WO2014177021A1 - 强分泌性信号肽增强小肽模序及其应用 - Google Patents

强分泌性信号肽增强小肽模序及其应用 Download PDF

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WO2014177021A1
WO2014177021A1 PCT/CN2014/076249 CN2014076249W WO2014177021A1 WO 2014177021 A1 WO2014177021 A1 WO 2014177021A1 CN 2014076249 W CN2014076249 W CN 2014076249W WO 2014177021 A1 WO2014177021 A1 WO 2014177021A1
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signal peptide
amino acid
small peptide
secretory signal
strong secretory
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PCT/CN2014/076249
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English (en)
French (fr)
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何冰芳
陈文华
米兰
吴珊珊
朱芸
陈珊珊
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南京工业大学
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Priority to US14/786,160 priority Critical patent/US20160145303A1/en
Priority to EP17200981.3A priority patent/EP3301106B1/en
Priority to EP14791458.4A priority patent/EP3000823B1/en
Publication of WO2014177021A1 publication Critical patent/WO2014177021A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • C12N15/625—DNA sequences coding for fusion proteins containing a sequence coding for a signal sequence
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2405—Glucanases
    • C12N9/2451—Glucanases acting on alpha-1,6-glucosidic bonds
    • C12N9/2454—Dextranase (3.2.1.11)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01011—Dextranase (3.2.1.11)

Definitions

  • the invention belongs to the field of protein engineering and genetic engineering, and relates to a class of strong secretory signal peptide enhancing small peptide motifs and applications thereof.
  • the protein secretion system is not only a prerequisite for protein sorting, localization and physiological functions, but also an important means for genetic engineering and protein engineering technology development.
  • a variety of prokaryotic and eukaryotic expression systems have been developed for the production of recombinant proteins.
  • E. coli expression system has attracted much attention because of its clear genetic background, simple operation, rapid growth in low-cost medium and easy large-scale fermentation culture. At present, E. coli system has been used to prepare various enzyme preparations, even some medicines. Proteins, such as interferons, insulin and human serum proteins, have achieved remarkable results in the field of biotechnology.
  • recombinant proteins can be produced in principle by the following methods: 1. Intracellular production of soluble proteins; 2. Intracellular production of inclusion bodies; 3. Secretion into periplasmic space or extracellular medium.
  • the E. coli system is suitable for the production of proteins that need not be modified after translation, and because E. coli lacks the cofactors required for protein folding, it often causes a large amount of intermediates to accumulate, which tends to form inclusion bodies, and must undergo tedious processes such as renaturation; The peroxidative redox environment of the periplasm is beneficial to the folding of proteins.
  • the downstream renaturation and separation and purification processes can be greatly simplified, and the toxicity of the expressed protein to the host strain can be reduced. Metabolic burden, significantly increased expression.
  • a signal peptide is a type of polypeptide of about 10-60 amino acids, usually located at the N-terminus of a secreted protein.
  • the signal peptide is used to secrete the expressed protein.
  • the general method is to clone the gene encoding the target protein to the rear end of the signal peptide sequence, and to achieve the fusion of the target gene and the signal peptide sequence at the transcriptional and translational levels in the host, relying on the N-terminal signal of the protein precursor.
  • the peptide directs binding, membrane localization or extracellular secretion of the entire polypeptide chain to the intracellular chaperone or secretion signal recognition particle.
  • Each recombinant protein has its own specific coding gene, the signal peptide and the recombinant protein have different transition point sequences, and the structure of the recombinant protein itself is also different, so the secreted water of the recombinant protein The level depends on the level of optimization of its signal peptide, the favorable transmembrane structure of the target protein, and the level of matching between them.
  • the basic secretory pathway of secreted proteins across the plasma membrane is dominated by Sec or Tat systems, such as the outer membrane protein (OmpA) signal peptide and the pectin lipolytic enzyme (PelB) signal peptide through the Sec pathway.
  • the trimethylamine N-oxide reductase (TorA) signal peptide passes through the Tat pathway, and both types of signal peptides have been successfully used to secrete expression of foreign proteins.
  • patent EP 0 461 165 B1 discloses a polypeptide structure in which a hydrophobic signal peptide, a hydrophilic peptide and a heterologous protein are fused.
  • the negatively charged amino acid is contained near the yeast processing site, which facilitates the full exposure of the processing site. , thereby increasing the efficiency of protease cleavage, increasing the expression level of the correctly processed (active) target protein, and then secreting it extracellularly.
  • a front end such as an ompA signal peptide or a pelB signal peptide
  • Another technical object of the present invention is to provide the above-mentioned application of a strong secretion signal peptide enhancing small peptide motif having a function of enhancing the secretion efficiency of a signal peptide, which is to construct a carrier for enhancing the secretion ability of a common signal peptide. To increase its ability to secrete expression of foreign proteins.
  • secretion in the present invention means that a molecule of a protein or peptide is transported to the outside of a bacterial cell, and it also includes a case where the protein or peptide molecule is finally placed in the medium in a completely free form, and Some of the proteins are outside the bacteria or some proteins are present in the periplasmic space of the bacteria.
  • a “variant" of a protein amino acid or polynucleotide refers to an amino acid sequence having one or more amino acid or nucleotide changes or a polynucleotide sequence encoding the same.
  • the "alteration” may include deletion, insertion or substitution of an amino acid sequence or a nucleotide sequence in an amino acid sequence or a nucleotide sequence.
  • Variants may have conservative changes in which the substituted amino acid has a structural or chemical similarity to the original amino acid, such as the replacement of isoleucine with leucine.
  • Variants may also have non-conservative changes, such as the replacement of glycine with tryptophan.
  • “Deletion” refers to the deletion of one or more amino acids or nucleotides in an amino acid sequence or nucleotide sequence.
  • “Insertion” or “addition” refers to an alteration in an amino acid sequence or nucleotide sequence that results in an increase in one or more amino acids or nucleotides as compared to the original molecule.
  • “Replacement” refers to the replacement of one or more amino acids or nucleotides by a different amino acid or nucleotide.
  • a strong secretory signal peptide enhances a small peptide motif having an amino acid sequence of the formula: ⁇ ( ⁇ / ⁇ ) ⁇ , wherein X represents an acidic amino acid;
  • ⁇ represents a basic amino acid
  • ⁇ is 0-2 neutral amino acids
  • ⁇ 0-2 neutral amino acids
  • ⁇ represents 1-10 neutral amino acids
  • ⁇ is 1-3.
  • the "/" in the formula means "or".
  • the acidic amino acid represented by X is preferably Glu or Asp.
  • the basic amino acid represented by Y is preferably Arg or Lys.
  • the neutral amino acid is preferably Ala, Cys, Leu, Val, lie or Phe.
  • ⁇ is 1 neutral amino acid
  • ⁇ is 0 neutral amino acid
  • ⁇ is 2-5 neutral amino acids
  • X is Glu
  • Y is Arg
  • the small peptide membrane has the best enhanced secretion effect.
  • the small peptide motif protected by the present invention according to the above formula, for example, the original small peptide motif is: MERACVAV; then the derivative derived therefrom can be changed as follows:
  • the present invention also claims a variant of the strong secretory signal peptide enhancing small peptide motif of the present invention having one or more amino acid residues that enhance the small peptide motif of the strong secretory signal peptide of the present invention.
  • One or more amino acid residues are substituted for an insertion or deletion of a group and/or an amino acid of similar nature. It is known from the common knowledge of those skilled in the art that such a variant still has the properties of a signal peptide or a function of enhancing secretion, and therefore, it should also fall within the scope of the strong secretory signal peptide enhancing small peptide motif claimed in the present invention.
  • a polynucleotide encoding a polypeptide, analog or derivative having a strong secretory signal peptide enhancing a small peptide motif as shown in the present invention It is known from the common knowledge of those skilled in the art that the polynucleotide of such an analog or derivative still has the property of a signal peptide or a function of enhancing secretion, and therefore, it should also belong to the enhancement of the strong secretory signal peptide claimed in the present invention.
  • the range of small peptide motifs are examples of small peptide motifs.
  • a recombinant vector comprising an exogenous polynucleotide which is a recombinant vector constructed by the polynucleotide of the third aspect of the present invention and a plasmid vector.
  • a genetic host cell comprising an exogenous polynucleotide which is a host cell transformed or transduced by the recombinant vector of the fourth aspect.
