WO2023045682A1 - 一种提高多肽可溶性表达产量的方法 - Google Patents

一种提高多肽可溶性表达产量的方法 Download PDF

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WO2023045682A1
WO2023045682A1 PCT/CN2022/114414 CN2022114414W WO2023045682A1 WO 2023045682 A1 WO2023045682 A1 WO 2023045682A1 CN 2022114414 W CN2022114414 W CN 2022114414W WO 2023045682 A1 WO2023045682 A1 WO 2023045682A1
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escherichia coli
polypeptide
recombinant
glucagon
glp
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French (fr)
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彭志恩
宋浩
柳学伟
杨晓瑜
巨晓芝
郭万成
信铭雁
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奥锐特药业(天津)有限公司
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1137—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against enzymes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/605—Glucagons
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    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/20—Type of nucleic acid involving clustered regularly interspaced short palindromic repeats [CRISPRs]
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    • C12N2800/00—Nucleic acids vectors
    • C12N2800/22—Vectors comprising a coding region that has been codon optimised for expression in a respective host

Definitions

  • the invention relates to the field of biotechnology, and more specifically relates to a method for increasing the soluble expression of polypeptides.
  • Peptides are a class of compounds that are simpler than proteins and have smaller molecular weights, and are connected by amino acids through peptide bonds. At present, more than 60 kinds of peptide drugs are on the market, and hundreds of peptide drugs are in the clinical research stage.
  • the structure of polypeptide drugs is similar to the protein structure of natural sources, and its properties are very close to normal physiological substances in the body, with high pharmacological activity, and its metabolites are small-molecule amino acids with high safety;
  • polypeptide drugs are less likely to be recognized by the immune system and cause allergic reactions. Therefore, in recent years, peptide drugs have shown a rapid development trend and have been used in different therapeutic fields, such as diabetes, arthritis, obesity, anti-infection, cardiovascular disease, diagnosis, allergy and tumors.
  • Chemical peptide synthesis is mainly realized by amino acid condensation reaction.
  • the groups that do not need to react should be temporarily protected, and then the connection reaction is carried out to ensure the directional synthesis, so it is necessary to consume More energy and more pollution.
  • Natural products have complex components and very little content of active components, which require a large amount of raw materials and are difficult to quickly separate and detect, which is not conducive to large-scale industrial production.
  • polypeptide biosynthesis is mainly expressed through prokaryotic or eukaryotic microbial expression systems.
  • direct expression of polypeptides has many limitations due to their small molecular weights, such as low expression levels and easy degradation by proteases. Fusion expression is a good choice.
  • the E. coli expression system has the advantages of short growth time, easy molecular manipulation, high-density fermentation, and low cost. It has become the main host for polypeptide production and is developing rapidly.
  • proteases are easily degraded by proteases in cells during microbial expression.
  • Protease is a general term for a class of enzymes that catalyze the hydrolysis of protein peptide chains and is widely distributed.
  • protease-deficient strains to reduce the degradation of peptides or other proteins.
  • protease-deficient strains tend to grow slowly, and it is difficult to quickly carry out high-density fermentation; at the same time, some proteases are required to degrade mutant proteins or provide nutrients, precursors and energy at the same time. Therefore, reducing rather than completely knocking out proteases is a more favorable option in this case.
  • CRISPR/Cas9 system is a powerful genome editing tool that has developed rapidly in recent years. Of course, in addition to being used for gene knockout, CRISPR/Cas9 will soon usher in further transformation. The researchers brought Cas9 to specific genomic sites to regulate the expression of target genes by initiating or stopping transcription.
  • CRISPR Inhibition (CRISPRi) is one such technology that is particularly well-suited for analyzing the specific functions of non-coding RNAs. CRISPRi technology inhibits the transcription of a given target, whether it is a coding or non-coding DNA segment.
  • CRISPRi uses catalytically inactive Cas9 (dCas9), which can reach the site specified by the guide RNA, but is unable to cut DNA.
  • CRISPRi can regulate multiple genes at the same time, and multiple sgRNAs are designed to target different genes.
  • the CRISPRi system can inhibit these genes at the same time, thereby achieving the purpose of using the CRISPRi system to simultaneously regulate multiple genes.
  • linker can reduce the degradation of the polypeptide by proteases.
  • the existence of the linker sequence (linker) is conducive to the correct folding of the protein, and its length and amino acid composition are the main considerations when selecting the linker.
  • Linkers with strong hydrophilicity and good flexibility are the first choice for constructing fusion proteins. Glycine has the smallest molecular weight among all amino acids and has no chiral carbon, so it is the most flexible. It is located between fusion proteins without affecting the conformation and function of the proteins on both sides, and has the least steric hindrance; serine is the most hydrophilic amino acid, which can Increase the hydrophilicity of the fusion protein. So glycine and serine are commonly used amino acids to build linkers.
  • the selection of an appropriate length linker is very important for the high-efficiency expression and solubility of the fusion protein.
  • the purpose of the present invention is to provide a method for increasing the soluble expression level of the polypeptide.
  • a method for improving the solubility and/or expression of exogenous proteins in recombinant bacteria comprising the step of inhibiting the endogenous protease gene in said recombinant bacteria, wherein said Proteases include: ClpA, ClpP, ClpX, or combinations thereof.
  • the protease further includes ClpQ, ClpY, PepD and/or HflB.
  • the recombinant bacterium is recombinant Escherichia coli.
  • the recombinant Escherichia coli includes a recombinant BL21(DE3) strain.
  • the foreign protein is selected from the group consisting of GLP-1, GLP-2, glucagon, glucagon-like peptide, enterokinase, adenosine deaminase, ⁇ / ⁇ - Glucosidase, glutathione reductase.
  • the glucagon-like peptides include glucagon-like peptide-1 and glucagon-like peptide-2.
  • the coding sequence of the foreign protein is a codon-optimized sequence for expression in Escherichia coli.
  • the foreign protein is GLP-1 and its analogs.
  • the GLP-1 analogs include GLP-1 truncated and/or N-terminal extended forms and/or partially amino acid mutated forms.
  • the GLP-1 analog includes Arg34GLP-1(7-31) or Arg34GLP-1(9-31).
  • the CRISPR/Cas9 system is used to suppress the endogenous protease gene of the recombinant E. coli strain.
  • the CRISPR/Cas9 system comprises sgRNA, and the sequence of sgRNA is shown in SEQ ID NO.:11.
  • the CRISPR/Cas9 system includes pdCas9 and pTarget containing the Multi-sgRNA unit, which have kanamycin resistance and ampicillin resistance respectively.
  • the Multi-sgRNA unit comprises the template strand-specific nucleotide sequence of the suppression object, dCas9 hindle, and terminator sequence.
