging.
  • Conventional TEV proteases and substrates are difficult to have both efficient cleavage activity and simultaneous dissolution under the same conditions.
  • Inclusion bodies are conducive to purification, and a relatively pure target protein can be obtained through simple processing.
  • inclusion body proteins require high concentration of urea or guanidine hydrochloride to dissolve.
  • Wild-type TEV protease is inactive or has very low activity under such conditions and cannot efficiently cleave fusion proteins.
  • the optimal digestion conditions are physiological conditions or non-denaturing conditions, usually with a certain degree of tolerance to low concentrations of denaturants (urea or guanidine hydrochloride), but it is extremely large
  • Most inclusion body proteins are insoluble under low-density denaturing conditions. Therefore, the conventional TEV protease is not suitable for this occasion.
  • the ideal production conditions for the fusion of TEV protease and polypeptide to prepare polypeptide drugs are: (1) the fusion protein is expressed in the form of inclusion bodies, which is conducive to purification; (2) the fusion protein does not undergo self-cleavage in the cell, otherwise it is not conducive to purification (3) After the fusion protein is dissolved in a high-density denaturant, it is diluted into the enzyme-cutting reaction system containing medium- and high-concentration denaturant, at this time, TEV protease restores its activity, efficiently cleaves itself, and releases the peptide; (4) The TEV protease variants screened from the high-capacity TEV protease library must have a broad spectrum of amino acids at the P1 'position of its cleavage site, so that it can be widely used to produce various natural N-terminal polypeptides or proteins.
  • US2010035300A1 provides a structure of a self-cleaving fusion protein that is usually prepared.
  • His tags are required to be tightly connected to the target protein to be expressed (such as EGFP).
  • the reason for this is that, when the fusion protein is expressed in vivo, the traditional wild-type TEVp or mutant TEVp has certain enzymatic cleavage activity in the cell and self-cleavage occurs.
  • the cleaved EGFP can be recovered through the His tag closely connected to it. Without this His tag, it is difficult to recover due to self-cleavage in the cell in advance, and EGFP cannot be efficiently produced. But this also brings a problem that the His tag of EGFP may be further removed according to specific needs. This is obviously more time-consuming and laborious.
  • the target protein in the fusion protein may be free of tags, such as His.
  • the fusion protein comprises the structure of Avi-TEVp-sTEV-ACTH, where Avi is an Avi tag protein and sTEV is a TEV protease cleavage site. Since TEVp variants (such as 12D variants) have very low intracellular enzymatic activity, the fusion protein that can be expressed will not substantially undergo intracellular cleavage. When collecting inclusion bodies, dissolving with 8M urea, and adjusting to the environment of 4M urea, 12D exerts its enzyme activity, and then cleaves its recognition site to sTEV.
  • TEV protease is easy to precipitate due to its poor solubility, and the biologically active peptides released are often easily dissolved in the buffer, so High-purity peptides can be obtained by centrifugation, which skillfully overcomes the need to increase the protease removal link, and greatly simplifies the later purification process.
  • inclusion bodies are more conducive to purification: 1) it is easy to harvest relatively concentrated and pure protein by centrifugation; 2) inclusion bodies protect the protein from protease hydrolysis.
  • toxic proteins are expressed in the form of inactive inclusion bodies and will not affect the growth of host bacteria.
  • the TEV-ACTH fusion protein involved in this method is expressed in the form of inclusion bodies, which greatly simplifies its purification steps, can achieve higher concentrations and purity, and will not be hydrolyzed by proteases, so it is easy to obtain stable high Yield.
  • a large-capacity TEV random mutant phage library is constructed, and its key gene structure is Avi-TEV-sTEV-Y1-PIII, which is displayed at the end of the bacteriophage PIII protein in this way.
  • Avitag is a short peptide tag composed of 15 amino acid residues (GLNDIFEAQKIEWHE), which can be linked to a biotin in the lysine residue (K) by biotin ligase in vivo or in vitro, thereby achieving protein Biotinylated, and the biotinylated protein can be specifically bound by streptavidin.
  • the TEV bacteriophage in the present invention can be biotinylated in vivo, and the biotinylated phage library can be chained Mycovidin magnetic beads are fixed.
  • TEV variants that have enzymatic cleavage activity when expressed in cells will self-digest during the expression process, resulting in the final assembled phage PIII protein end only containing the polypeptide Y1, without Avitag, and cannot be captured by magnetic beads; TEV variants that have no enzymatic cleavage activity during internal expression do not undergo self-digestion during expression.
  • the end of the final assembled phage PIII protein is Avi-TEV-sTEV-Y1, and the N-terminal Avi tag is biotinylated and can be contained Streptavidin magnetic beads capture.
  • the phages captured by the magnetic beads are incubated in medium and high concentration urea. Under these conditions, the phage that has been digested will fall off the magnetic beads due to self-cleavage and enter the solution. Collect the solution to obtain the initial target phage, and then enter the next round of screening after amplification. After several rounds of screening, TEV protease variants with weak enzyme digestion activity in vivo and enzyme digestion activity under in vitro denaturation conditions will be enriched. Through gene sequencing analysis, the enriched sequences can be obtained and cloned into an expression vector to be verified one by one, thereby screening the TEV variants of the present invention.
  • the enzyme digestion activity can be calculated according to the results of SDS-PAGE gel.
  • the specific process is: dilute the inclusion body of TEV protease variant by 10 times, add 5xSDS loading buffer containing 10% ⁇ -mercaptoethanol, and cook at 100 °C for 5min. After loading the sample, run the gel, put the gel in Coomassie Brilliant Blue Staining Solution for 30 minutes, and then put it in Coomassie Brilliant Blue Destaining Solution, and heat to decolorize it for about 20 minutes until the background is colorless. Then, put the gel into the gel imaging system to take pictures.
  • In vitro digestion efficiency 1-grayscale band value of fusion protein after digestion / band grayscale value of fusion protein before digestion.
  • urea above 8M or guanidine hydrochloride above 6M is regarded as a high-density denaturant, while 4M urea is a medium-density denaturant.
  • Medium to high denaturation conditions refer to 4M urea or 1.5M guanidine hydrochloride, 50 mM Tris-HCl (pH 8.0), 1 mM EDTA, 2 mM DTT, reaction temperature 4 °C ⁇ 37 °C, preferably 25 °C.
  • Phagemid vector pHEN1 purchased from BioVector NTCC plasmid vector strain cell gene deposit center, article number Biovector786623).
  • DNA polymerase, T4 DNA ligase, and restriction endonucleases were purchased from British Weijieji Trading Co., Ltd.
  • the plasmid extraction kit and agarose gel DNA recovery kit were purchased from Tiangen (Beijing) Biotechnology Co., Ltd.
  • Random mutation kit (GeneMorph II Random Mutagenesis Kit) was purchased from Agilent Technologies. Primer synthesis and gene sequencing were completed at Nanjing Kingsray Biological Technology Co., Ltd.
  • TEV-F 5-AAT CTCGAG GGATCTAAAGGTCCTGGAGAAAGCTTGTTTAAGGGACCAC-3
  • TEV-R 5-AAT GGATCC TTGCGAGTACACCAATTC-3
  • the specific steps are based on the instructions, control the number of PCR cycles and the amount of template, and set the PCR conditions to the conditions that produce moderate mutations.
  • the 50ul reaction system was prepared as follows: sequentially added to the PCR tube, 41.5 ⁇ l ddH2O, 5 ⁇ l 10 ⁇ Mutazyme II reaction buffer, 1 ⁇ l 40mM dNTP mixture (final concentration 200 ⁇ M each), 0.5 ⁇ l primer mixture (250ng / ⁇ l for each primer) , 1 ⁇ l Mutazyme II DNA polymerase (2.5U / ⁇ l), 1 ⁇ l TEVP template (template amount 10ng), PCR amplification conditions: 95 ° C pre-denaturation for 3min; 95 ° C, 30s; 60 ° C, 30s; 72 ° C, 50s, total 32 cycles, and finally extended for 10 minutes at 72 ° C.
  • the PCR product was extracted and purified with phenol and chloroform, and then double-digested with Xho1 and BamH1, and the digested product was recovered using an agarose gel DNA recovery kit and stored at -20 ° C.
  • phagemid vector pHEN1 purchased from BioVector NTCC Plasmid Vector Strain Cell Gene Collection Center, Catalog No. Biovector786623
  • pHEN1 purchased from BioVector NTCC Plasmid Vector Strain Cell Gene Collection Center, Catalog No. Biovector786623
  • an Avi tag sequence GLNDIFEAQKIEWHE is inserted downstream of the signal peptide.
  • the fusion protein thus produced can be biotinylated and then fixed by streptavidin magnetic beads.
  • the template for PCR amplification of Avi tag is pET28b-PD1-Avi (synthesized in Nanjing Kingsray, gene structure is Nco1-PD1 gene-Not1-BamHI-GGGS linker-avi tag-Xho1, Genebank accession number NM_005018 of PD1 gene, plasmid The map is shown in Figure 7).
  • the PCR primers are as follows:
  • the product was purified by phenol chloroform, double-digested with Nco1 and Xho1, and after recovery of the gel, it was ligated with the same pHEN1 vector digested to obtain pHEN1-Avi. Then, insert a tandem sequence containing sTEV (nucleotide sequence: GAAAATCTGTATTTTCAGAGC, amino acid sequence ENLYFQS) and human ACTH gene behind Avitag (cloning site Xho1 + Not1), PCR amplify the template of sTEV-ACTH tandem sequence It is pET28b-ACTH (synthesized in Nanjing Kingsray, the plasmid map is shown in Figure 8, the amino acid sequence is shown in SEQ ID NO. 2; the nucleotide sequence is shown in SEQ ID NO. 13), the PCR primers are as follows:
  • ACTH-F AAT CTCGAG GGATCT GGATCC GGAGGTGGCGGTAGCGAAAATCTGTATTTTCAGAGCTATAGCATGGAAC-3
  • ACTH-R 5-AAT GCGGCCGC AAATTCCAGCGGAAATGC-3
  • the BamH1 site was additionally introduced upstream of the primer, and the PCR product was purified by phenol chloroform, double digested with Not1 and Xho1, and then ligated with the same digested pHEN1-Avi vector to obtain pHEN1–Avi–sTEV-ACTH.
  • Vector linearization double digestion of pHEN1-Avi-sTEV-ACTH with Xho1 and BamH1, gel recovery of large fragments in the digestion product, storage at -20 °C.
  • the digested recovered vector (pHEN1-Avi-sTEV-ACTH) and the insert (randomly mutated TEVP gene) were connected in 1: 3, 1: 5, 1:10, the specific process: first prepare the vector pHEN1-Avi- sTEV-ACTH (see 1.2.2 Preparation of linearized vector), and then prepare the insert (see 1.2.1 Random mutation PCR to prepare TEVP DNA fragments), the two are connected by T4 DNA ligase (Thermo Fisher Scientific) to obtain pHEN1- Avi-TEVp-sTEV-ACTH.
  • connection system taking the 20ul reaction system as an example: add 150ng of linearized carrier in sequence to the PCR tube, the corresponding amount of insert (the molar ratio to the carrier is 1: 3 or 1: 5 or 1:10), 10 ⁇ T4 DNA Ligase buffer 2ul, T4 DNA Ligase 1ul, and finally make up the volume to 20ul with ddH2O.
  • the optimal ligation ratio was determined to be 1: 5.
  • the plasmid map is shown in Figure 9. Determine the best connection ratio is 1: 5.
  • the ligation products of different concentrations were used for transformation to determine the optimal amount of transformation product was 0.2 ⁇ g (10 ⁇ l).
  • the ligation product was purified by phenol chloroform extraction method to remove protein and salt ions, and dissolved in ddH2O.
  • the ligation product was transferred to the host strain TG1 by electroporation.
  • the preparation method of electrotransformed competent cells was in accordance with the molecular cloning experiment guide (third edition). Take 10 ⁇ l of the ligation product and 200 ⁇ l of electrocompetent cells and mix gently, ice bath for 2 min, transfer to a pre-cooled electrorotation cup with a pore size of 0.2 cm, and perform electrotransfer.
  • the parameters of the Bio-Rad Gene-Pulser were 2.5KV, 25 ⁇ F, 200 ⁇ .
  • Upstream primer 5-CCACCATGGCCGGTCTGAATGATATTTTTGAAGC-3
  • the PCR-positive recombinant plasmid was sent to sequencing (Kingsray Biotechnology Co., Ltd.), and the randomness, capacity and abundance of the constructed library were evaluated.
  • the molar ratio of the vector to the insert is 1: 5, and 10 ⁇ l of the ligation product is used for electrotransformation, and the conversion efficiency is 9 ⁇ 10 8 cfu / ⁇ g DNA.
  • After multiple conversions combined storage capacity of 2.02x10 9, to meet the needs for screening.
