WO2017120890A1 - 一种利用磷脂酶融合表达以提高外源蛋白表达量的方法 - Google Patents

一种利用磷脂酶融合表达以提高外源蛋白表达量的方法 Download PDF

Info

Publication number
WO2017120890A1
WO2017120890A1 PCT/CN2016/071043 CN2016071043W WO2017120890A1 WO 2017120890 A1 WO2017120890 A1 WO 2017120890A1 CN 2016071043 W CN2016071043 W CN 2016071043W WO 2017120890 A1 WO2017120890 A1 WO 2017120890A1
Authority
WO
WIPO (PCT)
Prior art keywords
fusion
protein
gene
sequence
seq
Prior art date
Application number
PCT/CN2016/071043
Other languages
English (en)
French (fr)
Inventor
喻晓蔚
徐岩
Original Assignee
江南大学
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 江南大学 filed Critical 江南大学
Priority to PCT/CN2016/071043 priority Critical patent/WO2017120890A1/zh
Publication of WO2017120890A1 publication Critical patent/WO2017120890A1/zh
Priority to US15/995,134 priority patent/US10323247B2/en

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/37—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi
    • C07K14/38—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi from Aspergillus
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
    • C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
    • 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)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/18—Carboxylic ester hydrolases (3.1.1)
    • C12N9/20—Triglyceride splitting, e.g. by means of lipase
    • 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)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y301/00—Hydrolases acting on ester bonds (3.1)
    • C12Y301/01—Carboxylic ester hydrolases (3.1.1)
    • C12Y301/01004—Phospholipase A2 (3.1.1.4)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
    • C07K2319/24—Fusion polypeptide containing a tag with affinity for a non-protein ligand containing a MBP (maltose binding protein)-tag
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/61—Fusion polypeptide containing an enzyme fusion for detection (lacZ, luciferase)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
    • C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
    • C12N15/815—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts for yeasts other than Saccharomyces