  • a recombinant Escherichia coli in the second aspect of the present invention, is provided, an expression cassette expressing a foreign protein is integrated in the genome of the recombinant Escherichia coli, and the endogenous protease gene of the recombinant Escherichia coli is suppressed;
  • the proteases include: ClpA, ClpP, ClpX, or a combination thereof.
  • the expression cassette for expressing foreign protein includes PET28a, fusion protein, optimized and screened flexible linker, enzyme cleavage site and polypeptide gene.
  • the recombinant Escherichia coli is prepared by the method described in the first aspect of the present invention.
  • the solubility and/or expression of the exogenous protein are increased by at least 50%. , preferably at least 100%, more preferably at least 200%.
  • a method for producing a polypeptide comprising the steps of:
  • the polypeptide is selected from the group consisting of GLP-1, GLP-2, glucagon, glucagon-like peptide, enterokinase, adenosine deaminase, ⁇ / ⁇ -glucose glucosidase, glutathione reductase.
  • a use of the recombinant Escherichia coli described in the second aspect of the present invention is provided, and the strain is used for producing polypeptides.
  • Figure 1 shows the strain construction diagram
  • Figure 2 shows the protein electropherogram.
  • Fig. 2A shows the electrophoresis diagram of shake flask fermentation protein
  • Fig. 2B shows the electrophoresis diagram of fermentation tank fermentation protein.
  • lane a recombinant bacterial protein
  • lane b supernatant after recombinant bacterial cell crushing
  • swimming lane c precipitate after recombinant bacterial cell crushing
  • They are respectively: Marker; whole bacterial protein after cell disruption before induction; whole bacterial protein after cell disruption after induction for 12 hours; whole bacterial protein after cell disruption after induction for 15 hours
  • the electrophoresis figure below is the supernatant after recombinant bacterial cell disruption
  • Marker supernatant after cell disruption before induction; supernatant after cell disruption for 12 hours
  • Figure 3 shows the separation and purification diagram of enzyme digestion. Among them, lane 2: purified fusion protein; lane 3: enzyme digestion at 25°C; lane 4: enzyme digestion at 4°C.
  • FIG. 4 shows the separation and purification results.
  • Figure 5 shows the liquid chromatography results.
  • Figure 6 shows the mass spectrometry results of the target polypeptide.
  • Figure 7 shows the soluble supernatant of intracellular fusion proteins expressed by recombinant bacteria containing different linkers; among them, the lanes 1-8 in the figure are respectively containing linkers (EAAAK) 4 , (EAAAK) 3 , (EAAAK) 2 , EAAAK , (GGGGS) 4 , (GGGGS) 3 , (GGGGS) 2 , and the soluble supernatant of the intracellular fusion protein expressed by recombinant bacteria of GGGGS (the part in the frame).
  • linkers EAAAK) 4 , (EAAAK) 3 , (EAAAK) 2 , EAAAK , (GGGGS) 4 , (GGGGS) 3 , (GGGGS) 2 , and the soluble supernatant of the intracellular fusion protein expressed by recombinant bacteria of GGGGS (the part in the frame).
  • the present invention uses E.coliBL21 (DE3) as the starting strain, and uses CRISPRi technology to inhibit the protease gene of the host cell, thereby reducing the formation of proteases that degrade the expressed polypeptides, thereby reducing the degradation of polypeptide fragments by proteases, and indirectly improving the expression of polypeptides.
  • the yield at the same time, using fusion expression, combining fusion tags and optimized linker to improve the soluble expression of polypeptides.
  • the invention provides the application of CRISPRi suppressor gene in improving the expression of polypeptide
  • the gene has any one of the following nucleotide sequences:
  • the polypeptide is glucagon, glucagon-like peptide-related polypeptide, glucagon-like peptide 1 and glucagon-like peptide 2, including truncated and/or Or in the form of N-terminal extension and site mutation, preferably glucagon-like peptide 1 truncated mutant sequence such as: Arg34GLP-1 (7-31) or Arg34GLP-1 (9-31).
  • the gene is a protease gene ClpA, ClpP, ClpQ, ClpX, ClpY, PepD, HflB;
  • said inhibition employs CRISPRi.
  • CRISPRi technology is used to inhibit the protease gene of the host cell and reduce the formation of protease that degrades the expressed polypeptide, thereby reducing the degradation of the polypeptide fusion protein by the protease, thereby increasing the production of the polypeptide.
  • the CRISPRi includes pdCas9 and pTarget containing Multi-sgRNAunit, respectively having kanamycin resistance and ampicillin resistance.
  • the present invention also provides a Multi-sgRNA unit, which includes the template strand-specific nucleotide sequence, dCas9 hindle, and terminator sequence of the inhibition target.
  • the selection method of the template strand-specific nucleotide sequence is: close to the 5' front end of the template strand, followed by NGG (N is any base ) is a protospacer adjacent motif (PAM) sequence characterized.
  • PAM protospacer adjacent motif
  • the present invention also provides an expression vector, which includes the coding nucleotide sequence of the fusion protein of the present invention.
  • the fusion protein includes a fusion tag, a polypeptide, a linker and an enzyme cutting site.
  • the fusion tag is selected from TrxA (thioredoxin), SUMO (small molecule ubiquitin-like modifying protein), UB (ubiquitin) or MBP (maltose binding protein); preferably TrxA.
  • the Linker includes a flexible linker or a rigid linker.
  • the flexible linker includes but not limited to GGGGS, (GGGGS) 2 , (GGGGS) 3 , (GGGGS) 4
  • the rigid linker includes but not limited to EAAAK, (EAAAK) 2 , (EAAAK) 3 , (EAAAK) 4 ; More preferred is (GGGGS) 3 .
  • the enzyme cleavage site includes but not limited to gene enterokinase, SUMO, ubiquitinase site.
  • the enzyme cleavage site is preferably an enterokinase site.
  • the expression vector is a PET28a plasmid.
  • the invention also provides strains, including the expression vector.
  • the present invention also provides the methods for the shake flask fermentation and high-density fermentation.
  • the application of the method in increasing the expression level.
  • the polypeptide is glucagon, glucagon-like peptide 1 and glucagon-like peptide 2, including truncated and/or N-terminal extended forms.
  • the glucagon-like peptide 1 truncated mutant sequence such as: Arg34GLP-1(7-31) or Arg34GLP-1(9-31).
  • the present invention uses E.coli BL21 (DE3) as the starting strain, uses CRISPRi technology to inhibit the protease gene, reduces the formation of protease, thereby reducing the degradation of polypeptide fragments by protease, and then indirectly increases the output of polypeptide, providing a theoretical basis for future industrial production .