  • Twenty clones were randomly selected from the peptide library and sequenced. The results show (Table 1) that the overall mutation frequency is medium to low (1.5-7 / kb). There is a certain deviation between the actual value and the theoretical value. The possible reason is that the synonymous mutations are not counted, and secondly, because of the small sample size, this difference will gradually decrease with the increase of sequencing samples.
  • the above results indicate that the constructed TEVP random phage library meets the design requirements in terms of basic framework, storage capacity and diversity.
  • the HRP-M13 antibody was purchased from Beijing Yiqiao Shenzhou Company, and the 96-well microplate and ELISA reagent were purchased from Shanghai Shengong. Streptavidin magnetic beads were purchased from NEB. D-biotin, IPTG and other reagents were purchased from Shanghai Shengong.
  • the bacteria were collected by centrifugation (2000xg for 20 minutes), and the supernatant was removed and discarded as much as possible.
  • -biotin resuspend and incubate at 25 rpm at 250 rpm overnight. The next day, the packaged phage was harvested and purified. Transfer the cell culture to a 750 ml centrifuge bottle and cool it slightly in ice. Centrifuge at 4500 rpm for 30 minutes at 4 ° C with a large bench-top centrifuge. If the supernatant is cloudy, transfer it to another Clean the bottle and repeat this step.
  • Avi tag is a short peptide tag (GLNDIFEAQKIEWHE) composed of 15 amino acid residues, which can be connected to a living body by a biotin ligase at a lysine residue in vivo or in vitro Biotinylation of proteins, and biotinylated proteins can be specifically bound by streptavidin. Based on these two reactions, the TEVp bacteriophage in the present invention can be biotinylated in vivo, biotin The phage library can be fixed with streptavidin magnetic beads.
  • TEVP variants with intracellular cleavage activity will self-digest during the expression process, resulting in the final assembled phage PIII protein terminal only ACTH, without Avitag, can not be captured by magnetic beads; and when expressed in cells
  • the enzymatically active TEVP variant does not undergo self-digestion during expression.
  • the final assembled phage PIII protein ends with Avi-TEVP-sTEV-ACTH.
  • the N-terminal Avi tag is biotinylated and can be captured by magnetic beads containing streptavidin.
  • the phage captured by the magnetic beads are incubated in medium and high concentration urea.
  • the TEVP variant that was originally insoluble under physiological conditions can be dissolved and restored to activity under this condition. It can be digested under this condition.
  • TEV protease variants with weak enzyme digestion activity in vivo and enzyme digestion activity under in vitro denaturation conditions will be enriched.
  • the enriched sequences can be obtained and cloned into an expression vector to be verified one by one. The rounds of panning are shown in Table 2 below.
  • Phage fixation In a clean 2ml EP tube, dilute 100-fold library capacity of phage to TBST buffer (50mM Tris-HCl PH7.5, 150mM NaCl, 0.1% [v / v] Tween-20) , Add appropriate amount of streptavidin magnetic beads to mix, rotate and incubate at 4 °C for 20min, precipitate the magnetic beads with a magnet, then wash the magnetic beads with TBST 5 times to remove unbound phage.
  • TBST buffer 50mM Tris-HCl PH7.5, 150mM NaCl, 0.1% [v / v] Tween-20
  • OD600 0.5
  • Sequencing analysis and screening for sequence enrichment After 3 rounds of panning, the phage eluted from the last round were infected with the host bacteria, spread on 2xYT-CG plates, and cultured at 30 ° C overnight. The next day, a single colony was picked, colony PCR was performed to identify whether it belonged to the library sequence, and the positive clone was sent for sequencing analysis. By analyzing the sequencing results and counting the frequency of mutation sites, the best 7 mutants were selected as candidate clones.
  • Bacterial treatment collecting bacteria by centrifugation, resuspending with ultrasonic wave, collecting inclusion body precipitate by centrifugation, washing the inclusion body, and finally dissolving the inclusion body with 8M urea (50mM Tris-HCl, 1mM EDTA, 2mM DTT, 8M urea, pH8.0) .
  • 8M urea 50mM Tris-HCl, 1mM EDTA, 2mM DTT, 8M urea, pH8.0
  • Inclusion body protein dilution and digestion test use TEVP digestion buffer (50 mM Tris-HCl, 1 mM EDTA, 2 mM DTT, pH 8.0) to dilute the inclusion body protein of each candidate clone so that the final concentration of urea is 3M or 4M, and then Digest at overnight at 25 ° C. The next day, run SDS-PAGE gel to check the digestion. The protein electrophoresis band was processed with Image J software to calculate the gray value of the band.
  • TEVP digestion buffer 50 mM Tris-HCl, 1 mM EDTA, 2 mM DTT, pH 8.0
  • positive clones obtained by the above digestion test should be further tested for digestion efficiency under different conditions, including: different urea concentration, different temperature, different guanidine hydrochloride concentration, DTT and EDTA in the reaction solution concentration.
  • the other was digested by self-digestion at 25 °C for 16h, and then added 5 ⁇ SDS-loading buffer, and boiled at 100 °C for 5min. This is the digested sample for 16h. Subsequently, SDS-PAGE denaturing gel was run to detect the digested samples of each variant at 0h and 16h. After electrophoresis, the gel was stained with Coomassie brilliant blue, decolorized, and photographed. The image is processed with Image J software to calculate the gray value of the electrophoretic band. The calculation method is as follows:
  • In vivo digestion activity 1-gray value of fusion protein band before digestion / (grey value of fusion protein band before digestion + Avi-TEVP band gray value x molecular weight of fusion protein / Avi-TEVP molecular weight).
  • In vitro digestion efficiency 1-grayscale band value of fusion protein after digestion / band grayscale value of fusion protein before digestion.
  • the TEV protease variant used in the preparation of ACTH polypeptide of the present invention is any one of Table 3.
  • the TEV protease variants screened by the present invention can be used for the preparation of other recombinant polypeptides and proteins.
  • Table 3 Yield of fusion protein of several protease variants and ACTH (calculation method: run fusion protein sample with BSA standard to run SDS-PAGE denaturing gel, after running the gel, put into Coomassie blue staining solution, and then transfer into Remove the background from the decolorizing solution and take a picture. Compare the gray value of the BSA standard strip in the picture with the sample gray value to estimate the concentration of the sample, and then calculate the concentration and yield of the fusion protein), in vivo activity and 4M in vitro The tolerance of urea (using S219V as a control) is based on the comparison of gray values of protein bands. The data are the statistical results of the yields of the fusion proteins of the TEVP variants expressed in three different batches and the ratio of in vivo and in vitro digestion.
  • the yield of fusion protein here refers to the fusion protein that has not been digested.
  • the digested part of the body is not counted because it has no use value.
  • S219V is the 219th amino acid of wild-type TEVP mutated from serine (S) to valine (V);
  • 32C mutation of amino acid 111 of S219V from leucine (L) to histidine (H), amino acid of 135 from serine (S) to glycine (G); amino acid of 187 from methionine (M) Mutation to isoleucine (I).
  • FIG. 1 shows that TEVp mutant 12D has the highest digestion efficiency in 4M urea solution
  • Figure 2 shows that TEVp mutant 4D has the highest digestion efficiency in 4M urea
  • Figure 3 shows that TEVp mutant 32C has the highest digestion efficiency in 4M urea
  • 4A and FIG. 4B show that TEVp variant 4D has the highest digestion efficiency under 4M urea at 25 ° C.
  • Fig. 5 shows that the in vivo digestion activity of the TEVp mutants 4D, 12D and 32C screened by the present invention is significantly lower than that of the S219V control (based on the 0 hour band comparison), and the in vitro digestion activity is slightly less than or equal to S219V.
  • Figure 6 shows that the fusion protein of each TEVp mutant has low solubility in 0.5M urea (the diluted supernatant has only a small amount of protein, most of which is in the diluted precipitate), proving that the TEVP mutant fusion protein is in Dilute to 0.5M urea in the form of precipitate.
  • the TEV protease mutant used in the preparation of ACTH polypeptide of the present invention is any one of Table 1.
  • the TEV protease mutants screened by the present invention can be used for the preparation of other recombinant polypeptides and proteins.
  • Example 1 the carboxy terminus of ACTH released by the fusion proteolytic cleavage bears a His tag, which is required to be removed in actual production. Therefore, it is necessary to introduce a stop codon downstream of the ACTH gene by PCR.
  • the specific process is: using the plasmid with TEV protease variant 12D with the best effect in step 3 of Example 1 as a template, the Avi-TEVP-sTEV-ACTH region of the vector open reading frame is amplified together by PCR (TEVP variant 12D sequence SEQ ID NO. 5, ACTH gene sequence SEQ ID NO. 13). KOD-plus DNA high fidelity polymerase (Toyobo) was used for amplification.
  • the amplification procedure was: pre-denaturation at 95 °C for 2min, denaturation at 98 °C for 10s, annealing at 60 °C for 30s, extension at 68 °C for 1min and 12s, and 30 cycles of amplification) ,
  • the stop codon was introduced downstream of the gene.
  • the primers are as follows:
  • TEV-ACTH-F 5-CCA CCATGG CCGGTCTGAATGATATTTTTGAAGC-3
  • TEV-ACTH-R 5-AGA GCGGCCGC TTATTAAAATTCCAGCGGAAATGCTTCTGC-3
  • the underlined parts are Nco1 and Not1, respectively.
  • PCR product and vector pET-28b Novagen
  • the fragments were recovered by gel recovery, and then ligated with T4 DNA ligase at 20 °C for 2h.
  • the ligation product was transformed into DH5 ⁇ competent cells, and then the transformed product was coated on kanamycin-resistant (50 ⁇ g / ml) LB plates and cultured until a single colony grew out, single colonies were picked, and plasmids were extracted for verification by enzyme digestion.
  • the recombinant plasmid was sent to Kingsley for sequencing to obtain the plasmid pET-28b-Avi-TEVP-sTEV-ACTH (the sTEV sequence is in the TEVP-ACTH plasmid).
  • the constructed expression vector is transformed. Specific process: take 50ng plasmid pET-28b-TEVP-sTEV-ACTH and add it to the corresponding chemically competent cells Rosetta2 (DE3), ice bath for 30 minutes, heat shock at 42 ° C for 45 seconds, ice bath for 2 minutes, add 1 ml without Antibiotic LB medium was incubated at 37 ° C for 1 hour. 100ul of bacterial solution was spread on LB plates containing kanamycin (50 ⁇ g / ml) + chloramphenicol (34 ⁇ g / ml) and incubated at 37 ° C until single colony length Out.
  • Rosetta2 Rosetta2
  • TEVP-ACTH After washing several times with washing buffer (composition: 50 mM Tris-HCl, 200 mM NaCl, 10 mM EDTA, 10 mM ⁇ -Mercaptoethanol, 0.5% Triton X-100), TEVP-ACTH is obtained
  • the crude fusion protein extract is added with a buffer solution containing the denaturant guanidine hydrochloride or urea (composition: 50mM Tris-HCl pH8.0, 1mM EDTA, 2mMDTT, 8M Urea or 6M GuHCl) to dissolve the TEVP-ACTH fusion protein to prepare a denaturing solution .
  • TEVP digestion buffer 3.556M Urea, 50mM Tris-HCl, 1mM EDTA, 2mM DTT, pH 8.0
  • TEV protease and undigested fusion protein will precipitate due to poor solubility (see Figure 6), while ACTH polypeptide can be dissolved into the solution Therefore, the supernatant was collected after centrifugation at 13,000g at 4 ° C and 13,000g for 30 minutes to obtain the ACTH stock solution.
  • ACTH stock solution was filtered with 0.22um filter membrane impurities, ultrafiltration concentrated with 1k ultrafiltration centrifuge tube (Millipore) while desalting, and then precipitated with 50% ammonium sulfate, ACTH will form a suspension on the upper layer of the solution, carefully collected, dissolved in PBS Concentration and desalting by ultrafiltration can obtain high-purity recombinant human or pig-based ACTH solution, which is then lyophilized and stored.
  • the molecular weight of the purified ACTH was measured using a mass spectrometer, and the measured molecular weight value was consistent with the theoretical value. The results are shown in Figure 10.
  • the mechanically dispersed cells were sucked several times with a pipette to form a suspension and filtered with a cell sieve Into a 50ml centrifuge tube, centrifuge at 1000xg for 10 minutes, carefully aspirate the supernatant, wash the precipitated cells twice with Hanks solution, and finally resuspend in DMEM / F12 medium (Gibco) containing 20% fetal bovine serum, adjust the concentration 2x10 5 / ml, and seeded into 90mm culture dishes, cultured at 37 °C, 5% CO2 conditions. Observe the growth process and morphological changes of adrenal cells under an inverted phase contrast microscope.
  • the adrenal cells grow adherently under the microscope, the cell volume increases, the cell body is round or polygonal, the cell body is large, and the cytoplasm is translucent. There are many particles of regular size in the cytoplasm, so as to obtain rat adrenal cortex cells.