Definitions

  • the present invention relates to a method for enhancing expression of a foreign protein by using phospholipase fusion expression, and particularly relates to a method for efficiently expressing a proline-specific endoprotease using PLA 2 as a fusion tag, GGGGSGGGGS as a linker peptide, and yeast Engineering bacteria and their applications belong to the field of bioengineering technology.
  • Prolyl endopeptidase [EC 3.4.21.26], which is not a typical serine protease family, is a special class of serine proteases, together with E. coli protease II (protease II). A family of aminoaceptidase. Most of the prolyl endopeptidases have a molecular weight of about 75 kDa, an optimum temperature of about 45 ° C, and an optimum pH of about 4.5.
  • a prolyl endopeptidase is a protease that specifically hydrolyzes a proline carboxy terminal peptide bond in a molecular polypeptide (the rate of hydrolysis of small peptides is much higher than that of a macropeptide), and is a site-specific protease. Prolyl endopeptidase also acts on alanine residues, but the hydrolysis efficiency is much lower than that of proline.
  • prolyl endopeptidase The most significant role of prolyl endopeptidase is in the treatment of biomedical diseases, and international research hotspots are also in this area.
  • PEP can degrade many peptide neurotransmitters and hormones, and its abnormal activity can cause mediation learning and spatial memory formation disorders.
  • Alzheimer's disease, glucose metabolism and regulation of pancreatic function therefore, the in-depth study of the physiological functions of the enzyme and the search for development inhibitors to treat memory disorders have potential social and economic benefits.
  • Celiac disease is a T cell-dominant bran intolerance to plants such as wheat.
  • the gluten-mediated T cell immunological epitope is rich in proline and therefore highly resistant to protein degradation in the gastrointestinal tract.
  • Ordinary prolyl endopeptidase oral agents have a number of limitations: for example, low stability in low pH environments, very sensitive to pepsin, and inability to completely digest all gluten in a normal diet per meal. Stepniak et al. obtained a novel prolyl endopeptidase (AN-PEP) from Aspergillus niger, which can effectively reduce gluten content in the simulated gastrointestinal environment in vitro and overcome many defects of appeal.
  • AN-PEP novel prolyl endopeptidase
  • the PEPase treatment has the advantage of processing compared to the silica gel and PVPP methods: no raw materials need to be processed.
  • the protein which is easy to cause turbid precipitation during beer storage is mainly a special polypeptide rich in proline fragment, which can be used to decompose the protein fragment causing macromolecular turbidity by using proline endoprotease, thereby improving the abiotic stability of beer. This replaces the currently used treatment methods for improving the stability of beer colloids.
  • PEP can be used as a molecular biology tool enzyme for protein sequencing, peptide mapping, specific site digestion, peptide chain modification and processing.
  • prolyl endopeptidase Since prolyl endopeptidase has such an important use, researchers have performed exogenous expression of prolyl endopeptidase. For example, in 1980, Japanese scholars discovered a strain of S. septicum that can produce a prolyl endothelium, and the enzyme activity after purification reached 34.5 U/g (wet cells). Subsequently, the research team introduced the PEP gene into E. coli for exogenous expression of 558 U/g (wet cells). In 1993, the prolyl endopeptidase derived from Aeromonas hydrophila was cloned and expressed in Escherichia coli JM83, and the enzyme activity reached 320 U/g. However, the exogenous expression activity of prolyl endopeptidase is not high. Therefore, it is important to develop effective strategies to increase the expression of PEP in foreign hosts.
  • the present invention provides a method of fusion expression with phospholipase to increase the expression level of a foreign protein, and a yeast engineering bacteria obtained based on the method and use thereof.
  • a first object of the present invention is to provide a fusion gene obtained by sequentially ligating a gene fragment encoding a fusion tag, a linker peptide fragment, and a gene fragment encoding a foreign protein.
  • the fusion tag is any one of the following: phospholipase A2 (PLA 2 ), cellulose binding domain (CBD), ubiquitin-related small tag regulator (SUMO), maltose binding Protein (MBP).
  • PHA 2 phospholipase A2
  • CBD cellulose binding domain
  • SUMO ubiquitin-related small tag regulator
  • MBP maltose binding Protein
  • the fusion tag is phospholipase A2, the amino acid sequence of which is:
  • nucleotide sequence encoding the amino acid sequence set forth in SEQ ID NO: 3 is set forth in SEQ ID NO: 8.
  • the amino acid sequence of the linker peptide is set forth in SEQ ID NO: 1 (GGGGSGGGGS).
  • the exogenous protein is a prolyl endopeptidase.
  • amino acid sequence of the prolyl endopeptidase is set forth in SEQ ID NO:4.
  • the fusion gene is directly linked to a linker peptide fragment encoding an amino acid sequence such as SEQ ID NO: 1 after encoding a gene fragment encoding the amino acid sequence of phospholipase A2, and then directly linked after ligation of the peptide fragment A fragment of a prolyl endopeptidase gene encoding an amino acid sequence such as SEQ ID NO: 4.
  • nucleotide sequence of the fusion gene is set forth in SEQ ID NO: 2.
  • a second object of the present invention is a genetically engineered bacteria expressing the fusion gene.
  • the yeast engineered bacteria is Pichia pastoris; and may be Pichia pastoris X33, GS115, KM71 or SMD1168, preferably Pichia pastoris GS115.
  • the vector used for expression may be pPIC9, pPIC3.5K, pPIC3.5, pPIC9K, PAO815 or pPICZ[alpha] series and the like; preferably pPICZ[alpha]A.
  • the Pichia pastoris engineering bacteria firstly ligates the fusion gene represented by the SEQ ID NO. 2 to the pPICZ ⁇ A to obtain the recombinant vector pPICZ ⁇ A-PLMH, and then recombines the recombinant plasmid pPICZ ⁇ A-PLMH.
  • the plasmid was linearized and transformed into Pichia pastoris.
  • the Pichia pastoris is Pichia pastoris GS115, or Pichia pastoris GS115 integrated with the pPIC9K plasmid (to overcome the histidine deficiency of Pichia pastoris, to facilitate culture).
  • the sequence of the fusion gene is followed by a 6x His tag for isolation and purification.
  • a third object of the present invention is to provide a method for producing a prolyl endopeptidase, which comprises using the fusion gene or an expression vector containing the fusion gene, or a genetically engineered bacteria expressing the fusion gene.
  • the genetically engineered bacteria (Pichia pastoris) expressing the fusion gene is activated and inoculated into BMGY medium, and cultured at 30 ° C, 200 r / min for 16-18 h.
  • the bacterial liquid was centrifuged, and the bacterial body weight was suspended in BMMY medium, and then cultured at 28 ° C, 250 r / min, and induced by adding 1% methanol every 24 hours.
  • the method is to activate a genetically engineered bacteria (Pichia pastoris) expressing the fusion gene, and inoculate the fermenter in a BSM basic salt medium (containing CaSO 4 ( Cp) 1.1 g/L, K 2 SO 4 (AR) 18.2 g/L, anhydrous magnesium sulfate (AR) 7.27 g/L, KOH (AR) 4.128 g/L, glycerol 40 g/L, 85% H 3 PO In 4 26.7ml/L, the glycerol growth phase was cultured at 30 ° C and pH 5.5.
  • a BSM basic salt medium containing CaSO 4 ( Cp) 1.1 g/L, K 2 SO 4 (AR) 18.2 g/L, anhydrous magnesium sulfate (AR) 7.27 g/L, KOH (AR) 4.128 g/L, glycerol 40 g/L, 85% H 3 PO
  • the glycerol growth phase was cultured at 30
  • a fourth object of the present invention is to provide use of a prolyl endopeptidase produced by using the fusion gene, an expression vector containing the fusion gene, or a genetically engineered strain expressing the fusion gene.
  • the application is in the fields of food, preparation of drugs, molecular biology and the like.
  • Such applications include reducing the level of sensitive proteins in the beer fermentation broth, or increasing the abiotic stability of the beer, or removing the bitter taste of the protein hydrolysate.
  • Such uses also include the preparation of a medicament for modulating glucose metabolism or pancreatic function.
  • the application further includes the use of prolyl endopeptidase as a molecular biological tool enzyme for protein sequence determination, peptide mapping, specific site digestion, peptide chain modification and processing.
  • a fifth object of the present invention is to provide a method for enhancing expression of a foreign protein by fusion expression using a phospholipase, which comprises cleavage of a gene fragment encoding phospholipase A2, and an amino acid sequence such as SEQ ID NO: 1 (GGGGSGGGGS)
  • the ligation peptide fragment and the gene fragment encoding the foreign protein are sequentially ligated to obtain a fusion gene, and then the fusion gene is expressed.
  • the fusion tag is phospholipase A2 having the amino acid sequence set forth in SEQ ID NO: 3 or a sequence having 90% or more homology to the sequence of SEQ ID NO: 3.
  • the exogenous protein is a prolyl endopeptidase, the amino acid sequence of which is set forth in SEQ ID NO:4.
  • nucleotide sequence of the fusion gene is set forth in SEQ ID NO: 2.
  • PLA 2 was the best expression when it was used as a fusion label, which was better than the original strain. 7.4 times.
  • PLA 2 derived from S. violaceoruber A-2688 has a high protein expression level (the highest extracellular protein concentration of 12 g/L when the 7 L fermenter is high-density fermentation) and a small molecular weight. The results of this study fully demonstrate that PLA 2, as a fusion tag, can effectively promote the secretion and expression of foreign proteins.
  • Figure 1 Partial indicators of strain shake flask fermentation, a: OD600; b: protein concentration; c: enzyme activity;
  • Figure 2 Figure of strain western blot results, a: extracellular protein; b: intracellular protein; M: marker; 1-6 are MOH, SLMH, CLMH, MLMH, PLMH, blank control;
  • Figure 3 PLMH fusion protein shake flask fermentation SDS-PAGE; wherein M: marker; 1-7 are shake flask fermentation 24, 48, 72, 84, 96, 120h fermentation broth;
  • Figure 4 Identification results of fusion protein PEP and fusion tag PLA2 mass spectrometry
  • Figure 5 Comparison of enzymatic properties of PLMH and MOH, a: optimum temperature and temperature stability; b: optimum pH and pH stability;
  • Figure 6 Effect of different enzyme dosages on sensitive proteins in beer fermentation broth; M:marker; 1-4 are beer fermentation broth samples after adding 0, 5, 15, 25 U/L unit enzyme treatment; Proline protease.
  • the reaction system consists of 10 ⁇ l of enzyme solution, 10 ⁇ l of 5 mM substrate (Ala-Ala-Pro-pNA), 80 ⁇ l of disodium hydrogen phosphate-citrate buffer, 40 ° C, reaction for 10 min, and absorbance by enzyme microplate at 410 nm (OD410). ).
  • enzyme unit (U) Under the conditions of the enzyme activity specified above, the amount of enzyme required to decompose the catalytic substrate per minute to produce 1 ⁇ mol of pNA is one prolyl endopeptidase enzyme unit.
  • ⁇ A absorbance change ODtest-ODblank
  • V total volume of reaction system (mL)
  • v1 sample amount (mL)
  • r molar extinction coefficient (cm 2 ⁇ ⁇ mol -1 )
  • b cuvette or enzyme label Plate optical path (cm);
  • t reaction time (min).
  • the fusion protein shake flask fermentation supernatant and intracellular protein were subjected to western blot analysis, and the western blot operation steps were as follows:
