WO2021139295A1 - 一种重组多肽连接酶原及其制备、激活方法与应用 - Google Patents

一种重组多肽连接酶原及其制备、激活方法与应用 Download PDF

Info

Publication number
WO2021139295A1
WO2021139295A1 PCT/CN2020/120817 CN2020120817W WO2021139295A1 WO 2021139295 A1 WO2021139295 A1 WO 2021139295A1 CN 2020120817 W CN2020120817 W CN 2020120817W WO 2021139295 A1 WO2021139295 A1 WO 2021139295A1
Authority
WO
WIPO (PCT)
Prior art keywords
recombinant polypeptide
ligase
recombinant
polypeptide ligase
protein
Prior art date
Application number
PCT/CN2020/120817
Other languages
English (en)
French (fr)
Inventor
胡松青
樊壬水
侯轶
Original Assignee
华南理工大学
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 华南理工大学 filed Critical 华南理工大学
Publication of WO2021139295A1 publication Critical patent/WO2021139295A1/zh

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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/93—Ligases (6)
    • 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
    • C12N15/70—Vectors or expression systems specially adapted for E. coli

Definitions

  • the present invention belongs to the field of biotechnology, and in particular relates to a recombinant polypeptide ligase proenzyme and its preparation, activation method and application.
  • Bioactive peptides have a variety of human metabolism and physiological regulation functions, are easy to digest and absorb, and have the effects of promoting immunity, hormone regulation, antibacterial, antiviral, lowering blood pressure, and lowering blood lipids.
  • cyclic peptides Compared with linear peptides, cyclic peptides not only have higher structural stability, thermal stability and bioavailability, but also can resist hydrolysis. They can maintain complete biological activity under the conditions of chemical denaturants. More importantly, cyclization can Enhance the original physiological activity of the polypeptide to varying degrees. Therefore, the cyclization of linear peptides into rings has always been a key issue in the international biochemistry and food and drug fields.
  • cyclic peptides are currently mainly used for the development of expensive drugs.
  • the main methods for preparing cyclic peptides are: separation and purification of natural cyclic peptides, chemical synthesis and microbial fermentation synthesis.
  • the content of cyclic peptides in natural products is very low, and separation and purification are difficult; chemical synthesis requires cumbersome group protection, which reduces the reaction yield and product purity; fermentation methods require special strains, and there are few types of cyclic peptides that can be produced.
  • the first objective of the present invention is to overcome the shortcomings and deficiencies of the prior art and provide a recombinant polypeptide ligase; the in vitro self-activation ability of the recombinant polypeptide ligase has been significantly improved.
  • the second object of the present invention is to provide a method for preparing recombinant polypeptide ligase, which realizes the high-efficiency and soluble expression of the recombinant polypeptide ligase.
  • the third object of the present invention is to provide a method for activating recombinant polypeptide ligase.
  • the activation product of the recombinant polypeptide ligase obtained by the activation method has strong activity and high catalytic efficiency.
  • the fourth object of the present invention is to provide the application of the recombinant polypeptide ligase proenzyme or its activation product.
  • the amino acid sequence of the recombinant polypeptide ligase is compared with the amino acid sequence SEQ ID NO: 2 of the wild-type butterfly bean-derived polypeptide ligase, and has the following amino acid mutations: 338th lysine is mutated to aspartic acid; 358th Proline was mutated to asparagine; glutamic acid at position 359 was mutated to aspartic acid, the signal peptide at positions 1-20 was truncated, and an MBP tag was fused to the N-terminus.
  • the DNA molecule encoding the recombinant polypeptide ligase proenzyme has the nucleotide sequence shown in SEQ ID NO: 3.
  • the nucleotide sequence is optimized based on the E. coli expression system to optimize the codons, which can significantly improve the expression efficiency of heterologous genes in host bacteria.
  • the recombinant expression vector contains the nucleotide sequence shown in SEQ ID NO: 3.
  • a preparation method of recombinant polypeptide ligase zymogen includes the following steps:
  • the nucleotide sequence encoding the recombinant polypeptide ligase pro-ligase is cloned into an expression vector to construct a recombinant expression vector; then the recombinant expression vector is transformed into a prokaryotic expression system to induce expression, and the bacterial pellet is collected by solid-liquid separation, After purification, the recombinant polypeptide ligase is obtained.
  • the prokaryotic expression system is preferably an E. coli expression system; more preferably E. coli ROSETTA (DE3).
  • the recombinant expression vector is preferably expressed through a T7 promoter.
  • the expression vector is preferably an expression vector containing an MBP tag, more preferably pMAL-c5x or pMAL-c5x-His, which can greatly increase the solubility of the target protein expression.
  • the specific operation steps for inducing expression are preferably: inoculating the positive transformant in LB culture broth for cultivation, and when the OD 600 of the bacterial broth reaches 0.5-0.9, add IPTG with a final concentration of 0.1-0.6 mmol/L, 15- It is induced at 30°C for 12-28 hours; the LB culture medium contains 30-50 ⁇ g/mL kanamycin and 50-100 ⁇ g/mL ampicillin.
  • the positive transformant is cultured in LB culture medium with shaking at 25-40°C overnight to obtain seed liquid;
  • the amount of inoculation described in step (2) is preferably inoculated at a volume ratio of 1:50 to 1:150 with respect to the LB culture solution.
  • the purification step is preferably: collecting the supernatant after lysing the cells, passing the supernatant through an affinity chromatography column, and eluting the purified recombinant polypeptide ligase.
  • the method for lysing the cells is preferably: lysing the cells in an ice bath with ultrasonic disruption.
  • the dosage of the buffer A is preferably 10 mL buffer A per gram of bacteria.
  • the conditions of the ultrasonic disintegration method are preferably: power of 50 to 200 W, ultrasound of 1 to 5 seconds, interval of 1 to 5 seconds, and duration of 2 to 30 minutes.
  • the operation of collecting the supernatant after lysing the cells is preferably: the lysed bacterial cells are frozen and centrifuged, the precipitate is discarded, and the membrane is passed to obtain the supernatant.
  • the conditions of the freezing centrifugation are preferably 12000 r/min centrifugation at 4° C. for 40 min.
  • the membrane is preferably filtered with a 0.22 ⁇ m microporous filter membrane.
  • the affinity chromatography column is preferably an MBP tag affinity chromatography column.
  • a method for activating recombinant polypeptide ligase including the following steps: the recombinant polypeptide ligase is dialyzed for the first time in an environment with a pH of 3.5-6, and then the second time is performed at a pH of 4.5-7. Dialysis, solid-liquid separation to remove the precipitate, to obtain the recombinant polypeptide ligase pro-ligase activation product.
  • the activation method is preferably carried out at 25-45°C.
  • the first dialysis is preferably performed in buffer I containing the following components: 20-50mM acetic acid-sodium acetate, 50-200mM NaCl, 1-5mM EDTA, 2-5mM DTT.
  • the time for the first dialysis is preferably 8-24 hours.
  • the second dialysis is preferably performed in buffer II containing the following components: 20-50mM Na 2 HPO 4 -NaH 2 PO 4 or Na 2 HPO 4 -citric acid, 50-200mM NaCl, 1-5mM EDTA , 0.5 ⁇ 1mM DTT.
  • the time for the second dialysis is preferably 2-8h.
  • the dialysis is preferably performed in a dialysis bag, and the dialysis bag is preferably a dialysis bag with a molecular weight cut-off of 3-10 kDa.
  • a recombinant polypeptide ligase pro-ligase activation product is obtained by the recombinant polypeptide ligase pro-ligase activation method.
  • the application of the recombinant polypeptide ligase proenzyme or the recombinant polypeptide ligase proenzyme activation product includes the cyclization of polypeptides or proteins, the connection between polypeptides and polypeptides or proteins, the connection between proteins and proteins, The protein or polypeptide is in connection with other compounds.
  • the C-terminus of the polypeptide or protein When used for the cyclization of a polypeptide or protein, the C-terminus of the polypeptide or protein contains an asparagine-histidine-valine (NHV) sequence, and the first amino acid at the N-terminus is excluding proline 19 common amino acids, the second amino acid residue at the N-terminal must be isoleucine, leucine, valine or cysteine (I, L, V or C); the polypeptide or protein is preferably Linear peptides or proteins.
  • NAV asparagine-histidine-valine
  • cyclization is that the histidine-valine (HV) sequence at the C-terminus of the polypeptide or protein is broken, and then the N-terminus and the C-terminus are connected by amide bonds to obtain a cyclic peptide.
  • HV histidine-valine
  • At least one polypeptide or protein When used for the connection between polypeptide and polypeptide or protein, between protein and protein, at least one polypeptide or protein contains an asparagine-histidine-valine (NHV) sequence at the C-terminus, and the other Peptides or proteins satisfy that the first amino acid at the N-terminus is 19 common amino acids other than proline, and the second amino acid residue at the N-terminus must be isoleucine, leucine, valine or cysteine ( I, L, V or C).
  • NHS asparagine-histidine-valine
  • connection form is that the histidine-valine (HV) sequence at the C-terminus of a polypeptide or protein is broken, and then forms an amide bond with the N-terminus of another polypeptide or protein to obtain a connection product.