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Abstract

本发明涉及筛选蛋白酶变体的方法以及获得的蛋白酶变体，所述蛋白酶变体具有特殊性能。所述筛选蛋白酶变体的方法包括以下步骤：（1）构建蛋白酶随机突变文库，优选蛋白酶随机突变病毒文库，优选蛋白酶随机突变噬菌体文库；（2）对所述蛋白酶随机突变文库，优选蛋白酶随机突变病毒文库，优选蛋白酶随机突变噬菌体文库筛选在宿主内或类似的条件下蛋白酶酶切活性弱且在体外变性条件下有蛋白酶酶切活性的蛋白酶变体；以及（3）任选地表征蛋白酶变体的步骤，其中所述蛋白酶优选是TEV蛋白酶或肠激酶。本发明的蛋白酶变体有利于蛋白质的工业化生产。

Description

筛选蛋白酶变体的方法以及获得的蛋白酶变体
本申请要求2018年10月10日提交的、发明名称“TEV蛋白酶变体、其融合蛋白及制备方法和用途”的中国发明专利申请No.201811177105.9和2018年12月17日提交的发明名称为“筛选蛋白酶变体的方法以及获得的蛋白酶变体”的中国发明专利申请No.201811540344.6的优先权权益，它们的全部内容通过引用并入。
技术领域
本发明涉及蛋白质领域，具体地涉及筛选TEV蛋白酶变体的方法以及TEV蛋白酶变体。
背景技术
TEV蛋白酶(TEV Protease)是来源于烟草蚀纹病毒(TEV)的Nla蛋白酶中27kDa的活性结构域，其氨基酸序列如SEQ ID NO.1所示。TEV蛋白酶具有很强的位点特异性，能够识别EXXYXQ(G/S)七氨基酸序列，最常用序列的是Glu-Asn-Leu-Tyr-Phe-Gln-Gly(或ENLYFQG)，其切割位点在谷氨酰胺Gln(P1)和甘氨酸Gly(P1′)之间(即P1和P1’之间)，其序列专一性远比凝血酶、因子Xa、肠激酶等蛋白酶高。
TEV蛋白酶能够耐受范围广泛的pH(pH 4-8.5)和温度(4-34℃)，对一些常见的增加蛋白可溶性或稳定性的添加剂(乙二醇、EGTA、去垢剂以及还原剂)也都有不同程度的耐受。有研究证明，TEV蛋白酶对乙二醇、EGTA和一些除垢剂(Triton X-100、Tween-20和NP-40)不敏感，当1％CHAPS存在时活性降低，在低浓度变性剂(2M尿素,1％SDS)和还原剂(0.7Mβ-巯基乙醇)中依旧可以保持大部分活性(C.Sun et al.,2012)。
野生型TEV酶在表达和溶解性等方面存在一定的缺陷，所以通过基因工程技术改良的突变体有大量报道。例如，天然的TEV蛋白酶会发生自我切割，在表达和纯化过程中，它会不断由于其它TEV蛋白酶的碰撞而发生构象变 化，在特定位点发生自我切割，从而使完整的蛋白酶被截短，活性大大降低。Lucast等人找到的S219N突变体，稳定性得到很大的提高，但是可溶性并不高，有大约95％的蛋白是以包涵体的形式存在于沉淀中。Kapust等人使用基因工程点突变了天然TEV蛋白酶的基因序列，得到S219V这一稳定性较高并且酶活性也有稍微提高的突变体，其稳定性比S219N要高出约100倍。其次，TEV蛋白酶表达产量不高，溶解度也非常低。大约只有5％的TEV蛋白酶存在于细胞破碎液的上清中，产量为12.5mg/L。Van den Berg等人通过基因改组(DNA shuffling)、易错PCR(error-prone PCR)发现了一种可以将产量提高到54mg/L的突变体TEVSH，其溶解度比S219N有很大提高，并且酶活性变化不大。Cabrita，L.D.等人使用PoPMuSiC软件设计，对TEV蛋白酶单点突变体进行稳定性分析，筛选出了五个突变体，它们的可溶性和酶活性相对于野生型TEV蛋白酶都得到提升，同时还得到一个双突变体，其可溶性及酶活性相对于单突变体有显著提高。针对TEV蛋白酶本身的缺点，研究者一直在寻找改良的突变体。例如，TEV Ser135Gly突变体比WT更稳定，可以耐受对更高的温度(>40℃)；还有一些突变(T17S，N68D，N177V)可以显著提高TEV蛋白酶的溶解性。
目前TEV蛋白酶的筛选方法主要关注提高TEV蛋白酶的酶活性、溶解度和产量等性质。尚未报道如本申请的TEV蛋白酶的筛选方法。
发明内容
本发明至少部分基于发明人在多肽药物生产过程中的发现：目前常规的TEV蛋白酶会在表达过程中大量切割融合蛋白(自我酶切)，导致多肽被提前释放，容易被胞内蛋白酶水解，不利于纯化，不适合工业化生产；常规TEV蛋白酶虽然在低浓度变性剂中(2M尿素,1％SDS)依旧可以保持大部分活性(C.Sun et al.,2012)，但是有很多蛋白在2M尿素等低浓度变性条件下往往是不能完全溶解的，这就限制了TEV蛋白酶的使用。对此，发明人提供了以下新的筛选方法来寻找适合于解决该问题的TEV蛋白酶变体。
本发明提供了筛选蛋白酶变体的方法，其包括以下步骤：
(1)构建蛋白酶随机突变文库，优选蛋白酶随机突变病毒文库，优选 蛋白酶随机突变噬菌体文库；
(2)对所述蛋白酶随机突变文库，优选蛋白酶随机突变病毒文库，优选蛋白酶随机突变噬菌体文库筛选在宿主内或类似的条件下蛋白酶酶切活性弱且在体外变性条件下有蛋白酶酶切活性的蛋白酶变体；以及
(3)任选地表征蛋白酶变体的步骤，
其中所述蛋白酶优选是TEV蛋白酶或肠激酶。
在一个实施方案中，方法还包括使用随机突变试剂盒产生所述突变文库。
在一个实施方案中，在步骤(1)中以合适的蛋白酶模板，例如TEV蛋白酶模板，例如具有以SEQ ID NO.10所示的氨基酸序列的S219V TEV蛋白酶为模板进行随机突变来构建所述文库。
在一个实施方案中，蛋白酶变体与初始蛋白酶相比在宿主内或类似的条件下具有更低的酶切活性且在体外变性条件下保留初始蛋白酶的酶切活性。
在一个实施方案中，TEV蛋白酶变体与具有以SEQ ID NO.10所示的氨基酸序列的S219V蛋白酶相比在宿主内或类似的条件下具有更低的酶切活性且在体外变性条件下保留以SEQ ID NO.10所示的氨基酸序列的S219V蛋白酶的酶切活性。
在一个实施方案中，以包含蛋白酶-蛋白酶切割序列-Y1，例如TEVp-sTEV-Y1的融合蛋白形式展示蛋白酶变体，优选TEV蛋白酶变体，优选在噬菌体表面展示蛋白酶变体，优选TEV蛋白酶变体，其中TEVp是TEV蛋白酶，sTEV为TEV蛋白酶切割序列，以及Y1是感兴趣的蛋白质；优选地其中所述TEV蛋白酶切割序列是EXXYXQG/S/H，其中X为任意氨基酸残基，优选所述TEV蛋白酶切割序列选自SEQ ID NO:7和8。
在一个实施方案中，方法包括在宿主内对所述蛋白酶变体，优选TEV蛋白酶变体的N端直接或通过接头间接添加部分A。
在一个实施方案中，部分A是以下一种：亲和标签、生物素、抗体、抗原、半抗原、聚链霉亲合素、配体、受体、核酸、酶、底物和适体。
在一个实施方案中，方法包括将添加有A的噬菌体文库在适合于所述蛋白酶的体外条件下与用特异性针对部分A的部分B固定化的固体支持物孵育一段时间，并且分离支持物与含有目的噬菌体的溶液，任选地对所述 目的噬菌体重复筛选一次或多次。
在一个实施方案中，部分B是以下一种：生物素、抗体、抗原、半抗原、聚链霉亲合素、配体、受体、核酸、酶、底物和适体，条件是部分B特异性结合部分A。
在一个实施方案中，以Avi-蛋白酶-蛋白酶切割位点-Y1-PIII，优选通过pHEN1-Avi-TEVp-sTEV-Y1构建体以Avi-TEVp-sTEV-Y1-PIII方式将蛋白酶，优选TEV蛋白酶展示于噬菌体PIII蛋白末端，其中Avi标签是由15个氨基酸残基GLNDIFEAQKIEWHE组成的短肽标签；优选地其中所述TEV蛋白酶切割序列是EXXYXQG/S/H，其中X为任意氨基酸残基，优选所述TEV蛋白酶切割序列选自SEQ ID NO:7和8。
在一个实施方案中，酶切活性是对包含所述蛋白酶及其切割序列，例如蛋白酶-蛋白酶切割序列-Y1，优选包含所述TEV蛋白酶变体及其切割序列的融合蛋白，优选TEVp-sTEV-Y1测定的。
在一个实施方案中，筛选包括在宿主内将所述蛋白酶，优选TEV蛋白酶随机突变噬菌体文库生物素化。
在一个实施方案中，筛选包括用链霉亲合素磁珠处理经生物素化的噬菌体文库，将被磁珠捕获的噬菌体置于中高浓度尿素中孵育一段时间，并且分离磁珠与含有目的噬菌体的溶液，任选地对所述目的噬菌体重复筛选一次或多次，
在一个实施方案中，通过菌落PCR进行验证所述文库。在一个实施方案中，所述菌落PCR使用的引物为：上游引物:5’-CCACCATGGCCGGTCTGAATGATATTTTTGAAGC-3’
下游引物：5’-TTGTTCTGCGGCCGCAAATTCCAGC-3’。
在一个实施方案中，文库容量达到1x10 9以上，优选2x10 9；优选整体突变频率为中低程度1.5-7个/kb。
在一个实施方案中，Y1是人或猪ACTH或GLP-1。在一个实施方案中，所述宿主是大肠杆菌。
在一个实施方案中，体外变性条件是中高程度变性条件，优选在3M-5M尿素，优选3.5M-4.5M尿素，更优选4M尿素或1M-2M盐酸胍，优选1.5M的盐酸胍的条件。
本发明提供了通过本发明的方法获得的蛋白酶变体。在一个实施方案 中，所述蛋白酶变体在宿主内表达过程中具有低酶切活性，优选所述TEV蛋白酶变体与具有以SEQ ID NO.10所示的氨基酸序列的S219V变体相比更低的酶切活性，所述TEV蛋白酶变体的切割序列选自EXXYXQG/S/H，其中X为任意氨基酸残基，优选所述TEV蛋白酶切割序列选自SEQ ID NO:7和8；优选地所述蛋白酶变体或TEV蛋白酶变体在中高程度变性条件下，优选在3M-5M尿素，优选3.5M-4.5M尿素，更优选4M尿素或1M-2M盐酸胍，优选1.5M的盐酸胍的体外环境下保留初始蛋白酶，例如S219V蛋白酶的体外酶切活性；优选地所述酶切活性是对包含所述蛋白酶变体及其切割序列，例如蛋白酶-蛋白酶切割序列-Y1，优选包含所述TEV蛋白酶变体及其切割序列的融合蛋白测定的，优选所述TEV蛋白酶切割序列是EXXYXQG/S/H，其中X为任意氨基酸残基，优选所述TEV蛋白酶切割序列选自SEQ ID NO:7和8。
在一个实施方案中，TEV蛋白酶变体包含选自下组的一种或多种突变：
与SEQ ID NO:1所示序列的第111位对应的位置处由亮氨酸(L)变为苯丙氨酸(F)或组氨酸(H)的突变；
与SEQ ID NO:1所示序列的第138位对应的位置处由异亮氨酸(I)变为赖氨酸(K)的突变；
与SEQ ID NO:1所示序列的第28位对应的位置处由组氨酸(H)变为亮氨酸(L)的突变；
与SEQ ID NO:1所示序列的第196位对应的位置处由谷氨酸(Q)变为组氨酸(H)的突变；
与SEQ ID NO:1所示序列的第135位对应的位置处由丝氨酸(S)变为甘氨酸(G)的突变；和
与SEQ ID NO:1所示序列的第187位对应的位置处蛋氨酸(M)变为异亮氨酸(I)的突变；
优选地，其中所述TEV蛋白酶变体包含选自以下的突变组合：
与SEQ ID NO:1所示序列的第111位对应的位置处由亮氨酸(L)变为苯丙氨酸(F)的突变和与SEQ ID NO:1所示序列的第138位对应的位置处由异亮氨酸(I)变为赖氨酸(K)的突变；
与SEQ ID NO:1所示序列的第28位对应的位置处由组氨酸(H)变为亮 氨酸(L)的突变、与SEQ ID NO:1所示序列的第111位对应的位置处由亮氨酸(L)变为苯丙氨酸(F)的突变和与SEQ ID NO:1所示序列的第196位对应的位置处由谷氨酸(Q)变为组氨酸(H)的突变；和