  • the protein gel is cut to an appropriate size and immersed in a transfer buffer to equilibrate.
  • the sealed PVDF membrane was immersed in a primary antibody solution diluted 1:1000 in PBST, and incubated at room temperature for 1 hour. The membrane was washed 5 times with PBST for 10 min each time.
  • the PVDF membrane was immersed in a secondary antibody solution diluted 1:400 in PBST, and the antibody pre-stained with Marker was added at 1:10000.
  • the bleaching shaker was shaken for 1 h.
  • the membrane was washed 5 times with PBST for 10 min each time.
  • the fermentation broth was centrifuged at 6000 ⁇ g for 30 min, the cells were discarded, and the supernatant was collected.
  • the 30-kDa ultrafiltration tube was concentrated by ultrafiltration. After concentration, the fermentation broth was treated with 60% ammonium sulfate (NH 4 ) 2 SO 4 . Precipitate for 4 h.
  • the protein pellet was dissolved in 20 mM phosphate buffer (pH 5.0) and dialyzed overnight. This process is operated on ice at 4 °C throughout. The dialyzed protease solution was subjected to Ni column purification.
  • Ni-NTA nickel column purification step 500 mL of fermentation broth obtained by shake flask fermentation was centrifuged at 6300 rpm for 30 min at 4 ° C, the supernatant was taken, filtered through a 0.22 ⁇ m aqueous phase microfiltration membrane, and concentrated to 50 mL with a 30-kDa ultrafiltration tube.
  • the AKTApurifier protein purification system routinely performs protein purification.
  • the system and the 5 mL Ni-NTA Superflow Cartridge column were pre-equilibrated with the A solution before loading, and the sample was collected, paying attention to the collection of the breakthrough peak.
  • the system and the Ni column were washed again to equilibrium with an equilibration solution.
  • the protein was officially washed with solution B and washed with 5% of solution B to elute the hybrid protein attached to the column. 0-0.05mol ⁇ L -1 eluent was linearly eluted. Note that the elution peak was collected, the flow rate was controlled at 2 mL ⁇ min -1 , and the elution peak was detected by SDS-PAGE gel electrophoresis. The purified protein was subjected to SDS-PAGE analysis.
  • Optimum pH detection method prepare buffers of different pH, 0.02mol ⁇ L-1 citrate phosphate buffer (pH 2.0-8.0), 0.05mol ⁇ L-1 Tris-HCl buffer (pH 9.0-10.5), glycine/NaOH buffer (pH 11.0-12.0), and determine the enzyme activity of the prolyl endopeptidase in these pH ranges, with the highest enzyme activity being 100% relative enzyme activity.
  • the phospholipase A 2 gene is derived from Streptomyces violaceoruber, the amino acid sequence is SEQ ID NO: 3, and the sequence can be amplified from the pPIC9K-PLA 2 plasmid previously constructed in our laboratory; prolyl endopeptide
  • the enzyme (PEP) gene MO is derived from Aspergillus oryzae, the amino acid sequence is SEQ ID NO: 4, and the sequence can be amplified from the previously constructed pPIC9K-MO plasmid; maltose binding protein (MBP), cellulose binding domain
  • the amino acid sequences of the (CBD) and ubiquitin-related small tag regulator (SUMO) genes are SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7, respectively.
  • the above genes were optimized according to Pichia pastoris codon preference and synthesized by Bioengineering Engineering (Shanghai) Co., Ltd.
  • fusion gene fusion gene (phospholipase A 2, MBP, CBD, SUMO ) PEP gene fragment and gene fragments, overlap extension PCR to give after column purification (fusion gene fusion gene
  • the proteins are sequentially named PLMH, MLMH, CLMH, SLMH, wherein the fusion gene contains a fusion tag fragment-linker peptide-PEP gene fragment in turn, wherein the linker peptide sequence (GGGGSGGGGS) is shown as SEQ ID NO. 1.
  • the obtained phospholipase is contained.
  • the nucleotide sequence of the fusion gene PLMH of A 2 (nucleotide sequence as shown in SEQ ID NO: 8) is shown in SEQ ID NO. 2.
  • the primers used in the present invention are shown in Table 1.
  • the underlined portion is an enzyme cleavage site
  • the black bold is a ligated peptide base sequence
  • the fusion gene fragment PLMH obtained after purification of the column of Example 1 was subjected to addition of A tail (aKaRa rTaq 0.5 ⁇ L, 10 ⁇ PCR Buffer 5 ⁇ L, dNTP Mixture 4 ⁇ L, purified product 40.5 ⁇ L at 72 ° C for 15-20 min) Then, it was ligated to the pMD-19T vector and transformed into E. coli.
  • the plasmid of the transformant was extracted, sequenced, and the recombinant plasmid confirmed to be correct was named pMD-19T-PLMH.
  • pPIC9K is designed to overcome the host's histidine deficiency to facilitate culture).
  • Electrotransfer method Take a tube of competent cells and transfer them to the linearized plasmid after enzymatic digestion and concentration, and then mix them evenly, transfer them to a 2mm pre-cooled electric rotor, place them on ice for 3-5 minutes, and then carry out electrospinning; It is: voltage 1500V, resistance 200 ⁇ , capacitance 25 ⁇ F.
  • 1 mL of 1 mol/L sorbitol solution was added, mixed, transferred to 1.5 mL EP tube, and resuscitated at 30 ° C for 1 h. After centrifugation, the supernatant was removed to 100 ⁇ L, and the cells were resuspended and coated with YPD+Zeocin plate.
  • a series of single colony transformants were obtained after 3 days of culture in a 30 ° C incubator.
  • a monoclonal antibody was selected for positive verification, and the correct strain was verified to be a successful fusion expression protein of Pichia pastoris genetically engineered strain GS115/pPIC9K/pPICZ ⁇ A-PLMH, and the secreted proline protease was named PLMH.
  • Example 4 Shake flask fermentation of four proline protease recombinant strains
  • the recombinant strain was streaked on the YPD plate for about 3 days, and the monoclonal and 25 mL flasks containing 25 ml of BMGY medium were picked and cultured at 30 ° C, 200 r / min for 16-18 h, so that the OD 600 reached 2-6.
  • the bacterial solution was centrifuged, and the bacteria were suspended in 50 mL of BMMY medium, cultured at 28 ° C, 250 r / min, and induced by adding 1% methanol every 24 hours.
  • the bacterial concentration, protein concentration and enzyme activity were determined by timed sampling.
  • the inventors used a GGGGSGGGGSKR (amino acid sequence such as SEQ ID NO: 19) linker peptide containing a KEX2 protease cleavage site and a LEVLFQGPENLYFQS (amino acid sequence such as SEQ ID NO: 20) linker peptide containing two protease cleavage sites, respectively.
  • GGGGSGGGGSKR amino acid sequence such as SEQ ID NO: 19
  • LEVLFQGPENLYFQS amino acid sequence such as SEQ ID NO: 20
  • Example 7 SDS-PAGE analysis and mass spectrometric identification of fusion protein PLMH
  • the purified PLMH and MOH were studied for enzymatic properties, including optimum temperature, temperature stability, pH optimum, pH stability, k m , k cat .
  • PLMH and MOH have consistent enzymatic properties.
  • the optimum temperature for both is 40 °C.
  • the remaining enzyme activity was almost zero.
  • the optimum pH of both is 5.5, and at pH 6.0, the stable phase is the best.
  • the remaining enzyme activity was still higher than 40%, indicating that PEP was better tolerant to pH.
  • the K m , k cat , k cat /K m of the fusion protein PLMH were 0.23 ⁇ 0.01 mM, 112.51 ⁇ 0.02 S -1 , 489.17 s -1 mM -1
  • K m of the MOH, k cat , k cat / K m were 0.28 ⁇ 0.01mM, 139.4 ⁇ 0.02S -1 , 496.4s -1 mM -1, little difference between the two.
  • the sensitive protein causing the turbidity of the beer fermentation broth is about 40 kDa and 12 kDa, compared with the blank control.
  • Proline protease can reduce the content of sensitive protein in beer fermentation broth, and the content of sensitive protein decreases with the increase of enzyme amount.
  • the prolyl endopeptidase PLMH was added to the filtered beer fermentation broth according to the concentration gradient (5 U/L, 15 U/L, 25 U/L), and stored at 4 ° C in cold storage. A turbidity measurement was taken every other week. Each group is three parallels and lasts for 6 weeks.
  • proline protease The effect of proline protease on the non-biological stability of beer after fermentation was determined.
  • the accumulation of protein in beer during storage leads to an increase in turbidity (EBC).
  • EBC turbidity
  • the addition of proline protease can reduce the turbidity during beer storage, and the effect is increased with the increase of enzyme amount.
  • Add 15U/ The post-L EBC was reduced to 0.3, indicating that the proline protease developed by the present invention can be effectively applied to the beer industry.
  • High-density fermentation of 7L fermentor was carried out using the genetically engineered strain GS115/pPIC9K/pPICZ ⁇ A-PLMH containing the fusion gene PLMH.
  • the activity of PLMH enzyme was measured after fermentation, and the highest enzyme activity reached 1800 U/L.
  • the fermentation method is as follows:
  • the recombinant bacteria were placed in YPD medium, cultured for 16-18 h, and then inoculated into a 7 L fermentor containing 2.1 L of BSM basic salt medium, and the glycerol growth phase was cultured at 30 ° C and pH 5.5.
  • the glycerol is consumed in the basal medium, the dissolved oxygen value increases greatly, and then enters the glycerol transition phase, and 50% (V/V) glycerol (including 12 mL/L PTM1) is added to make the OD 600 value of the cells reach 90-110.
  • V/V 50%
  • glycerin supplementation was stopped, starvation for 0.5 h, and then the induction phase was introduced, and methanol was added thereto.
  • the methanol concentration was maintained at 0.08 to 0.12%, and the temperature was controlled at 26 to 28 °C.
  • YPD protein powder 20g / L, yeast powder 10g / L, glucose 20g / L;
  • BSM CaSO 4 (cp) 1.1 g / L, K 2 SO 4 (AR) 18.2 g / L, anhydrous magnesium sulfate (AR) 7.27 g / L, KOH (AR) 4.128 g / L, glycerol 40 g / L, 85% H 3 PO 4 26.7 ml/L;
  • PTM1 6g/L copper sulfate pentahydrate, 0.089g/L potassium iodide, 3.0g/L sulfuric acid monohydrate, 0.2g/L sodium molybdate, 0.02g/L boric acid, 42.2g/L zinc sulfate heptahydrate, heptahydrate Ferrous sulfate 65g / L, biotin 0.2g / L, cobalt chloride hexahydrate 0.5g / L, sulfuric acid 5ml / L.
  • Example 12 Effect of phospholipase A2 gene sequence on fusion protein
  • phospholipase A2 Since phospholipase A2 has phospholipase activity, if it is directly applied to the fermentation supernatant of genetically engineered bacteria, the phospholipase activity contained therein may affect the application effect. Therefore, two sets of mutations were designed to investigate the effect of mutations on phospholipase A2 activity and fusion protein expression levels.
  • truncation mutation which is the amino acid sequence of SEQ ID NO: 3, which truncates the four amino acids from position 1 to position 4; the result shows that the fusion of phospholipase A2 with a truncated amino acid sequence is expressed.
  • the recombinant strain of the gene still detected phospholipase A2 activity in the fermentation supernatant, but did not affect the expression of proline protease, and the enzyme activity was comparable to that without truncation.
  • the second group of mutations is a point mutation, which is based on the amino acid sequence of SEQ ID NO: 3, and amino acid mutations at position 69, 70, 73, 74, 77 or 90, respectively.
  • alanine As alanine (Ala, A), it was found that a recombinant strain containing a fusion gene of six phospholipase A2 mutant sequences did not detect phospholipase activity in the fermentation supernatant, but did not affect proline protease expression. Enzyme activity is comparable to no mutation.