  • the use of the strong secretory signal peptide of the present invention to enhance the small peptide motif that is, the strong secretory signal peptide-enhanced small peptide motif of the present invention is used to construct a vector for enhancing the secretion ability of common signal peptides. To increase its secretion of foreign protein expression methods.
  • the small peptide motif is enhanced by the fusion of the strong secretory signal peptide of the present invention at the front end of the signal peptide, and the secreted vector thus constructed is transformed into the large intestinal expression host to induce expression to achieve a strong secretory signal peptide. Enhance the enhanced secretion function of the small peptide motif.
  • a fructosidase FRU6 derived from A/ /zrakzc er ⁇ ' ⁇ and a glucanase BGL derived from Bacillus subtilis S te'fe are used as target proteins, common signal peptides OmpA selected from the outer membrane protein and pelB signal peptide derived from pectate lyase.
  • the small peptide module of the present invention is fused at the front end of the ompA or pelB signal peptide, and the secreted vector thus constructed is transformed into the large intestine expression host to induce expression, and the enhanced secretion function of the small peptide motif of the present invention is verified by a detection method.
  • the host used in the present invention is £ Y BL21 (DE3), and the basic framework of E. coli pET-22b as a vector is constructed to remove the pelB signal peptide sequence in the original pET-22b.
  • the ompA signal peptide/pelB signal peptide and fructosidase FRU6 or glucanase BGL were fused by overlapping PCR, and the upstream primer was designed to add the small peptide motif gene to the signal peptide gene sequence.
  • the secreted enhanced expression vectors pET-EompA-FRU6 and pET-EpelB-FRU6 and pET-EompA-BGL and pET-EpelB-BGL were constructed by restriction enzyme digestion and ligation.
  • Recombinant plasmid vectors pET-EompA-FRU6 and pET-EpelB-FRU6 (or pET-EompA-BGL and pET-EpelB-) containing the fusion-enhancing secreted signal peptide and fructosidase FRU6 (or glucanase BGL) gene
  • E. coli BL21, BGL were designated BL21/pET-EompA-FRU6 and PBL21/pET-EpelB-FRU6 (or BL21/pET-EompA-BGL and BL21/pET-EpelB-BGL), respectively.
  • Induction was induced in LB medium, and the inducer was IPTG.
  • Examples of secretions employed in the present invention are fructosidase FRU6 and ⁇ -1,3-1,4 glucanase BGL.
  • Fructosidase FRU6 molecular weight approximately 55 kDa, derived from Arthrobacter arilaitensis NJEM01, species accession number CCTCC M 2012155.
  • the fructosidase system has been classified as EC 3.2.1.80 and is an extracellular enzyme that specifically catalyzes the hydrolysis of non-reducing terminal 2, 1- ⁇ -glycosidic bonds in fructan molecules composed of ⁇ -D-fructose.
  • 2,6- ⁇ -glycosidic bond in addition, it can also hydrolyze inulin, sucrose, raffinose and the like. It has a wide range of uses in the fields of biology, medicine, and food.
  • Glucanase BGL molecular weight approx. 28 kDa, EC 3.2.1.73, ⁇ -glucanase, derived from Bacillus subtilis ⁇ .
  • s rife is an endohydrolase that efficiently and metastatically hydrolyzes ⁇ - ⁇ -1,4 glycosidic bond in the glucan adjacent to the ⁇ -1,3 glycosidic bond, thereby reducing the negative impact of ⁇ -glucan in the grain on industrial production, in the beer brewing industry, the word industry, etc.
  • the field has important applications.
  • the beneficial effects of the present invention are as follows:
  • the small peptide motif which enhances the secretory function is linked to the terminal end of the signal peptide, and the effect of enhancing secretion of the target protein is remarkable.
  • Experiments using the secretory expression example of fructosidase FRU6 showed that the addition of small peptide motifs before the ompA signal peptide was up to 5.1 times higher than the unadded secretion efficiency, and the addition of small peptide motifs before the pelB signal peptide was not added. The secretion efficiency was increased by up to 5.4 times.
  • the small peptide motif increased the ompA signal peptide by a factor of 2.3 and the PelB signal peptide by a factor of 2.5.
  • the polypeptide structure disclosed in EP 0461165 B1 is intended to promote the correct processing of the target protein by modifying the amino acid sequence between the C-terminus of the peptide and/or the N-terminus of the heterologous protein to fully expose the yeast processing site. Increase the amount of active protein expressed.
  • the secretory expression vector constructed by the present invention can be applied to a variety of recombinant protein production.
  • Figure 1 is a map of the recombinant vector pET-EompA-FRU6;
  • ompA SP outer membrane protein A signal peptide coding sequence
  • FRU6 fructosidase FRU6 coding sequence
  • Amp sequence ampicillin resistance gene coding sequence
  • T7 promoter T7 promoter.
  • Figure 3 Comparison of enzyme activities of BL21/pET-EompA-FRU6 and BL21/pET-EpelB-FRU6 with negative control fermentation supernatant in LB medium.
  • Figure 4 SDS-PAGE electrophoresis pattern of BL21/pET-EompA-FRU6 fermentation broth in LB medium;
  • M protein Marker
  • 1 and 2 negative control, fructosidase FRU6 before the signal peptide sequence of BL21 strain fermentation supernatant and bacterial cell disruption supernatant
  • 3 and 4 BL21/pET-ompA-FRU6 strain The supernatant of the fermentation broth and the supernatant of the bacteria are broken.
  • Wo P 6 BL21/pET-EompA-FRU6 strain fermentation supernatant and bacterial cell disrupted supernatant.
  • the small peptide motif sequence added before the ompA signal peptide is MERACALA.
  • Arrow direction is fructosidase FRU6 Mature peptide molecular weight size position.
  • FIG. 5 SDS-PAGE electrophoresis pattern of BL21/pET-EpelB-FRU6 fermentation broth in LB medium;
  • M protein Marker
  • 1 and 2 negative control, fructosidase FRU6 before the signal peptide sequence of BL21 strain fermentation supernatant and bacterial cell disrupted supernatant
  • 3 and 4 BL21/pET-pelB-FRU6 strain The supernatant of the fermentation broth and the supernatant of the bacteria are broken.
  • 5 and 6 BL21/pET-EpelB-FRU6 strain fermentation supernatant and bacterial cell disrupted supernatant.
  • the small peptide motif sequence added before the pelB signal peptide is MERACALA).
  • the direction of the arrow is the molecular size of the fructosidase FRU6 mature peptide.
  • Figure 6 SDS-PAGE electrophoresis pattern of BL21/pET-EompA-BGL fermentation broth in LB medium;
  • M protein Marker
  • 1 and 2 negative control, glucanase BGL without signal peptide sequence of BL21 strain fermentation supernatant and bacterial cell disrupted supernatant
  • 3 and 4 BL21/pET-ompA-BGL The supernatant of the fermentation broth and the supernatant of the bacterial cell are broken.
  • Wo P 6 BL21/pET-EompA-BGL strain fermentation supernatant and bacterial cell disrupted supernatant.
  • the small peptide motif sequence added before the ompA signal peptide is MERACALA).
  • the direction of the arrow is the position of the molecular weight of the glucanase BGL mature peptide.
  • Figure 7 SDS-PAGE electrophoresis pattern of BL21/pET-EpelB-BGL fermentation broth in LB medium;
  • M is a protein Marker
  • 1 and 2 a negative control, a fermentation supernatant of the BL21 strain containing no signal peptide sequence before the glucanase BGL, and a bacterial cell disrupted supernatant
  • 3 and 4 BL21/pET-pelB-BGL The supernatant of the fermentation broth and the supernatant of the bacterial cell were disrupted
  • 5 and 6 the supernatant of the fermentation broth of BL21/pET-EpelB-BGL strain and the supernatant of the bacterial cell.
  • the small peptide motif sequence added before the pelB signal peptide is MERACALA.
  • the direction of the arrow is the molecular weight position of the glucanase BGL mature peptide.
  • the source of the fructosidase Fru6 gene described in the present example is: Arthrobacter arilaitensis NJEM01, which is the inventor's prior Chinese patent application CN102732456A, and the accession number of the strain is: CCTCC NO: M 2012155.
  • primers involved in this example were synthesized by Yingjun Company, as shown in Table 1.
  • the following primer numbers are uniformly indicated by the "P" addition number.