与SEQ ID NO:1所示序列的第111位对应的位置处由亮氨酸(L)变为组氨酸(H)的突变、与SEQ ID NO:1所示序列的第135位对应的位置处由丝氨酸(S)变为甘氨酸(G)的突变和与SEQ ID NO:1所示序列的第187位对应的位置处蛋氨酸(M)变为异亮氨酸(I)的突变；优选地，所述TEV蛋白酶变体包含与SEQ ID NO:1所示序列的第219位对应的位置处由丝氨酸(S)变为缬氨酸(V)的突变；优选其中所述TEV蛋白酶进一步包含一个或多个除上述突变以外的突变，条件是所述TEV蛋白酶变体在宿主内表达过程中具有低酶切活性，优选地与具有以SEQ ID NO.10所示的氨基酸序列的S219V变体相比更低的酶切活性和/或所述TEV蛋白酶变体在中高程度变性条件下，优选在3M-5M尿素，优选3.5M-4.5M尿素，更优选4M尿素或1M-2M盐酸胍，优选1.5M的盐酸胍的体外环境下具有高酶切活性，优选保留S219V变体的体外酶切活性。
在一个实施方案中，同源物包含与SEQ ID NO:4、5或6具有至少90％，优选至少95％，更优选至少98％，最优选至少99％序列同一性的氨基酸序列；优选地，其中所述同源物包含与SEQ ID NO:4、5或6具有至少1个，优选至少2个，更优选至少3个，最优选至少4个氨基酸位点的取代、缺失或添加的氨基酸序列；优选地其中所述同源物在宿主内表达过程中具有低酶切活性，优选与具有以SEQ ID NO.10所示的氨基酸序列的S219V变体相比更低的酶切活性和/或所述TEV蛋白酶变体在中高程度变性条件下，优选在3M-5M尿素，优选3.5M-4.5M尿素，更优选4M尿素或1M-2M盐酸胍，优选1.5M的盐酸胍的体外环境下保留S219V变体的体外酶切活性。
本发明提供了融合蛋白。融合蛋白包含根据本发明的蛋白酶变体；优选地所述融合蛋白包含结构蛋白酶-蛋白酶切割序列-Y1，优选TEVp-sTEV-Y1，其中Y1为感兴趣的多肽；TEVp为根据本发明的TEV蛋白酶变体；sTEV为TEV蛋白酶切割序列，其是EXXYXQG/S/H，其中X为任意氨基酸残基，优选所述切割序列选自SEQ ID NO:7和8；优选地其中感兴趣的多肽选自ACTH、GLP-1/GLP-2、IFN-α、IFN-γ、Histatin、CCL5、SDF-1α、IGF-1、Leptin、BNP、Ex-4，优选ACTH，优选人ACTH，更优 选SEQ ID NO.2所示的氨基酸序列的人ACTH；优选其中蛋白酶变体与蛋白酶变体切割序列，例如TEVp与sTEV是直接连接的或者相隔一个或多个氨基酸残基，条件是蛋白酶变体，例如TEVp能识别并切割蛋白酶变体切割序列，例如sTEV；优选其中蛋白酶变体切割序列，例如sTEV与Y1是直接连接的或者相隔一个或多个氨基酸残基，条件是蛋白酶变体，例如TEVp能识别并切割蛋白酶变体切割序列，例如sTEV，优选地蛋白酶变体切割序列，例如sTEV与Y1是直接连接的；优选所述融合蛋白还包含标签，优选其中所述标签为纯化标签；优选所述标签选自下组：His标签、麦芽糖结合蛋白(MBP)标签、谷胱甘肽转移酶(GST)标签、NusA标签、SUMO标签、Avi标签、T7标签、S标签、Flag标签、HA标签、c-myc标签、或StrepⅡ标签；优选其中所述标签在融合蛋白的N端和/或C端，或者TEVp的N端无标签。
本发明提供了蛋白酶变体或融合蛋白的多核苷酸序列，优选选自SEQ ID NO.14-16。
本发明提供了多核苷酸构建体，其包含本发明的多核苷酸序列。
本发明提供了表达载体，其包含根据本发明的多核苷酸序列或本发明的多核苷酸构建体。优选地，所述表达载体为真核生物表达载体或原核生物表达载体；优选地选自下组：pRS314、pYES2、杆状病毒-S2表达系统和pcDNA3.1或优选选自下组：pET系列表达载体、pQE系列表达载体和pBAD系列表达载体。
本发明提供了细胞，其包含根据本发明的多核苷酸序列、根据本发明的多核苷酸构建体或根据本发明的表达载体；优选为真核细胞或原核细胞；优选所述真核细胞选自下组：酿酒酵母、昆虫细胞表达系统；优选所述原核细胞选自下组：BL21、BL21(DE3)、BL21(DE3)pLysS、Rosetta2、Rosetta2pLysS、Tuner(DE3)、或Origami 2。
本发明提供了制备根据本发明的蛋白酶变体，优选TEV蛋白酶变体的方法，其包括：
(1)在适合于培养本发明的细胞的条件下在培养基中培养所述细胞；
(2)收获培养基，或裂解细胞以收获裂解物；
(3)纯化获得蛋白酶变体，优选所述TEV蛋白酶变体。
本发明提供了根据本发明的蛋白酶变体，优选TEV蛋白酶变体用于制 备感兴趣的多肽的用途，其中蛋白酶变体，优选所述TEV蛋白酶变体与所述感兴趣的多肽以融合蛋白形式表达，优选地，所述融合蛋白为本发明的融合蛋白。
本发明提供了制备感兴趣的多肽的方法，其包括：
(1)在合适的条件下在培养基中培养本发明的融合蛋白；
(2)获得融合蛋白的包涵体；
(3)在高变性条件下，例如约8M尿素或约6M盐酸胍溶解包涵体；
(4)在中高程度变性条件下，优选在3M-5M尿素，优选3.5M-4.5M尿素，更优选4M尿素或1M-2M盐酸胍，优选1.5M的盐酸胍的条件下在一定的温度，例如20-40℃，优选25℃温育一段时间，例如10-24小时，例如12小时；
(5)用缓冲液例如Tris-HCl，优选50mM Tris-HCl稀释后沉淀蛋白酶变体，优选TEV蛋白酶；
(6)离心除去蛋白酶变体，优选TEV蛋白酶沉淀物，获得所述感兴趣的多肽；并且
(7)纯化所述感兴趣的多肽，优选其中所述纯化用选自下组的技术进行：盐析、超滤、有机溶剂沉淀、凝胶过滤、离子交换层析柱、反相高效液相色谱。
通过本发明的筛选方法，发明人获得了新的TEV蛋白酶变体4D、12D和32C，表明使用本发明的方法可以获得在宿主内TEV蛋白酶酶切活性弱且在体外变性条件下有TEV蛋白酶酶切活性的TEV蛋白酶变体。另外，当使用TEV蛋白酶识别序列EXXYXQH进行筛选时，还可以获得高效切割ENLYFQ↓H的TEV蛋白酶突变体。对于许多N端以组氨酸开头的多肽来说是期望的。
1.本发明的TEV蛋白酶变体可以广泛用于生产各种具有天然N端的多肽或蛋白。
2.本发明的TEV蛋白酶变体是通过筛选获得的，但是直接获得的突变体无法满足要求或部分满足要求。通过一系列的突变点组合，能获得具有独特的获得性能进一步提高的改良型突变体，例如L111F与其他位点组合，其特点是体内无或极低活性，但是体外中等变性条件下有较高活性。
3.本发明的方法采用DNA重组技术和原核表达系统表达目的蛋白， 具有广泛的适用性。
4.相比于猪脑垂体提取ACTH的方法，采用本发明的方法制备人源或猪源ACTH的生产成本比传统猪脑提取生产技术降低能达上百倍，极大地缩短了生产周期，节省了生产时间和成本，且不再依赖于大量生猪屠宰获得猪脑垂体，避免了使用猪源ACTH产生的免疫原性过敏反应等风险，大大提升药物安全性。而且本方法制备的ACTH与人体自身分泌的ACTH完全一样，具有极高氨基酸序列精准度和产品纯度，优异的生物活性，提高了药物疗效，降低了不良反应的发生。相比于化学有机合成的方法，本方法制备全长人源ACTH的难度和成本都显著降低，操作简单，不需要昂贵的催化剂和高压设备，产率高，适合大规模生产。总体而言，本方法生产工艺明确、简单，具有较好的可重复性，易于实现规模化生产，且降低了对环境的污染。
附图说明
图1：TEVP突变体12D在不同浓度尿素中酶切效率的SDS-PAGE凝胶考马斯亮蓝染色图。箭头表示目标蛋白。
图2：TEVP突变体4D在不同浓度尿素中酶切效率的SDS-PAGE凝胶考马斯亮蓝染色图。箭头表示目标蛋白。
图3：TEVP突变体32C在不同浓度尿素中酶切效率的SDS-PAGE凝胶考马斯亮蓝染色图。箭头表示目标蛋白。
图4A：TEVP突变体4D在不同浓度尿素中酶切效率的SDS-PAGE凝胶考马斯亮蓝染色图。图4B：电泳条带灰度值比较。
图5：TEVP突变体4D、12D和32C的体内酶切活性、体外酶切活性与S219V的比较。箭头表示目标蛋白。
图6：TEVP突变体4D、12D、32C和S219V的融合蛋白在0.5M尿素中的溶解度检测。
图7：质粒pET-28b-PD1-Avi图谱。
图8：质粒pET-28b-ACTH图谱。
图9：质粒Avi-TEV-sTEV-ACTH基因结构图。
图10：质谱仪测定纯化后的ACTH的分子量。
图11：体外法测定ACTH活性。
具体实施方式
为使本发明的目的、技术方案和优点更加清楚，下面将对本发明实施方式作进一步地详细描述，但本发明的实施方式不限于此。
如本文中使用，术语“宿主”可以是任何适合表达蛋白质的真核或原核细胞。在一个实施方案中，宿主细胞是大肠杆菌细胞。
如本文中使用，术语“体外变性条件”是指使含有TEV蛋白酶的融合蛋白溶解而具有酶切活性的条件，例如在3M-5M尿素，优选3.5M-4.5M尿素，更优选4M尿素或1M-2M盐酸胍，优选1.5M的盐酸胍的条件。
如本文中使用，TEV蛋白酶是来源于烟草蚀纹病毒(TEV)的Nla蛋白酶中27kDa的活性结构域。本文中使用以SEQ ID NO.10所示的氨基酸序列的S219V TEV蛋白酶作为起始TEV蛋白酶。然而，本领域技术人员应当理解各种其它TEV蛋白酶可以用作本发明方法的起始TEV蛋白酶。TEV蛋白酶具有很强的位点特异性，能够识别EXXYXQ(G/S)七氨基酸序列，最常用序列的是Glu-Asn-Leu-Tyr-Phe-Gln-Gly(或ENLYFQG)，其切割位点在谷氨酰胺Gln(P1)和甘氨酸Gly(P1’)之间(即P1和P1’之间)，其序列专一性远比凝血酶、因子Xa、肠激酶等蛋白酶高。
如本文中使用，“TEV蛋白酶变体”是指与筛选所用的起始TEV蛋白酶相比具有氨基酸残基差异的TEV蛋白酶，其中变体并不暗示TEV蛋白酶具有特定的序列和/或结构。
如本文中使用，“TEV蛋白酶酶切活性”是指TEV蛋白酶自身切割包含TEV蛋白酶及其切割位点的融合蛋白的酶切活性。在一个实施方案中，本发明的TEV蛋白酶酶切活性是对含有EXXYXQ(G/S/H)的融合蛋白的切割活性。
在本文中使用噬菌体展示技术展示融合蛋白。噬菌体展示技术是将外源蛋白或多肽的DNA序列插入到噬菌体外壳蛋白结构基因的适当位置，使外源基因随外壳蛋白的表达而表达，同时，外源蛋白随噬菌体的重新组装而展示到噬菌体表面的生物技术。本领域技术人员应当理解使用其它TEV蛋白酶随机突变文库的选择对于本领域技术人员来进行本发明的方 法。
噬菌体展示系统可以是单链丝状噬菌体展示系统、λ噬菌体展示系统和T4噬菌体展示系统。在单链丝状噬菌体展示系统的情况下，可以是PIII展示系统和PVIII展示系统。在λ噬菌体展示系统的情况下，可以是PV展示系统和D蛋白展示系统。优选地，在一个实施方案中，噬菌体展示系统是PIII展示系统。
如本文中使用，“部分A”和“部分B”指一对结合配偶体，例如选自生物素、抗体、抗原、半抗原、聚链霉亲合素、配体、受体、核酸、酶、底物和适体，条件是“部分A”和“部分B”彼此特异性结合。
在一个实施方案中，将含有部分B的固体支持物用于捕获含有部分A的融合蛋白。在一个实施方案中，当标签为生物素部分时，固体支持物可以是经过链霉亲合素涂覆的珠或树脂，诸如Dynabeads M-280链霉亲合素、Dynabeads MyOne链霉亲合素、Dynabeads M-270链霉亲合素(Invitrogen)、链霉亲合素琼脂糖树脂(Pierce)、链霉亲合素超联树脂、MagnaBind链霉亲合素珠(ThermoFisher Scientific)、BioMag链霉亲合素、ProMag链霉亲合素、二氧化硅链霉亲和素(Bangs Laboratories)、高效链霉亲合素琼脂糖(Streptavidin Sepharose High Performance)(GE Healthcare)、链霉亲合素聚苯乙烯微球体(Microspheres-Nanospheres)、经过链霉亲合素涂覆的聚苯乙烯颗粒(Spherotech)或本领域技术人员常用于捕获生物素标记的分子的任何其它经过链霉亲合素涂覆的珠或树脂。