Abstract

一种利用磷脂酶融合表达以提高外源蛋白表达量的方法，属于生物工程技术领域。以PLA 2，MBP，CBD，SUMO四种蛋白作为融合标签，构建融合表达菌株。与出发菌株MOH相比，四种融合蛋白的胞外蛋白表达量及酶活均有不同程度的提高，其中，以PLA 2作为融合标签时的表达效果最好，比出发菌株提高了7.4倍。与其它融合标签相比，来源于S.violaceoruber A-2688的PLA 2具有蛋白表达量高(7L发酵罐高密度发酵时，胞外最高蛋白浓度可达到12g/L)及分子量小等特点。PLA 2作为融合标签，能有效的促进外源蛋白的分泌表达。

Description

一种利用磷脂酶融合表达以提高外源蛋白表达量的方法 技术领域
本发明涉及一种利用磷脂酶融合表达以提高外源蛋白表达量的方法，具体涉及一种利用PLA2作为融合标签，以GGGGSGGGGS为连接肽，高效表达脯氨酸特异性内切蛋白酶方法、酵母工程菌及其应用，属于生物工程技术领域。
背景技术
脯氨酰内肽酶(prolyl endopeptidase，简称PEP)[EC 3.4.21.26]，不属于典型的丝氨酸蛋白酶家族，是一类特殊的丝氨酸蛋白酶，和大肠杆菌蛋白酶II(protease II)等一起称为脯氨酰内肽酶家族。大多数脯氨酰内肽酶的分子量在75KDa左右，最适作用温度约为45℃，最适pH约为4.5。脯氨酰内肽酶能特异性水解分子多肽中脯氨酸羧基端肽键的蛋白酶(水解小肽段的速率要远远高于大分子肽段)，属于位点专一性蛋白酶。脯氨酰内肽酶也能作用于丙氨酸残基，但是水解效率要远远低于作用于脯氨酸。
脯氨酰内肽酶最显著的作用为在生物医药疾病治疗方面，目前国际上的研究热点也在于此方面。PEP能降解许多多肽类神经递质和激素，其活性的异常会引起调解的学习和空间记忆形成的障碍。阿兹海默症，糖代谢及胰腺功能的调节，因此，深入研究该酶的生理功能和寻找开发抑制剂来治疗记忆障碍性疾病具有潜在的社会效益和经济效益。
几乎所有的蛋白质水解液都会产生不同程度的苦味，主要原因是因为疏水氨基酸(如脯氨酸)的存在，苦味的程度与疏水性氨基酸的含量正相关。Edens等研究发现，从黑曲霉胞外提取纯化的脯氨酰内肽酶，能有效的除去苦味酪蛋白水解液的苦味。
乳糜泻是一种T细胞主导的对小麦等植物麸皮不耐受症。由谷蛋白介导的T细胞免疫表位富含脯氨酸，因此对胃肠道的蛋白质降解有很高的的抗性。普通的脯氨酰内肽酶口服剂具有诸多局限性：例如，在低pH环境下的低稳定性、对胃蛋白酶非常敏感、不能够完全酶解每餐正常饮食中的所有谷蛋白。Stepniak等从黑曲霉中得到了一种新型的脯氨酰内肽酶(AN-PEP)，可以在体外模拟的胃肠道环境中有效的降低谷蛋白含量，且能克服上诉诸多缺陷。
Lopez等发现，在瓶装啤酒发酵过程中，即使添加入极少量的特异性酸性脯氨酰内肽酶，也可以有效的预防啤酒生产过程中的冷浑浊问题，同时啤酒的泡沫稳定性亦不受影响。另外，PEPase处理法与硅胶和PVPP法相比，还有加工过程的优势：不需要处理原材料。啤酒贮存期间容易引起混浊沉淀的蛋白主要是含有丰富脯氨酸片段的特殊多肽，可以通过利用脯氨酸内切蛋白酶来分解引起大分子混浊的蛋白片段，从而提高啤酒的非生物稳定性，以此代替目前常用的提高啤酒胶体稳定性的处理方法。
此外，PEP可以作为一种分子生物学的工具酶，应用于蛋白质序列测定，肽谱分析、特异位点的酶切、肽链的修饰以及加工等。
由于脯氨酰内肽酶具有如此重要的用途，已有研究者对脯氨酰内肽酶进行外源表达。比如，1980年，日本学者发现了可以产脯氨酰内胎的脑膜脓毒性黄杆菌，纯化后酶活达到34.5U/g(湿菌体)。随后，该研究团队将该PEP基因导入到大肠杆菌中外源表达558U/g(湿菌体)。1993年，来源于嗜水气单孢菌的脯氨酰内肽酶被克隆表达于大肠杆菌JM83，酶活达到了320U/g。然而，脯氨酰内肽酶的外源表达活力并不高。因此，开发有效的策略以提高PEP在外源宿主中的表达量至关重要。
发明内容
为了克服上述问题，本发明提供了用磷脂酶融合表达以提高外源蛋白表达量的方法，以及基于该方法得到的酵母工程菌及其应用。
本发明的第一个目的是提供一种融合基因，所述融合基因是将编码融合标签的基因片段、连接肽片段、编码外源蛋白的基因片段依次连接得到的。
在本发明的一种实施方式中，所述融合标签为以下任意一种：磷脂酶A2(PLA2)、纤维素结合域(CBD)、泛素相关的小标签调节器(SUMO)、麦芽糖结合蛋白(MBP)。
在本发明的一种实施方式中，所述融合标签为磷脂酶A2，其氨基酸序列是：
(a)SEQ ID NO:3的序列；或者
(b)在SEQ ID NO:3的序列的的基础上截去了第1位至第4位的4个氨基酸的序列；或者
(c)在SEQ ID NO:3的序列的的基础上，将第69位、第70位、第73位、第74位、第77位或者第90位的氨基酸突变为丙氨酸后的氨基酸序列；或者
(d)与(a)的序列具有90％以上同源性的序列。
在本发明的一种实施方式中，编码SEQ ID NO:3所示的氨基酸序列的核苷酸序列如SEQ ID NO:8所示。
在本发明的一种实施方式中，所述连接肽的氨基酸序列如SEQ ID NO:1(GGGGSGGGGS)所示。
在本发明的一种实施方式中，所述外源蛋白为脯氨酰内肽酶。
在本发明的一种实施方式中，所述脯氨酰内肽酶的氨基酸序列如SEQ ID NO:4所示。
在本发明的一种实施方式中，所述融合基因是在编码磷脂酶A2氨基酸序列的基因片段后直接连接编码氨基酸序列如SEQ ID NO:1的连接肽片段，然后在连接肽片段后直接连接编码氨基酸序列如SEQ ID NO:4的脯氨酰内肽酶基因片段。
在本发明的一种实施方式中，所述融合基因的核苷酸序列如SEQ ID NO:2所示。
本发明的第二个目的一种表达所述融合基因的基因工程菌。
在本发明的一种实施方式中，所述酵母工程菌是毕赤酵母工程菌；可以是毕赤酵母X33，GS115，KM71或SMD1168，优选毕赤酵母GS115。
在本发明的一种实施方式中，所述表达所使用的载体可以是pPIC9，pPIC3.5K，pPIC3.5，pPIC9K，PAO815或pPICZα系列及其类似载体；优选pPICZαA。
在本发明的一种实施方式中，所述毕赤酵母工程菌，是先将核苷酸序列如SEQ ID NO.2所示的融合基因连接到pPICZαA上得到重组载体pPICZαA-PLMH，再将重组质粒线性化后转化入宿主毕赤酵母中得到的。
在本发明的一种实施方式中，所述宿主毕赤酵母为毕赤酵母GS115，或者整合有pPIC9K质粒的毕赤酵母GS115(以克服毕赤酵母的组氨酸缺陷，便于培养)。
在本发明的一种实施方式中，所述融合基因的序列后加上了一段6x His标签，以便于分离纯化。
本发明的第三个目的是提供一种生产脯氨酰内肽酶的方法，是利用所述融合基因或含有所述融合基因的表达载体，或者是表达所述融合基因的基因工程菌。
在本发明的一种实施方式中，所述方法，是将表达所述融合基因的基因工程菌(毕赤酵母工程菌)活化后接种至BMGY培养基，30℃、200r/min培养16-18h，当OD600达2-6时将菌液离心，菌体重 悬于BMMY培养基中，再于28℃、250r/min培养，每24h添加1％甲醇诱导表达。
在本发明的一种实施方式中，所述方法是将表达所述融合基因的基因工程菌(毕赤酵母工程菌)活化后接种至发酵罐中，在BSM基础盐培养基(含有CaSO4(cp)1.1g/L，K2SO4(AR)18.2g/L，无水硫酸镁(AR)7.27g/L，KOH(AR)4.128g/L，甘油40g/L，85％H3PO426.7ml/L)中，于30℃、pH 5.5条件下进行甘油生长相培养，当BSM基础盐培养基中甘油消耗完，溶氧值大幅度上升，然后进入甘油过渡相，流加50％(V/V)甘油(含12mL/L PTM1，其中PTM1含有五水硫酸铜6g/L、碘化钾0.089g/L、一水硫酸猛3.0g/L、钼酸钠0.2g/L、硼酸0.02g/L、七水硫酸锌42.2g/L、七水硫酸亚铁65g/L、生物素0.2g/L、六水氯化钴0.5g/L、硫酸5ml/L)，使菌体OD600值达到90～110之间，停止甘油补加，饥饿0.5h，然后进入诱导相，进行甲醇流加，甲醇浓度维持于0.08～0.12％，温度控制于26～28℃。
本发明的第四个目的是提供利用述融合基因、含有所述融合基因的表达载体或者是表达所述融合基因的基因工程菌生产得到的脯氨酰内肽酶的应用。
所述应用，是用于食品、制备药物、分子生物学等领域。
所述应用包括用于降低啤酒发酵液中敏感蛋白的含量，或者提高啤酒的非生物稳定性，或者去除蛋白质水解液的苦味。
所述应用也包括用来制备调节糖代谢或者胰腺功能的药物。
所述应用，还包括将脯氨酰内肽酶作为一种分子生物学的工具酶，应用于蛋白质序列测定，肽谱分析、特异位点的酶切、肽链的修饰以及加工等。
本发明的第五个目的是提供一种利用磷脂酶融合表达以提高外源蛋白表达量的方法，所述方法是将编码磷脂酶A2的基因片段、氨基酸序列如SEQ ID NO:1(GGGGSGGGGS)的连接肽片段、编码外源蛋白的基因片段依次连接得到融合基因，然后再表达融合基因。
在本发明的一种实施方式中，所述融合标签为磷脂酶A2，其氨基酸序列如SEQ ID NO:3所示，或者与SEQ ID NO:3的序列具有90％以上同源性的序列。
在本发明的一种实施方式中，所述外源蛋白为脯氨酰内肽酶，其氨基酸序列如SEQ ID NO:4所示。
在本发明的一种实施方式中，所述融合基因的核苷酸序列如SEQ ID NO:2所示。
本发明的有益效果：
本发明以PLA2，MBP，CBD，SUMO四种蛋白作为融合标签，构建融合表达菌株。结果表明，与出发菌株MOH相比，四种融合蛋白的胞外蛋白表达量及酶活均有不同程度的提高，其中，以PLA2作为融合标签时的表达效果最好，比出发菌株提高了7.4倍。与其它融合标签相比，来源于S.violaceoruberA-2688的PLA2具有蛋白表达量高(7L发酵罐高密度发酵时，胞外最高蛋白浓度可达到12g/L)及分子量小等特点。此研究结果充分表明，PLA2作为融合标签，能有效的促进外源蛋白的分泌表达。