  • HV histidine-valine
  • the application conditions are preferably: the temperature is 25-60°C, and the pH is 4.5-7.
  • the application is preferably carried out in a buffer containing the following components: 20-50mM Na 2 HPO 4 -NaH 2 PO 4 , Na 2 HPO 4 -citric acid or 2-morpholineethanesulfonic acid, 50-200mM NaCl, 1 ⁇ 5mM EDTA, 0.5 ⁇ 1mM DTT.
  • the buffer solution also contains 0.05% to 0.3% Triton X-100.
  • the present invention has the following advantages and effects:
  • the present invention adopts the E. coli prokaryotic expression method to prepare a soluble and efficiently activated recombinant polypeptide ligase.
  • the content of the target protein accounts for 60 to 70% of the total protein, the expression is high, and only one isolation and purification is required.
  • Affinity chromatography simple and efficient, easy to express in large quantities, suitable for industrial production.
  • the present invention optimizes codons for the prokaryotic expression system, and at the same time, site-directed mutations of the three amino acids of wild-type Butelase 1 can enhance the in vitro self-activation ability of the recombinant polypeptide ligase, and at the same time improve the activation product of the recombinant polypeptide ligase. Catalytic efficiency.
  • the present invention uses an expression vector with an MBP tag (for example, pMAL-c5x-His vector) to express the target protein, so that the expressed protein has an MBP solubility-promoting tag at the N-terminus. It not only increases the expression and solubility of the target protein, but also simplifies the separation and purification of the recombinant polypeptide ligase by MBP tag affinity chromatography.
  • MBP tag for example, pMAL-c5x-His vector
  • the present invention adopts a low-pH activation method, and the obtained activation product has the same function as the naturally extracted polypeptide ligase Butelase 1, and can carry out the cyclization of polypeptides or proteins, the connection between polypeptides and polypeptides or proteins, and the protein The connection between proteins, proteins or polypeptides and other compounds; moreover, compared with other E. coli prokaryotic expression polypeptide ligase Butelase 1 in the prior art, it has higher catalytic efficiency and more specificity Cyclization activity.
  • the method of the present invention does not require butterfly pea as a raw material, only a simple culture medium as a raw material, and the process of preparing polypeptide ligase is not limited by raw materials. , Medicine, food and other fields have broad prospects; compared with the recombinant expression of Butelase 1 obtained by yeast expression system in the prior art, the method of the present invention has a shorter expression cycle, higher expression amount, and simpler separation and purification.
  • Figure 1 is the SDS-PAGE image of MBP tag affinity chromatography purification of recombinant polypeptide ligase; in which, lanes 1 to 4 are total protein, superalbumin, penetrating protein and eluted protein in sequence.
  • Figure 2 is an SDS-PAGE diagram of the activation of recombinant polypeptide ligase; in which, lanes 1 and 2 are the recombinant polypeptide ligase and the activation product of the recombinant polypeptide ligase, respectively.
  • Figure 3 is a diagram of relative enzyme activity before and after activation of recombinant polypeptide ligase.
  • Figure 4 is a liquid chromatogram of the cyclized polypeptide KB1-NHV, an activation product of recombinant polypeptide ligase.
  • Figure 5 is a MALDI-TOF diagram of the product peak of the circularized polypeptide KB1-NHV, an activation product of recombinant polypeptide ligase.
  • Figure 6 is a MALDI-TOF diagram of the cyclized antimicrobial peptide SoD6-NHV, an activation product of recombinant polypeptide ligase.
  • Figure 7 is a MALDI-TOF diagram of the cyclized antimicrobial peptide P113-NHV, an activation product of recombinant polypeptide ligase.
  • Fig. 8 is a MALDI-TOF diagram of the intermolecular ligation of the recombinant polypeptide ligase activation product.
  • Figure 9 is a MALDI-TOF diagram of the cyclized sunflower trypsin inhibitor, an activation product of recombinant polypeptide ligase.
  • Example 10 is a diagram showing the results of relative enzyme activity analysis of recombinant polypeptide ligase activation products expressed by different strains in Example 11.
  • FIG. 11 is a diagram showing the relative enzyme activity analysis results of the wild-type recombinant polypeptide ligase activation product and the mutant type recombinant polypeptide ligase activation product in Example 12.
  • the various reagents and raw materials used in the present invention are all commercially available products or products that can be prepared by known methods.
  • Example 1 Construction of a recombinant vector containing a nucleotide sequence encoding a recombinant polypeptide ligase
  • the construction of a recombinant vector containing a nucleotide sequence encoding a recombinant polypeptide ligase includes the following steps:
  • the synthetic primer sequence is as follows, Nde I-FP: ACGCCATATGATTCGTGATGATTTTCTGCG; Sal I-RP: ACGCGTCGACTCACACGCTAAACCCCG.
  • Nde I-FP ACGCCATATGATTCGTGATGATTTTCTGCG
  • Sal I-RP ACGCGTCGACTCACACGCTAAACCCCG.
  • the amplification conditions were: 98°C for 30s; 98°C for 10s, 58°C for 30s, 72°C for 30s, a total of 30 cycles; 72°C for 5 minutes.
  • Agarose gel electrophoresis was used to recover the target gene fragment with a size of about 1.4kb.
  • the target gene fragment and the vector pMAL-c5x-His were digested with Nde I and Sal I (purchased from NEB), and T4 DNA enzyme (purchased from Tiangen Bio Technology Co., Ltd.) performs ligation reaction, and the ligation product is transformed into DH5 ⁇ competent cells (purchased from Tiangen Biotechnology Co., Ltd.).
  • the obtained plasmid is the recombinant vector containing the DNA molecule encoding the recombinant polypeptide ligase.
  • the recombinant vector obtained in Example 1 was transformed into the host cell Escherichia coli ROSETTA (DE3) (purchased from Shanghai Weidi Biotechnology Co., Ltd.), and the positive transformants of Escherichia coli were selected and inoculated with a small amount of kanamycin 30 ⁇ g/mL and Ampicillin 50 ⁇ g/mL LB culture medium, shake culture overnight at 25°C in a shaker as a seed solution, and inoculate the seed solution at a volume ratio of 1:150 to contain 30 ⁇ g/mL of kanamycin and 50 ⁇ g of ampicillin /mL of LB broth, culture at 25°C and 150r/min until the OD 600 of the bacterial solution reaches 0.5, add IPTG to each bottle of bacterial solution to a final concentration of 0.6 mmol/L, and induce expression at 15°C for 28 hours. Centrifuge to collect the bacterial pellet.
  • E3 obtained from Shanghai Weidi Biotechnology Co., Ltd.
  • the recombinant vector obtained in Example 1 was transformed into the host cell Escherichia coli ROSETTA (DE3), and the positive transformants of Escherichia coli were selected and inoculated into LB culture medium containing a small amount of kanamycin 40 ⁇ g/mL and ampicillin 75 ⁇ g/mL.
  • microfiltration membrane filtrate was separated with MBPTrap TM HP 5mL chromatography column. Connect the chromatography column to the column valve of the fast protein purification instrument, clean the system and the column with ultrapure water, balance with buffer A, then load the filtrate with a sample pump, and clean the column with buffer A.
  • the protein or impurities that are not bound to the column are washed away; then use buffer B (20mM NaH 2 PO 4 -Na 2 HPO 4 , 100 mM NaCl, 1 mM EDTA, 10 mM maltose, pH 7.0) for one-step elution, and protein denaturation electrophoresis detection
  • the elution peak is to collect the elution peak containing the recombinant polypeptide ligase of the target protein, and the collected volume is 8 mL, and the concentration is 4.0 mg/mL, that is, 1L of fermentation broth is purified to obtain 32 mg of recombinant polypeptide ligase.
  • the protein concentration of the eluate was measured by ultraviolet spectrophotometry, the measurement wavelength was 280nm, the molar absorption coefficient was 133620M -1 cm -1 , and the molecular weight was 94239Da. Take a sample of 20 ⁇ L and detect it by SDS-PAGE protein electrophoresis. The results are shown in Figure 1. Lanes 1 to 4 are total protein, superalbumin, penetrating protein, and eluted protein in sequence.
  • the eluted protein with a molecular weight of 94.3 kDa is the band of the target protein recombinant polypeptide ligase; It was unexpectedly discovered that the present invention only needs to pass MBP tag affinity chromatography once to obtain electrophoresis-pure recombinant polypeptide ligase progenitor, and no other purification steps (such as Ni ion affinity chromatography, etc.) are required; at the same time, through SDS-PAGE gray-scale analysis method confirms that the content of the target protein in the present invention accounts for 60-70% of the total protein, and the expression level is high.
  • the recombinant vector obtained in Example 1 was transformed into the host cell Escherichia coli ROSETTA (DE3), and the positive transformants of Escherichia coli were selected and inoculated into LB culture medium containing a small amount of kanamycin 50 ⁇ g/mL and ampicillin 100 ⁇ g/mL. Incubate with shaking at 37°C overnight in a shaker as a seed solution.