  • CRISPRi technology inhibits protease genes of host cells, reducing the formation of proteases that degrade expressed polypeptides.
  • Escherichia coli BL21(DE3) was used for polypeptide expression.
  • the polypeptide gene is connected to the PET28a vector containing fusion protein, linker and restriction site.
  • the digested DNA fragment was connected to pTarget digested with the same enzyme, transformed into Escherichia coli DH5 ⁇ by conventional methods, screened, and after sequencing and verification, it was named pTarget (the construction process of the strain is shown in Figure 1).
  • Competent cells of Escherichia coli BL21 (DE3) (both purchased from Life Technologies) were prepared according to the calcium chloride method provided in the fourth edition of "Molecular Cloning Experiment Guide” published by Cold Spring Harbor Laboratory, USA. 1 ⁇ L of the sgRNA expression vector and the dCas9 expression vector pdCas9 were simultaneously transformed into Escherichia coli BL21(DE3) competent cells, and the transformation method was also carried out according to the calcium chloride method of the fourth edition of the "Molecular Cloning Experiment Guide”. The transformation solution was applied to LB solid medium added with kanamycin and ampicillin (final concentration: 50 ⁇ g/ml), and cultured upside down at 37°C until a single colony appeared, that is, the mutant strain intended to suppress the target gene was obtained.
  • the genes suppressed in this embodiment include ClpA, ClpP and ClpX, and the sequence of the sgRNA used is shown in SEQ ID NO.:11.
  • the recombinant expression plasmid PET28a-TrxA-(GGGGS) 3 -DDDDK-Arg34GLP-1(7-31) was transformed into its competent cells by the calcium chloride method, and the transformation method was the same
  • the transformation method was the same
  • PET28a was transformed from kanamycin resistance to chloramphenicol resistance through enzyme-cut ligation.
  • the transformation solution was applied to LB solid medium supplemented with chloramphenicol (final concentration: 50 ⁇ g/ml), cultured upside down at 37° C. until a single colony appeared, and sequenced to verify that the recombinant expression strain was obtained.
  • Embodiment 2 Shake flask fermentation
  • the recombinant expression strain containing CRISPRi ClpA-ClpP-ClpX prepared in Example 1 was prepared in a single cell containing appropriate antibiotics (100 ⁇ g/ml ampicillin, 50 ⁇ g/ml chloramphenicol, 100 ⁇ g/ml kanamycin) in LB liquid medium, cultivate overnight at 37°C and 220rpm; transfer the culture to 50mL fresh LB medium (250mL shake flask) at a ratio of 1:100, and incubate at 37 grow at °C.
  • appropriate antibiotics 100 ⁇ g/ml ampicillin, 50 ⁇ g/ml chloramphenicol, 100 ⁇ g/ml kanamycin
  • IPTG Isopropylthiogalactopyranoside
  • the separation and purification of polypeptides first adopts ion exchange adsorption fusion protein, Buffer A 25mMCH3COONa, pH4.5; Buffer B 25mM PBS, 1M NaCl pH7.5; The fusion protein was exchanged by ultrafiltration for enzyme digestion buffer, and then further passed through the ion exchange column balanced by Buffer A after enzyme digestion, and the flow-through part was the target polypeptide product.
  • the electrophoresis results are shown in Figure 3.
  • Embodiment 4 high-density fermentation
  • the fermentation culture is divided into two stages. In the first stage, after the inoculation, the culture is about 4 hours, the carbon source in the medium is completely consumed, and the feedback feeding is carried out according to DO. After feeding, the temperature dropped to 30°C, and the dissolved oxygen was kept above 30%. After 8 hours of feeding, isopropylthiogalactopyranoside (IPTG) was added for induction, and after 12 hours of induction, the tank was placed.
  • IPTG isopropylthiogalactopyranoside
  • Comparative example 1 CRISPRi technology inhibits different protease genes
  • CRISPRi technology was used to suppress ClpA and/or ClpP and/or ClpX genes, as well as ClpQ, ClpY, PepD, and HflB genes in Escherichia coli.
  • the NCBIGene IDs of ClpA, ClpP, ClpX, ClpQ, ClpY, PepD, and HflB sequences are: 945764, 945082, 945083, 948429, 948430, 945013, and 947690; at the same time, select the appropriate gene segment according to the target gene sequence to be edited As a component of sgRNA.
  • Multi-sgRNA unit based on the protease sequence, including the template strand-specific nucleotide sequence, dCas9 hindle, and terminator sequence of the target to be inhibited.
  • the name of the gene targeted by the sgRNA, the nucleotide sequence of the sgRNA, and the sequence number (SEQID NO.) are shown below:
  • HflB CTTTCGCTACGCTACGGCCGGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTTTT (SEQ ID NO.: 6);
  • Fermentation was carried out using a method similar to that of Example 2, and the yield of the target product was detected, and the expression strain not inhibited by CRISPRi was used as a control strain, and the percentage change in yield of each protease-inhibited recombinant expression strain compared to the control strain was calculated.
  • the volumetric yield of the E. coli recombinant expression strain containing the CRISPRi ClpA plasmid can reach 156%; the fermentation culture of the E. coli recombinant expression strain containing the CRISPRi ClpX plasmid The volumetric yield can reach 183%; the fermentation volume yield of the E. coli recombinant expression strain containing CRISPRi ClpP plasmid can reach 200%; the fermentation culture volume yield of the E. coli recombinant expression strain containing CRISPRi ClpA-ClpP-ClpX can reach 230%.
  • CRISPRi plasmid and expression plasmid When the co-expression of CRISPRi plasmid and expression plasmid was carried out, cell growth was basically not affected, but the protein expression level was greatly increased, indicating that this method can effectively inhibit the expression of protease genes, that is, CRISPRi can effectively regulate gene expression.
  • ClpP protease is an important heat shock protein in cells, and it mainly acts as a proteolytic enzyme in the body to degrade abnormal proteins or short-lived proteins.
  • ClpP often combines with ATPase subunits (ClpA/ClpX, etc.) to form a Clp complex.
  • the formation of the complex can more effectively degrade complex polypeptides and folded proteins.
  • ClpA/ClpX can specifically recognize the substrate and unfold it, and the unfolded polypeptide is transported to the central hydrolysis cavity of the ClpP protein for hydrolysis.
  • Lanes 1-8 in the protein electrophoresis map contain linker(EAAAK) 4 , (EAAAK) 3 , (EAAAK) 2 , EAAAK, (GGGGS) 4 , (GGGGS) 3 , (GGGGS) 2 , GGGGS respectively
  • the soluble supernatant of the intracellular fusion protein expressed by the recombinant bacteria the part in the frame; the optical density comparison shows that the 6th lane has the highest soluble ratio of the fusion protein containing linker (GGGGS) 3 .