附图说明
图1：菌株摇瓶发酵部分指标，a：OD600；b：蛋白浓度；c：酶活；
图2：菌株western blot结果图，a：胞外蛋白；b：胞内蛋白；M：marker；1-6分别为MOH，SLMH，CLMH，MLMH，PLMH，空白对照；
图3：PLMH融合蛋白摇瓶发酵SDS-PAGE；其中M：marker；1-7分别为摇瓶发酵24、48、72、84、96、120h发酵液；
图4：融合蛋白PEP及融合标签PLA2质谱鉴定结果；
图5：PLMH及MOH酶学性质比较，a：最适温度及温度稳定性；b：最适pH及pH稳定性；
图6：不同加酶量对啤酒发酵液中敏感蛋白的影响；其中，M：marker；1-4分别为添加0，5，15，25U/L单位酶活处理后啤酒发酵液样品；5：脯氨酸蛋白酶。
具体实施方式
1、蛋白浓度测定方法
将20μl发酵液上清加到96孔板的孔中，再加入200μl G250染色液，室温放置3-5分钟。用酶标仪测定A595波长的吸光度，根据标准曲线计算出样品中的蛋白浓度。
2、蛋白酶活力测定方法
反应体系组成为10μl酶液，5mM底物(Ala-Ala-Pro-pNA)10μl，磷酸氢二钠-柠檬酸缓冲液80μl，40℃，反应10min，410nm波长下用酶标仪测吸光度(OD410)。
酶活单位(U)的定义：在上述指定的酶活测定条件下，每分钟催化底物分解生成相当于1μmol pNA所需的酶量为一个脯氨酰内肽酶酶活单位。
酶活计算公式：酶活(U·mL-1)＝△A*V/(v1rbt)，
其中△A：吸光度变化ODtest-ODblank；V：反应体系总体积(mL)；v1：样品量(mL)；r：摩尔消光系数(cm2·μmol-1)；b：比色杯或酶标板光程(cm)；t：反应时间(min)。
3、western blot分析
将融合蛋白摇瓶发酵上清液和胞内蛋白进行western blot分析，western blot操作步骤如下：
(1)常规蛋白电泳结束后将蛋白胶割至合适大小，浸入转膜缓冲液中平衡。
(2)裁剪与凝胶同样大小的PVDF膜和滤纸(1张膜需要8张滤纸)，先浸纯甲醇、再浸入转膜缓冲液中处理10min。
(3)将PVDF膜、滤纸和凝胶整齐叠放在转印盒中。由下到上的顺序依次为4层滤纸、PVDF膜、凝胶、4层滤纸。每贴一层，用滚轮轻轻滚压，赶走多余气泡和缓冲液，最后用纸巾吸干周围的缓冲液，合上盖子，放入转印仪内。
(4)转印：打开电源；依次选择List、User Defined(此为编辑好的程序25V，1.0A，10min)、Run。
(5)转印完后取出PVDF膜浸于封闭液中，脱色摇床轻轻振荡1h。
(6)将封闭后的PVDF膜浸于按1:1000用PBST稀释的一抗溶液中，振荡室温孵育1h。用PBST清洗膜5次，每次10min。
(7)将PVDF膜浸于按1:400用PBST稀释的二抗溶液中，预染Marker的抗体按1:10000加入。脱色摇床振荡1h。再用PBST洗膜5次，每次10min。
(8)洗涤后的PVDF膜置于化学发光底板中。显色液淋洗(A/B液1:1混合)，凝胶成像仪拍照。胞内蛋白提取使用生物工程(上海)股份有限公司购买的一步法酵母活性蛋白提取试剂盒。
4、融合蛋白的纯化
将摇瓶发酵后84h的发酵液6000×g离心30min，弃菌体，收集上清液，30-kDa超滤管超滤浓缩，浓缩后发酵液用60％硫酸铵(NH4)2SO4沉淀4h。蛋白沉淀用20mM的磷酸缓冲液(pH5.0)溶解，透析过夜。此过程全程在4℃冰上操作。透析后的蛋白酶液进行Ni柱纯化。Ni-NTA镍柱纯化步骤：将 摇瓶发酵所得的500mL发酵液4℃ 6300rpm离心30min，取上清，0.22μm水相微孔滤膜过滤后用30-kDa超滤管浓缩至50mL。AKTApurifier蛋白纯化系统常规方法进行蛋白纯化。上样前先用A液将系统和5mL Ni-NTA Superflow Cartridge层析柱预平衡，上样，注意穿透峰的收集。再次用平衡液将系统和Ni层析柱洗至平衡。正式用B液洗脱收集蛋白前先用5％的B液冲洗，目的是将附着在柱子上的杂蛋白洗脱下来。0-0.05mol·L-1洗脱液线性洗脱，注意洗脱峰的收集，控制流速为2mL·min-1，洗脱峰进行SDS-PAGE凝胶电泳检测纯度。纯化后的蛋白进行SDS-PAGE分析。
5、融合蛋白PLMH的质谱鉴定
重组蛋白样品经过常规SDS-PAGE分离、染色、脱色后，将需要鉴定的目的条带切下，采用肽质量指纹图谱进行酶蛋白的验证。具体操作步骤如下：
(1)用手术刀片切下胶上目标条带，置于EP管中(胶块切成约1mm3大小)
(2)加入200–400μL 100mM NH4HCO3/30％脱色，清洗至透明，去除上清。
(3)每管加入90μL 100mM NH4HCO3，10μL 100mM DTT，56℃孵化30min，还原蛋白质。
(4)去上清，每管加入100μL100％CAN，5min后吸去。
(5)每管加入70μL 100mM NH4HCO3，30μL 200mM IAA，暗处理20min。
(6)去上清，每管加入100μL 100mM NH4HCO3，室温15min。
(7)去上清，加入100μL 100％ACN，5min后吸去，冻干。
(8)冻干后，各加入5μL 2.5–10ng·μL-1trypsin置于4℃ 30-60min，使胶块充分吸胀。
(9)再加入20–30μL左右25mM NH4HCO3缓冲液，37℃反应过夜，20h左右。
(10)吸出酶解液，转移至新EP管冻干。
(11)样品制备完成，加入0.1％TFA复溶，点样，进行质谱分析。
6、融合蛋白PLMH性质研究
(1)最适温度测定方法：按照酶活测定方法所述，将反应体系分别置于25-80℃下，反应5min。
(2)温度稳定性测定方法：将纯化后的融合蛋白酶液在25-80℃下放置120h，按照常规酶活测定方法检测剩余酶活。
(3)最适pH检测方法：配制不同pH的缓冲液，0.02mol·L-1的柠檬酸磷酸盐缓冲液(pH 2.0-8.0)、0.05mol·L-1的Tris-HCl缓冲液(pH 9.0-10.5)、甘氨酸/NaOH缓冲液(pH 11.0-12.0)，并测定脯氨酰内肽酶在这些pH范围的酶活力，以最高酶活为相对酶活100％。
(4)pH稳定性检测方法：将酶液置于上述缓冲液中室温下孵育120h，于pH 5.0,40℃检测剩余酶活。
(5)以Ala-Ala-Pro-pNA为底物，配制底物终浓度为0.1-1.0mM底物溶液，在40℃测定脯氨酰内肽酶活力，双倒数作图，计算相应的动力学参数。所有实验均设置3个平行。
实施例1：融合基因的获得
(1)基因来源
磷脂酶A2基因是来源于紫红链霉菌(Streptomyces violaceoruber)，氨基酸序列如SEQ ID NO:3，序列可从本实验室先前构建的pPIC9K-PLA2质粒上扩增的到；脯氨酰内肽酶(PEP)基因MO来源于米曲霉，氨基酸序列如SEQ ID NO:4，序列可从本实验室先前构建的pPIC9K-MO质粒上扩增的到；麦芽糖结合蛋白(MBP)、纤维素结合域(CBD)、泛素相关的小标签调节器(SUMO)基因的氨基酸 序列分别为SEQ ID NO:5、SEQ ID NO:6、SEQ ID NO:7。上述基因，根据毕赤酵母密码子偏好性进行优化，由生工生物工程(上海)有限公司合成。
(2)采用PCR扩增或者化学合成的方法得到融合标签(磷脂酶A2、MBP、CBD、SUMO)的基因片段和PEP基因片段，柱纯化后进行重叠延伸PCR得到融合基因(融合基因表达的蛋白依次命名为PLMH、MLMH、CLMH、SLMH，其中融合基因中依次含有融合标签片段-连接肽-PEP基因片段，其中连接肽序列(GGGGSGGGGS)如SEQ ID NO.1所示。得到的含有磷脂酶A2(核苷酸序列如SEQ ID NO:8所示)融合标签的融合基因PLMH的核苷酸序列如SEQ ID NO.2所示。本发明用到的引物如表1所示。
表1本发明用到的引物
Figure PCTCN2016071043-appb-000001
其中，下划线部分为酶切位点，黑色加粗为连接肽碱基序列。
实施例2：重组表达载体的构建
以融合基因PLMH为例：
(1)将实施例1柱纯化后得到的融合基因片段PLMH进行加A尾(aKaRa rTaq 0.5μL、10×PCR Buffer 5μL、dNTP Mixture 4μL、纯化产物40.5μL在72℃条件下反应15-20min)，然后连接到pMD-19T载体上，再转化大肠杆菌。提取转化子的质粒，测序，得到验证正确的重组质粒命名为pMD-19T-PLMH。
(2)将测序正确的质粒pMD-19T-PLMH和pPICZαA同时用EcoR I和Not I酶切，然后连接得到含有融合基因PLMH的重组表达载体pPICZαA-PLMH(重组质粒构建过程中，在脯氨酰内肽酶基因序列后添加6x His标签以便于分离纯化)。
采用类似的方法，得到重组表达表达pPICZαA-MLMH、pPICZαA-CLMH、pPICZαA-SLMH、pPICZαA-MOH。其中MOH是在构建重组质粒过程中，在原始的脯氨酰内肽酶MO基因序列后加上了一段6x His标签而得到的。
实施例3：基因工程菌的构建
以重组表达载体pPICZαA-PLMH为例：
将pPICZαA-PLMH用Sac I线性化后电转入P.pichia GS115/pPIC9K酵母感受态中(宿主中导入 pPIC9K是为了克服宿主的组氨酸缺陷，以方便培养)。
电转方法：取一管感受态细胞转移至酶切浓缩后的线性化质粒中，均匀混合，再转移至2mm预冷的电转杯中，冰上放置3-5min后进行电转；电转化仪设置参数为：电压1500V，电阻200Ω，电容25μF。电转化后立即加入1mL 1mol/L的山梨醇溶液，混匀后转移至1.5mL EP管中，30℃复苏1h，离心后除上清至剩余100μL，重悬菌体后涂布YPD+Zeocin平板，30℃培养箱中培养3d后就可得到一系列单菌落转化子。挑取单克隆进行阳性验证，验证正确的菌株即为构建成功的融合表达蛋白的毕赤酵母基因工程菌GS115/pPIC9K/pPICZαA-PLMH，其分泌的脯氨酸蛋白酶命名为PLMH。