  • the recombinant polypeptide ligase was dialyzed in buffer I (20mM acetic acid-sodium acetate, 100mM NaCl, 1mM EDTA, 5mM DTT, pH 4.5) with a 10kDa dialysis bag at 37°C for 12h after activation, the protein will be loaded Transfer the dialysis bag to buffer II (20mM Na 2 HPO 4 -NaH 2 PO 4 , 100 mM NaCl, 1 mM EDTA, 0.5 mM DTT, pH 6.0) and dialyze for 6 h, centrifuge to remove the precipitate, and obtain a recombinant with cyclization activity. Polypeptide ligase pro-activation product.
  • Lanes 1 and 2 are the activation products of recombinant polypeptide ligase and recombinant polypeptide ligase respectively.
  • the activation product has protein bands around 40kDa, indicating that under low pH conditions, recombinant polypeptide ligase Can self-activate well.
  • HCl with a final concentration of 100 mM was added to terminate the reaction.
  • the reaction solution was filtered with a 0.22 ⁇ m microporous membrane, and then 20 ⁇ L was analyzed by HPLC.
  • the molar concentration of the enzyme solution was measured by ultraviolet spectrophotometry, the measurement wavelength was 280nm, and the molar absorption coefficient was 133620M -1 cm -1 .
  • the yield of the cyclized product is calculated by the liquid phase peak area, and the yield of the cyclized KB1-NHV polypeptide product of the recombinant polypeptide ligase pro-activation product is set as 100% of the enzyme activity to calculate the relative enzyme activity.
  • the results are shown in Figure 3, the recombinant polypeptide ligase that has not been activated has no activity, and the activation product obtained after activation is active; it further illustrates that the method can successfully activate the recombinant polypeptide ligase.
  • the recombinant polypeptide ligase obtained in Example 3 was dialyzed in buffer I (50mM acetic acid-sodium acetate, 200mM NaCl, 5mM EDTA, 2mM DTT, pH 6.0) with a 3kDa dialysis bag at 45°C for 24h after activation Transfer the dialysis bag containing the protein solution to buffer II (50mM Na 2 HPO 4 -NaH 2 PO 4 , 200 mM NaCl, 5 mM EDTA, 1 mM DTT, pH 7.0) and dialyze for 8 h, and centrifuge to remove the precipitate. The active recombinant polypeptide is ligated to the proenzyme activation product.
  • buffer I 50mM acetic acid-sodium acetate, 200mM NaCl, 5mM EDTA, 2mM DTT, pH 6.0
  • buffer II 50mM Na 2 HPO 4 -NaH 2 PO 4 , 200
  • reaction buffer (20mM NaH 2 PO 4 -Na 2 HPO 4 , 50mM NaCl, 1mM EDTA, 0.5mM DTT, 0.05% TritonX-100, pH 6.0
  • 50 ⁇ M recombinant polypeptide ligase proactivator and 50 ⁇ M KalataB1-NHV polypeptide (amino acid sequence is GLPVCGETCVGGTCNTPGCTCSWPVCTRNHV).
  • HCl with a final concentration of 100 mM was added to terminate the reaction.
  • the reaction solution was filtered with a 0.22 ⁇ m microporous membrane, and then 20 ⁇ L was analyzed and identified by HPLC.
  • Cyclization determination the molecular weight of the linear polypeptide KB1-NHV is 3152.65 Da, and the molecular weight of the cyclic-KB1 obtained after the reaction is 2898.33 Da.
  • the recombinant polypeptide ligase obtained in Example 3 was dialyzed in buffer I (30mM acetic acid-sodium acetate, 50mM NaCl, 3mM EDTA, 4mM DTT, pH 3.5) with a 5kDa dialysis bag at 25°C for 8h after activation , Transfer the dialysis bag containing the protein solution to buffer II (30mM Na 2 HPO 4 -citric acid, 50mM NaCl, 2mM EDTA, 0.75mM DTT, pH 4.5) and dialyze for 2h, centrifuge to remove the precipitate, and then obtain the recombinant polypeptide connection Enzyme activation product.
  • buffer I (30mM acetic acid-sodium acetate, 50mM NaCl, 3mM EDTA, 4mM DTT, pH 3.5
  • buffer II (30mM Na 2 HPO 4 -citric acid, 50mM NaCl, 2mM EDTA, 0.
  • reaction buffer 50mM NaH 2 PO 4 -Na 2 HPO 4 , 200mM NaCl, 5mM EDTA, 1mM DTT, 0.3% Triton X-100, pH 7.0
  • reaction buffer 50mM NaH 2 PO 4 -Na 2 HPO 4 , 200mM NaCl, 5mM EDTA, 1mM DTT, 0.3% Triton X-100, pH 7.0
  • 50 ⁇ M recombinant polypeptide ligase proactivator 50 ⁇ M spinach leaf cell wall antimicrobial peptide SoD6-NHV polypeptide
  • amino acid sequence is GIFSNMYARTPAGYFRGPAGYAANHV
  • fragment of human salivary histamine 5 P113-NHV polypeptide amino acid sequence is GLAKRHHGYKRKFHNHV.
  • Cyclization determination the linear polypeptide SoD6-NHV has a molecular weight of 2789.12 Da, and the cyclic product cyclic SoD6 obtained after the reaction has a molecular weight of 2534.83 Da.
  • Cyclization determination The molecular weight of the linear polypeptide P113-NHV is 2085.38 Da, and the molecular weight of the cyclic P113 obtained after the reaction is 1831.12 Da.
  • the recombinant polypeptide ligase obtained in Example 3 was dialyzed in buffer I (20mM acetic acid-sodium acetate, 100mM NaCl, 2mM EDTA, 5mM DTT, pH 5.0) with a 10kDa dialysis bag at 37°C for 16h after activation , Transfer to buffer II (20mM Na 2 HPO 4 -NaH 2 PO 4 , 100 mM NaCl, 1 mM EDTA, 0.5 mM DTT, pH 6.5) and dialyze for 4 h, and centrifuge to remove the precipitate to obtain the recombinant polypeptide ligase activation product.
  • buffer I 20mM acetic acid-sodium acetate, 100mM NaCl, 2mM EDTA, 5mM DTT, pH 5.0
  • buffer II 20mM Na 2 HPO 4 -NaH 2 PO 4 , 100 mM NaCl, 1 mM EDTA,
  • reaction buffer (30 mM NaH 2 PO 4 -citric acid, 100 mM NaCl, 2 mM EDTA, 0.75 mM DTT, 0.1% Triton X-100, pH 4.5
  • 0.5 ⁇ M recombinant polypeptide ligase pro-ligase activation product 50 ⁇ M
  • the octapeptide KALVINHV (abbreviated as K)
  • 1mM hexapeptide GIGGIR (abbreviated as G).
  • the reaction solution was filtered with a 0.22 ⁇ m microporous membrane, and after desalting by a Ziptip desalting column, a 1 ⁇ L dot plate was analyzed and identified by MALDI-TOF.
  • the results are shown in Figure 8.
  • the recombinant polypeptide ligase activation product can well connect the octapeptide KALVINHV and the hexapeptide GIGGIR intermolecularly to obtain the product dodecapeptide KALVINGIGGIR (abbreviated as KG), which is calculated based on the peak intensity of MALDI-TOF
  • KG dodecapeptide KALVINGIGGIR
  • the linear octapeptide KALVINHV (abbreviated as K) has a molecular weight of 893.03Da
  • the linear hexapeptide GIGGIR (abbreviated as G) has a molecular weight of 571.66Da
  • the linkage product KALVINGIGGIR (abbreviated as KG) has a molecular weight of 1210.47Da.
  • reaction buffer (20mM 2-morpholineethanesulfonic acid, 100mM NaCl, 1mM EDTA, 0.5mM DTT, pH 5.5)
  • 1mg/mL of the recombinant polypeptide ligase activation product obtained in Example 4 250 ⁇ M polypeptide Substrate sunflower trypsin inhibitor SFTI-NHV linear peptide (amino acid sequence: GRCTKSIPPICFPNHV).
  • HCl with a final concentration of 100 mM was added to terminate the reaction.
  • the reaction solution was filtered with a 0.22 ⁇ m microporous membrane, and after desalting by a Ziptip desalting column, a 1 ⁇ L dot plate was analyzed and identified by MALDI-TOF.
  • the result is shown in Fig. 9.
  • the recombinant polypeptide ligase activation product can circularize the polypeptide substrate SFTI-NHV. According to the peak intensity of the MALDI-TOF mass spectrum, the yield of the cyclization product was 75%, and no hydrolysis product was detected, indicating that the enzyme preparation has excellent catalytic efficiency and cyclization specificity.
  • Cyclization determination The molecular weight of the linear polypeptide SFTI-NHV is 1769.11 Da, and the molecular weight of the cyclic SFTI obtained after the reaction is 1514.83 Da.
  • Butelase 1 Compared with Butelase 1 (referred to as Butelase 1 (2019) ) heterologously expressed by Australian scholar Amy M. James in 2019, the recombinant polypeptide ligase activation product obtained in the present invention has higher catalytic efficiency and a more specific loop. ⁇ activity.