  • the primer No. 45 in Table 2 the code is P45, which is numbered SEQ ID NO: 45 in the sequence listing.
  • Fructosidase FRU6 gene acquisition Using the genome of Afthmbactef arilaitensis NJEM01 as a template, primers P1 and P2 were used for PCR to amplify the gene fragment of fructosidase FRU6 (without the enzyme's own signal peptide sequence). After cloning into the cloning vector pMD 18-T vector, lj pMD-T-FRU6 was obtained and transformed into the cloning host DH5a, and the DNA sequence was determined to verify its correctness.
  • the amino acid sequence of the small peptide motif is MERACALA, and the more small peptide motifs are shown in Table 2 and Table 3.
  • the upstream primers P45 and P46 were designed, and P45 and P46 were respectively carried with Nde I restriction sites.
  • the combination of primer P45 and P6 can make the small peptide motif gene before the ompA signal peptide gene; the primer P46 and P10 can make the small peptide motif gene (such as the small peptide motif amino acid sequence is MERACALA).
  • the pET-22b vector (purchased from Yingjun) was also subjected to restriction enzyme digestion with restriction enzymes Nde I and EcoR I to recover a large fragment of the plasmid from which the pelB signal peptide sequence was removed. Then, the large fragment of the recovered plasmid and the digested product of the secreted signal peptide and the fructosidase fusion gene are ligated, and then ligated with T4 ligase to obtain pET-EompA-FRU6 (plasmid map shown in Fig. 1) and P ET-EpelB-FRU6. (The plasmid map is similar to the former, so it is omitted). Transformation into the cloning host E. coli DH5a, DNA sequencing to verify its correctness.
  • the primer number in this table corresponds to P or SEQ ID NO.
  • the OmpA and PelB signal peptide sequences are as follows:
  • the primer number in this table corresponds to P or SEQ ID NO.
  • fructosidase FRU6 The recombinant bacteria were inoculated into LB liquid medium containing 100 g/mL ampicillin, cultured at 37 ° C, 200 rpm overnight, and then inoculated to fresh LB medium at 2% inoculum. Medium (containing 100 g/mL ampicillin), 37 ° C, 200 rpm culture OD 600 was 0.6-0.9, the inducer IPTG (final concentration 1 mmol / L) was added, and samples were collected every 1 hour.
  • the enzyme activity detection method was as follows: 1 Preparation of the substrate: O. lg puerarin, 2.8 g of sucrose was fully dissolved in 100 mL of 0.05 mol/L, pH 6 phosphate buffer. 2 reaction system: 50 ⁇ L of phosphate buffer (0.05 mol / L, pH 6) was added to 950 ⁇ L of the substrate, placed at 35 ° C for 10 min, immediately removed 100 to add 900 methanol to terminate the reaction, as Blank control. At the same time, 50 ⁇ L of enzyme solution was added to 950 ⁇ L ⁇ substrate, and reacted at 35 °C for 10 min. Immediately after taking 100 and adding 900 ⁇ m of methanol, the reaction was terminated as a sample. The measurement was carried out by HPLC. 3 The enzyme activity unit is defined as: the amount of enzyme required to consume 1 ⁇ of ⁇ puerarin per minute at 35 V is one unit of activity (U).
  • Example 1 In the same manner as in Example 1, the fructosidase in Example 1 was replaced with the glucanase gene in this example.
  • the source of the sequence of glucanase BGL Bacillus subtilis subsp. subtilis 6051-HGW, GenBank SEQ ID NO: CP003329.1, range: 4011849 to lj 4012490.
  • Primers P79 and P80 were PCR-polymerized to amplify the glucanase BGL gene fragment and prepared into the T vector pMD-T-BGL.
  • the overlapping PCR primers P81-P84 or P85-P88 were fused to the glucanase BGL and the signal peptides ompA or pelB, respectively, and both P84 and P88 were ligated with EcoR I.
  • the P89-P96 upstream primer is combined with the downstream primer P84 or P88, and different enhancement motifs can be designed to be fused to the ompA or pelB signal peptide, respectively.
  • Table 5 required primers for Example 2 80 TTATTTTTTTGTATAGCGCACCCA
  • the primer number in this table corresponds to P or SEQ ID NO.
  • the constructed glucanase BGL secretory expression vector was transformed into the expression host BL21 (DE3), and finally the recombinant strains were named BL21/pET-EompA-BGL and BL21/pET-EpelB-BGL. Transformants were screened on LB plates containing 100 g/mL ampicillin and plasmid validation was performed therefrom, as described in the Molecular Cloning Manual.
  • the recombinant bacteria were inoculated into LB liquid medium containing 100 g/mL ampicillin, and cultured at 37 ° C, 200 rpm overnight, and then inoculated into fresh LB medium (containing 100 g/mL ampicillin) at a 2% inoculation amount.
  • OD 6 (X) was cultured at 37 ° C and 200 rpm from 0.6 to 0.9
  • the inducer IPTG final concentration 1 mmol/L;
  • Table 4 corresponds to the enzymatically involved small peptide sequence amino acid sequence and enzyme activity involved in glucanase
  • the primer number in this table corresponds to P or SEQ ID NO.
  • the variant of the small peptide motif is enhanced by the strong secretory signal peptide of the present invention, which has one or more of the strong secretory signal peptide enhancing the small peptide motif.
  • the insertion or deletion of an amino acid residue and/or the substitution of one or more amino acid residues with a similar amino acid are a variant or a reasonable extension based on the invention and should also fall within the scope of the invention.
  • a polynucleotide encoding a polypeptide, analog or derivative having a strong secretory signal peptide enhancing small peptide motif according to the present invention; a recombinant vector containing an exogenous polynucleotide, which is the present invention
  • a recombinant vector constructed by the polynucleotide and the plasmid vector A recombinant vector constructed by the polynucleotide and the plasmid vector; a genetic host cell containing the exogenous polynucleotide, which is a host cell transformed or transduced by the recombinant vector of the present invention.