  • proteases can degrade proteins and peptides, so reducing protein degradation by proteases is also an indirect way to increase protein production. Therefore, we investigated the effect of proteases on the expression of polypeptides in E. coli.
  • Lon is a DNA-binding ATP-dependent protease, which belongs to serine protease; OmpT, also known as outer membrane protease VII, belongs to serine protease; Escherichia coli BL21 (DE3) has knocked out Lon and OmpT.
  • OmpT also known as outer membrane protease VII
  • Escherichia coli BL21 (DE3) has knocked out Lon and OmpT.
  • the knockout of too many protease genes will change the growth state of the strain, so we tried to interfere with the transcription of protease genes through CRISPRi, and reduce the expression of some proteases without affecting the growth of the strain.
  • ClpA, ClpP and ClpX have a significant effect on reducing the degradation of polypeptide fusion proteins of recombinant engineering bacteria, thereby indirectly increasing the yield of polypeptides.
  • the present invention also screens out a more suitable linker, adopts fusion expression, and combines the fusion tag and the preferred linker to increase the soluble expression of the polypeptide.

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Abstract

一种提高多肽可溶性表达产量的方法，包括以E.coliBL21(DE3)为出发菌株，利用CRISPRi技术抑制蛋白酶基因，减少降解所表达多肽的蛋白酶的形成，从而减少蛋白酶对多肽片段的降解，进而提高多肽的产量；同时，筛选出更适宜的linker，采用融合表达，结合融合标签和优选的linker提高多肽的可溶性表达量。

Description

一种提高多肽可溶性表达产量的方法 技术领域
本发明涉及生物技术领域，更具体地涉及一种提高多肽可溶性表达量的方法。
背景技术
多肽是比蛋白质简单、分子量小，由氨基酸通过肽键相连的一类化合物。目前已有60种以上的多肽药物上市，数百种多肽药物处于临床研究阶段。与传统的小分子药物相比，多肽类药物的结构与天然来源的蛋白质结构类似，性质与体内正常生理物质十分接近，药理活性较高，且其代谢产物为小分子氨基酸，安全性高；较大分子蛋白药物相比，多肽药物不易被免疫系统识别而引起过敏反应。因此，近年来多肽药物呈现快速发展的趋势，己经被用在不同的治疗领域，如糖尿病、关节炎、肥胖症、抗感染、心血管疾病、诊断、过敏和肿瘤等。
合成多肽有以下三种途径：化学多肽合成、天然提取和生物多肽合成。
1、化学多肽合成主要通过氨基酸缩合反应来实现。为得到具有特定顺序的合成多肽，当合成原料中包含两个以上氨基酸单体时，应将不需要反应的基团暂时保护起来，然后再进行连接反应，以保证合成的定向进行，因此需要消耗较多能量且产生较大污染。
2、从天然产物中提取活性组分。天然产物成分复杂、活性组分含量非常少，则需要消耗大量原材料，且难以进行快速分离和检测，不利于大规模工业生产。
3、生物合成利用基因重组技术，将多肽的基因序列构建到载体上，形成重组DNA表达载体，在原核或真核细胞中进行多肽分子表达，并进一步分离纯化。随着基因工程生产多肽的技术趋于完善，加快了基因工程类多肽药物的研制及临床应用的步伐，具有巨大的潜力。
目前，多肽生物合成主要是通过原核或真核微生物表达系统进行表达。然而，多肽由于分子量较小直接表达有诸多限制，例如，表达量低、易被蛋白酶降解等。融合表达是一种很好的选择。而与真核表达系统相比，大肠杆菌表达系统具有生长时间短、易于进行分子操作、可以实现高密度发酵、成本低廉等优势，成为生产多肽的主要宿主，并快速发展。目前，已有多种在大肠杆菌中生产的多肽或蛋白被美国FDA批准上市：阿斯利康公司的艾塞纳肽(通用名Exenatide)、礼来公司生产的人胰岛素(通用名Humulin)、赛诺菲公司生产的甘精胰岛素(通用名Lantus)和Genentech公司生产的Ranibizumab(Lucentis)等。
微生物表达过程中多肽易被细胞中的蛋白酶降解。蛋白酶是催化蛋白质肽链水解的一类酶的总称，分布广泛。现有的一些研究开发了蛋白酶缺陷菌株来减少多肽或其他蛋白质的降解。但是蛋白酶缺陷菌株往往生长缓慢，难以快速进行高密度发酵；同时有些蛋白酶同时是降解变异蛋白或提供营养、前体和能量所需要的。因此，这种情况下减少而不是完全敲除蛋白酶是一种更有利的选择。
CRISPR/Cas9系统是近些年发展迅猛的强大的基因组编辑工具。当然，除了用作基因敲除外，CRISPR/Cas9很快迎来了进一步的改造。研究者们将Cas9带到特定的基因组位点，通过起始或停止转录来调控靶基因的表达。CRISPR抑制(CRISPRi)就是这样的技术，它特别适合分析非编码RNA的具体功能。CRISPRi技术能抑制指定目标的转录，不论是编码还是非编码的DNA片段。CRISPRi使用催化失活的Cas9(dCas9)，dCas9能到达引导RNA指定的位点，但无力对DNA进行剪切。当dCas9结合到基因组的时候，会阻断转录机器的结合，阻止这一过程的进行。CRISPRi能够同时调节多个基因，设计多个sgRNA分别靶向不同基因，CRISPRi系统可将这几个基因同时抑制，由此达到利用CRISPRi系统同时对多基因进行调节的目的。