在一个实施方案中，可以以Avi-TEVp-sTEV-Y1-PIII方式将TEV蛋白酶展示于噬菌体PIII蛋白末端(N端)，其中Avi标签是由15个氨基酸残基GLNDIFEAQKIEWHE组成的短肽标签。通过在宿主表达过程中，通过Avi标签将Avi-TEVp-sTEV-Y1-PIII生物素化。
如本文中使用，“肠激酶”是存在于哺乳动物十二指肠内的一种异源二聚体丝氨酸蛋白酶。肠激酶的酶切位点是公知的例如DDDDK，肠激酶的核苷酸序列和氨基酸序列也是本领域技术人员可获得的，例如GenBank:AAC50138.1，AAB40026.1等等。
TEV蛋白酶
TEV蛋白酶虽然经常被用于高效切割融合蛋白，但是仅限于在二者都可溶状态下切割。目前大部分多肽药物是溶解性较差的，通常以包涵体形式表达，这与TEV蛋白酶的酶切条件不匹配。包涵体需要用高浓度尿素或盐酸胍溶解，但在这种条件下酶切是极具挑战性的，常规TEV蛋白酶与底物很难在同一条件下既有高效切割活性又同时溶解。
包涵体有利于纯化，通过简单处理即可得到相对较纯的目的蛋白。但是包涵体蛋白需要用高浓度尿素或盐酸胍才能溶解，野生型TEV蛋白酶在这种条件下是无活性或活性极低，无法高效切割融合蛋白。目前，改良型TEV蛋白酶有大量报道，但是其最佳酶切条件都是生理条件或非变性条件，通常对低浓度的变性剂(尿素或盐酸胍)具有一定程度的耐受性，但是绝大多数包涵体蛋白在低浓度变性条件下是不溶解的，因此，常规TEV蛋白酶不适用于这种场合。
发明人发现TEV蛋白酶与多肽融合制备多肽药物的理想生产条件是：(1)该融合蛋白以包涵体形式表达，利于纯化；(2)该融合蛋白在胞内不发生自我切割，否则不利于纯化；(3)该融合蛋白在高浓度变性剂中溶解后，稀释到含中高等浓度变性剂的酶切反应体系中，此时TEV蛋白酶恢复活性，高效地自我切割，释放多肽；(4)从高容量的TEV蛋白酶文库中筛选进化得到的TEV蛋白酶变体对其酶切位点的P1’位置的氨基酸要具有广谱性，这样就可以广泛用于生产各种具有天然N端的多肽或蛋白。
US2010035300A1中提供了通常制备的具有自我切割的融合蛋白的结构。在融合蛋白的制备中，均需要His标签与要表达的目标蛋白(例如EGFP)紧密相连。这样的原因在于，当融合蛋白体内表达时，传统的野生型TEVp或突变体TEVp在胞内具有一定的酶切活性，会发生自我切割。切割后的EGFP可以通过与其紧密相连的His标签回收。若没有该His标签，则由于在胞内提前发生自我切割而难以回收，无法高效生产EGFP。但这样同样带来一个问题，即可能根据具体需要会进一步除去EGFP的His标签。这样做显然较为费时费力。
在本发明中，融合蛋白中的目标蛋白可以不含标签，例如His。在一个实施方案中，融合蛋白包含Avi-TEVp-sTEV-ACTH的结构，其中，Avi为Avi标签蛋白，sTEV为TEV蛋白酶酶切位点。由于TEVp变体(如12D变体)在胞内的酶活性极低，因此可以所表达的融合蛋白基本上不会在胞内 发生裂解的情况。在收集包涵体，用8M尿素溶解，并调整到4M尿素的环境下时，12D发挥其酶活性，进而切割其识别位点为sTEV。酶切结束后，通过简单的稀释，使尿素或盐酸胍终浓度降至0.5M，TEV蛋白酶因溶解性很差，容易沉淀下来，而释放的具有生物活性的多肽往往易于溶于缓冲液，因此通过离心就可以得到高纯度的多肽，巧妙克服了原本需要增加的蛋白酶去除环节，大幅精简后期的纯化工艺。
形成包涵体较有利于纯化：1)容易通过离心收获浓度高而相对纯净的蛋白；2)包涵体保护蛋白免受蛋白酶水解。另外，毒性蛋白以无活性的包涵体形式表达，不会影响宿主菌的生长。例如，本方法中涉及的TEV-ACTH融合蛋白是以包涵体的形式表达，大大地简化了其纯化步骤，能够达到较高浓度和纯度，且不会受到蛋白酶的水解，因此容易获得稳定的高的产量。
本发明的筛选方法
在本发明中，构建了一个大容量的TEV随机突变噬菌体文库，其关键基因结构为Avi-TEV-sTEV-Y1-PⅢ，即通过这个方式展示于噬菌体PⅢ蛋白末端。其中Avi tag是一个由15个氨基酸残基组成的短肽标签(GLNDIFEAQKIEWHE)，在体内或体外都能被生物素连接酶在赖氨酸残基(K)连接上一个生物素，从而实现蛋白的生物素化，而生物素化的蛋白可以专一地被链霉亲和素结合，基于这两个反应，本发明中的TEV噬菌体可在体内被生物素化，生物素化的噬菌体文库可用链霉亲和素磁珠固定。理想条件下，在细胞内表达时有酶切活性的TEV变体会在表达过程中自我酶切，导致最终组装的噬菌体PⅢ蛋白末端只有多肽Y1，无Avi tag，无法被磁珠捕获；而在细胞内表达时无酶切活性的TEV变体在表达过程中不发生自我酶切，最终组装的噬菌体PⅢ蛋白的末端是Avi-TEV-sTEV-Y1，N端的Avi tag被生物素化，可被含有链霉亲和素磁珠捕获。被磁珠捕获的噬菌体置于中高浓度尿素中孵育，在此条件下发生酶切的噬菌体会因自我切割从磁珠上掉下来，进入溶液。收集该溶液即获得初始目的噬菌体，经扩增后就可进入下一轮筛选。经过数轮的筛选，体内酶切活性弱同时在体外变性条件下有酶切活性的TEV蛋白酶变种会被富集。通过基因测序分析，得到富集的序列，克隆到表达载体即可逐一验证，从而筛选到本发明的TEV变种。
酶切活性或效率的测定
酶切活性可以根据SDS-PAGE胶的结果计算，具体过程是：将TEV蛋白酶变体的包涵体稀释10倍后加含10％β-巯基乙醇的5xSDS上样缓冲液，100℃煮样5min，上样跑胶，结束后将胶放入考马斯亮蓝染色液中染色30min，随后放入考马斯亮蓝脱色液中，加热脱色20min左右，至背景无色为止。然后，将胶放入凝胶成像系统拍照。获取的图片用Image J软件处理，将条带灰度值量化，体内酶切活性＝1-融合蛋白条带灰度值/(融合蛋白条带灰度值+Avi-TEV条带灰度值*融合蛋白分子量/Avi-TEV分子量)。
在本文中，用Image J软件分析电泳条带灰度值的方法为目前的通用方法。
在本文中，体外酶切效率的计算方法如下：
体外酶切效率＝1-酶切后融合蛋白的条带灰度值/酶切前融合蛋白的条带灰度值。
在本文中，8M以上尿素或6M以上盐酸胍视为高浓度的变性剂，而4M尿素为中等浓度的变性剂。中高程度变性条件是指4M尿素或1.5M盐酸胍、50mM Tris-HCl(pH8.0)、1mM EDTA,2mM DTT，反应温度4℃～37℃，优选25℃。
提供以下实施例以例示本发明。
实施例
实施例1：TEV蛋白酶变体的获得
1.大容量TEV蛋白酶随机突变文库构建
1.1实验材料：
大肠杆菌TG1：supE hsd Δ5thiΔ(lac-proAB)F’[traD36proAB+lacIq lacZΔM15]，购买自北京宝科食维安公司。噬菌粒载体pHEN1(购自BioVector NTCC质粒载体菌种细胞基因保藏中心，货号Biovector786623)。DNA聚合酶、T4 DNA连接酶、限制性内切酶购自英潍捷基贸易有限公司。质粒抽提试剂盒及琼脂糖凝胶DNA回收试剂盒购自天根(北京)生物科技有限公司。随机突变试剂盒(GeneMorph II Random Mutagenesis Kit)购自Agilent Technologies。引 物合成和基因测序在南京金斯瑞生物科技有限公司完成。
1.2.TEVP随机突变文库构建
1.2.1随机突变PCR制备TEVP DNA片段
以pQE30-TEV(S219V)(此27kDa TEV NIa蛋白酶变体基因由南京金斯瑞合成，克隆到pQE30载体的BamH1和HindIII之间，S219V氨基酸序列见SEQ ID NO.10，核苷酸序列见SEQ ID NO.3)为模板，用随机突变试剂盒(GeneMorph II Random Mutagenesis Kit)对TEVP S219V基因进行大范围随机突变。PCR引物为：
TEV-F：5-AAT CTCGAGGGATCTAAAGGTCCTGGAGAAAGCTTGTTTAAGGGACCAC-3
TEV-R：5-AAT GGATCCTTGCGAGTACACCAATTC-3
具体步骤依据说明书操作，控制好PCR循环数及模板用量，将PCR条件设置为产生中等程度突变的条件。50ul的反应体系配制如下：在PCR管中依次加入，41.5μl ddH2O，5μl 10×Mutazyme II反应缓冲液，1μl 40mM dNTP混合物(终浓度各200μM)，0.5μl引物混合物(每种引物250ng/μl)，1μl Mutazyme II DNA聚合酶(2.5U/μl)，1μl TEVP模板(模板量10ng)，PCR扩增条件：95℃预变性3min；95℃，30s；60℃，30s；72℃，50s，共32个循环，最后72℃延伸10min。PCR结束后，取5ul产物跑1％琼脂糖凝胶电泳检测，分子量标准用试剂盒里的1.1-kb凝胶标准品，染色后比较目的条带与标准品的亮度，计算PCR产量，除以模板量得出扩增倍数，以此计算PCR过程中的d值(计算公式：2 d＝PCR产量/初始模板量)。本发明的TEVP随机突变PCR最后的d值＝7.5，对应的突变频率约为9个突变/kb(参考说明书)，符合预期要求。PCR产物用酚氯仿抽提纯化，然后用Xho1和BamH1进行双酶切，利用琼脂糖凝胶DNA回收试剂盒回收酶切产物，-20℃保存。
1.2.2线性化载体的制备
在线性化噬菌粒载体pHEN1(购自BioVector NTCC质粒载体菌种细胞基因保藏中心，货号Biovector786623)之前，先对它进行改造。首先，要将一个Avi tag序列GLNDIFEAQKIEWHE插入到信号肽下游，如此产生的融合蛋白能被生物素化，进而被链霉亲合素磁珠固定。PCR扩增Avi标签的模板用 pET28b-PD1-Avi(在南京金斯瑞合成，基因结构为Nco1-PD1基因-Not1-BamHI-GGGS接头-avi标签-Xho1，PD1基因的Genebank登录号NM_005018，质粒图谱见图7)，PCR引物如下：
Avi-F:5-ACT CCATGGCCGGTCTGAATGATATTTTTGAAGC-3
Avi-R:5-AAT CTCGAGCTCGTGCCACTCGATTTTCTG-3
产物经过酚氯仿纯化，用Nco1和Xho1进行双酶切，胶回收后与同样酶切过的pHEN1载体连接，得到pHEN1-Avi。然后，将一段包含sTEV(核苷酸序列为：GAAAATCTGTATTTTCAGAGC，氨基酸序列ENLYFQS)与人ACTH基因的串联序列插入到Avi tag后面(克隆位点Xho1+Not1)，PCR扩增sTEV-ACTH串联序列的模板为pET28b-ACTH(在南京金斯瑞合成，质粒图谱见图8，氨基酸序列见SEQ ID NO.2；核苷酸序列见SEQ ID NO.13)，PCR引物如下：
ACTH-F:AAT CTCGAGGGATCT GGATCCGGAGGTGGCGGTAGCGAAAATCTGTATTTTCAGAGCTATAGCATGGAAC-3
ACTH-R:5-AAT GCGGCCGCAAATTCCAGCGGAAATGC-3
引物上游额外引入BamH1位点，PCR产物经过酚氯仿纯化，用Not1和Xho1进行双酶切，然后与同样酶切的pHEN1-Avi载体连接，得到pHEN1–Avi–sTEV-ACTH。载体线性化：用Xho1和BamH1进行双酶切pHEN1-Avi-sTEV-ACTH，胶回收酶切产物中的大片段，-20℃保存。