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Abstract

提供了一类强分泌性信号肽增强小肽模序及其应用，所述小肽模序具有如下通式的氨基酸序列：M(αΧβΥγ/αΥβΧγ)n，其中X代表酸性氨基酸；Υ代表碱性氨基酸；α为0-2个中性氨基酸；β代表0-2个中性氨基酸；γ代表1-10个中性氨基酸；n为1-3。本发明的小肽模序可用于构建增强普通信号肽分泌能力的载体，以提高外源蛋白的分泌表达。

Description

强分泌性信号肽增强小肽模序及其应用
技术领域
本发明属于蛋白质工程和基因工程领域， 涉及一类强分泌性信号肽增强小肽模序及其应 用。
背景技术
蛋白质的分泌系统不仅是蛋白质分选、定位和实现生理功能的前提， 更是基因工程及蛋白 质工程技术开发的重要手段。人们已经研究出多种原核和真核表达系统用以生产重组蛋白。大 肠杆菌表达系统以其遗传背景清楚、操作简单、能在廉价培养基中迅速生长及易于大规模发酵 培养等特点而备受关注； 目前大肠杆菌体系已用于制备多种酶制剂， 甚至一些药用蛋白质， 如 干扰素、 胰岛素和人血清蛋白等， 在生物技术领域取得了令人瞩目的成果。
在大肠杆菌中，重组蛋白在原则上可以被以下几种方式生产： 1、可溶性蛋白胞内生产； 2、 包涵体胞内生产； 3、 分泌至周质空间或胞外培养基中。 大肠杆菌体系适合于生产翻译后无需 修饰加工的蛋白质， 且由于大肠杆菌缺乏蛋白折叠过程中需要的辅助因子，往往使中间体大量 积聚， 易形成包涵体沉淀， 还须经过复性等繁琐过程； 而周质氧化型的氧化还原环境有利于蛋 白质的折叠。如果蛋白质跨过细胞的内膜定位于周质空间，甚至穿过细胞外膜直接分泌至胞外 培养基中，可以极大简化下游复性及分离纯化工艺，减少表达蛋白对宿主菌的毒性和代谢负担， 显著提高表达量。
现有的分泌表达体系大多数是分泌到周质空间， 获取重组蛋白仍然需要细胞破碎和纯化。 少数分泌到胞外的则由于大肠杆菌本身的蛋白质分泌系统不够完善，或人类对其分泌系统认识 的局限性， 导致了分泌效率极低及融合蛋白的后加工问题。
信号肽是一类 10-60个氨基酸左右的多肽，一般位于分泌蛋白的 N端。利用信号肽分泌表达 蛋白，一般操作方法是将目的蛋白的编码基因克隆到信号肽序列后端，在宿主中实现目的基因 和信号肽序列在转录和翻译水平的融合， 依靠蛋白质前体 N端信号肽引导整个多肽链与胞内分 子伴侣或分泌信号识别颗粒的结合、 膜定位或分泌出胞外。
尽管胞外分泌表达策略相比其他表达方式有着显著的优势，但是目前在大肠杆菌体系中广 泛运用此种策略仍有诸多限制。因此，拥有能够携带目的蛋白分泌出胞外的信号肽是先决条件， 并且即使信号肽具有介导蛋白胞外分泌的能力，也并不表明特定的信号肽对所有的重组蛋白具 有这种介导能力或者具有同样水平的介导能力。每个重组蛋白具有自己特定的编码基因， 信号 肽和重组蛋白的过渡点序列不同， 而且重组蛋白自身的结构也有差异， 因而重组蛋白的分泌水 平取决于其信号肽的最优化水平、 目标蛋白有利的跨膜结构以及它们之间的匹配度水平。 大肠杆菌表达系统中， 分泌蛋白穿越质膜的基本分泌途径以 Sec或 Tat这两种系统为主， 例如外膜蛋白 （OmpA)信号肽、 果胶酸脂裂解酶 （PelB)信号肽通过 Sec途径， 三甲胺 N氧化 物还原酶 （TorA) 信号肽通过 Tat途径， 这两种类型的信号肽都有成功运用于分泌表达外源蛋 白的案例。
针对酵母加工系统中目标蛋白水解加工不正确或不完全问题， 专利 EP 0461165 B1公开了 一个由疏水的信号肽、亲水的导肽和异源蛋白融合而成的多肽结构。通过对导肽 C端和 /或异源 蛋白 N端之间 (也就是邻近酵母加工位点)的氨基酸序列进行修饰， 使酵母加工位点附近包含带 负电荷氨基酸， 利于加工位点的充分暴露， 从而提高了蛋白酶剪切效率， 增加了正确加工（有 活性） 目标蛋白的表达量， 进而分泌到了胞外。
发明内容
本发明的技术目的在于提供一种具有增强信号肽分泌效率的功能的强分泌性信号肽增强 小肽模序， 使得通过在诸如 ompA信号肽或 pelB信号肽前端融合添加本发明的小肽模序， 可以 实现上述信号肽在大肠杆菌体系中的分泌表达外源蛋白的能力。
本发明的另一个技术目的在于提供上述具有增强信号肽分泌效率的功能的强分泌信号肽 增强小肽模序的应用， 即将此小肽模序用于构建一种增强普通信号肽分泌能力的载体， 以提高 其分泌表达外源蛋白的方法。
本发明中的术语 "分泌"， 是指蛋白质或肽的分子被转运到细菌细胞的外部， 而且它也包 括这样的情况： 其中蛋白质或肽分子最终以完全游离的形式置于培养基中， 以及其中部分蛋白 质处在细菌外部或部分蛋白质存在于细菌的周质空间。
本发明中使用的下列术语除非特别说明具有如下的含义：
蛋白质氨基酸或多核苷酸的 "变体"是指具有一个或多个氨基酸或核苷酸改变的氨基酸序 列或编码它的多核苷酸序列。所述 "改变"可包括氨基酸序列或核苷酸序列中氨基酸或核苷酸 的缺失、插入或替换。变体可具有保守性改变， 其中替换的氨基酸具有与原氨基酸相类似的结 构或化学性质， 如用亮氨酸替换异亮氨酸。变体也可以具有非保守性改变， 如用色氨酸替换甘 氨酸。
"缺失"是指氨基酸序列或核苷酸序列中的一个或多个氨基酸或核苷酸的缺失。 "插入" 或 "添加"是指氨基酸序列或核苷酸序列中的改变导致与原先分子相比， 一个或多个氨基酸或 核苷酸的增加。 "替换"是指由不同的氨基酸或核苷酸替换一个或多个氨基酸或核苷酸。 为了实现本发明的技术目的， 本发明的技术方案如下：
一、 强分泌性信号肽增强小肽模序， 其具有如下通式的氨基酸序列： Μ(αΧβΥγ/αΥβΧγ)η， 其中 X代表酸性氨基酸；
Υ代表碱性氨基酸；
α为 0-2个中性氨基酸；
β代表 0-2个中性氨基酸；
γ代表 1-10个中性氨基酸；
η为 1-3。
其中， 通式中的 "/"为 "或者"之意。
进一步的， X代表的酸性氨基酸优选为 Glu或 Asp。
进一步的， Y代表的碱性氨基酸优选为 Arg或 Lys。
进一步的， 中性氨基酸优选为 Ala、 Cys、 Leu、 Val、 lie或者 Phe。
进一步的， 当 n=l时， 本发明的小肽膜序的增强分泌效果较佳。
进一步的，作为本发明的一个最优选的实施例，当 α为 1个中性氨基酸， β为 0个中性氨基酸， γ为 2-5个中性氨基酸， X为 Glu， Y为 Arg时， 该小肽膜序的增强分泌效果最佳。
因此， 根据上述通式得出的本发明所保护的小肽模序， 其例如原始小肽模序为： MERACVAV; 则其衍生的类似物可以如下变化：
MREACVAV
MAERACVAV;
MEAEACVAV;
MDKACVAV;
MERLIVFAV; …。
或小肽模序的叠加如 MERACVA+VEARLIVFAV等， 更多衍化类型详见本发明具体实施方 式。
二、本发明还要求保护本发明所述的强分泌性信号肽增强小肽模序的变体，其具有对本发 明的强分泌性信号肽增强小肽模序的其中 1个或多个氨基酸残基的插入或缺失和 /或性质相似 的氨基酸取代 1个或多个氨基酸残基。 根据本领域技术人员的公知常识可知， 这种变体仍具有 信号肽的特性或增强分泌的功能， 因此，其也应当属于本发明要求保护的强分泌性信号肽增强 小肽模序的范围。 三、编码具有本发明所示的强分泌性信号肽增强小肽模序的多肽、类似物或衍生物的多核 苷酸。根据本领域技术人员的公知常识可知，这种类似物或衍生物的多核苷酸仍具有信号肽的 特性或增强分泌的功能， 因此，其也应当属于本发明要求保护的强分泌性信号肽增强小肽模序 的范围。
四、一种含有外源多核苷酸的重组载体， 其是由本发明第三点所述的多核苷酸与质粒载体 构建而成的重组载体。
五、 一种含有外源多核苷酸的遗传宿主细胞， 它是由第四点所述的重组载体转化或转导的 宿主细胞。
六、本发明所述的强分泌性信号肽增强小肽模序的应用， 即将本发明所述的强分泌性信号 肽增强小肽模序用于构建一种增强普通信号肽分泌能力的载体，以提高其分泌表达外源蛋白的 方法。
具体的， 是指通过在信号肽前端融合添加本发明所述的强分泌性信号肽增强小肽模序， 以 此构建的分泌载体转化进大肠表达宿主后诱导表达，以实现强分泌性信号肽增强小肽模序的增 强分泌功能。
在本发明的具体实施方式中， 采用 A/ /zrakzc er ΩΠ'ΖΩΖΥ^Ζ 来源的果糖苷酶 FRU6和来源于 枯草芽孢杆菌 S te'fe的葡聚糖酶 BGL作为目标蛋白， 普通的信号肽选自外膜蛋白的 ompA和 来自果胶酸脂裂解酶的 pelB信号肽。 通过在 ompA或 pelB信号肽前端融合添加本发明的小肽模 序， 以此构建的分泌载体转化进大肠表达宿主后诱导表达，通过检测方法验证本发明的小肽模 序的增强分泌功能。