  • the recombinant polypeptide ligase activation product can catalyze the cyclization of sunflower trypsin inhibitor, and the yield of the cyclization product of the enzyme reaction for 1 h is 75%, and no hydrolysate is detected.
  • the Butelase 1 (2019) catalyzed the sunflower trypsin inhibitor cyclization reaction although there is no direct catalytic efficiency data, but it is estimated based on the reported peak intensity of the MALDI-TOF mass spectrum. After the catalytic reaction for 24 hours, the ring The yield of the chemical product is only about 10%, and about 1% of the substrate is hydrolyzed.
  • the recombinant polypeptide ligase activation product has higher catalytic efficiency and more specific cyclization activity than Butelase 1 (2019) (Document: James AM, Haywood J, Leroux J, IgnasiakK, Elliott AG, Schmidberger JW, Fisher MF, Nonis SG, Fenske R, Bond CS, Mylne JS.
  • the macrocyclizing protease butelase 1 remains auto-catalytic and reveals the structural basis for ligase activity.
  • the recombinant polypeptide ligase obtained in the present invention has the advantages of shorter expression cycle, higher expression, and simpler separation and purification.
  • the expression cycle of the recombinant polypeptide ligase progenin of the present invention is 15 hours, the expression amount is 32 mg of target protein per liter of fermentation broth, and only one MBP tag column affinity chromatography is required for separation and purification.
  • the expression cycle of rBTase is 96h, and the expression amount is 16mg of target protein per liter of fermentation broth.
  • the constructed pET-28a recombinant vector, the expressed recombinant protein is histidine tag + full-length polypeptide ligase, and the amino acid sequence is shown in SEQ ID NO: 4.
  • the pET-28a recombinant vector is transferred into Escherichia coli ROSETTA (DE3) Express in. The result is no target protein expression.
  • the constructed pHUE-ubiquitin recombinant vector the expressed recombinant protein is histidine tag + ubiquitin tag + full-length polypeptide ligase, and the amino acid sequence is shown in SEQ ID NO: 5.
  • the pHUE-ubiquitin recombinant vector is transferred into E. coli It was expressed in ROSETTA (DE3), and the result was that a small amount of the target protein was soluble expression, most of which were expressed in inclusion bodies.
  • the constructed pET-28a-SUMO recombinant vector, the expressed recombinant protein is histidine tag + SUMO tag + full-length polypeptide ligase, and the amino acid sequence is shown in SEQ ID NO: 6.
  • the pHUE-ubiquitin recombinant vector is transferred into the large intestine It is expressed in Bacillus ROSETTA (DE3). The result is that the target protein is soluble and expressed, but the target protein cannot be effectively combined with the nickel ion affinity chromatography column, that is, the recombinant polypeptide ligase cannot be purified.
  • the constructed pMAL-c5x-His recombinant vector, the expressed recombinant protein is MBP tag + full-length polypeptide ligase, and the amino acid sequence is shown in SEQ ID NO:1.
  • the pMAL-c5x-His recombinant vector was transformed into Escherichia coli ROSETTA( DE3). The result is that the target protein is soluble and expressed, and the recombinant polypeptide ligase can be obtained after purification with a MBPTrap TM HP 5mL column.
  • the present invention preferably uses an expression vector containing an MBP tag, and more preferred expression vectors such as pMAL-c5x, pMAL-c5x-His and other vectors.
  • E. coli BL21 (DE3) (purchased from Tiangen Biotechnology Co., Ltd.) and E. coli ROSETTA (DE3) expression strains.
  • the recombinant vector obtained in Example 1 was transformed into the host cells E. coli BL21 (DE3) and E. coli ROSETTA (DE3), and the positive transformants of E. coli were selected to induce expression, and the results were all soluble expression of recombinant polypeptide ligase.
  • E. coli BL21 (DE3) and E. coli ROSETTA (DE3) the positive transformants of E. coli were selected to induce expression, and the results were all soluble expression of recombinant polypeptide ligase.
  • MBPTrap TM HP 5mL chromatographic column for purification, the recombinant polypeptide ligase can be obtained, and the activation and activity test were performed under the activation conditions and enzyme activity test conditions described in Example 4.
  • the results are shown in Figure 10, E. coli