1.2.3连接及连接产物的回收处理
对酶切回收的载体(pHEN1-Avi-sTEV-ACTH)与插入片段(随机突变的TEVP基因)按1∶3，1∶5，1∶10进行连接，具体过程：先准备载体pHEN1-Avi-sTEV-ACTH(见1.2.2线性化载体的制备)，再准备插入片段(见1.2.1随机突变PCR制备TEVP DNA片段)，二者通过T4 DNA连接酶(Thermo Fisher Scientific)连接，得到pHEN1-Avi-TEVp-sTEV-ACTH。连接体系参考说明书，以20ul反应体系为例：在PCR管中依次加入线性化的载体150ng，对应量的插入片段(与载体的摩尔比为1:3或1:5或1:10)，10×T4 DNA Ligase buffer 2ul，T4 DNA Ligase 1ul，最后用ddH2O补足体积至20ul。通过计算转化产生的克隆数，确定最佳连接比例为1∶5。质粒图谱见图9。确定最佳连接比例为1∶5。 用不同浓度的连接产物进行转化以确定最佳转化产物量为0.2μg(10μl)。连接产物经过酚氯仿抽提法纯化，除去蛋白和盐离子，溶于ddH2O中。
1.2.4电转化
连接产物通过电穿孔法，转到宿主菌TG1中。电转化感受态细胞的制备方法依照分子克隆实验指南(第三版)。取10μl连接产物与200μl电转感受态细胞轻柔混匀，冰浴2min，转入预冷的孔隙为0.2cm的电转杯，进行电转。电转仪(Bio-Rad Gene-Pulser)参数为2.5KV，25μF，200Ω。电击后，立即加入1ml SOC培养基，吸出后再用1ml SOC培养基漂洗电击杯2次，合并3ml菌液，于37℃，200rpm振摇45～60min。将菌液梯度稀释：10 -2，10 -3,10 -4，每个梯度取100ul菌体涂布SOC(含25μg/ml羧苄青霉素)小平板，其余5000xg离心5min收集菌体，重悬后涂布SOC(含25μg/ml羧苄青霉素)方形大平板(25x25cm)，37℃倒置培养12～16h，以小平板进行计数，大平板上的菌落用2YT培养基冲洗，轻轻用涂布棒均匀刮下来，加甘油至终浓度50％，分装成小管，-80℃冻存。文库容量计算方法：容量板上的克隆数*稀释倍数*稀释前菌液总体积。重复多次电转，直到文库容量达到1x10 9以上。
1.2.5序列测定及分析
随机挑取若干原始细菌克隆，通过做菌落PCR对重组质粒进行验证，PCR验证引物：
上游引物:5-CCACCATGGCCGGTCTGAATGATATTTTTGAAGC-3
下游引物：5-TTGTTCTGCGGCCGCAAATTCCAGC-3
PCR阳性的重组质粒，将其送去测序(金斯瑞生物科技有限公司)，并对构建的文库的随机性、库容及丰度进行评价。
1.2.6文库的库容及多样性评价
载体与插入片段摩尔比为1∶5，取10μl的连接产物进行电转，转化效率为9x10 8cfu/μg DNA。将多次转化合并后，库容为2.02x10 9，满足用于筛选的需要。从肽库中随机挑取20个克隆，进行序列测定。结果显示(表1)，整体突变频率为中低程度(1.5～7个/kb)。实际值与理论值存在一定的偏差，可能原因一是同义突变未统计，二是由于样本较小的缘故，随着测序样本的增加，这种差异将逐渐减小。上述结果表明，所构建的TEVP随机噬菌体文库不论从基本框架，还是从库容及多样性方面均满足设计要求。
表1随机挑选克隆氨基酸突变的分布
Figure PCTCN2019071417-appb-000001
2.从TEVP随机突变噬菌体文库中筛选目标克隆
2.1实验材料
HRP－M13抗体购自北京义翘神州公司，96孔酶标板和ELISA试剂购自上海生工。链霉亲合素磁珠购自NEB。D-biotin，IPTG等试剂均购自上海生工。
2.2噬菌体文库培养
取至少一个文库容量的菌加到含2xYT-CG(2xYT+100微克/毫升羧苄青霉素及2％葡萄糖)的2L锥形瓶中，使初始OD600值＝0.1左右，于37℃，200rpm 振摇，直到OD600达到0.5左右，加入辅助噬菌体M130K07(购自北京宝科食为安公司))侵染(MOI＝20)，在100转/分的缓慢转动中继续37℃培养一小时。随后，离心收菌(2000xg离心20分钟)，尽量去除并丢弃上清液,菌体重新用1L 2xYT-CK(2xYT+100微克/毫升羧苄青霉素和50微克/毫升卡那霉素+200uM D-biotin)重悬，在25℃中用250转/分钟培养过夜。第二天，收获并纯化包装的噬菌体。把细胞培养物转移到一个750毫升离心瓶中并在冰中稍微冷却，用大型台式离心机以4500转/分钟在4℃离心30分钟，如果上清液是混浊的,把它转入另一个干净瓶子里并重复这一步骤。把上层80％的上清液(不要搅动细胞沉定物)转入到另一个新的750毫升离心瓶里,加1/6体积的PEG/NaCl溶液(20％[w/v]PEG-8000，2.5MNaCl)，混合,然后在冰中静止至少两小时，在4℃下用4500转/分钟离心30分钟，小心地去除所有的上清液，加10毫升PBS,用移液器重新溶解沉淀物，然后转入2ml Ep管中。4℃下用15000xg离心20分钟,以去掉残留的菌体或碎片。小心吸取上清液到新的Ep管，加入于加1/6体积的PEG/NaCl溶液，在冰中放置1小时再次沉淀噬菌体，然后4℃下15000xg离心20分钟收集噬菌体沉淀，小心吸去并丢弃所有的上清液。随后，用适量PBS重新溶解噬菌体沉淀，彻底溶解后，4℃下15000xg离心10分钟以去掉剩余的不溶杂质。把上清液分装到新的EP管子里，同时加入50％甘油，放-80℃长期保存。
2.3测定文库滴度
参考Carol M Y Lee等(M Y Lee,Carol&Iorno,Niccoló&Sierro,Frederic&Christ,Daniel.(2007).Selection of human antibody fragments by phage display.Nature protocols.2.3001-8.10.1038/nprot.2007.448.)的方法，用2×YT梯度稀释噬菌体文库，取10 -9，10 -10，10 -11的稀释液各10ul，加入到已经培养到对数期的新鲜TG1菌液200ul，混匀，37℃温育30min，全部涂含2×YT-CG平板。30℃温箱培养过夜，次日计单菌落数，计算滴度。
2.4文库淘选
本发明淘选TEVP变体的原理：Avi tag是一个由15个氨基酸残基组成的短肽标签(GLNDIFEAQKIEWHE)，在体内或体外都能被生物素连接酶在赖氨酸残基连接上一个生物素，从而实现蛋白的生物素化，而生物素化的蛋白可以专一地被链霉亲和素结合，基于这两个反应，本发明中的TEVp噬菌体可在 体内被生物素化，生物素化的噬菌体文库可用链霉亲和素磁珠固定。理想条件下，胞内有酶切活性的TEVP变体会在表达过程中自我酶切，导致最终组装的噬菌体PⅢ蛋白末端只有ACTH，无Avi tag，无法被磁珠捕获；而在细胞内表达时无酶切活性的TEVP变体在表达过程中不发生自我酶切，最终组装的噬菌体PⅢ蛋白末端是Avi-TEVP-sTEV-ACTH，N端的Avi tag被生物素化，可被含有streptavidin磁珠捕获。被磁珠捕获的噬菌体置于中高浓度尿素中孵育，本来在生理条件下不溶解的TEVP变种，在此条件下能溶解并恢复活性，进行酶切，在此条件下发生酶切的噬菌体会因自我切割从磁珠上掉下来，进入溶液。收集该溶液即获得初始目的噬菌体，经扩增后就可进入下一轮筛选。经过数轮的筛选，体内酶切活性弱同时在体外变性条件下有酶切活性的TEV蛋白酶变种会被富集。通过基因测序分析，得到富集的序列，克隆到表达载体即可逐一验证。各轮的淘选情况如下表2所示。
表2.各轮淘选数据
Figure PCTCN2019071417-appb-000002
噬菌体淘选具体流程如下：
(1)噬菌体固定：在一个干净的2ml EP管中，将100倍文库容量的噬菌体稀释到TBST缓冲液(50mM Tris-HCl PH7.5，150mM NaCl，0.1％[v/v]Tween-20)，加入适量链霉亲和素磁珠混合，4℃旋转孵育20min，用磁铁沉淀磁珠，然后用TBST清洗磁珠5次，去掉未结合的噬菌体。
(2)酶切筛选：在磁珠中加入含3M尿素的TBS缓冲液，室温孵育1-2h，用磁铁沉淀磁珠，尽量收集全部上清。
(3)噬菌体扩增：将洗脱下来的噬菌体加入50ml OD600＝0.5的TG1宿主菌中，37℃缓慢摇晃1小时，加入100微克/毫升羧苄青霉素及2％葡萄糖，继续培养2小时，加入辅助噬菌体(M130K07(购自北京宝科食为安公司))侵染(MOI＝20)，低转速继续37℃培养1小时。随后离心收菌，尽量去除并丢弃上清液,菌体重新用100ml 2xYT-CK(2xYT+100微克/毫升羧苄青霉素和50微克/毫升卡那霉素+200uM D-biotin)重悬，在25℃中用250转/分钟培养过夜。第 二天，收获及纯化包装好的噬菌体。将培养物转入干净的50ml离心管中，4℃13,000g离心20min。将上清的上部80％转入新鲜离心管中，加入1/6体积的PEG/NaCl溶液，4℃沉淀1小时以上。然后再次离心收集沉淀，加适量PBS重悬噬菌体沉淀，再离心一次去除细菌碎片等杂质，收集上清，加入1/6体积的PEG/NaCl溶液，冰上静置1小时，然后再次离心收集沉淀，将其溶于适量PBS，随后13,000g离心10分钟，去除不溶解的杂质，将上清转移至另一新鲜Ep管中，即为扩增后的洗脱物。
(4)取出1μl纯化好的噬菌体用于滴度测定，其他用来进行下一轮淘选或保存。
(5)测序分析筛选序列富集情况：3轮淘选后，将最后一轮洗脱下来的噬菌体感染宿主菌后铺在2xYT-CG板上，30℃培养过夜。次日挑取单克隆菌落，先进行菌落PCR鉴定是否属于文库序列，阳性克隆再送测序分析。通过分析测序结果，统计突变位点出现的频率，最终挑选出其中最佳的7个突变体，作为候选克隆。
3.候选克隆表征
上述7个候选序列经酶切(Nco1/Not1)克隆到表达载体pET28b(购自Novagen)，并转入Rosetta2(DE3)(购自湖南优宝生物)表达。表达条件：在LB培养基中，37℃培养，至OD600＝0.6左右。诱导条件：37℃下250rpm摇，2mM IPTG，诱导4小时。菌处理：离心收菌，重悬后用超声波破碎，离心收集包涵体沉淀，洗涤包涵体，最后用8M尿素(50mM Tris-HCl,1mM EDTA,2mM DTT,8M尿素,pH8.0)溶解包涵体。
包涵体蛋白稀释及酶切测试：用TEVP酶切缓冲液(50mM Tris-HCl,1mM EDTA,2mM DTT,pH8.0)稀释各候选克隆的包涵体蛋白，使尿素终浓度为3M或4M，然后在25℃酶切过夜。次日，跑SDS-PAGE胶检测酶切情况。蛋白电泳条带用Image J软件处理，计算条带灰度值。
酶切条件优化：通过上面的酶切试验得到的阳性克隆，要进一步测试在不同条件下的酶切效率，这些条件包括：不同尿素浓度，不同温度，不同盐酸胍浓度，反应液中DTT及EDTA浓度。
本发明通过上述筛选，最终获取体内酶切活性弱但是体外活性正常的突变体3个。测试结果见图1、2、3、4A-B。