本发明采用的宿主为 £ Y BL21(DE3)， 以大肠杆菌 pET-22b作为载体构建的基本框架， 去 除原 pET-22b中的 pelB信号肽序列。通过重叠 PCR的方法融合 ompA信号肽 /pelB信号肽和果糖苷 酶 FRU6或葡聚糖酶 BGL， 设计上游引物将小肽模序基因添加到信号肽基因序列前。 通过酶切 位点的酶切和连接操作，构建成分泌增强型表达载体 pET-EompA-FRU6和 pET-EpelB-FRU6以及 pET-EompA-BGL禾口 pET-EpelB-BGL。
将含有融合的增强分泌型信号肽与果糖苷酶 FRU6 (或葡聚糖酶 BGL)基因的重组质粒载 体 pET-EompA-FRU6和 pET-EpelB-FRU6 (或 pET-EompA-BGL和 pET-EpelB-BGL)转化进宿 主大肠杆菌 BL21 后， 分别命名为 BL21/ pET-EompA-FRU6 禾 P BL21/pET-EpelB-FRU6 (或 BL21/pET-EompA-BGL和 BL21/pET-EpelB-BGL )。在 LB培养基进行诱导表达，诱导剂为 IPTG。
上述两种目标蛋白分泌表达后的结果通过检测培养基细胞内外的酶活以及 SDS-PAGE分 析。
本发明采用的分泌实例为果糖苷酶 FRU6以及 β-1,3-1,4葡聚糖酶 BGL。 果糖苷酶 FRU6 (分子量约为 55kDa， 来源于 Arthrobacter arilaitensis NJEM01， 菌种保藏号 CCTCC M 2012155 )。 果糖苷酶系统分类号为 EC 3.2.1.80, 是一种胞外酶， 特异性地催化水解由 β-D- 果糖组成的果聚糖分子中的非还原性末端 2, 1-β-糖苷键或 2,6-β-糖苷键， 此外， 还可水解菊糖、 蔗糖、 棉子糖等。 在生物、 医药、 食品等领域均有着广泛的利用价值。 葡聚糖酶 BGL (分子量 约为 28 kDa， EC 3.2.1.73 , β-葡聚糖酶， 来源于枯草芽孢杆菌 β. s rife)是一种内切水解酶， 能高效、 转移性地水解 β-葡聚糖中与 β-1,3糖苷键毗邻的 β-1,4糖苷键， 从而减少谷物中 β-葡 聚糖给工业生产带来的负面影响， 在啤酒酿造业、 词料业等领域有着十分重要的应用。
除上述以外，本发明的有益效果还在于： 本发明中可增强分泌功能的小肽模序为与信号肽 Ν端相连， 其对目标蛋白的增强分泌效果显著。运用在果糖苷酶 FRU6的分泌表达实例的实验 显示， 在 ompA信号肽前添加小肽模序比未添加的分泌效率最高提高了达 5.1倍， 在 pelB信 号肽前添加小肽模序比未添加的分泌效率最高提高了达 5.4倍。 运用在葡聚糖酶 BGL的分泌 表达实例显示，小肽模序对 ompA信号肽最高提高了 2.3倍，对 PelB信号肽最高提高了 2.5倍。 而 EP 0461165 B 1公开的多肽结构， 其意在通过对导肽 C端和 /或异源蛋白 N端之间的氨基酸 序列进行修饰， 充分暴露酵母加工位点， 从而促进目标蛋白的正确加工， 提高有活性蛋白表达 量。 另外， 本发明所构建的分泌表达载体可以运用到多种重组蛋白生产上。
附图说明
图 1 重组载体 pET-EompA-FRU6的图谱；
其中， ompA SP: 外膜蛋白 A信号肽编码序列； FRU6: 果糖苷酶 FRU6编码序列； Amp sequence: 氨苄霉素抗性基因编码序列； T7 promoter: T7 启动子。
图 2 小肽模序增强分泌效率与发酵时间的关系。
图 3 LB培养基中 BL21/pET-EompA-FRU6和 BL21/pET-EpelB-FRU6与阴性对照发酵上清 液酶活力比较。
图 4 LB培养基中 BL21/pET-EompA-FRU6发酵液的 SDS-PAGE电泳图谱；
其中， M: 蛋白质 Marker; 1和 2: 阴性对照， 果糖苷酶 FRU6前不含信号肽序列的 BL21 菌株发酵上清液和菌体破碎上清； 3和 4: BL21/pET-ompA-FRU6菌株发酵液上清和菌体破碎 上清。 5禾 P 6: BL21/pET-EompA-FRU6菌株发酵液上清和菌体破碎上清。 （ pET-Eomp A-FRU6 质粒中， ompA信号肽前添加的小肽模序序列为 MERACALA)。 箭头方向为果糖苷酶 FRU6 成熟肽分子量大小位置。
图 5 LB培养基中 BL21/pET-EpelB-FRU6发酵液的 SDS-PAGE电泳图谱；
其中， M: 蛋白质 Marker; 1和 2: 阴性对照， 果糖苷酶 FRU6前不含信号肽序列的 BL21 菌株发酵上清液和菌体破碎上清； 3和 4: BL21/pET-pelB-FRU6菌株发酵液上清和菌体破碎上 清。 5和 6: BL21/pET-EpelB-FRU6菌株发酵上清和菌体破碎上清。 （pET-EpelB-FRU6质粒中， pelB信号肽前添加的小肽模序序列为 MERACALA)。 箭头方向为果糖苷酶 FRU6成熟肽分子 量大小位置。
图 6 LB培养基中 BL21/pET-EompA-BGL发酵液的 SDS-PAGE电泳图谱；
其中， M: 蛋白质 Marker; 1和 2: 阴性对照， 葡聚糖酶 BGL前不含信号肽序列的 BL21 菌株发酵上清液和菌体破碎上清； 3和 4: BL21/pET-ompA-BGL菌株发酵液上清和菌体破碎上 清。 5禾 P 6: BL21/pET-EompA-BGL菌株发酵液上清和菌体破碎上清。 （pET-EompA-BGL质粒 中， ompA信号肽前添加的小肽模序序列为 MERACALA)。 箭头方向为葡聚糖酶 BGL成熟肽 分子量大小位置。
图 7 LB培养基中 BL21/pET-EpelB-BGL发酵液的 SDS-PAGE电泳图谱；
其中， M为蛋白质 Marker; 1和 2: 阴性对照， 葡聚糖酶 BGL前不含信号肽序列的 BL21 菌株发酵上清液和菌体破碎上清； 3和 4: BL21/pET-pelB-BGL菌株发酵液上清和菌体破碎上 清； 5和 6: BL21/pET-EpelB-BGL菌株发酵液上清和菌体破碎上清。（pET-EpelB-BGL质粒中， pelB信号肽前添加的小肽模序序列为 MERACALA)。箭头方向为葡聚糖酶 BGL成熟肽分子量 大小位置。
具体实施方式
下述实施例中如无特殊说明所用方法均为常规方法， 所用涉及质粒、试剂等材料均可从商 业途径获得。
实施例 1 . 小肽增强模序在果糖苷酶分泌表达上的应用
首先， 本实施例所述的果糖苷酶 Fru6基因的来源是： Arthrobacter arilaitensis NJEM01菌， 为本发明人的在先中国专利申请 CN102732456A , 该菌株的保藏编号为： CCTCC NO : M 2012155。
本实施例所涉及的所有引物均由英骏公司合成， 见表 1。 以下引物编号统一用 "P "加序 号来表示， 例如， 表 2中的第 45号引物， 其代码即 P45， 其在序列表的编号为 SEQ ID NO: 45。
表 1 实施例 1所需的引物和合成序列的核苷酸序列 SEQ ID NO 引物核苷酸序列 （5'- 3' )
1 GCCACCGAACCAGTGCCTGG
2 TTACTTTGCTACTGCTTTGCC
3 ATGAAAAAGACAGCTATCGCG
4 CTGGTTCGGTGGCAGCTTGGGCTACGGTAGCGAAA
5 ACCGTAGCCCAAGCTGCCACCGAACCAGTGCCTGG
6 CCGGAATTC TTACTTTGCTACTGCTTTGCC
7 ATGAAATACCTATTGCCTACG
8 ACTGGTTCGGTGGCAGCCATGGCTGGTTGGGCAGC
9 AACCAGCCATGGCTGCCACCGAACCAGTGCCTGGC
10 CCGGAATTC TTACTTTGCTACTGCTTTGCC
注： 表中下划线部分所示为限制性酶切位点。
( 1 ) 果糖苷酶 FRU6基因获取： 以 Afthmbactef arilaitensis NJEM01菌的基因组为模板， 采用引物 P1和 P2进行 PCR反应扩增出果糖苷酶 FRU6的基因片段 （不含该酶自身信号肽序 列）。 克隆到克隆载体 pMD 18-T载体中， 得至 lj pMD-T-FRU6， 转化进克隆宿主 DH5a中， DNA 序列测定以验证其正确性。