此外，融合表达能减少蛋白酶对多肽的降解。接头序列(linker)的存在有利于蛋白的正确折叠，其长度和氨基酸组成是选择linker时主要考虑的问题。亲水性强和柔性好的linker是构建融合蛋白的首选。甘氨酸是所有氨基酸中分子量最小，没有手性碳，所以柔性最好，位于融合蛋白之间不会影响两边蛋白的构象和功能，且空间位阻最小；丝氨酸是亲水性最强的氨基酸，能增加融合蛋白的亲水性。所以甘氨酸和丝氨酸是构建linker的常用氨基酸。Linker长度过长，融合蛋白在生产过程中对蛋白酶比较敏感，导致活性融合蛋白的产量降低；linker长度太短，可能使两个分子距离太近导致蛋白功能丧失。因此，合适长度的linker选择对融合蛋白的高效表达和可溶性非常重要。
发明内容
本发明的目的在于提供一种提高多肽可溶性表达量的方法。
在本发明的第一方面，提供了一种提高重组菌中外源蛋白可溶性和/或表达量的方法，所述方法包括抑制所述重组菌中内源的蛋白酶基因的步骤，其中，所述的蛋白酶包括：ClpA、ClpP、ClpX、或其组合。
在另一优选例中，所述蛋白酶还包括ClpQ、ClpY、PepD和/或HflB。
在另一优选例中，所述重组菌为重组大肠杆菌。
在另一优选例中，所述的重组大肠杆菌包括重组BL21(DE3)菌株。
在另一优选例中，所述的外源蛋白选自下组：GLP-1、GLP-2、胰高血糖素、胰 高血糖素样肽、肠激酶、腺苷脱氨酶、α/β-葡萄糖苷酶、谷胱甘肽还原酶。
在另一优选例中，所述的胰高血糖素样肽包括胰高血糖素样肽1和胰高血糖素样肽2。
在另一优选例中，所述外源蛋白的编码序列是经密码子优化以在大肠杆菌中表达的序列。
在另一优选例中，所述的外源蛋白为GLP-1及其类似物。
在另一优选例中，所述GLP-1类似物包括GLP-1截短和/或N-末端延伸的形式和/或部分氨基酸突变形式。
在另一优选例中，所述GLP-1类似物包括Arg34GLP-1(7-31)或Arg34GLP-1(9-31)。
在另一优选例中，利用CRISPR/Cas9系统抑制所述重组大肠杆菌菌株的内源的蛋白酶基因。
在另一优选例中，所述的CRISPR/Cas9系统包含sgRNA，且sgRNA的序列如SEQ IDNO.:11所示。
在另一优选例中，所述的CRISPR/Cas9系统包括pdCas9和含有Multi-sgRNA unit的pTarget，分别具有卡那霉素抗性和氨苄青霉素抗性。
在另一优选例中，所述Multi-sgRNA unit包含抑制对象的模板链特异核苷酸序列、dCas9 hindle、terminator sequence。
在本发明的第二方面，提供了一种重组大肠杆菌，所述重组大肠杆菌的基因组中整合有表达外源蛋白的表达盒，并且，所述重组大肠杆菌的内源的蛋白酶基因被抑制；所述的蛋白酶包括：ClpA、ClpP、ClpX、或其组合。
在另一优选例中，所述的表达外源蛋白的表达盒包括PET28a、融合蛋白、优化筛选后的柔性linker、酶切位点和多肽基因。
在另一优选例中，所述的重组大肠杆菌利用本发明第一方面所述的方法制备。
在另一优选例中，与整合相同外源蛋白表达盒的野生型大肠杆菌相比，所述内源蛋白酶基因被抑制的重组大肠杆菌中，外源蛋白可溶性和/或表达量提高至少50％，较佳地至少100％，更佳地至少200％。
在本发明的第三方面，提供了一种生产多肽的方法，所述方法包括步骤：
(i)培养如本发明第二方面所述的重组大肠杆菌，从而获得含所述多肽的发酵产物；和
(ii)从所述的发酵产物中分离出所述多肽。
在另一优选例中，所述的多肽选自下组：GLP-1、GLP-2、胰高血糖素、胰高血糖素样肽、肠激酶、腺苷脱氨酶、α/β-葡萄糖苷酶、谷胱甘肽还原酶。
在本发明的第四方面，提供了一种本发明第二方面所述的重组大肠杆菌的用途， 所述菌株用于生产多肽。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
附图说明
图1显示了菌株构建图。
图2显示了蛋白电泳图。其中，图2A显示了摇瓶发酵蛋白电泳图；图2B显示了发酵罐发酵蛋白电泳图。图2A中，泳道a：重组菌全菌蛋白；泳道b：重组菌细胞破碎后上清；泳道c：重组菌细胞破碎后沉淀；图2B中，上方电泳图为重组菌全菌蛋白，各泳道分别为:Marker；诱导前细胞破碎后全菌蛋白；诱导12小时细胞破碎后全菌蛋白；诱导15小时细胞破碎后全菌蛋白；下方电泳图为重组菌细胞破碎后上清；各泳道分别为:Marker；诱导前细胞破碎后上清；诱导12小时细胞破碎后上清；诱导15小时细胞破碎后上清。
图3显示了酶切分离纯化图。其中，泳道2：纯化融合蛋白；泳道3：25℃酶切；泳道4：4℃酶切。
图4显示了分离纯化结果。
图5显示了液相色谱结果。
图6显示了目的多肽的质谱结果。
图7显示了含不同linker的重组菌表达后的胞内融合蛋白可溶上清；其中，图中1-8泳道分别为含linker(EAAAK) 4、(EAAAK) 3、(EAAAK) 2、EAAAK、(GGGGS) 4、(GGGGS) 3、(GGGGS) 2、GGGGS的重组菌表达后的胞内融合蛋白可溶上清(框内部分)。
具体实施方式
本发明人经过广泛而深入地研究，发现了抑制蛋白酶基因在提高多肽可溶性表达产量中的应用。具体地，本发明以E.coliBL21(DE3)为出发菌株，利用CRISPRi技术抑制宿主细胞的蛋白酶基因，从而减少降解所表达多肽的蛋白酶的形成，从而减少蛋白酶对多肽片段的降解，进而间接提高多肽的产量；同时，采用融合表达，结合融合标签和优化的linker来提高多肽的可溶性表达。
为了实现上述发明目的，本发明提供以下技术方案：
本发明提供了CRISPRi抑制基因在提高多肽的表达量中的应用；
所述基因具有如下所示的核苷酸序列中的任意一项：
I、具有基因ClpA、ClpP、ClpQ、ClpX、ClpY、PepD、HflB的核苷酸序列；优选ClpA和/或ClpP和/或ClpX的核苷酸序列；
II、具有如I所示的核苷酸序列经修饰、取代、缺失或添加一个或多个碱基获得的核苷酸序列；
III、与如I所示的核苷酸序列具有至少80％同源性的序列或翻译后所得蛋白与基因ClpA和/或ClpP和/或ClpX表达的蛋白功能相同或相近的核苷酸序列；
IV、如I、II或III所示序列的互补序列。
在本发明的一些具体实施方案中，所述多肽为胰高血糖素、胰高血糖素样肽相关多肽、胰高血糖素样肽1和胰高血糖素样肽2，其中包括截短和/或N-末端延伸及位点突变的形式，优选胰高血糖素样肽1截短突变序列如：Arg34GLP-1(7-31)或Arg34GLP-1(9-31)。
在本发明的一些具体实施方案中，所述基因为蛋白酶基因ClpA、ClpP、ClpQ、ClpX、ClpY、PepD、HflB；
在本发明的一些具体实施方案中，所述抑制采用CRISPRi。利用CRISPRi技术抑制宿主细胞的蛋白酶基因，减少降解所表达多肽的蛋白酶的形成，从而减少蛋白酶对多肽融合蛋白的降解，进而提高多肽的产量。
在本发明的一些具体实施方案中，所述CRISPRi包括pdCas9和含有Multi-sgRNAunit的pTarget，分别具有卡那霉素抗性和氨苄青霉素抗性。