  • the activated product of the recombinant protein expressed by BL21 (DE3) has no cyclization activity; the product activated by the recombinant protein expressed by E
  • the preferred expression strain is Escherichia coli ROSETTA (DE3).
  • the amino acid sequence of the recombinant polypeptide ligase of mutation scheme 1 is shown in SEQ ID NO:1. Compared with the amino acid sequence of wild-type butterfly bean-derived polypeptide ligase shown in SEQ ID NO: 2, it has the following amino acid mutations: position 338 Lysine was mutated to aspartic acid; proline at position 358 was mutated to asparagine; glutamic acid at position 359 was mutated to aspartic acid.
  • the amino acid sequence of the recombinant polypeptide ligase of mutation scheme 2 is shown in SEQ ID NO: 7, and compared with the amino acid sequence of the wild-type butterfly bean-derived polypeptide ligase shown in SEQ ID NO: 2, it has the following amino acid mutations: position 368 Lysine was mutated to glutamic acid; Lysine at position 369 was mutated to glutamic acid.
  • the wild-type recombinant polypeptide ligase, the mutation scheme one recombinant polypeptide ligase and the mutation scheme two recombinant polypeptide ligase were expressed under the expression conditions described in Example 2, as described in Example 3. Purification was performed under purification conditions, and activation was performed under the activation conditions described in Example 4. The corresponding product 1, product 2 and product 3 were obtained. The activity test was performed under the enzyme activity test conditions described in Example 4. The results are shown in Figure 11. The product 2 obtained by mutation program 1 has better relative enzyme activity than product 1, indicating that its catalytic efficiency is higher and it is a forward mutation; there is no significant difference between the activity of product 3 and product 1 obtained by mutation program 2. .
  • the first mutation scheme can effectively improve the catalytic efficiency of the activation product of the recombinant polypeptide ligase proenzyme, and the effect is significant, so it can be used as a preferred scheme.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Organic Chemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Wood Science & Technology (AREA)
  • General Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • Molecular Biology (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Biophysics (AREA)
  • Plant Pathology (AREA)
  • Enzymes And Modification Thereof (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

提供了一种重组多肽连接酶原及其制备、激活方法与应用。提供了氨基酸序列如SEQ ID NO:1所示的重组多肽连接酶原，其体外自激活能力获得了显著的提高。还提供了一种重组多肽连接酶原的制备方法，实现了所述的重组多肽连接酶原的高效可溶表达，所述方法简便高效，原料简单，表达量高，分离纯化易行，易于大量表达，适合工业化生产；还提供了一种重组多肽连接酶原的激活方法，实现了重组多肽连接酶原的高效激活，激活产物催化多肽环化或连接活性显著；所述的激活产物可用于多肽或蛋白质的环化、多肽与多肽或蛋白质之间的连接、蛋白质与蛋白质之间的连接、蛋白质或多肽与其它化合物的连接中，具有广阔的应用前景。

Description

一种重组多肽连接酶原及其制备、激活方法与应用 技术领域
本发明属于生物技术领域，特别涉及一种重组多肽连接酶原及其制备、激活方法与应用。
背景技术
生物活性肽具有多种人体代谢和生理调节功能，易消化吸收，有促进免疫、激素调节、抗菌、抗病毒、降血压、降血脂等作用。与线性多肽相比，环肽不但具有较高的结构稳定性、热稳定性和生物利用度，而且能够抗水解，在化学变性剂条件下能保持完整生物活性，更为重要的是环化能够不同程度地增强多肽原有生理活性。因此，将线性多肽首尾环化成环状一直是国际生物化学与食品药品领域致力解决的关键问题。
受制备技术的限制，目前环肽主要用于价格昂贵的药物开发。目前来说，制备环肽的主要方法有：天然环肽分离精制、化学合成及微生物发酵合成。然而，天然产物中环肽含量非常低，分离提纯困难；化学合成需要繁琐的基团保护，降低了反应产率和产品纯度；发酵法则需要特殊菌株，能生产的环肽种类少。相对于上述环肽合成方式，近年来环肽的酶法合成表现出极高的应用前景，可克服其它制备方法产量和纯度低、副产物多、分离纯化复杂等缺点。
2014年，新加坡学者首次报道了从药用植物蝶豆(Clitoria ternatea)豆荚中分离纯化出特异性天冬酰氨内肽酶Butelase 1，发现其能高效催化线性多肽分子内环化，具有底物范围广、识别基序简单、催化速度快等特点。但是，植物提取容易受原料限制，提取工艺耗时长、产率低，耗时3天从1kg豆荚中仅得到的5mg天然Butelase 1。因此，通过重组异源表达技术生产和纯化活性Butelase 1是研究热点。截止目前，在原核表达方面仅有澳大利亚学者Amy M.James通过大肠杆菌异源表达全长氨基酸序列的Butelase 1，但其活性远不及天然提取的Butelase 1。也有学者尝试利用酵母表达系统重组表达Butelase 1，但是，与大肠杆菌原核表达系统相比，真核的酵母表达体系表达量低，表达周期长，分离纯化成本高。
发明内容
本发明的第一目的在于克服现有技术的缺点与不足，提供一种重组多肽连接酶原；所述的重组多肽连接酶原的体外自激活能力获得了显著的提高。
本发明的第二目的在于提供一种重组多肽连接酶原的制备方法，实现了所述的重组多肽连接酶原的高效可溶表达。
本发明的第三目的在于提供一种重组多肽连接酶原的激活方法，所述激活方法获得的重组多肽连接酶原激活产物活性强，催化效率高。
本发明的第四目的在于提供所述的重组多肽连接酶原或其激活产物的应用。
本发明的目的通过下述技术方案实现：
一种重组多肽连接酶原，具有SEQ ID NO:1所示氨基酸序列。所述重组多肽连接酶原的氨基酸序列与野生型蝶豆源多肽连接酶原的氨基酸序列SEQ ID NO:2相比，具有以下氨基酸突变：338位赖氨酸突变为天冬氨酸；358位脯氨酸突变为天冬酰胺；359位谷氨酸突变为天冬氨酸，将1～20位信号肽截短，以及N末端融合有MBP标签。
编码所述的重组多肽连接酶原的DNA分子，具有SEQ ID NO:3所示的核苷酸序列。所述的核苷酸序列基于大肠杆菌表达系统对密码子进行了优化，能显著提高异源基因在宿主菌中的表达效率。
一种重组表达载体，含有编码所述的重组多肽连接酶原的核苷酸序列。
优选的，所述的重组表达载体含有如SEQ ID NO:3所示的核苷酸序列。
一种重组多肽连接酶原的制备方法，包括如下步骤：
将编码所述的重组多肽连接酶原的核苷酸序列克隆入表达载体中构建重组表达载体；再将所述的重组表达载体转化原核表达系统中进行诱导表达，固液分离收集菌体沉淀，纯化后得到所述的重组多肽连接酶原。
所述的原核表达系统优选为大肠杆菌表达系统；更优选为大肠杆菌ROSETTA(DE3)。
所述的重组表达载体优选为通过T7启动子进行表达。
所述的表达载体优选为含有MBP标签的表达载体，更优选为pMAL-c5x或pMAL-c5x-His，可以极大地增加目的蛋白表达的可溶性。
所述的诱导表达的具体操作步骤优选为：将阳性转化体接种于LB培养液进行培养，当菌液OD 600达到0.5～0.9时，加入终浓度为0.1～0.6mmol/L的IPTG，15～30℃诱导12～28h；所述的LB培养液中含有卡那霉素30～50μg/mL和氨苄青霉素50～100μg/mL。