( 2 )信号肽与果糖苷酶基因的融合：人工合成 ompA和 pdB信号肽基因序列 (即引物 P11 和引物 P12)， 提取第 (1)步中的质粒 pMD-T-FRU6。 通过设计重叠 PCR引物 P3-P6或 P7-P10, 分别将果糖苷酶 FRU6和信号肽 ompA或 pelB融合，其中 P6和 P10均带有 EcoR I酶切位点。
( 3 ) 增强分泌型载体的设计：
以小肽模序氨基酸序列为 MERACALA, 更多小肽模序见表 2和表 3。
以第二步中的融合基因为模板， 通过设计上游引物 P45与 P46， P45与 P46分别均带有 Nde I酶切位点。通过普通 PCR反应，引物 P45与 P6联用可以使得小肽模序基因添加到 ompA 信号肽基因前； 引物 P46 和 P10 联用可以使得小肽模序基因 （如小肽模序氨基酸序列为 MERACALA) 添加到 pelB信号肽基因前。 经 Nde I和 EcoR I双酶切获得黏性末端的目的片 段。 将 pET-22b载体 （英骏公司购得） 也用限制酶 Nde I和 EcoR I进行酶切降解， 回收去除 pelB 信号肽序列的质粒大片段。 然后将回收的质粒大片段和增强分泌型信号肽与果糖苷酶融 合基因的酶切产物， 经 T4 连接酶连接后得到 pET-EompA-FRU6 (质粒图谱见图 1 ) 和 PET-EpelB-FRU6 (质粒图谱与前者类似， 故略）。 转化进克隆宿主大肠杆菌 DH5a 中， DNA 序列测定以验证其正确性。
表 2对应果糖苷酶涉及的增强分泌的小肽模序氨基酸序列及引物和酶活 n M α X (或 Υ) β Υ (或 X) Υ 引物 信号肽 酶活
13 ompA 1279
1 M Ε R
14 pelB 1521 15 ompA 801
M R E
16 pelB 917
17 ompA 1831
M E R A
18 pelB 1734
M E A R AA 19 ompA 1678
20 pelB 1530
M AA E R AC 21 ompA 2169
22 pelB 1781
M R E IV 23 ompA 907
24 pelB 862
M E R LC 25 ompA 2380
26 pelB 1972
M T R T E ACA 27 ompA 2024
28 pelB 1976
M C R C D ACAL 29 ompA 2175
30 pelB 2005
M E R ACAL 31 ompA 2169
32 pelB 1781
M V E LT R ACALA 33 ompA 2513
34 pelB 2100
M E R ACALA 35 ompA 1877
36 pelB 1762
M CL E R ACALA 37 ompA 2395
38 pelB 2230
M V E T R ACALA 39 ompA 2460
40 pelB 2195
M VA E LT R ACALA 41 ompA 2380
42 pelB 2120
M E A R ACVAV 43 ompA 2390
44 pelB 2275
M E R ACALA 45 ompA 2016
46 pelB 1979
M D K ACVAV 47 ompA 1193
48 pelB 1272
M L D V R ACALAA 49 ompA 1754
50 pelB 1644
M E R ACALAA 51 ompA 1980
52 pelB 1642
M AA D LT K ACALAAA 53 ompA 1766
54 pelB 1790
M E R ACALAAA 55 ompA 1987
56 pelB 1755
M E R ACALAAAA 57 ompA 2485 58 IpelB 1643
1 M TC K CL D ACALAAAAA 59 ompA 1906
60 pelB 1560
1 M E R LLCCTTTTT 61 ompA 1986
62 pelB 1756
1 M E R ACALAAAAA 63 olm A 1762
64 pelB 1882
1 M LT K CL E CATACCCCCC 65 ompA 1883
66 pelB 1986
1 M E R TTLTCCCCCC 67 ompA 1880
68 pelB 1670
2 MERACVAV+MERACVAV 69 ompA 1787
70 pelB 1754
2 MERACALA+VERACAL 71 ompA 2460
72 pelB 1845
3 MERACAL+VERACAL+VERACAL 73 ompA 1680
74 pelB 1855
3 MERCLATL+VERLCVAV+VERACALA 75 ompA 2260
76 pelB 2042
0 空白 77 ompA 420
78 pelB 495 注： 此表中引物编号对应于 P或者 SEQ ID NO。
OmpA与 PelB信号肽序列如下：
OmpA :
(SEQ ID NO:
PelB:
(SEQ ID NO:
表 3表 2所述的膜序小肽对应设计的上游引物
引物 序列 （5'→3' )
13 CGCCATATGGAGAGAATGAAAAAGACAGCTATCGCG
14 CGCCATATGGAGAGAATGAAATACCTATTGCCTACG
15 CGCCATATGAGAGAGATGAAAAAGACAGCTATCGCG
16 CGCCATATGAGAGAGATGAAATACCTATTGCCTACG
17 CGCCATATGGAGAGAGCGATGAAAAAGACAGCTATCGCG
18 CGCCATATGGAGAGAGCGATGAAATACCTATTGCCTACG
19 CGCCATATGGAGGCGAGAGCGGCGATGAAAAAGACAGCTATCGCG
20 CGCCATATGGAGGCGAGAGCGGCGATGAAATACCTATTGCCTACG
21 CGCCATATGGCGGCGGAGAGAGCGTGT ATGAAAAAGACAGCTATCGCG
22 CGCCATATGGCGGCGGAGAGAGCGTGTATGAAATACCTATTGCCTACG
23 CGCCATATGAGAGAGATTGTGATGAAAAAGACAGCTATCGCG CGCCATATGAGAGAGATTGTGATGAAATACCTATTGCCTACG
CGCCATATGGAGAGACTCTGT ATGAAAAAGACAGCTATCGCG
CGCCATATGGAGAGACTCTGTATGAAATACCTATTGCCTACG
CGCCATATGACCAGAACCGAGGCGTGTGCGATGAAAAAGACAGCTATCGCG
CGCCATATGACCAGAACCGAGGCGTGTGCGATGAAATACCTATTGCCTACG
CGCCATATGTGTAGATGTGACGCGTGTGCGCTC ATGAAAAAGACAGCTATCGCG
CGCCATATGTGTAGATGTGACGCGTGTGCGCTCATGAAATACCTATTGCCTACG
CGCCATATGGAGAGAGCGTGCGCGCTC ATGAAAAAGACAGCTATCGCG
CGCCATATGGAGAGAGCGTGCGCGCTCATGAAATACCTATTGCCTACG
CGCCATATGGTGGAGCTCACCAGAGCGTGCGCGCTCGCGATGAAAAAGACAGCTATCGCG
CGCCATATGGTGGAGCTCACCAGAGCGTGCGCGCTCGCGATGAAATACCTATTGCCTACG
CGCCATATGGTGGCGGAGCTCACCAGAGCGTGCGCGCTCGCGATGAAAAAGACAGCTATCGCG
CGCCATATGGTGGCGGAGCTCACCAGAGCGTGCGCGCTCGCGATGAAATACCTATTGCCTACG
CGCCATATGGAGAGAGCGTGTGCGCTCGCGATGAAAAAGACAGCTATCGCG
CGCCATATGGAGAGAGCGTGTGCGCTCGCGATGAAATACCTATTGCCTACG
CGCCATATGTGTCTCGAGAGAGCGTGCGCGCTCGCGATGAAAAAGACAGCTATCGCG
CGCCATATGTGTCTCGAGAGAGCGTGCGCGCTCGCGATGAAATACCTATTGCCTACG
CGCCATATGGTGGCGGAGCTCACCAGAGCGTGCGCGCTCGAGATGAAAAAGACAGCTATCGCG