在此基础上，本发明还提供了Multi-sgRNA unit，包含抑制对象的模板链特异核苷酸序列、dCas9 hindle、terminator sequence。
在本发明的一些具体实施方案中，所述Multi-sgRNA unit，所述的模板链特异核苷酸序列的选定方法为：靠近模板链5‘前端，后面为以NGG(N为任意碱基)为特点的protospacer adjacent motif(PAM)序列。
本发明还提供了表达载体，所述表达载体包括本发明的融合蛋白的编码核苷酸序列。所述融合蛋白包括融合标签、多肽、linker和酶切位点。
基于本发明的教导，本领域技术人员可以知晓并选择各种适用于本发明的融合蛋白中的融合标签、多肽、linker和酶切位点。在具体的实施方式中，所述融合标签选自TrxA(硫氧还蛋白)、SUMO(小分子泛素样修饰蛋白)、UB(泛素)或MBP(麦芽糖结合蛋白)；优选TrxA。在一个具体的实施方式中，所述Linker包括柔性linker或刚性linker。所述柔性linker包括但不限于GGGGS、(GGGGS) 2、(GGGGS) 3、(GGGGS) 4，所述刚性linker包括但不限于EAAAK、(EAAAK) 2、(EAAAK) 3、(EAAAK) 4；更优选(GGGGS) 3。在一个具体的实施方式中，所述酶切位点包括但不限于基因肠激酶、SUMO、泛素酶位点。在一个优选的实施方式中，所述酶切位点是优选肠激酶位点。
基于本发明的教导，本领域技术人员可以知晓并选择各种适用于本发明的表达载体。例如，所述表达载体是PET28a质粒。
本发明还提供了菌株，包括所述的表达载体。
在上述研究的基础上，本发明还提供了所述的摇瓶发酵及高密度发酵的方法。所述方法在提高表达量中的应用。在本发明的一些具体实施方案中，所述多肽为胰高血糖素、胰高血糖素样肽1和胰高血糖素样肽2，其中包括截短和/或N-末端延伸的形式。优选胰高血糖素样肽1截短突变序列如：Arg34GLP-1(7-31)或Arg34GLP-1(9-31)。
本发明以E.coli BL21(DE3)为出发菌株，利用CRISPRi技术抑制蛋白酶基因，减少蛋白酶的形成，从而减少蛋白酶对多肽片段的降解，进而间接提高多肽的产量，为将来的工业化生产提供理论依据。
本发明的主要优点包括：
(a)融合标签结合优化的linker提高多肽可溶性表达比例。
(b)CRISPRi技术抑制宿主细胞的蛋白酶基因，减少降解所表达多肽的蛋白酶的形成。
(c)多肽降解减少，可溶性多肽表达比例提高。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件，例如Sambrook等人，分子克隆：实验室手册(New York:Cold Spring HarborLaboratory Press，1989)中所述的条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数是重量百分比和重量份数。
实施例1重组工程菌的构建
在本实施例中，大肠杆菌BL21(DE3)用于多肽表达。多肽基因连接到包含融合蛋白、linker、酶切位点的PET28a载体中。
1.抑制基因CRISPRi菌株的构建
首先根据拟编辑靶标基因序列选择合适的基因区段作为sgRNA的组成部分委托基因合成服务公司合成完整的sgRNA基因序列，合成好后使用Thermo公司的限制性内切酶SspI和EcoR I对sgRNA基因序列进行酶切，同样用Ssp I和EcoR I对质粒pTarget(购自Addgene公司)进行酶切。将酶切后的DNA片段连接至经同样酶酶切的pTarget，经常规的方法转化大肠杆菌DH5α，筛选，测序验证正确后，命名为pTarget(菌株的构建过程如图1所示)。
按照美国冷泉港实验室出版的《分子克隆实验指南》第四版提供的氯化钙法，制备大肠杆菌BL21(DE3)(均购自Life Technologies公司)感受态细胞。分别取1μL sgRNA表达载体以及dCas9表达载体pdCas9同时转化至大肠杆菌BL21(DE3)感受态细胞，转化方法同样按照《分子克隆实验指南》第四版的氯化钙法进行。将转化液涂布至添加了卡那霉素和氨苄青霉素(终浓度为50μg/ml)的LB固体培养基，37℃倒置培养 直到出现单菌落，即获得了拟抑制靶标基因的突变菌株。
具体地，本实施例抑制的基因包括ClpA、ClpP和ClpX，使用的sgRNA的序列如SEQID NO.:11所示。
2.重组多肽表达载体的构建
以已抑制靶标基因的突变菌株为宿主菌，将重组表达质粒PET28a-TrxA-(GGGGS) 3-DDDDK-Arg34GLP-1(7-31)用氯化钙法转化至其感受态细胞，转化方法同样按照《分子克隆实验指南》第四版的氯化钙法进行。PET28a经过酶切连接改造将卡那霉素抗性改为氯霉素抗性。将转化液涂布至添加了氯霉素(终浓度为50μg/ml)的LB固体培养基，37℃倒置培养直到出现单菌落，测序验证，即获得了重组表达菌株。
实施例2摇瓶发酵
将实施例1制备的含CRISPRi ClpA-ClpP-ClpX的重组表达菌株(抑制ClpA、ClpP和ClpX的重组表达菌株)以单个细胞在含有适当抗生素(100μg/ml氨苄青霉素、50μg/ml氯霉素、100μg/ml卡那霉素)的LB液体培养基中培养，37℃和220rpm下过夜培养；将培养物以1:100比例转接入50mL新鲜LB培养基(250mL摇瓶)中，并在37℃下生长。当600nm处的光密度(OD600)达到约0.6时，培养箱温度降至25℃，平衡温度20分钟。加入异丙基硫代半乳糖苷(IPTG)，使其终浓度为1mM，细胞在25℃下生长16小时。
摇瓶发酵的电泳结果如图2A所示，表明融合蛋白成功表达。
实施例3多肽的分离纯化
将实施例2获得的发酵后的培养物，6500rpm和4℃收集并离心。细胞用0.01M PBS(pH＝7.2-7.4)洗涤。用PBS重悬细胞并进行高压破碎处理。然后，将细胞裂解物在10，000rpm和4℃下离心30分钟，收集上清液，使用0.45μm滤膜进行过滤。
在本实施例中，多肽分离纯化先采用离子交换吸附融合蛋白，Buffer A 25mMCH3COONa，pH4.5；Buffer B 25mM PBS，1M NaCl pH7.5；Buffer A平衡上样，Buffer B洗脱；洗脱后的融合蛋白使用超滤进行酶切缓冲液交换，酶切后再进一步通过经Buffer A平衡的离子交换柱，流穿部分即为目的多肽产物，电泳结果如图3所示。