所述的将阳性转化体接种于LB培养液进行培养的具体操作为：
(1)将所述的阳性转化体在LB培养液中于25～40℃振荡培养过夜得到种子液；
(2)将种子液接种到LB培养液中，于25～40℃，150～250r/min条件下培养。
步骤(2)中所述的接种量优选按与LB培养液的体积比为1:50～1:150进行接种。
所述的纯化的步骤优选为：裂解细胞后收集上清液，将上清液过亲和层析柱，洗脱得到纯化后的重组多肽连接酶原。
所述的裂解细胞的方法优选为：用超声破碎法冰浴裂解细胞。
所述的超声破碎法中，优选将菌体于缓冲液A中重悬后进行；所述的缓冲液A的配方为20mM NaH 2PO 4-Na 2HPO 4、200mM NaCl、1mM EDTA、1mM DTT，pH＝7。
所述的缓冲液A的用量优选为每克菌体加入10mL缓冲液A。
所述的超声破碎法的条件优选为：功率为50～200W，超声1～5s，间隔1～5s，持续2～30min。
所述的裂解细胞后收集上清液的操作优选为：裂解后的菌体进行冷冻离心后弃沉淀，过膜，得到所述的上清液。
所述的冷冻离心的条件优选在4℃条件下12000r/min离心40min。
所述的过膜优选为用0.22μm微孔滤膜过滤。
所述的亲和层析柱优选为MBP标签亲和层析柱。
所述的洗脱优选为先用缓冲液A平衡，再用缓冲液B洗脱；所述的缓冲液B的配方为：20mM NaH 2PO 4-Na 2HPO 4、100mM NaCl、1mM EDTA、10mM麦芽糖，pH＝7。
一种重组多肽连接酶原的激活方法，包括如下步骤：将重组多肽连接酶原先在pH为3.5～6的环境下进行第一次透析，再于pH为4.5～7的环境下进行第二次透析，固液分离去除沉淀，得到所述的重组多肽连接酶原激活产物。
所述的激活方法优选在25～45℃下进行。
所述的第一次透析优选在包含如下组分的缓冲液Ⅰ中进行：20～50mM乙酸-乙酸钠、50～200mM NaCl、1～5mM EDTA、2～5mM DTT。
所述的第一次透析的时间优选为8～24h。
所述的第二次透析优选在包含如下组分的缓冲液Ⅱ中进行：20～50mM Na 2HPO 4-NaH 2PO 4或Na 2HPO 4-柠檬酸、50～200mM NaCl、1～5mM EDTA、0.5～1mM DTT。
所述的第二次透析的时间优选为2～8h。
所述的透析优选在透析袋中进行，所述的透析袋优选为截留分子量为3～10kDa的透析袋。
一种重组多肽连接酶原激活产物，通过所述的重组多肽连接酶原激活方法得到。
所述的重组多肽连接酶原或所述的重组多肽连接酶原激活产物的应用，包括用于多肽或蛋白质的环化、多肽与多肽或蛋白质之间的连接、蛋白质与蛋白质之间的连接、蛋白质或多肽与其它化合物的连接中。
当用于多肽或蛋白质的环化时，所述的多肽或蛋白质的C端含有天冬酰胺-组氨酸-缬氨酸(NHV)序列，N端第一位氨基酸是除了脯氨酸以外的19种常见氨基酸，N端第二位氨基酸残基必须是异亮氨酸、亮氨酸、缬氨酸或者半胱氨酸(I、L、V或C)；所述的多肽或蛋白质优选为线性多肽或蛋白质。
所述的环化的形式为，多肽或蛋白质的C端的组氨酸-缬氨酸(HV)序列断裂，然后N端和C端首尾以酰胺键相连即得到环肽。
当用于多肽与多肽或蛋白质之间的连接、蛋白质与蛋白质之间的连接时，至少有一个多肽或蛋白质的C端含有天冬酰胺-组氨酸-缬氨酸(NHV)序列，另一个多肽或蛋白质满足N端第一位氨基酸是除了脯氨酸以外的19种常见氨基酸，N端第二位氨基酸残基必须是异亮氨酸、亮氨酸、缬氨酸或者半胱氨酸(I、L、 V或C)。
所述的连接的形式为，一条多肽或蛋白质的C端的组氨酸-缬氨酸(HV)序列断裂，然后和另一条多肽或蛋白质的N端形成酰胺键即得到连接产物。
所述的应用的条件优选为：温度为25～60℃，pH为4.5～7。
所述的应用优选在含有如下成分的缓冲液中进行：20～50mM Na 2HPO 4-NaH 2PO 4、Na 2HPO 4-柠檬酸或2-吗啉乙磺酸，50～200mM NaCl，1～5mM EDTA，0.5～1mM DTT。
所述的缓冲液优选还含有0.05％～0.3％的Triton X-100。
本发明相对于现有技术具有如下的优点及效果：
1、本发明采用大肠杆菌原核表达的方法，制备了可溶性的且能被高效激活的重组多肽连接酶原，目的蛋白含量占总蛋白的60～70％，表达量高，且分离纯化只需一次亲和层析，简单高效，易于大量表达，适合工业化生产。
2、本发明针对原核表达体系进行了密码子优化，同时定点突变了野生型Butelase 1的三个氨基酸，可以增强重组多肽连接酶原的体外自激活能力，同时提高重组多肽连接酶原激活产物的催化效率。
3、本发明利用带有MBP标签的表达载体(例如pMAL-c5x-His载体)来进行目的蛋白的表达，使得表达的蛋白在N端具有一个MBP促溶标签。不但增加了目的蛋白的表达量和可溶性，而且，经MBP标签亲和层析，可以简化重组多肽连接酶原的分离纯化。
4、本发明采用低pH激活的方法，获得的激活产物具有和天然提取的多肽连接酶Butelase 1相同的功能作用，可以进行多肽或蛋白质的环化、多肽与多肽或蛋白质之间的连接、蛋白质与蛋白质之间的连接、蛋白质或多肽与其它化合物的连接；而且，与现有技术中其它的大肠杆菌原核表达得到的多肽连接酶Butelase 1相比，具有更高的催化效率和更专一的环化活性。
5、相对于现有技术中通过天然提取方法获得Butelase 1，本发明的方法无需以蝶豆作为原料，只需要简单的培养基作为原料，制备多肽连接酶的过程不受原料限制，在生物化工、医药和食品等领域具有广阔的前景；相对于现有技术中通过酵母表达系统重组表达获得Butelase 1，本发明的方法表达周期更短，表达量更高，分离纯化更简单。
附图说明
图1为MBP标签亲和层析纯化重组多肽连接酶原的SDS-PAGE图；其中，泳道1～4依次为总蛋白、上清蛋白、穿透蛋白和洗脱蛋白。
图2为重组多肽连接酶原激活的SDS-PAGE图；其中，泳道1和2分别是重组多肽连接酶原和重组多肽连接酶原激活产物。
图3为重组多肽连接酶原激活前后的相对酶活图。
图4为重组多肽连接酶原激活产物环化多肽KB1-NHV的液相色谱图。
图5为重组多肽连接酶原激活产物环化多肽KB1-NHV的产物峰的MALDI-TOF图。
图6为重组多肽连接酶原激活产物环化抗菌肽SoD6-NHV的MALDI-TOF图。
图7为重组多肽连接酶原激活产物环化抗菌肽P113-NHV的MALDI-TOF图。
图8为重组多肽连接酶原激活产物进行多肽分子间连接的MALDI-TOF图。
图9为重组多肽连接酶原激活产物环化向日葵胰蛋白酶抑制剂的MALDI-TOF图。
图10是实施例11中不同菌株表达得到的重组多肽连接酶原激活产物的相对酶活结果分析图。
图11是实施例12中野生型重组多肽连接酶原激活产物和突变型重组多肽连接酶原激活产物的相对酶活结果分析图。
具体实施方式
下面结合实施例及附图对本发明作进一步详细的描述，但本发明的实施方式不限于此。
除无特别说明，本发明中用到的各种试剂、原料均为可以从市场上购买的商品或者可以通过公知的方法制得的产品。
实施例1构建含编码重组多肽连接酶原的核苷酸序列的重组载体
构建含编码重组多肽连接酶原的核苷酸序列的重组载体，包括如下步骤：
(1)根据所述重组多肽连接酶原的氨基酸序列，进行大肠杆菌表达系统的密码子优化后，获得能在大肠杆菌中进行高效表达的DNA分子，利用拼接PCR的方法，人工合成编码所述重组多肽连接酶原的DNA分子，具体如SEQ ID NO:3所示。
(2)pMAL-c5x-His载体的构建
通过Nde I：CATATG和BamH I：GGATCC两个酶切位点，将基因片段“GTCGACAAGCTTGCGGCCGCACTCGAGCACCACCACCACCACCACTGAGATCCGGCTGCTAACGGATCC”接入pMAL-c5x载体(由广州英赞生物科技有限公司提供)，得到的pMAL-c5x-His载体能够表达带有His标签的目的蛋白。
(3)将编码重组多肽连接酶原的DNA分子与表达载体pMAL-c5x-His进行重组
合成引物序列如下，Nde I-FP：ACGCCATATGATTCGTGATGATTTTCTGCG；Sal I-RP：ACGCGTCGACTCACACGCTAAACCCCG。利用PCR技术对重组多肽连接酶原的DNA分子进行扩增，以人工合成编码所述重组多肽连接酶原的DNA分子为模板，加入25μL 2×PfuMax HiFi PCR ProMix(由广州英赞生物科技有限公司生产，EnzyValley，货号P217)、上下游引物Nde I-FP和Sal I-RP各1μL以及适量的灭菌水，进行PCR扩增。扩增条件为：98℃30s；98℃10s，58℃30s，72℃30s，共30个循环；72℃5min。琼脂糖凝胶电泳回收大小约1.4kb的目的基因片段，以Nde I和Sal I(购于NEB)双酶切目的基因片段和载体pMAL-c5x-His，并用T4 DNA酶(购于天根生物科技有限公司)进行连接反应，连接产物转化至DH5α感受态细胞(购于天根生物科技有限公司)中。
(4)挑取单菌落进行菌落PCR鉴定，并将阳性单克隆送往测序公司测序验证，证实编码重组多肽连接酶原的DNA分子的正确性，培养验证正确的感受态细胞，并提取质粒，所获得的质粒即为含有编码重组多肽连接酶原的DNA分子的重组载体。
实施例2重组多肽连接酶原在大肠杆菌中的表达和鉴定
将实施例1获得的重组载体转化到宿主细胞大肠杆菌ROSETTA(DE3)(购于上海唯地生物技术有限公司)中，挑选大肠杆菌阳性转化体，接种于含有少量卡那霉素30μg/mL和氨苄青霉素50μg/mL的LB培养液中，在摇床中于25℃振荡培养过夜作为种子液，取种子液按体积比1:150的比例接种至含有卡那霉素30μg/mL和氨苄青霉素50μg/mL的LB培养液中，于25℃，150r/min条件下培养至菌液OD 600达到0.5，向每瓶菌液加入IPTG至终浓度为0.6mmol/L，于15℃诱导表达28h后，离心收集菌体沉淀。
取IPTG诱导前后菌液各1mL，12000r/min离心1min后取沉淀，加入150μL缓冲液A(20mM NaH 2PO 4-Na 2HPO 4、200mM NaCl、1mM EDTA、1mM DTT，pH7.0)，震荡均匀，冰浴超声破碎(超声条件为：功率50W，超声1s，间隔1s，持续2min)，离心取上清液，沉淀物加入150μL缓冲液A，震荡重悬。取上清及沉淀组分悬液进行SDS-PAGE分析，观察蛋白表达情况。SDS-PAGE电泳检测发现，诱导后的大肠杆菌菌体中表达有可溶性的重组多肽连接酶原。
实施例3重组多肽连接酶原纯化