CGCCATATGGTGGCGGAGCTCACCAGAGCGTGCGCGCTCGAGATGAAATACCTATTGCCTACG
CGCCATATGGAGGCGAGAGCGTGTGTGGCGGTGATGAAAAAGACAGCTATCGCG
CGCCATATGGAGGCGAGAGCGTGTGTGGCGGTGATGAAATACCTATTGCCTACG
CGCCATATGGAGAGAGCGTGTGCGCTCGCGATGAAAAAGACAGCTATCGCG
CGCCATATGGAGAGAGCGTGTGCGCTCGCGATGAAATACCTATTGCCTACG
CGCCATATGGACAAAGCGTGCTGTGTGGCGGTGATGAAAAAGACAGCTATCGCG
CGCCATATGGACAAAGCGTGCTGTGTGGCGGTGATGAAATACCTATTGCCTACG
CGCCATATGCTCGACGTGAGAGCGTGTGCGCTCGCGGCGATGAAAAAGACAGCTATCGCG
CGCCATATGCTCGACGTGAGAGCGTGTGCGCTCGCGGCGATGAAATACCTATTGCCTACG
CGCCATATGGAGAGAGCGTGCGCGCTCGCGGCG ATGAAAAAGACAGCTATCGCG
CGCCATATGGAGAGAGCGTGCGCGCTCGCGGCGATGAAATACCTATTGCCTACG
CGCCATATGGCGGCGGACCTCACCAAAGCGTGCGCGCTCGCGGCGGCGATGAAAAAGACAGCT ATCGCG
CGCCATATGGCGGCGGACCTCACCAAAGCGTGCGCGCTCGCGGCGGCGATGAAATACCTATTGC CTACG
CGCCATATGGAGAGAGCGTGTGCGCTCGCGGCGGCGATGAAAAAGACAGCTATCGCG
CGCCATATGGAGAGAGCGTGTGCGCTCGCGGCGGCGATGAAATACCTATTGCCTACG
CGCCATATGGAGAGAGCGTGCGCGCTCGCGGCGGCGGCGATGAAAAAGACAGCTATCGCG
CGCCATATGGAGAGAGCGTGCGCGCTCGCGGCGGCGGCGATGAAATACCTATTGCCTACG
CGCCATATGACCTGCAAATGCCTCGACGCGTGCGCGCTCGCGGCGGCGGCGGCGATGAAAAAG ACAGCTATCGCG
CGCCATATGACCTGCAAATGCCTCGACGCGTGCGCGCTCGCGGCGGCGGCGGCGATGAAATAC CTATTGCCTACG
CGCCATATGGAGAGACTCCTCTGTTGTACCACCACCACCACCACCATGAAAAAGACAGCTATCG CG 62 CGCCATATGGAGAGACTCCTCTGTTGTACCACCACCACCACCACCATGAAATACCTATTGCCTAC G
63 CGCCATATGGAGAGAGCGTGCGCGCTCGCGGCGGCGGCGGCGATGAAAAAGACAGCTATCGCG
64 CGCCATATGGAGAGAGCGTGCGCGCTCGCGGCGGCGGCGGCGATGAAATACCTATTGCCTACG
65 CGCCATATGCTCACCAAATGCCTCGAGTGCGCGACCGCGTGTTGTTGTTGTTGTATGAAAAAGA CAGCTATCGCG
66 CGCCATATGCTCACCAAATGCCTCGAGTGCGCGACCGCGTGTTGTTGTTGTTGTATGAAATACCT ATTGCCTACG
67 CGCCATATGGAGAGAACCACCCTCACCTGCTGCTGCTGCTGC ATGAAAAAGACAGCTATCGCG
68 CGCCATATGGAGAGAACCACCCTCACCTGCTGCTGCTGCTGCATGAAATACCTATTGCCTACG
69 CGCCATATGGAGAGAGCGTGCGTGGCGGTGGAGAGAGCGTGCGTGGCGGTGATGAAAAAGAC AGCTATCGCG
70 CGCCATATGGAGAGAGCGTGCGTGGCGGTGGAGAGAGCGTGCGTGGCGGTGATGAAATACCTA TTGCCTACG
71 CGCCATATGGAGAGAGCGTGCGCGCTCGCGGTGGAGAGAGCGTGCGCGCTCATGAAAAAGAC AGCTATCGCG
72 CGCCATATGGAGAGAGCGTGCGCGCTCGCGGTGGAGAGAGCGTGCGCGCTCATGAAATACCTA TTGCCTACG
73 CGCCATATGGAGAGAGCGTGCGCGCTCGTGGAGAGAGCGTGCGCGCTCGTGGAGAGAGCGTG TGCGCTC ATGAAAAAGACAGCTATCGCG
74 CGCCATATGGAGAGAGCGTGCGCGCTCGTGGAGAGAGCGTGCGCGCTCGTGGAGAGAGCGTG TGCGCTCATGAAATACCTATTGCCTACG
75 CGCCATATGGAGAGATGCCTCGCGACCCTCGTGGAGAGACTCTGCGTGGCGGTGGTGGAGAGA GCGTGCGCGTGCGCGCTCGCG ATGAAAAAGACAGCTATCGCG
76 CGCCATATGGAGAGATGCCTCGCGACCCTCGTGGAGAGACTCTGCGTGGCGGTGGTGGAGAGA GCGTGCGCGTGCGCGCTCGCGATGAAATACCTATTGCCTACG
77 CGCCATATGATGAAAAAGACAGCTATCGCG
78 CGCCATATGATGAAATACCTATTGCCTACG
注： 此表中引物编号对应于 P或者 SEQ ID NO。
(4) 增强分泌型表达菌株的构建： 将 pET-EompA-FRU6和 pET-EpelB-FRU6载体分别转 化进表达宿主 BL21里， 操作方法见《分子克隆手册》， 在含 lOO g/mL的氨苄 LB平板上筛选 得到含 pET-EompA-FRU6 和 pET-EpelB-FRU6 载体的大肠杆菌 BL21 重组菌株， 命名为 BL2 l/pET-EompA-FRU6和 BL21/pET-EpelB-FRU6。
( 5 ) 果糖苷酶 FRU6的分泌表达： 将重组菌接种至含 100 g/mL氨苄青霉素的 LB液体 培养基， 37°C， 200 rpm培养过夜后， 按 2%接种量接种至新鲜 LB培养基中 (含 100 g/mL氨 苄青霉素)， 37°C， 200 rpm培养 OD600为 0.6-0.9时， 加入诱导剂 IPTG (终浓度 1 mmol/L), 每隔 1小时收集取样。
( 6 ) 酶活检测： 重组菌株摇瓶发酵后， 离心取上清和破碎细胞取无细胞破碎液。 检测 BL2 l/pET-EompA-FRU6和 BL21/pET-EpelB-FRU6发酵上清以及胞内的酶活，并且以无增强小 肽模序的重组菌作为阴性对照， 结果见图 2 ( IPTG诱导 0-24小时， 含小肽模序、 不含小肽模 序以及无信号肽的 BL21宿主发酵后上清果糖苷酶分泌效率的比较） 和图 3 (LB培养基中 2% 接种量 6 小时摇瓶发酵取样。 单位菌体质量 （mg ) 条件下， BL21/pET-EompA-FRU6 或 BL21/pET-EpelB-FRU6与阴性对照发酵上清液酶活力比较） （更多小肽模序增强效果见表 2， 其中每个小肽模序均选取发酵 6小时取样检测酶活， 其他时间取样品不在此一一列举）。
酶活检测方法如下：①底物的配制： O. lg葛根素， 2.8g蔗糖充分溶解在 100 mL 0.05 mol/L, pH6的磷酸盐缓冲液中。②反应体系： 取 50 μL的磷酸盐缓冲（0.05 mol/L, pH 6 )加入 950 μL 底物中， 置于 35 °C中反应 lO min后， 立即取出 100 加入 900 的甲醇中终止反应， 作为 空白对照。同时另取 50 μL·酶液加入 950 μL·底物中，置于 35 °C中反应 10 min,后立即取出 100 加入 900 μ 的甲醇中终止反应，作为样品。 HPLC进行测定。③酶活力单位定义为：在 35 V 条件下， 每分钟反应消耗 1 μ§葛根素所需酶的量为一个活力单位 （U)。