具体过程：将各个已溶解到8M尿素缓冲液中的TEV蛋白酶变体-ACTH融合蛋白分别以1:10比例加到稀释液中，稀释液为含不同浓度的尿素的50mM Tris-HCl pH8.0，1mM EDTA，2mMDTT。对于尿素终浓度4M时，稀释液为3.56M Urea，50mM Tris-HCl pH8.0，1mM EDTA，2mMDTT。每个样品混匀后立即等分到两支EP管中，一支立即加入5×SDS-上样缓冲液，100℃煮样5min，此为酶切0h的样品。另一支放25℃自我酶切16h，然后加入5×SDS-上样缓冲液，100℃煮样5min，此为酶切16h的样品。随后，跑SDS-PAGE变性胶检测各个变体的酶切0h、16h的样品。电泳结束后，凝胶经过考马斯亮蓝染色，脱色，拍照。图片用Image J软件处理，计算电泳条带灰度值。计算方法如下：
体内酶切活性＝1-酶切前融合蛋白条带灰度值/(酶切前融合蛋白条带灰度值+Avi-TEVP条带灰度值x融合蛋白分子量/Avi-TEVP分子量)。
体外酶切效率＝1-酶切后融合蛋白的条带灰度值/酶切前融合蛋白的条带灰度值。
如表3所示，4D，12D和32C蛋白酶变种体内酶切活性显著下降，体外4M尿素中切割效率都在30％以上。本发明制备ACTH多肽所用的TEV蛋白酶变种为表3中任一种。本发明筛选到的TEV蛋白酶变种可用于其他重组多肽和蛋白的制备。
表3：几种蛋白酶变种与ACTH的融合蛋白产量(计算方法：将融合蛋白样品与BSA标准品一起跑SDS-PAGE变性凝胶，跑胶结束后放入考马斯亮蓝染色液染色，然后转入脱色液中脱去背景，拍照。比较图片中BSA标准品条带的灰度值和样品灰度值即可估算样品的浓度，进而计算出融合蛋白浓度和产量)、体内活性及在体外对4M尿素的耐受能力(以S219V为对照)，基于蛋白条带灰度值比较。数据为3个不同批次表达的TEVP变体的融合蛋白的产量和体内、外酶切比例统计结果。
Figure PCTCN2019071417-appb-000003
Figure PCTCN2019071417-appb-000004
注：此处的融合蛋白产量是指未发生酶切的融合蛋白，体内已酶切的部分因无利用价值故不计算进去。S219V为野生型TEVP的第219个氨基酸由丝氨酸(S)突变为缬氨酸(V)；
4D：为S219V的第111位氨基酸由亮氨酸(L)突变为苯丙氨酸(F)，第138位氨基酸由异亮氨酸(I)突变为赖氨酸(K)；
12D：S219V的第28位氨基酸由组氨酸(H)突变为亮氨酸(L)，第111位氨基酸由亮氨酸(L)突变为苯丙氨酸(F)，第196位氨基酸由谷氨酸(Q)突变为组氨酸(H)；
32C：为S219V的第111位氨基酸由亮氨酸(L)突变为组氨酸(H)，第135位氨基酸由丝氨酸(S)突变为甘氨酸(G)；第187位氨基酸由蛋氨酸(M)突变为异亮氨酸(I)。
图1表明TEVp突变体12D在4M尿素溶液中酶切效率最高；图2表明TEVp突变体4D在4M尿素中酶切效率最高；图3表明TEVp突变体32C在4M尿素中酶切效率最高；图4A和图4B表明TEVp变体4D在4M尿素、25℃条件下的酶切效率最高。当尿素浓度超出4M时，各种突变体的酶切效率逐渐下降。
图5表明本发明筛选到的TEVp突变体4D、12D和32C的体内酶切活性显著低于S219V对照(根据0小时的条带比较)，体外酶切活性略小于或等于S219V。
图6表明各TEVp突变体的融合蛋白在0.5M尿素中的溶解度都较低(稀释后的上清中只有少量蛋白，大部分都在稀释后的沉淀中)，证明了TEVP突变体融合蛋白在稀释到0.5M尿素后以沉淀形式存在。
本发明制备ACTH多肽所用的TEV蛋白酶突变体为表1中任一种。本发明筛选到的TEV蛋白酶突变体可用于其他重组多肽和蛋白的制备。
实施例2：以TEVP-ACTH融合蛋白制备ACTH多肽
1.TEVP-ACTH融合蛋白表达载体构建
在实施例1中，融合蛋白酶切释放的ACTH的羧基端带有His标签，在实际生产中要求去掉该标签。因此，需要通过PCR在ACTH基因下游引入终止密码子。具体过程是：以在实施例1步骤3中效果最佳的带TEV蛋白酶变体12D的质粒为模板，通过PCR将该载体开放阅读框中的Avi-TEVP-sTEV-ACTH区域一起扩增出来的(TEVP变体12D序列SEQ ID NO.5，ACTH基因序列SEQ ID NO.13)。用KOD-plus DNA高保真聚合酶(东洋纺)进行扩增，扩增程序为：95℃预变性2min，98℃变性10s，60℃退火30s，68℃延伸1min 12s，扩增30个循环)，基因下游引入终止密码子。引物如下：
TEV-ACTH-F:5-CCA CCATGGCCGGTCTGAATGATATTTTTGAAGC-3
TEV-ACTH-R:5-AGA GCGGCCGCTTATTAAAATTCCAGCGGAAATGCTTCTGC-3
其中下划线部分别为酶切位点Nco1和Not1。PCR产物和载体pET-28b(Novagen)用Nco1和Not1在37℃酶切3h之后，胶回收酶切片段，然后用T4 DNA连接酶在20℃连接2h。将连接产物转化进入DH5α感受态细胞，然后将转化产物涂布在卡那霉素抗性(50μg/ml)LB平板上培养直至单菌落长出，挑取单菌落，提取质粒进行酶切验证，将重组质粒送金斯瑞测序，得到质粒pET-28b-Avi-TEVP-sTEV-ACTH(其中的sTEV序列在TEVP-ACTH质粒中)。
2.TEVP-ACTH融合蛋白的诱导表达、纯化、酶切及验证
将构建的表达载体转化。具体过程：取50ng质粒pET-28b-TEVP-sTEV-ACTH加到相应的化学感受态细胞Rosetta2(DE3)中，冰浴30分钟，42℃热激45秒，冰浴2分钟，加入1毫升无抗生素的LB培养基，37℃培养1小时，取100ul菌液涂布在含卡那霉素(50μg/ml)+氯霉素(34μg/ml)的LB平板上，37℃培养直至单菌落长出。
TEVP-ACTH融合蛋白的诱导表达：挑取单菌落于含卡那霉素(50μg/ml)和氯霉素(34μg/ml)的LB液体培养基中培养至OD600＝0.5～0.8。将培养物以体积比1:50的比例接种于LB液体培养基，37℃剧烈震荡培养至OD600＝0.5～0.8，加入终浓度为2mM的IPTG于37℃诱导4h。在表达过程中，TEVP-ACTH融合蛋白以不可溶的包涵体存在。发酵完成后，收集菌体超声破碎后，经洗涤缓冲液(组成：50mM Tris-HCl，200mM NaCl，10mM EDTA，10mM β -Mercaptoethanol，0.5％Triton X-100)多次洗涤后，得到TEVP-ACTH融合蛋白粗提物，加入含变性剂盐酸胍或尿素(组成：50mM Tris-HCl pH8.0，1mM EDTA，2mMDTT，8M Urea或6M GuHCl)的缓冲液溶解TEVP-ACTH融合蛋白，制备成变性溶液。
将融合蛋白变性溶液用TEVP酶切缓冲液(3.556M Urea，50mM Tris-HCl,1mM EDTA,2mM DTT,pH8.0)稀释十倍，使尿素终浓度为4M，放25℃自我酶切过夜，酶切产物经过8倍稀释(稀释液为50mM Tris-HCl pH8.0)后，TEV蛋白酶和未酶切的融合蛋白因溶解性差，会沉淀出来(见图6)，而ACTH多肽可以溶解到溶液中，因此经4℃，13000g低温高速离心30分钟后收集上清液，即获得ACTH原液。
3.ACTH的纯化与保存
ACTH原液用0.22um滤膜过滤杂质，用1k的超滤离心管(Millipore)超滤浓缩同时脱盐，然后用50％硫酸铵沉淀，ACTH会形成悬浮物在溶液上层，小心收集，用PBS溶解，超滤浓缩脱盐，即可获得高纯度的重组人源或猪源ACTH溶液，再经冻干后保存。
4.ACTH的结构鉴定
采用质谱仪测定纯化后的ACTH的分子量，测定的分子量值与理论值一致。结果见图10。
5.ACTH的活性测定
取健康2周龄的SD大鼠，用1％戊巴比妥钠(40mg/kg)麻醉，在无菌条件下取出肾上腺，去除被摸和髓质，放入Hanks平衡盐溶液中，剪成1mm 3大小的碎块，转入含Ⅰ型胶原酶和DNA酶的消化液中消化1小时，间隔5分钟振摇混匀，用吸管吹吸数次机械离散细胞形成悬液，用细胞筛过滤到50ml离心管中，1000xg离心10分钟，小心吸掉上清，沉淀的细胞用Hanks液洗涤2次，最后用含20％胎牛血清的DMEM/F12培养基(Gibco)重悬，调整浓度为2x10 5个/ml，接种到90mm培养皿，在37℃、5％CO2条件下培养。倒置相差显微镜下观察肾上腺细胞的生长过程及其形态变化,培养48小时后，在显微镜下可见肾上腺细胞贴壁生长，细胞体积增大，胞体呈圆形或多角形，胞体大，胞浆透亮，胞浆内有许多大小较为规则的颗粒，从而获得大鼠肾上腺皮质细胞。
将纯化后的ACTH与离体的大鼠肾上腺皮质细胞孵育。具体地，分别在 各组肾上腺细胞中加入不同浓度(0.1μU、0.2μU、2μU、20μU、200μU，1U＝10μg蛋白)的ACTH(细胞与ACTH的体积比400:1)，37℃孵育24小时后取培养液，离心去掉细胞碎片后用酶联免疫分析法测定其中皮质酮浓度(酶联免疫分析法可以参见何上进等，"PCNA在大鼠肾上腺细胞培养中的表达及其意义."临床泌尿外科杂志21.8(2006):625-626.)，结果显示随着ACTH浓度的增加，皮质酮浓度也逐步增加。ACTH刺激细胞分泌甾体类激素，通过测定生成的皮质酮的浓度来定量ACTH的活性(ELISA法)。结果显示，纯化后的多个批次的ACTH具有很高的生物活性和活性稳定性。结果见图11。
以上所述，仅是本发明的较佳实施例而已，并非对本发明做任何形式上的限制，故凡未脱离本发明技术方案的内容，依据本发明的技术实质对以上实施例所做的任何简单修改、等同变化与修饰，均仍属于本发明技术方案的范围内。
Figure PCTCN2019071417-appb-000005
Figure PCTCN2019071417-appb-000006
Figure PCTCN2019071417-appb-000007
Figure PCTCN2019071417-appb-000008

Claims (20)

  1. 筛选蛋白酶变体的方法，其包括以下步骤：
    (1)构建蛋白酶随机突变文库，优选蛋白酶随机突变病毒文库，优选蛋白酶随机突变噬菌体文库；
    (2)对所述蛋白酶随机突变文库，优选蛋白酶随机突变病毒文库，优选蛋白酶随机突变噬菌体文库筛选在宿主内或类似的条件下蛋白酶酶切活性弱且在体外变性条件下有蛋白酶酶切活性的蛋白酶变体；以及
    (3)任选地表征蛋白酶变体的步骤，
    其中所述蛋白酶优选是TEV蛋白酶或肠激酶。
  2. 根据权利要求1所述的方法，所述方法还包括使用随机突变试剂盒产生所述突变文库。
  3. 根据权利要求1或2所述的方法，其中在步骤(1)中以合适的蛋白酶模板，例如TEV蛋白酶模板，例如具有以SEQ ID NO.10所示的氨基酸序列的S219V TEV蛋白酶为模板进行随机突变来构建所述文库，
    优选地其中蛋白酶变体与初始蛋白酶相比在宿主内或类似的条件下具有更低的酶切活性且在体外变性条件下保留初始蛋白酶的酶切活性；
    优选地，所述TEV蛋白酶变体与具有以SEQ ID NO.10所示的氨基酸序列的S219V蛋白酶相比在宿主内或类似的条件下具有更低的酶切活性且在体外变性条件下保留以SEQ ID NO.10所示的氨基酸序列的S219V蛋白酶的酶切活性。
  4. 根据前述权利要求中任一项的方法，其中以包含蛋白酶-蛋白酶切割序列-Y1，例如TEVp-sTEV-Y1的融合蛋白形式展示蛋白酶变体，优选TEV蛋白酶变体，优选在噬菌体表面展示蛋白酶变体，优选TEV蛋白酶变体，其中TEVp是TEV蛋白酶，sTEV为TEV蛋白酶切割序列，以及Y1是感兴趣的蛋白质；优选地其中所述TEV蛋白酶切割序列是EXXYXQG/S/H，其中X为任意氨基酸残基，优选所述TEV蛋白酶切割序列选自SEQ ID NO:7和8。