将实施例1获得的重组载体转化到宿主细胞大肠杆菌ROSETTA(DE3)中，挑选大肠杆菌阳性转化体，接种于含有少量卡那霉素40μg/mL和氨苄青霉素75μg/mL的LB培养液中，在摇床中于40℃振荡培养过夜作为种子液，取种子液按体积比1:50的比例接种至含有卡那霉素40μg/mL和氨苄青霉素75μg/mL的LB培养液中，于40℃，250r/min条件下培养至菌液OD 600达到0.9，向每瓶菌液加入IPTG至终浓度为0.3mmol/L，于28℃诱导表达15h后，离心收集菌体沉淀，-20℃冻存备用。
取1L发酵液发酵得到的冻存菌体，按每克菌体加入10mL缓冲液A(20mM NaH 2PO 4-Na 2HPO 4、200mM NaCl、1mM EDTA、1mM DTT，pH 7.0)重悬菌体，用超声波细胞破碎仪冰浴裂解菌体，超声条件为：功率200W，超声5s，间隔5s，持续30min。将裂解后菌体放入高速冷冻离心机，4℃下12000r/min离心30min，取上清液用0.22μm微孔滤膜过滤，备用。
微滤膜滤过液用MBPTrap TMHP 5mL层析柱进行分离。将层析柱接入快速蛋白质纯化仪柱位阀中，用超纯水清洗系统和柱子，再用缓冲液A平衡，然后用样品泵将滤过液上样，用缓冲液A清洗柱子，将没有结合上柱子的蛋白或者是杂质冲洗干净；再用缓冲液B(20mM NaH 2PO 4-Na 2HPO 4、100mM NaCl、1mM EDTA、10mM麦芽糖，pH 7.0)进行一步洗脱，蛋白变性电泳检测洗脱峰，收集含有目的蛋白重组多肽连接酶原的洗脱峰，收集得到的体积为8mL，浓度为4.0mg/mL，即1L发酵液纯化得到32mg重组多肽连接酶原。洗脱液的蛋白浓度采用紫外分光光度法，测量波长为280nm，摩尔吸光系数133620M -1cm -1，分子量为94239Da。取样20μL，用SDS-PAGE蛋白电泳检测。结果如图1所示，泳道1～4依次为总蛋白、上清蛋白、穿透蛋白和洗脱蛋白，洗脱蛋白中分子量为94.3kDa位置是目的蛋白重组多肽连接酶原的条带；出乎意料地发现，本发明只需要通过一次MBP标签亲和层析，就得到了电泳纯的重组多肽连接酶原，无需再进行 其他纯化步骤(如Ni离子亲和层析等)；同时，通过SDS-PAGE灰度分析法，确定本发明中目的蛋白含量占总蛋白的60～70％，表达量高。
实施例4重组多肽连接酶原的激活
将实施例1获得的重组载体转化到宿主细胞大肠杆菌ROSETTA(DE3)中，挑选大肠杆菌阳性转化体，接种于含有少量卡那霉素50μg/mL和氨苄青霉素100μg/mL的LB培养液中，在摇床中于37℃振荡培养过夜作为种子液，取种子液按体积比1:100的比例接种至含有卡那霉素50μg/mL和氨苄青霉素100μg/mL的LB培养液中，于37℃，200r/min条件下培养至菌液OD 600达到0.8，向每瓶菌液加入IPTG至终浓度为0.1mmol/L，于18℃诱导表达18h后，离心收集菌体沉淀。按实施例3所述步骤分离纯化制备重组多肽连接酶原，4℃冷藏备用。
将重组多肽连接酶原在37℃条件下，用10kDa的透析袋在缓冲液Ⅰ(20mM乙酸-乙酸钠、100mM NaCl、1mM EDTA、5mM DTT，pH 4.5)中透析12h激活后，将装有蛋白液的透析袋转移至缓冲液Ⅱ(20mM Na 2HPO 4-NaH 2PO 4、100mM NaCl、1mM EDTA、0.5mM DTT，pH 6.0)中透析6h，离心去除沉淀，即得到具有环化活性的重组多肽连接酶原激活产物。取样20μL，用SDS-PAGE蛋白电泳检测。结果如图2所示，泳道1和2分别是重组多肽连接酶原和重组多肽连接酶原激活产物，激活产物在约40kDa左右出现蛋白条带，说明在低pH条件下，重组多肽连接酶原能够很好地自激活。
进一步对激活前的重组多肽连接酶原与激活产物的相对酶活进行测试。在50μL的反应缓冲液(20mM NaH 2PO 4-Na 2HPO 4、50mM NaCl、1mM EDTA、0.5mM DTT、0.05％Triton X-100，pH 6.0)中，加入0.5μM激活前的重组多肽连接酶原或激活产物和50μM Kalata B1-NHV多肽(氨基酸序列为GLPVCGETCVGGTCNTPGCTCSWPVCTRNHV)。37℃温育30min后，加入终浓度为100mM的HCl来终止反应。反应液用0.22μm微孔滤膜过滤，然后取20μL通过HPLC进行分析。酶溶液的摩尔浓度采用紫外分光光度法，测量波长为280nm，摩尔吸光系数133620M -1cm -1。
通过液相峰面积计算环化产物的得率，将重组多肽连接酶原激活产物环化KB1-NHV多肽产物的得率定为酶活100％，从而来计算相对酶活。结果如图3所示，未进行激活的重组多肽连接酶原没有活性，激活后得到的激活产物活性显著；进一步说明了所述方法可以成功激活所述的重组多肽连接酶原。
实施例5重组多肽连接酶原激活产物的活性测试
将实施例3获得的重组多肽连接酶原在45℃条件下，用3kDa的透析袋在缓冲液Ⅰ(50mM乙酸-乙酸钠、200mM NaCl、5mM EDTA、2mM DTT，pH 6.0)中透析24h激活后，将装有蛋白液的透析袋转移至缓冲液Ⅱ(50mM Na 2HPO 4-NaH 2PO 4、200mM NaCl、5mM EDTA、1mM DTT，pH 7.0)中透析8h，离心去除沉淀，即得到具有环化活性的重组多肽连接酶原激活产物。
在50μL的反应缓冲液(20mM NaH 2PO 4-Na 2HPO 4、50mM NaCl、1mM EDTA、0.5mM DTT、0.05％TritonX-100，pH 6.0)中，加入0.5μM重组多肽连接酶原激活产物和50μM KalataB1-NHV多肽(氨基酸序列为GLPVCGETCVGGTCNTPGCTCSWPVCTRNHV)。37℃温育20min后，加入终浓度为100mM的HCl来终止反应。反应液用0.22μm微孔滤膜过滤，然后取20μL通过HPLC进行分析鉴定。结果如图4所示，Kalata B1-NHV多肽的环化产物约在线性多肽1min后出峰，约有70％的产率；为了鉴定产物峰的身份，接取产物峰液相的流出液，取1μL点板通过MALDI-TOF进行分析鉴定，结果如图5所示，产物的分子量为2898.33Da，和理论的环化产物分子量吻合，即该产物峰就是环化产物峰。所以本发明利用大肠杆菌原核表达系统生产的重组多肽连接酶原激活产物具有良好的环化多肽的活性。
环化判定：线性多肽KB1-NHV分子量为3152.65Da，反应之后得到环化产物cyclic-KB1的分子量为2898.33Da。
实施例6重组多肽连接酶原激活产物合成环状抗菌肽的应用
将实施例3获得的重组多肽连接酶原在25℃条件下，用5kDa的透析袋在缓冲液Ⅰ(30mM乙酸-乙酸钠、50mM NaCl、3mM EDTA、4mM DTT，pH 3.5)中透析8h激活后，将装有蛋白液的透析袋转移至缓冲液Ⅱ(30mM Na 2HPO 4-柠檬酸、50mM NaCl、2mM EDTA、0.75mM DTT，pH 4.5)中透析2h，离心去除沉淀，即得到重组多肽连接酶原激活产物。
在50μL的反应缓冲液(50mM NaH 2PO 4-Na 2HPO 4、200mM NaCl、5mM EDTA、1mM DTT、0.3％Triton X-100，pH 7.0)中，加入0.5μM重组多肽连接酶原激活产物和50μM菠菜叶细胞壁抗菌肽SoD6-NHV多 肽(氨基酸序列为GIFSNMYARTPAGYFRGPAGYAANHV)或人唾液组胺素5的片段P113-NHV多肽(氨基酸序列为GLAKRHHGYKRKFHNHV)。60℃温育2h后，加入终浓度为100mM的HCl来终止反应。反应液用0.22μm微孔滤膜过滤，经Ziptip脱盐柱脱盐后，取1μL点板通过MALDI-TOF进行分析鉴定。结果如图6和7，所述重组多肽连接酶原激活产物能够很好地将线性的SoD6-NHV或P113-NHV抗菌肽进行首尾连接环化，并且都是表现出专一性的环化作用，达到了合成环状抗菌肽的目的，根据MALDI-TOF质谱峰强度计算环化产物cyclic-SoD6的得率为95％，环化产物cyclic-P113的得率为95％。
环化判定：线性多肽SoD6-NHV分子量为2789.12Da，反应之后得到环化产物cyclic SoD6的分子量为2534.83Da。
环化判定：线性多肽P113-NHV分子量为2085.38Da，反应之后得到环化产物cyclic P113的分子量为1831.12Da。
实施例7重组多肽连接酶原激活产物对多肽进行分子间连接的应用
将实施例3获得的重组多肽连接酶原在37℃条件下，用10kDa的透析袋在缓冲液Ⅰ(20mM乙酸-乙酸钠、100mM NaCl、2mM EDTA、5mM DTT，pH 5.0)中透析16h激活后，转移至缓冲液Ⅱ(20mM Na 2HPO 4-NaH 2PO 4、100mM NaCl、1mM EDTA、0.5mM DTT，pH 6.5)中透析4h，离心去除沉淀，即得到重组多肽连接酶原激活产物。
在50μL的反应缓冲液(30mM NaH 2PO 4-柠檬酸、100mM NaCl、2mM EDTA、0.75mM DTT、0.1％Triton X-100，pH 4.5)中，加入0.5μM重组多肽连接酶原激活产物、50μM八肽KALVINHV(简称K)和1mM六肽GIGGIR(简称G)。25℃温育1h后，加入终浓度为100mM的HCl来终止反应。反应液用0.22μm微孔滤膜过滤，经Ziptip脱盐柱脱盐后，取1μL点板通过MALDI-TOF进行分析鉴定。结果如图8，所述重组多肽连接酶原激活产物能够很好地将八肽KALVINHV和六肽GIGGIR进行分子间连接，得到产物十二肽KALVINGIGGIR(简称KG)，根据MALDI-TOF质谱峰强度计算连接产物的得率为80％。
连接判定：线性八肽KALVINHV(简称K)分子量为893.03Da，线性六肽GIGGIR(简称G)分子量为571.66Da，反应之后得到连接产物十二肽KALVINGIGGIR(简称KG)的分子量为1210.47Da。
实施例8重组多肽连接酶原激活产物环化向日葵胰蛋白酶抑制剂
在50μL的反应缓冲液(20mM 2-吗啉乙磺酸、100mM NaCl、1mM EDTA、0.5mM DTT，pH 5.5)中，加入1mg/mL实施例4获得的重组多肽连接酶原激活产物、250μM多肽底物向日葵胰蛋白酶抑制剂SFTI-NHV线形肽(氨基酸序列：GRCTKSIPPICFPNHV)。37℃温育1h后，加入终浓度为100mM的HCl来终止反应。反应液用0.22μm微孔滤膜过滤，经Ziptip脱盐柱脱盐后，取1μL点板通过MALDI-TOF进行分析鉴定。结果如图9，所述的重组多肽连接酶原激活产物能够将多肽底物SFTI-NHV进行环化。根据MALDI-TOF质谱峰强度计算环化产物的得率为75％，未检测到水解产物，说明该酶制剂的催化效率和环化专一性优良。