( 7 ) SDS-PAGE聚丙烯酰胺凝胶电泳： 操作方法见 《分子克隆手册》， 结果见图 4和图 根据上述酶活检测方法，综合表 2和表 3可知，在果糖苷酶 FRU6分泌表达所采用的 ompA 或 pelB等信号肽的前端融合了表 2所述的本发明所述的小肽后， 该酶的酶活明显增强， 说明 本发明所述的小肽增强模序具有极强的增强分泌表达的能力。
实施例 2小肽增强模序在葡聚糖酶分泌表达上的应用
( 1 ) 葡聚糖酶 BGL的 N端不同信号肽前添加小肽增强模序表达载体构建
与实施例 1相同， 本实施例中将实施例 1中的果糖苷酶替换成葡聚糖酶基因。
其中，葡聚糖酶 BGL的序列的来源： Bacillus subtilis subsp. subtilis 6051-HGW, GenBank 序列号： CP003329.1 , 范围 ： 4011849 至 lj 4012490。
引物 P79 和 P80 进行 PCR反应扩增出葡聚糖酶 BGL 的基因片段， 制备成 T 载体 pMD-T-BGL。重叠 PCR引物 P81-P84或 P85-P88 ,分别将葡聚糖酶 BGL和信号肽 ompA或 pelB 融合， 其中 P84和 P88均带有 EcoR I酶切位点。 P89-P96上游引物与下游引物 P84或 P88联 用， 可以分别设计不同的增强模序融合在 ompA或 pelB信号肽前。
本实施例所涉及的所有引物均由英骏公司合成， 见表 4。
表 5实施例 2所需的引物
Figure imgf000014_0001
80 TTATTTTTTTGTATAGCGCACCCA
81 ATGAAAAAGACAGCTATCGCG
82 AAAAAACGATCCACCTGTTTGAGCTTGGGCTACGGT
83 TACCGTAGCCCAAGCTCAAACAGGTGGATCGTTTTTT
84 CCGGAATTTTATTTTTTTGTATAGCGCACCCA
85 ATGAAATACCTATTGCCTACG
86 AAAAAACGATCCACCTGTTTGAGCCATGGCTGGTTGGGCAGC
87 CCAACCAGCCATGGCTCAAACAGGTGGATCGTTTTTT
88 CCGGAATTTTATTTTTTTGTATAGCGCACCCA
89 CGCCATATGGAACGAGCATGTGTTGCAATGAAAAAGACAGCTATCGCG
90 CGCCATATGGAACGAGCATGTGTTGCAATGAAATACCTATTGCCTACG
CGCCATATGGTGGAGAGACTATGTGTGGCAGTGGTTGAAAGGGCGTGTGCGCTAGCGATGAAA
91
AAGACAGCTATCGCG
CGCCATATGGTGGAGAGACTATGTGTGGCAGTGGTTGAAAGGGCGTGTGCGCTAGCGATGAAAT
92
ACCTATTGCCTACG
CGCCATATGGTTGAAAGGTGTCTCGCGACCCTCGTGGAGAGACTATGTGTGGCAGTGGTTGAA
93
AGGGCGTGTGCGCTAGCG ATGAAAAAGACAGCTATCGCG
CGCCATATGGTTGAAAGGTGTCTCGCGACCCTCGTGGAGAGACTATGTGTGGCAGTGGTTGAA
94
AGGGCGTGTGCGCTAGCG ATGAAATACCTATTGCCTACG
95 CGCCATATGATGAAAAAGACAGCTATCGCG
96 CGCCATATGATGAAATACCTATTGCCTACG
注： 此表中引物编号对应于 P或者 SEQ ID NO。
(2) 在 LB培养基中分泌表达葡聚糖酶 BGL
将构建的葡聚糖酶 BGL分泌表达载体转化进表达宿主 BL21 (DE3 ), 最终得到重组菌株 命名为 BL21/pET-EompA-BGL和 BL21/pET-EpelB-BGL。在含 100 g/mL氨苄霉素的 LB平板 上筛选转化子并从中提取质粒验证， 方法见 《分子克隆手册》。
将重组菌接种至含 lOO g/mL氨苄青霉素的 LB液体培养基， 37°C， 200 rpm培养过夜后， 按 2%接种量接种至新鲜 LB培养基中（含 100 g/mL氨苄青霉素）， 37°C， 200 rpm培养 OD6(X) 为 0.6〜0.9时， 加入诱导剂 IPTG(终浓度 1 mmol/L；)， 诱导 6 h后收集。
(3 ) 葡聚糖酶酶活测定方法
重组菌株摇瓶发酵后， 离心取上清和破碎细胞取无细胞破碎液。 检测 BL21/pET-EompA-BGL和 BL21/pET-EpelB-BGL发酵上清以及胞内的酶活，并且以无增强小肽 模序的重组菌作为阴性对照， 小肽模序的增强效果见表 5 (选取部分结果， 均为发酵 6小时取 样检测酶活）
DNS 法测定酶活力： 取 1% ( W/V) 的大麦 β-葡聚糖 （溶解于 ρΗ 6.5， 20 mmol/L Na2HP04-Citrate缓冲液） 1.0 mL作为底物， 加入 0.5 mL适当稀释的酶液， 在 45°C条件下反应 10 min, 用 DNS法测定还原糖， 以灭活的酶作为空白对照。 酶活力单位定义： 将在上述条件 下， 每分钟由底物产生 1 μιηοΐ还原糖所需的酶量定义为一个活力单位 （U)。
表 4对应葡聚糖酶涉及的增强分泌的小肽模序氨基酸序列和酶活
Figure imgf000016_0001
注： 此表中引物编号对应于 P或者 SEQ ID NO。
( 4 )重组菌 BL21/pET-EompA-BGL和 BL21/pET-EpelB-BGL表达产物的 SDS-PAGE检测， 操作方法见 《分子克隆手册》， 结果见图 6和图 7。
根据本领域技术人员的公知常识可知，通过本发明的所述的强分泌性信号肽增强小肽模序 的变体， 其具强分泌性信号肽增强小肽模序的其中 1 个或多个氨基酸残基的插入或缺失和 /或 性质相似的氨基酸取代 1个或多个氨基酸残基， 属于基于本发明的一种变形或合理扩展， 也应 当属于本发明的保护范围。
同理，编码具有本发明所述的强分泌性信号肽增强小肽模序的多肽、类似物或衍生物的多 核苷酸；一种含有外源多核苷酸的重组载体，其是由本发明所述的多核苷酸与质粒载体构建而 成的重组载体； 一种含有外源多核苷酸的遗传宿主细胞， 它是由本发明所述的重组载体转化或 转导的宿主细胞。 上述变形都应属于本发明所保护的范围。

Claims

权利要求
1. 强分泌性信号肽增强小肽模序， 其特征在于其具有如下通式的氨基酸序列： Μ(αΧβΥγ/αΥβΧγ)η,
其中 X代表酸性氨基酸；
Υ代表碱性氨基酸；
α为 0-2个中性氨基酸；
β代表 0-2个中性氨基酸；
γ代表 1-10个中性氨基酸；
η为 1-3。
2. 根据权利要求 1所述的强分泌性信号肽增强小肽模序， 其特征在于所述的 X代表的酸 性氨基酸为 Glu或 Asp。
3. 根据权利要求 1所述的强分泌性信号肽增强小肽模序， 其特征在于所述的 Y代表的碱 性氨基酸为 Arg或 Lys。
4. 根据权利要求 1所述的强分泌性信号肽增强小肽模序， 其特征在于所述的中性氨基酸 为 Ala、 Cys、 Leu、 Val、 lie或者 Phe。
5. 根据权利要求 1所述的强分泌性信号肽增强小肽模序， 其特征在于所述的 n为 1。
6. 根据权利要求 1所述的强分泌性信号肽增强小肽模序， 其特征在于所述的 α为 1个中 性氨基酸， β为 0个中性氨基酸， γ为 2-5个中性氨基酸， X为 Glu， Y为 Arg。
7. 权利要求 1所述的强分泌性信号肽增强小肽模序的变体， 其具有权利要求 1的强分泌 性信号肽增强小肽模序的其中 1个或多个氨基酸残基的插入或缺失和 /或性质相似的氨基酸取 代 1个或多个氨基酸残基。
8. 编码具有权利要求 1所述的强分泌性信号肽增强小肽模序的多肽、 类似物或衍生物的 多核苷酸。
9. 一种含有外源多核苷酸的重组载体， 其是由权利要求 8所述的多核苷酸与质粒载体构 建而成的重组载体。
10. 一种含有外源多核苷酸的遗传宿主细胞， 它是由权利要求 9所述的重组载体转化或转 导的宿主细胞。
11. 权利要求 1所述的强分泌性信号肽增强小肽模序的应用， 其特征在于将强分泌性信号 肽增强小肽模序用于构建一种增强普通信号肽分泌能力的载体，以提高其分泌表达外源蛋白的 方法。
PCT/CN2014/076249 2013-05-03 2014-04-25 强分泌性信号肽增强小肽模序及其应用 WO2014177021A1 (zh)

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