本实施例中通过酶切融合蛋白后Arg34GLP-1(7-31)的分离纯化及液相色谱检测结果分别见图4(具体是横坐标300ml所对应的峰)和图5(具体是保留时间为15.273min时的峰)；质谱结果见图6(Spectrum from 20180206_JXZ_biaopin.wiff(sample 1)-20180206_JXZ_biaopin，Experiment 1，+TOF MS(100-2000)from 21.249min)，图6中显著的质荷比数值分别为*677.3414(5)、677.5425(5)、677.7428(5)、677.9432(5)、678.1436(5)、678.3440(5)、*846.4247(4)、846.6759(4)、846.9271(4)、847.1770(4)、847.4281(4)、847.6772(4)、847.9279(4)、852.1695(4)、852.4241(4)、*1128.2299(3)、 1128.5651(3)、1128.8998(3)、1129.2339(3)、1129.5664(3)、1129.9031(3)、*1135.5584(3)、1136.2275(3)、1136.5614(3)、1136.8936(3)、1692.3404(2)、1692.8494(2)。
结果表明，成功获得Arg34GLP-1(7-31)多肽。
实施例4高密度发酵
接种实施例1获得的含CRISPRi ClpA-ClpP-ClpX的重组表达菌株于3mL液体LB培养基中，37℃、250rpm震荡过夜培养后，按1％左右的比例接种于400mL液体LB培养基中，培养至OD600达到4时作为种子液，接入2L的发酵培养基中进行高密度发酵。初始温度37℃，搅拌速度300rpm，通气量1.5vvm/L/min，pH为6.8，之后不断提高搅拌转速最大至1000rpm。高密度发酵需氧量大，如氧气缺乏，会产生乙酸，甚至造成菌体裂解释放有害物质，对重组工程菌的表达产生不可逆的影响。因此，后期需补充一定的纯氧。发酵培养分为两个阶段，第一阶段接种后，培养4小时左右，培养基中的碳源消耗完全，按照DO进行反馈补料。补料后温度降为30℃，溶氧保持30％以上，补料8小时后加入异丙基硫代半乳糖苷(IPTG)进行诱导，诱导12小时后放罐。
结果显示，目的产物产量可达2g/L，与对照菌株(未采用CRISPRi)相比产量可提高2倍以上。发酵罐表达电泳结果如图2B所示。从表1可以看出，未采用CRISPRi的工程菌的融合蛋白可溶体积产量为100％，采用CRISPRi的工程菌融合蛋白可溶体积产量最高可达253％。
对比例1 CRISPRi技术抑制不同蛋白酶基因
采用实施例1类似的方法，利用CRISPRi技术抑制不同种类的蛋白酶基因，减少蛋白酶的形成，从而减少蛋白酶对多肽片段的降解，进而间接提高多肽的产量。
具体地，利用CRISPRi技术在大肠杆菌中抑制ClpA和/或ClpP和/或ClpX基因，以及ClpQ、ClpY、PepD、HflB基因。其中，ClpA、ClpP、ClpX、ClpQ、ClpY、PepD、HflB序列的NCBIGene ID分别为:945764、945082、945083、948429、948430、945013、947690；同时，根据拟编辑靶标基因序列选择合适的基因区段作为sgRNA的组成部分。
根据蛋白酶序列设计Multi-sgRNA unit，包含抑制对象的模板链特异核苷酸序列、dCas9 hindle、终止子序列。
以下分别显示了sgRNA所靶向的基因名称、sgRNA的核苷酸序列以及序列编号(SEQID NO.)：
ClpA:CTTGATCTCTTCCATCGCATGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTT(SEQ ID NO.:1)；
ClpX:GTACATGGTATCGAGCAGTGGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTT TT(SEQ ID NO.:2)；
ClpP:GTATTCCACCGCTTCAGGGGGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTT(SEQ ID NO.:3)；
ClpQ:AAGTTTGCGCAGCATGCGATGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTT(SEQ ID NO.:4)；
ClpY:TCAGCGCCTGCAGTTCAACGGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTT(SEQ ID NO.:5)；
HflB:CTTTCGCTACGCTACGGCCGGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTT(SEQ ID NO.:6)；
PepD:ATCACCGGAGAATTAGCGTCGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTT(SEQ ID NO.:7)；
ClpA-ClpP:CTTGATCTCTTCCATCGCATGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTTGTATTCCACCGCTTCAGGGGGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTT(SEQID NO.:8)；
ClpP-ClpX:GTATTCCACCGCTTCAGGGGGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTTGTACATGGTATCGAGCAGTGGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTT(SEQID NO.:9)；
ClpA–ClpX:CTTGATCTCTTCCATCGCATGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTTGTACATGGTATCGAGCAGTGGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTT(SEQID NO.:10)；
ClpA-ClpP-ClpX:CTTGATCTCTTCCATCGCATGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTTGTATTCCACCGCTTCAGGGGGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTTGTACATGGTATCGAGCAGTGGTTTTAGAGCTAGAAATAGCAAGTTAAAATAA GGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTT(SEQ ID NO.:11)。
采用实施例2类似的方法进行发酵，检测目的产物的产量，并以未经CRISPRi抑制的表达菌株作为对照菌株，计算各个蛋白酶被抑制的重组表达菌株相比对照菌株的产量变化百分比。