采用类似的方法，可制备表达其他融合基因的重组菌株GS115/pPIC9K/pPICZαA-MLMH，GS115/pPIC9K/pPICZαA-CLMH，GS115/pPIC9K/pPICZαA-SLMH及出发菌株GS115/pPIC9K/pPICZαA-MOH。上述菌株分泌的脯氨酸蛋白酶分别命名为MLMH，CLMH，SLMH，MOH。
实施例4：表达四种脯氨酸蛋白酶重组菌的摇瓶发酵
将重组菌划线与YPD平板上培养3天左右，挑取单克隆与含25ml BMGY培养基的250mL三角瓶中，30℃、200r/min培养16-18h，使OD600达2-6，将菌液离心，菌体重悬于50mL BMMY培养基中，28℃、250r/min培养，每24h添加1％甲醇诱导表达。定时取样测定其菌体浓度、蛋白浓度及酶活力。
四种表达融合蛋白重组菌与对照MOH菌株摇瓶发酵结果如图1所示。结果显示，在120h发酵周期内，五种菌株的菌体浓度(OD600)几乎完全一致(图1a)。蛋白浓度方面，96h时，菌体胞外蛋白浓度达到最高(图1b)，PLMH，MLMH，CLMH，SLMH，MOH分别为0.22±0.05mg/ml，0.20±0.05mg/ml，0.17±0.03mg/ml，0.15±0.05mg/ml，0.12±0.04mg/ml，最高酶活出现于84h(图1c)，分别为280±7.1U/L，187±6.9U/L，146±5.5U/L，105±6.5U/L，38±7.2U/L。在摇瓶发酵期间，不论是蛋白浓度还是酶活，PLMH一直高于其它三种融合蛋白及MOH亲本菌株。
实施例5：不同连接肽对融合基因的影响
发明人使用了含有KEX2蛋白酶切位点的GGGGSGGGGSKR(氨基酸序列如SEQ ID NO:19)连接肽和含有两个蛋白酶切位点的LEVLFQGPENLYFQS(氨基酸序列如SEQ ID NO:20)连接肽，分别替代本发明的融合基因PLMH中的连接肽GGGGSGGGGS，构建了另外两种含有不同连接肽的以磷脂酶A2为融合标签的脯氨酸蛋白酶融合基因。并且将这两种融合基因采用类似的方法构建了重组菌并进行发酵培养。
结果显示，以本发明的GGGGSGGGGS为连接肽的融合酶表达水平如图1所示，胞外发酵液酶活力达到280U/L，而使用另外两种连接肽的融合酶在毕赤酵母中无法实现分泌表达，发酵上清液中无法检测到酶活。
实施例6：融合蛋白的western blot检测
对五种重组菌株的胞内、胞外蛋白进行western blot检测分析。结果显示，胞外western blot图中(图2a)目的条带出现在80kDa附近，且只有单一条带，同时，western blot结果与胞外蛋白含量，酶活相一致，融合蛋白含量均高于MOH。胞内western blot结果(图2b)说明毕赤酵母细胞合成的PEP蛋白全部分泌至胞外，胞内并无积聚。
实施例7：融合蛋白PLMH的SDS-PAGE分析和质谱鉴定
GS115/pPIC9K/pPICZαA-PLMH重组菌株摇瓶发酵液SDS-PAGE图实验结果显示，80kDa左右的目的条带随着发酵时间的增加，条带亮度明显变量的同时，24,18,14kDa的条带也明显变量。说明随着甲醇诱导时间的增加，目的蛋白的分泌量也在不断增加。将图3箭头所指的a，b，c，d四条带切胶进行质谱鉴定。
质谱鉴定结果显示，图3a箭头指向的蛋白为PEP，b、c、d箭头指向的为PLA2蛋白，d为原始蛋白，b，c为发生糖基化的结果(仅列举a，c两条带的质谱鉴定结果，如图4)。质谱结果表明，融合蛋白PLMH在细胞内发生了酶原自切割，以PLA2和PEP两种独立的蛋白形式分泌至细胞外。
实施例8：融合蛋白PLMH及MOH蛋白的纯化
蛋白纯化各步骤收率及比活如表2。经过超滤浓缩，硫酸铵沉淀，透析，Ni柱纯化等步骤后获得电泳纯度的PLMH及MOH蛋白。
表2PLMH和MOH的纯化
Figure PCTCN2016071043-appb-000002
实施例9：融合蛋白PLMH及MOH蛋白的酶学性质比较
将纯化后的PLMH及MOH进行酶学性质研究，包括最适温度，温度稳定性，最适pH，pH稳定性，km，kcat。如图5所示，PLMH和MOH有着一致的酶学性质。温度方面，两者的最适温度均为40℃。但是，在40℃孵育120min后，剩余酶活仅剩下30％左右，当温度达到45℃时，剩余酶活几乎为零。pH方面，两者最适pH均为5.5，在pH 6.0时，稳定相最好。在pH 5.0-7.5孵育120min，剩余酶活仍高于40％，说明PEP对pH的耐受性较好。
此外，融合蛋白PLMH的Km,kcat，kcat/Km分别为0.23±0.01mM，112.51±0.02S-1，489.17s-1mM-1，MOH的Km，kcat，kcat/Km分别为0.28±0.01mM，139.4±0.02S-1，496.4s-1mM-1，两者间相差不大。
实施例10：脯氨酰内肽酶PLMH的应用
(1)不同浓度脯氨酸蛋白酶加酶量对啤酒敏感蛋白的影响
将500ml啤酒后酵液过滤后，加入一定量的PEP，40℃恒温水浴1h，100％的饱和硫酸铵沉淀过夜后，4℃，10000rpm离心30min，用柠檬酸盐缓冲液溶解沉淀，4℃透析获得啤酒总蛋白，然后梯度浓度添加PLMH(5U/L，15U/L，25U/L)，40℃酵解3h后进行SDS-PAGE电泳检测。
从图6可以发现，引起啤酒发酵液浑浊的敏感蛋白大小约为40kDa及12kDa，与空白对照相比， 脯氨酸蛋白酶能够降低啤酒发酵液中敏感蛋白的含量，且随着加酶量的增大，敏感蛋白含量逐渐减少。
(2)不同脯氨酸蛋白酶加酶量对啤酒后酵液贮存期间的非生物稳定性研究
按浓度梯度(5U/L，15U/L，25U/L)将脯氨酰内肽酶PLMH加入至过滤除菌后的啤酒发酵液中，4℃冷藏保存。每隔一星期取样进行浊度测定。每组三个平行，持续6周。
测定了脯氨酸蛋白酶对啤酒后酵液非生物稳定性影响。啤酒在贮存过程中会有蛋白值的积聚导致浑浊度(EBC)的增加，加入脯氨酸蛋白酶后能够降低啤酒贮存期间的浑浊度，且随着加酶量的增加效果愈加明显，加入15U/L后EBC降低到0.3，说明本发明开发的脯氨酸蛋白酶能够有效应用于啤酒行业。
实施例11：利用基因工程菌生产脯氨酰内肽酶
使用含有融合基因PLMH的基因工程菌株GS115/pPIC9K/pPICZαA-PLMH进行7L发酵罐高密度发酵。发酵后测定PLMH酶活力，最高酶活达到1800U/L。
发酵方法如下：
将重组菌置于YPD培养基中，培养16-18h，然后接种于7L发酵罐中，发酵罐中含有BSM基础盐培养基2.1L，在30℃、pH5.5条件下进行甘油生长相培养。当基础培养基中甘油消耗完，溶氧值大幅度上升，然后进入甘油过渡相，流加50％(V/V)甘油(含12mL/L PTM1)，使菌体OD600值达到90～110之间，停止甘油补加，饥饿0.5h，然后进入诱导相，进行甲醇流加，甲醇浓度维持于0.08～0.12％，温度控制于26～28℃。
其中
YPD：蛋白粉20g/L，酵母粉10g/L，葡萄糖20g/L；
BSM：CaSO4(cp)1.1g/L，K2SO4(AR)18.2g/L，无水硫酸镁(AR)7.27g/L，KOH(AR)4.128g/L，甘油40g/L，85％H3PO426.7ml/L；
PTM1：五水硫酸铜6g/L，碘化钾0.089g/L，一水硫酸猛3.0g/L，钼酸钠0.2g/L，硼酸0.02g/L，七水硫酸锌42.2g/L，七水硫酸亚铁65g/L，生物素0.2g/L，六水氯化钴0.5g/L，硫酸5ml/L。
实施例12：磷脂酶A2基因序列对融合蛋白的影响
由于磷脂酶A2具有磷脂酶活性，若直接利用基因工程菌的发酵上清液进行应用，其含有的磷脂酶活性有可能会影响应用效果。因此设计两组突变，来研究突变对磷脂酶A2活性以及融合蛋白表达水平的影响。
一组突变为截断突变，是在氨基酸序列如SEQ ID NO:3的基础上截去了第1位至第4位的4个氨基酸；结果显示表达有截短了氨基酸序列的磷脂酶A2的融合基因的重组菌，在发酵上清液中仍旧检测到磷脂酶A2活性，但是不影响脯氨酸蛋白酶表达，酶活与没有截短的相当。
第二组突变为点突变，是在氨基酸序列如SEQ ID NO:3的基础上，分别将第69位、第70位、第73位、第74位、第77位或者第90位的氨基酸突变为丙氨酸(Ala，A)，结果发现含有6种磷脂酶A2突变体序列的融合基因的重组菌，在发酵上清液中检测不到磷脂酶活性，但是不影响脯氨酸蛋白酶表达，酶活与不发生突变的相当。
基于以上研究结果，建议应用时，1)采用纯化的未突变磷脂酶A2和截断突变磷脂酶A2融合的脯氨酸蛋白酶；2)或采用未纯化的6个点突变磷脂酶A2融合的脯氨酸蛋白酶；从而达到最佳的应用效果。
虽然本发明已以较佳实施例公开如上，但其并非用以限定本发明，任何熟悉此技术的人，在不脱离本发明的精神和范围内，都可做各种的改动与修饰，因此本发明的保护范围应该以权利要求书所界定的为准。
Figure PCTCN2016071043-appb-000003
Figure PCTCN2016071043-appb-000004
Figure PCTCN2016071043-appb-000005
Figure PCTCN2016071043-appb-000006
Figure PCTCN2016071043-appb-000007
Figure PCTCN2016071043-appb-000008
Figure PCTCN2016071043-appb-000009
Figure PCTCN2016071043-appb-000010
Figure PCTCN2016071043-appb-000011