  5. 根据前述权利要求中任一项的方法，所述方法包括在宿主内对所述蛋白酶变体，优选TEV蛋白酶变体的N端直接或通过接头间接添加部分A，
    其中优选地所述部分A是以下一种：亲和标签、生物素、抗体、抗原、半抗原、聚链霉亲合素、配体、受体、核酸、酶、底物和适体。
  6. 根据前述权利要求中任一项的方法，所述方法包括将添加有A的噬菌体文库在适合于所述蛋白酶的体外条件下与用特异性针对部分A的部分B固定化的固体支持物孵育一段时间，并且分离支持物与含有目的噬菌体的溶液，任选地对所述目的噬菌体重复筛选一次或多次，
    其中优选地所述部分B是以下一种：生物素、抗体、抗原、半抗原、聚链霉亲合素、配体、受体、核酸、酶、底物和适体，条件是部分B特异性结合部分A。
  7. 根据前述权利要求中任一项的方法，其中以Avi-蛋白酶-蛋白酶切割位点-Y1-PIII，优选通过pHEN1-Avi-TEVp-sTEV-Y1构建体以Avi-TEVp-sTEV-Y1-PIII方式将蛋白酶，优选TEV蛋白酶展示于噬菌体PIII蛋白末端，其中Avi标签是由15个氨基酸残基GLNDIFEAQKIEWHE组成的短肽标签；优选地其中所述TEV蛋白酶切割序列是EXXYXQG/S/H，其中X为任意氨基酸残基，优选所述TEV蛋白酶切割序列选自SEQ ID NO:7和8；
    优选地其中所述酶切活性是对包含所述蛋白酶及其切割序列，例如蛋白酶-蛋白酶切割序列-Y1，优选包含所述TEV蛋白酶变体及其切割序列的融合蛋白，优选TEVp-sTEV-Y1测定的；
    优选地其中所述筛选包括在宿主内将所述蛋白酶，优选TEV蛋白酶随机突变噬菌体文库生物素化；
    优选地其中所述筛选包括用链霉亲合素磁珠处理经生物素化的噬菌体文库，将被磁珠捕获的噬菌体置于中高浓度尿素中孵育一段时间，并且分离磁珠与含有目的噬菌体的溶液，任选地对所述目的噬菌体重复筛选一次或多次，
    优选地其中通过菌落PCR进行验证所述文库，优选地其中所述菌落PCR使用的引物为：上游引物:5’-CCACCATGGCCGGTCTGAATGATATTTTTGAAGC-3’
    下游引物：5’-TTGTTCTGCGGCCGCAAATTCCAGC-3’；
    优选地其中文库容量达到1x10 9以上，优选2x10 9；优选整体突变频率为中低程度1.5-7个/kb。
  8. 根据前述权利要求中任一项的方法，其中Y1是人或猪ACTH或GLP-1，优选地其中所述宿主是大肠杆菌。
  9. 根据前述权利要求中任一项的方法，其中所述体外变性条件是中高程度变性条件，优选在3M-5M尿素，优选3.5M-4.5M尿素，更优选4M尿素或1M-2M盐酸胍，优选1.5M的盐酸胍的条件。
  10. 通过前述权利要求中任一项的方法获得的蛋白酶变体，其中所述蛋白酶变体在宿主内表达过程中具有低酶切活性，优选所述TEV蛋白酶变体与具有以SEQ ID NO.10所示的氨基酸序列的S219V变体相比更低的酶切活性，所述TEV蛋白酶变体的切割序列选自EXXYXQG/S/H，其中X为任意氨基酸残基，优选所述TEV蛋白酶切割序列选自SEQ ID NO:7和8；优选地所述蛋白酶变体或TEV蛋白酶变体在中高程度变性条件下，优选在3M-5M尿素，优选3.5M-4.5M尿素，更优选4M尿素或1M-2M盐酸胍，优选1.5M的盐酸胍的体外环境下保留初始蛋白酶，例如S219V蛋白酶的体外酶切活性；优选地所述酶切活性是对包含所述蛋白酶变体及其切割序列，例如蛋白酶-蛋白酶切割序列-Y1，优选包含所述TEV蛋白酶变体及其切割序列的融合蛋白测定的，优选所述TEV蛋白酶切割序列是EXXYXQG/S/H，其中X为任意氨基酸残基，优选所述TEV蛋白酶切割序列选自SEQ ID NO:7和8。
  11. 根据权利要求10所述的蛋白酶变体，其中所述TEV蛋白酶变体包含选自下组的一种或多种突变：
    与SEQ ID NO:1所示序列的第111位对应的位置处由亮氨酸(L)变为苯丙氨酸(F)或组氨酸(H)的突变；
    与SEQ ID NO:1所示序列的第138位对应的位置处由异亮氨酸(I)变为赖氨酸(K)的突变；
    与SEQ ID NO:1所示序列的第28位对应的位置处由组氨酸(H)变为亮氨酸(L)的突变；
    与SEQ ID NO:1所示序列的第196位对应的位置处由谷氨酸(Q)变为组氨酸(H)的突变；
    与SEQ ID NO:1所示序列的第135位对应的位置处由丝氨酸(S)变为甘氨酸(G)的突变；和
    与SEQ ID NO:1所示序列的第187位对应的位置处蛋氨酸(M)变为异 亮氨酸(I)的突变；
    优选地，其中所述TEV蛋白酶变体包含选自以下的突变组合：
    与SEQ ID NO:1所示序列的第111位对应的位置处由亮氨酸(L)变为苯丙氨酸(F)的突变和与SEQ ID NO:1所示序列的第138位对应的位置处由异亮氨酸(I)变为赖氨酸(K)的突变；
    与SEQ ID NO:1所示序列的第28位对应的位置处由组氨酸(H)变为亮氨酸(L)的突变、与SEQ ID NO:1所示序列的第111位对应的位置处由亮氨酸(L)变为苯丙氨酸(F)的突变和与SEQ ID NO:1所示序列的第196位对应的位置处由谷氨酸(Q)变为组氨酸(H)的突变；和
    与SEQ ID NO:1所示序列的第111位对应的位置处由亮氨酸(L)变为组氨酸(H)的突变、与SEQ ID NO:1所示序列的第135位对应的位置处由丝氨酸(S)变为甘氨酸(G)的突变和与SEQ ID NO:1所示序列的第187位对应的位置处蛋氨酸(M)变为异亮氨酸(I)的突变；优选地，所述TEV蛋白酶变体包含与SEQ ID NO:1所示序列的第219位对应的位置处由丝氨酸(S)变为缬氨酸(V)的突变；优选其中所述TEV蛋白酶进一步包含一个或多个除上述突变以外的突变，条件是所述TEV蛋白酶变体在宿主内表达过程中具有低酶切活性，优选地与具有以SEQ ID NO.10所示的氨基酸序列的S219V变体相比更低的酶切活性和/或所述TEV蛋白酶变体在中高程度变性条件下，优选在3M-5M尿素，优选3.5M-4.5M尿素，更优选4M尿素或1M-2M盐酸胍，优选1.5M的盐酸胍的体外环境下具有高酶切活性，优选保留S219V变体的体外酶切活性。
  12. 根据权利要求10或11所述的蛋白酶变体，其中所述同源物包含与SEQ ID NO:4、5或6具有至少90％，优选至少95％，更优选至少98％，最优选至少99％序列同一性的氨基酸序列；优选地，其中所述同源物包含与SEQ ID NO:4、5或6具有至少1个，优选至少2个，更优选至少3个，最优选至少4个氨基酸位点的取代、缺失或添加的氨基酸序列；优选地其中所述同源物在宿主内表达过程中具有低酶切活性，优选与具有以SEQ ID NO.10所示的氨基酸序列的S219V变体相比更低的酶切活性和/或所述TEV蛋白酶变体在中高程度变性条件下，优选在3M-5M尿素，优选3.5M-4.5M尿素，更优选4M尿素或1M-2M盐酸胍，优选1.5M的盐酸胍的体外环境下保留S219V变体的体外酶切活性。
  13. 融合蛋白，其包含根据权利要求10-12中任一项所述的蛋白酶变体；优选地所述融合蛋白包含结构蛋白酶-蛋白酶切割序列-Y1，优选TEVp-sTEV-Y1，其中Y1为感兴趣的多肽；TEVp为根据权利要求10-12中任一项所述的TEV蛋白酶变体；sTEV为TEV蛋白酶切割序列，其是EXXYXQG/S/H，其中X为任意氨基酸残基，优选所述切割序列选自SEQ ID NO:7和8；优选地其中感兴趣的多肽选自ACTH、GLP-1/GLP-2、IFN-α、IFN-γ、Histatin、CCL5、SDF-1α、IGF-1、Leptin、BNP、Ex-4，优选ACTH，优选人ACTH，更优选SEQ ID NO.2所示的氨基酸序列的人ACTH；优选其中蛋白酶变体与蛋白酶变体切割序列，例如TEVp与sTEV是直接连接的或者相隔一个或多个氨基酸残基，条件是蛋白酶变体，例如TEVp能识别并切割蛋白酶变体切割序列，例如sTEV；优选其中蛋白酶变体切割序列，例如sTEV与Y1是直接连接的或者相隔一个或多个氨基酸残基，条件是蛋白酶变体，例如TEVp能识别并切割蛋白酶变体切割序列，例如sTEV，优选地蛋白酶变体切割序列，例如sTEV与Y1是直接连接的；优选所述融合蛋白还包含标签，优选其中所述标签为纯化标签；优选所述标签选自下组：His标签、麦芽糖结合蛋白(MBP)标签、谷胱甘肽转移酶(GST)标签、NusA标签、SUMO标签、Avi标签、T7标签、S标签、Flag标签、HA标签、c-myc标签、或StrepⅡ标签；优选其中所述标签在融合蛋白的N端和/或C端，或者TEVp的N端无标签。
  14. 编码根据权利要求10-12中任一项所述的蛋白酶变体或根据权利要求13所述的融合蛋白的多核苷酸序列，优选选自SEQ ID NO.14-16。
  15. 多核苷酸构建体，其包含根据权利要求14所述的多核苷酸序列。
  16. 表达载体，其包含根据权利要求14所述的多核苷酸序列或权利要求15所述的多核苷酸构建体；优选其中所述表达载体为真核生物表达载体或原核生物表达载体；优选选自下组：pRS314、pYES2、杆状病毒-S2表达系统和pcDNA3.1或优选选自下组：pET系列表达载体、pQE系列表达载体和pBAD系列表达载体。
  17. 细胞，其包含根据权利要求14所述的多核苷酸序列、根据权利要求15所述的多核苷酸构建体或根据权利要求16所述的表达载体；优选为真核细胞或原核细胞；优选所述真核细胞选自下组：酿酒酵母、昆虫细胞表达系统；优选所述原核细胞选自下组：BL21、BL21(DE3)、BL21(DE3) pLysS、Rosetta2、Rosetta2 pLysS、Tuner(DE3)、或Origami 2。
  18. 制备根据权利要求10-12中任一项所述的蛋白酶变体，优选TEV蛋白酶变体的方法，其包括：
    (1)在适合于培养权利要求17所述的细胞的条件下在培养基中培养所述细胞；
    (2)收获培养基，或裂解细胞以收获裂解物；
    (3)纯化获得蛋白酶变体，优选所述TEV蛋白酶变体。
  19. 根据权利要求10-12中任一项所述的蛋白酶变体，优选TEV蛋白酶变体用于制备感兴趣的多肽的用途，其中蛋白酶变体，优选所述TEV蛋白酶变体与所述感兴趣的多肽以融合蛋白形式表达，优选地，所述融合蛋白为根据权利要求13所述的融合蛋白。
  20. 制备感兴趣的多肽的方法，其包括：
    (1)在合适的条件下在培养基中培养根据权利要求13所述的融合蛋白；
    (2)获得融合蛋白的包涵体；
    (3)在高变性条件下，例如约8M尿素或约6M盐酸胍溶解包涵体；
    (4)在中高程度变性条件下，优选在3M-5M尿素，优选3.5M-4.5M尿素，更优选4M尿素或1M-2M盐酸胍，优选1.5M的盐酸胍的条件下在一定的温度，例如20-40℃，优选25℃温育一段时间，例如10-24小时，例如12小时；
    (5)用缓冲液例如Tris-HCl，优选50mM Tris-HCl稀释后沉淀蛋白酶变体，优选TEV蛋白酶；
    (6)离心除去蛋白酶变体，优选TEV蛋白酶沉淀物，获得所述感兴趣的多肽；并且
    (7)纯化所述感兴趣的多肽，优选其中所述纯化用选自下组的技术进行：盐析、超滤、有机溶剂沉淀、凝胶过滤、离子交换层析柱、反相高效液相色谱。
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