结果如下表1所示，与未进行基因编辑的对照组相比，含有CRISPRi ClpA质粒的大肠杆菌重组表达菌株发酵培养体积产率可达156％；含有CRISPRi ClpX质粒的大肠杆菌重组表达菌株发酵培养体积产率可达183％；含有CRISPRi ClpP质粒的大肠杆菌重组表达菌株发酵培养体积产率可达200％；含有CRISPRi ClpA-ClpP-ClpX大肠杆菌重组表达菌株发酵培养体积产率可达230％。在进行CRISPRi质粒和表达质粒的共表达时，细胞生长基本不受影响，但蛋白表达水平大幅提高，表明该方法可以有效的抑制蛋白酶基因的表达，即CRISPRi可以有效的调控基因表达。
表1.不同大肠杆菌重组表达菌株发酵培养体积
Figure PCTCN2022114414-appb-000001
ClpP蛋白酶是细胞内一种重要的热休克蛋白，在体内主要发挥蛋白水解酶作用，降解异常蛋白或短寿期蛋白。ClpP作为蛋白酶亚基常与ATP酶亚基(ClpA/ClpX等)结合成Clp复合物，复合物的形成能更为有效地降解复杂的多肽和折叠的蛋白。ClpA/ClpX作为分子伴侣能特异性地识别底物并将其去折叠，去折叠的多肽被运输到ClpP蛋白中心水解腔进行水解。
对比例2
合成包含柔性linker：GGGGS、(GGGGS) 2、(GGGGS) 3、(GGGGS) 4；刚性linker：EAAAK、(EAAAK) 2、(EAAAK) 3、(EAAAK) 4的不同重组表达载体PET28a-TrxA-linker-DDDDK-Arg34GLP-1(7-31)；不同重组表达载体导入宿主感受态后，分别进行摇瓶表达筛选，筛选结果显示，含linker：(GGGGS) 3的重组菌可溶性表达比例最佳。请见图7:蛋白电泳图中1-8泳道分别为含linker(EAAAK) 4、(EAAAK) 3、(EAAAK) 2、EAAAK、(GGGGS) 4、(GGGGS) 3、(GGGGS) 2、GGGGS的重组菌表达后的胞内融合蛋白可溶上清(框内部分)；通过光密度比对显示第6泳道，即含linker(GGGGS) 3的融合蛋白可溶性比例最高。
讨论
蛋白酶对大肠杆菌中多肽表达的影响
蛋白酶可以降解蛋白质和肽，所以减少蛋白酶降解蛋白质也是一种间接提高蛋白质产量的方法。因此，我们研究了蛋白酶对大肠杆菌表达多肽的影响。
Lon是DNA结合ATP依赖性蛋白酶，属于丝氨酸蛋白酶；OmpT又名外膜蛋白酶VII，属于丝氨酸蛋白酶；大肠杆菌BL21(DE3)已敲除了Lon和OmpT。但过多蛋白酶基因的敲除会改变菌株的生长状态，因此我们尝试通过CRISPRi对蛋白酶基因的转录进行干扰，在不影响菌株生长的情况下，减少部分蛋白酶的表达。基于实验筛选ClpA、ClpP和ClpX对减少重组工程菌多肽融合蛋白降解效果显著，进而间接提高了多肽的产量。
同时，本发明还筛选出了更适宜的linker，采用融合表达，结合融合标签和优选的linker来提高多肽的可溶性表达量。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (19)

  1. 一种提高重组菌中外源蛋白可溶性和/或表达量的方法，所述方法包括抑制所述重组菌中内源的蛋白酶基因的步骤，其中，所述的蛋白酶包括：ClpA、ClpP、ClpX、或其组合。
  2. 如权利要求1所述的方法，其特征在于，所述蛋白酶还包括ClpQ、ClpY、PepD和/或HflB。
  3. 如权利要求1所述的方法，其特征在于，所述重组菌为重组大肠杆菌；优选重组BL21(DE3)菌株。
  4. 如权利要求1所述的方法，其特征在于，所述的外源蛋白选自下组：GLP-1、GLP-2、胰高血糖素、胰高血糖素样肽、肠激酶、腺苷脱氨酶、α/β-葡萄糖苷酶、谷胱甘肽还原酶。
  5. 如权利要求4所述的方法，其特征在于，所述的胰高血糖素样肽包括胰高血糖素样肽1和胰高血糖素样肽2。
  6. 如权利要求1所述的方法，其特征在于，所述外源蛋白的编码序列是经密码子优化以在大肠杆菌中表达的序列。
  7. 如权利要求1所述的方法，其特征在于，所述的外源蛋白为GLP-1及其类似物；
    优选地，所述GLP-1类似物包括GLP-1截短和/或N-末端延伸的形式和/或部分氨基酸突变形式；
    更优选地，所述GLP-1类似物包括Arg34GLP-1(7-31)或Arg34GLP-1(9-31)。
  8. 如权利要求3所述的方法，其特征在于，利用CRISPR/Cas9系统抑制所述重组大肠杆菌菌株的内源的蛋白酶基因。
  9. 如权利要求8所述的方法，其特征在于，所述的CRISPR/Cas9系统包含sgRNA，且sgRNA的序列如SEQ ID NO.:11所示。
  10. 如权利要求8所述的方法，其特征在于，所述的CRISPR/Cas9系统包括pdCas9和含有Multi-sgRNA unit的pTarget，分别具有卡那霉素抗性和氨苄青霉素抗性；
    优选地，所述Multi-sgRNA unit包含抑制对象的模板链特异核苷酸序列、dCas9 hindle、terminator sequence。
  11. 一种重组大肠杆菌，所述重组大肠杆菌的基因组中整合有表达外源蛋白的表达盒，并且，所述重组大肠杆菌的内源的蛋白酶基因被抑制；所述的蛋白酶包括：ClpA、ClpP、ClpX、或其组合。
  12. 如权利要求11所述的重组大肠杆菌，其特征在于，所述的表达外源蛋白的表达盒包括PET28a、融合蛋白、优化筛选后的柔性linker、酶切位点和多肽基因。
  13. 如权利要求12所述的重组大肠杆菌，其特征在于，所述柔性linker选自：GGGGS、(GGGGS) 2、(GGGGS) 3、(GGGGS) 4；
    优选地，所述柔性linker是(GGGGS) 3。
  14. 如权利要求11-13中任一项所述的重组大肠杆菌，其特征在于，所述的重组大肠杆菌利用权利要求1-12中任一项所述的方法制备。
  15. 如权利要求11所述的重组大肠杆菌，其特征在于，与整合相同外源蛋白表达盒的野生型大肠杆菌相比，所述内源蛋白酶基因被抑制的重组大肠杆菌中，外源蛋白可溶性和/或表达量提高至少50％，较佳地至少100％，更佳地至少200％。
  16. 一种生产多肽的方法，所述方法包括步骤：
    (i)培养如权利要求11-15中任一项所述的重组大肠杆菌，从而获得含所述多肽的发酵产物；和
    (ii)从所述的发酵产物中分离出所述多肽。
  17. 如权利要求16所述的方法，其特征在于，所述的多肽选自下组：GLP-1、GLP-2、胰高血糖素、胰高血糖素样肽、肠激酶、腺苷脱氨酶、α/β-葡萄糖苷酶、谷胱甘肽还原酶。
  18. 权利要求11-15中任一项所述的重组大肠杆菌的用途，所述菌株用于生产多肽。
  19. 如权利要求18所述的用途，其特征在于，所述的多肽选自下组：GLP-1、GLP-2、胰高血糖素、胰高血糖素样肽、肠激酶、腺苷脱氨酶、α/β-葡萄糖苷酶、谷胱甘肽还原酶。
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