Claims (10)

  1. 一种融合基因，所述融合基因是将编码融合标签的基因片段、连接肽片段、编码外源蛋白的基因片段依次连接得到的；所述融合标签为以下任意一种：磷脂酶A2(PLA2)、纤维素结合域(CBD)、泛素相关的小标签调节器(SUMO)、麦芽糖结合蛋白(MBP)。
  2. 根据权利要求1所述的融合基因，其特征在于，所述磷脂酶A2的氨基酸序列是
    (a)SEQ ID NO:3的序列；或者
    (b)在SEQ ID NO:3的序列的的基础上截去了第1位至第4位的4个氨基酸的序列；或者
    (c)在SEQ ID NO:3的序列的的基础上，将第69位、第70位、第73位、第74位、第77位或者第90位的氨基酸突变为丙氨酸后的氨基酸序列；或者
    (d)与(a)的序列具有90％以上同源性的序列。
  3. 根据权利要求2所述的融合基因，其特征在于，所述外源蛋白是脯氨酰内肽酶，优选氨基酸序列是SEQ ID NO:4的脯氨酰内肽酶。
  4. 根据权利要求1所述的融合基因，其特征在于，所述融合基因是在编码磷脂酶A2氨基酸序列的基因片段后直接连接编码氨基酸序列如SEQ ID NO:1的连接肽片段，然后在连接肽片段后直接连接编码氨基酸序列如SEQ ID NO:4的脯氨酰内肽酶基因片段。
  5. 含有权利要求1所述融合基因的核苷酸片段/载体、表达所述融合基因的基因工程菌。
  6. 一种酵母基因工程菌，其特征在于，所述酵母基因工程菌先将权利要求1所述的融合基因连接到pPICZαA上得到重组载体pPICZαA-PLMH，再将重组质粒线性化后转化入宿主毕赤酵母中得到的。
  7. 一种生产脯氨酰内肽酶的方法，包括利用权利要求1所述的融合基因，或权利要求5所述的核苷酸片段/载体，或者权利要求5或6所述的基因工程菌。
  8. 根据权利要求7所述的方法得到的脯氨酰内肽酶。
  9. 权利要求8所述脯氨酰内肽酶的应用，是用于食品、制备药物、分子生物学等领域；可选地，用于降低啤酒发酵液中敏感蛋白的含量、或者提高啤酒的非生物稳定性、或者去除蛋白质水解液的苦味；也可选地，是用来制备调节糖代谢或者胰腺功能的药物，还可选地，是应用于蛋白质序列测定，肽谱分析、特异位点的酶切、肽链的修饰以及加工等。
  10. 一种利用磷脂酶融合表达以提高外源蛋白表达量的方法，所述方法是将编码磷脂酶A2的基因片段、氨基酸序列如SEQ ID NO.1的连接肽片段、编码外源蛋白的基因片段依次连接得到融合基因，然后再表达融合基因。
PCT/CN2016/071043 2016-01-15 2016-01-15 一种利用磷脂酶融合表达以提高外源蛋白表达量的方法 WO2017120890A1 (zh)

Priority Applications (2)

Application Number Priority Date Filing Date Title
PCT/CN2016/071043 WO2017120890A1 (zh) 2016-01-15 2016-01-15 一种利用磷脂酶融合表达以提高外源蛋白表达量的方法
US15/995,134 US10323247B2 (en) 2016-01-15 2018-06-01 Methods for improving expression levels of foreign proteins by means of phospholipase fusion expression

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/CN2016/071043 WO2017120890A1 (zh) 2016-01-15 2016-01-15 一种利用磷脂酶融合表达以提高外源蛋白表达量的方法

Related Child Applications (1)

Application Number Title Priority Date Filing Date
US15/995,134 Continuation US10323247B2 (en) 2016-01-15 2018-06-01 Methods for improving expression levels of foreign proteins by means of phospholipase fusion expression

Publications (1)

Publication Number Publication Date
WO2017120890A1 true WO2017120890A1 (zh) 2017-07-20

Family

ID=59310650

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2016/071043 WO2017120890A1 (zh) 2016-01-15 2016-01-15 一种利用磷脂酶融合表达以提高外源蛋白表达量的方法

Country Status (2)

Country Link
US (1) US10323247B2 (zh)
WO (1) WO2017120890A1 (zh)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108048339A (zh) * 2017-12-07 2018-05-18 王艺璇 一种重组表达磷脂酶的菌株及其应用

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
RU2716087C1 (ru) * 2018-10-19 2020-03-05 Федеральное государственное бюджетное учреждение "Государственный научно-исследовательский институт генетики и селекции промышленных микроорганизмов Национального исследовательского центра "Курчатовский институт" (НИЦ "Курчатовский институт" - ГосНИИгенетика) Фосфолипаза А2 для экспрессии в дрожжах (варианты)

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009023270A2 (en) * 2007-08-15 2009-02-19 Amunix, Inc. Compositions and methods for modifying properties of biologically active polypeptides
WO2010064748A1 (en) * 2008-12-04 2010-06-10 Korea Research Institute Of Bioscience And Biotechnology Screening of abundantly secreted proteins and their use as fusion partners for the production of recombinant proteins
CN102654504A (zh) * 2012-03-18 2012-09-05 生工生物工程(上海)有限公司 一种快速检测重组蛋白表达量的方法
CN102977206A (zh) * 2012-11-19 2013-03-20 中国农业科学院生物技术研究所 细胞色素结合结构域蛋白作为助分泌因子提高外源基因在毕赤酵母中分泌表达量的用途

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009023270A2 (en) * 2007-08-15 2009-02-19 Amunix, Inc. Compositions and methods for modifying properties of biologically active polypeptides
WO2010064748A1 (en) * 2008-12-04 2010-06-10 Korea Research Institute Of Bioscience And Biotechnology Screening of abundantly secreted proteins and their use as fusion partners for the production of recombinant proteins
CN102654504A (zh) * 2012-03-18 2012-09-05 生工生物工程(上海)有限公司 一种快速检测重组蛋白表达量的方法
CN102977206A (zh) * 2012-11-19 2013-03-20 中国农业科学院生物技术研究所 细胞色素结合结构域蛋白作为助分泌因子提高外源基因在毕赤酵母中分泌表达量的用途

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
JIANG TING: "High-level expression of prolyl endopeptidase in Pichia pastoris using PLA2 as a fusion partner", JOURNAL OF MOLECULAR CATALYSIS B:ENZYMATIC, vol. 125, 15 January 2016 (2016-01-15) - March 2016 (2016-03-01), pages 81 - 87, XP055598863, ISSN: 1381-1177, DOI: 10.1016/j.molcatb.2016.01.005 *
KANG CHAO: "Cloning and expression of a novel prolyl endopeptidase from Aspergillus oryzae and its application in beer stabilization", JOURNAL OF INDUSTRIAL MICROBIOLOGY AND BIOTECHNOLOGY, vol. 42, no. 2015, 30 December 2014 (2014-12-30) - 2015, pages 263 - 272, XP035426876, ISSN: 1367-5435, DOI: 10.1007/s10295-014-1571-8 *
LI NINGHUAN ET AL.: "Secreted Expression of prolyl endopeptidase from aspergillus fumigatus in pichia pastoris and characterization of the recombinant enzyme", MICROBIOLOGY, vol. 40, no. 3, 20 March 2013 (2013-03-20), pages 391 - 399 *
LIU AIXIA: "Streptomyces violaceoruber Phospholipase A2: Expression in Pichia pastoris, Properties, and Application in Oil Degumming", APPLIED BIOCHEMISTRY AND BIOTECHNOLOGY, vol. 175, 25 January 2015 (2015-01-25), pages 3195 - 3206, XP002762314 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108048339A (zh) * 2017-12-07 2018-05-18 王艺璇 一种重组表达磷脂酶的菌株及其应用
CN108048339B (zh) * 2017-12-07 2021-12-28 无锡蔚蓝生物科技有限公司 一种重组表达磷脂酶的菌株及其应用

Also Published As

Publication number Publication date
US20180265876A1 (en) 2018-09-20
US10323247B2 (en) 2019-06-18

Similar Documents

Publication Publication Date Title
CN104039345B (zh) 来自放线菌放线异壁酸菌属(Actinoallomurus)的能在酸性pH水解谷蛋白肽和蛋白的新蛋白酶
US7575920B2 (en) Yeast expression vectors for production of ITF
CN111206025B (zh) 一种比活提高的溶菌酶突变体
CN109486800B (zh) 一种新型赖氨酰肽链内切酶及其制备方法
CA1340970C (en) Lipase from gastric mucosa
Sørensen et al. pH‐dependent processing of yeast procarboxypeptidase Y by proteinase A in vivo and in vitro
WO2017120890A1 (zh) 一种利用磷脂酶融合表达以提高外源蛋白表达量的方法
CN116333957A (zh) 一种展示猪链球菌前噬菌体裂解酶的重组芽孢杆菌及其构建方法和应用
CN104232611B (zh) 一种重组布氏白僵菌蛋白酶k及其工业化生产与纯化方法
JP3957630B2 (ja) 組換えヒト副甲状腺ホルモンを生産する形質転換酵母及び該ホルモンの生産方法
CN109439643B (zh) 一种新型赖氨酸特异性内切酶及其制备方法
US11447780B2 (en) Preparation of wheat cysteine protease triticain-alpha produced in soluble form and method of producing same
WO2019062709A1 (zh) 一种重组牛血清蛋白成熟肽及其制备方法和应用
Hofmann [49] Penicillocarboxypeptidases S-1 and S-2
Jiang et al. High-level expression of prolyl endopeptidase in Pichia pastoris using PLA2 as a fusion partner
WO2014177021A1 (zh) 强分泌性信号肽增强小肽模序及其应用
CN103602605B (zh) 一种高效表达米曲霉脯氨酰内肽酶的基因工程菌及其应用
RU2740319C1 (ru) Способ повышения активности тритикаина-альфа
CN106632683B (zh) 具有pNPPC水解酶活性的多肽，其编码基因、制备方法及应用
WO2016197296A1 (zh) 一种催化活性和比酶活提高的脯氨酰内肽酶突变体
CN103589741B (zh) 一种新的脯氨酸特异性内切蛋白酶基因及其应用
Niu et al. Enhanced expression of a novel trypsin from Streptomyces fradiae in Komagataella phaffii GS115 through combinational strategies of propeptide engineering and self-degredation sites modification
CN116731144B (zh) 一种重组大米抗氧化肽及其制备方法和应用
CN116144687B (zh) 酸性蛋白酶agp及其制备方法和应用
Kim et al. Modulation of Kex2p Cleavage Site for In Vitro Processing of Recombinant Proteins Produced by Saccharomyces cerevisiae

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 16884452

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 16884452

Country of ref document: EP

Kind code of ref document: A1