环化判定：线性多肽SFTI-NHV分子量为1769.11Da，反应之后得到环化产物cyclic SFTI的分子量为1514.83Da。
实施例9本发明中重组多肽连接酶原激活产物与现有Butelase 1的比较
2014年新加坡学者从1kg药用植物蝶豆(Clitoria ternatea)豆荚中分离纯化得到的5mg天然Butelase 1，工艺耗时长且复杂，提取产率也较低，大量生产时会受到蝶豆原料来源的限制。本发明采用大肠杆菌原核表达的方法，获得了体外自激活能力显著提高的重组多肽连接酶原，表达量高，且分离纯化只需一次亲和层析，简单高效，易于大量表达，适合工业化生产。激活所述的重组多肽连接酶原后得到的重组多肽连接酶原激活产物的生物活性及催化效率出众。
本发明所得的重组多肽连接酶原激活产物与2019年澳大利亚学者Amy M.James等异源表达的Butelase 1(简称Butelase 1 (2019))相比，具有更高的催化效率和更专一的环化活性。如实施例8中所述，所述重组多肽连接酶原激活产物能催化向日葵胰蛋白酶抑制剂环化，酶反应1h环化产物的得率为75％，未检测到水解产物。在相同的酶反应条件下，Butelase 1 (2019)催化向日葵胰蛋白酶抑制剂环化反应虽然没有直接的催化效率数据，但是，根据报道的MALDI-TOF质谱峰强度进行估算，催化反应24h后，环化产物的得率仅约为10％，并且约1％的底物被水解。由此可见，所述重组多肽连接酶原激活产物比Butelase 1 (2019)有更高的催化效率和更专一的环化活性(文献：James AM,Haywood J,Leroux J,IgnasiakK,Elliott AG,Schmidberger JW,Fisher MF,Nonis SG,Fenske R,Bond CS,Mylne JS.The macrocyclizing protease butelase 1 remains auto-catalytic and reveals the structural basis for ligase activity.The Plant Journal,2019,98(6):988-999.DOI: 10.1111/tpj.14293.)。
本发明所得的重组多肽连接酶原与2019年Ni Pi等采用酵母表达系统重组表达获得的Butelase 1(简称rBTase)相比，具有表达周期更短，表达量更高，分离纯化更简单的优势。本发明的重组多肽连接酶原表达周期为15h，表达量为每升发酵液得到32mg目的蛋白，分离纯化只需一次MBP标签柱亲和层析。rBTase表达周期为96h，表达量为每升发酵液得到16mg目的蛋白，分离纯化需联用凝胶排阻层析和Ni离子亲和层析才能达到纯度符合要求的目的蛋白(文献：Pi N,Gao M,Cheng X,LiuH,Kuang Z,Yang Z,Yang J,Zhang B,Chen Y,Liu S,Huang Y,Su Z.Recombinant butelase-mediated cyclization of the p53-binding domain of the oncoprotein MdmX-stabilized protein conformation as a promising model for structural investigation.Biochemistry,2019,58(27):3005-3015.DOI:10.1021/acs.biochem.9b00263.)。
实施例10表达载体的筛选
在本发明的研究过程中，曾尝试用pET-28a、pHUE-ubiquitin、pET-28a-SUMO和pMAL-c5x-His四种载体(载体均由广州英赞生物科技有限公司提供)来表达重组多肽连接酶原。
构建的pET-28a重组载体，表达的重组蛋白为组氨酸标签+全长多肽连接酶原，氨基酸序列如SEQ ID NO:4所示，将pET-28a重组载体转入大肠杆菌ROSETTA(DE3)中进行表达。结果为无目标蛋白的表达。
构建的pHUE-ubiquitin重组载体，表达的重组蛋白为组氨酸标签+泛素标签+全长多肽连接酶原，氨基酸序列如SEQ ID NO:5所示，将pHUE-ubiquitin重组载体转入大肠杆菌ROSETTA(DE3)中进行表达，结果为少量目标蛋白为可溶表达，大部分均为包涵体表达。
构建的pET-28a-SUMO重组载体，表达的重组蛋白为组氨酸标签+SUMO标签+全长多肽连接酶原，氨基酸序列如SEQ ID NO:6所示，将pHUE-ubiquitin重组载体转入大肠杆菌ROSETTA(DE3)中进行表达。结果为目标蛋白呈可溶表达，但是目标蛋白未能和镍离子亲和层析柱有效结合，即不能纯化得到重组多肽连接酶原。
构建的pMAL-c5x-His重组载体，表达的重组蛋白为MBP标签+全长多肽连接酶原，氨基酸序列如SEQ ID NO:1所示，将pMAL-c5x-His重组载体转入大肠杆菌ROSETTA(DE3)中进行表达。结果为目标蛋白为可溶表达，利用一次MBPTrap TMHP 5mL层析柱纯化即能得到重组多肽连接酶原。
综上，经过多种载体的表达筛选研究，本发明优选采用含有MBP标签的表达载体，更优选的表达载体如pMAL-c5x、pMAL-c5x-His等载体。
实施例11表达菌株的筛选
在本发明研究过程中，曾尝试过用大肠杆菌BL21(DE3)(购于天根生物科技有限公司)和大肠杆菌ROSETTA(DE3)表达菌株，来表达重组多肽连接酶原。
将实施例1获得的重组载体转化到宿主细胞大肠杆菌BL21(DE3)和大肠杆菌ROSETTA(DE3)中，分别挑选大肠杆菌阳性转化体进行诱导表达，结果均为重组多肽连接酶原可溶表达。利用一次MBPTrap TMHP 5mL层析柱纯化均能得到重组多肽连接酶原，并在实施例4所描述的激活条件和酶活测试条件下进行激活和活性测试，结果如图10所示，大肠杆菌BL21(DE3)表达的重组蛋白激活得到产物没有环化活性；大肠杆菌ROSETTA(DE3)表达的重组蛋白激活得到的产物则具有良好的环化活性。
综上，经过表达菌株的筛选研究，得到优选的表达菌株为大肠杆菌ROSETTA(DE3)。
实施例12突变位点的优化研究
在本发明的研究过程中，曾尝试过两种突变方案。
突变方案一的重组多肽连接酶原的氨基酸序列如SEQ ID NO:1所示，与野生型蝶豆源多肽连接酶原的氨基酸序列如SEQ ID NO:2相比，具有以下氨基酸突变：338位赖氨酸突变为天冬氨酸；358位脯氨酸突变为天冬酰胺；359位谷氨酸突变为天冬氨酸。
突变方案二的重组多肽连接酶原的氨基酸序列如SEQ ID NO:7所示，与野生型蝶豆源多肽连接酶原的氨基酸序列如SEQ ID NO:2相比，具有以下氨基酸突变：368位赖氨酸突变为谷氨酸；369位赖氨酸突变为谷氨酸。
野生型重组多肽连接酶原、所述突变方案一重组多肽连接酶原和所述突变方案二重组多肽连接酶原，在实施例2所记载的表达条件下进行表达，在实施例3所记载的纯化条件下进行纯化，在实施例4所记载的激活条件下进行激活。得到相应的产物1、产物2和产物3。在实施例4所记载的酶活测试条件下进行活性测试。结果如图11所示，突变方案一所获得的产物2相对酶活比产物1好，说明其催化效率更高，为正向突变；突变方案二所获得的产物3和产物1活性无明显差别。
综上，突变方案一可以有效地提高重组多肽连接酶原激活产物的催化效率，效果显著，可以作为优选方案。
上述实施例为本发明较佳的实施方式，但本发明的实施方式并不受上述实施例的限制，其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化，均应为等效的置换方式，都包含在本发明的保护范围之内。
Figure PCTCN2020120817-appb-000001
Figure PCTCN2020120817-appb-000002
Figure PCTCN2020120817-appb-000003
Figure PCTCN2020120817-appb-000004
Figure PCTCN2020120817-appb-000005
Figure PCTCN2020120817-appb-000006
Figure PCTCN2020120817-appb-000007
Figure PCTCN2020120817-appb-000008
Figure PCTCN2020120817-appb-000009
Figure PCTCN2020120817-appb-000010
Figure PCTCN2020120817-appb-000011
Figure PCTCN2020120817-appb-000012
Figure PCTCN2020120817-appb-000013
Figure PCTCN2020120817-appb-000014
Figure PCTCN2020120817-appb-000015
Figure PCTCN2020120817-appb-000016
Figure PCTCN2020120817-appb-000017
Figure PCTCN2020120817-appb-000018
Figure PCTCN2020120817-appb-000019
Figure PCTCN2020120817-appb-000020
Figure PCTCN2020120817-appb-000021
Figure PCTCN2020120817-appb-000022

Claims (10)

  1. 一种重组多肽连接酶原，其特征在于：
    具有SEQ ID NO:1所示氨基酸序列。
  2. 一种DNA分子，其特征在于：
    所述的DNA分子编码权利要求1所述的重组多肽连接酶原。
  3. 一种重组表达载体，其特征在于：
    含有编码权利要求1所述的重组多肽连接酶原的核苷酸序列。
  4. 一种重组多肽连接酶原的制备方法，其特征在于，包括如下步骤：
    将编码权利要求1所述的重组多肽连接酶原的核苷酸序列克隆入表达载体中构建重组表达载体；再将所述的重组表达载体转化原核表达系统中进行诱导表达，固液分离收集菌体沉淀，纯化后得到所述的重组多肽连接酶原。
  5. 根据权利要求4所述的重组多肽连接酶原的制备方法，其特征在于：
    所述的原核表达系统为大肠杆菌表达系统和大肠杆菌ROSETTA(DE3)；
    所述的表达载体为含有MBP标签的表达载体和pMAL-c5x或pMAL-c5x-His；
    所述的诱导表达的具体操作步骤为：将阳性转化体接种于LB培养液进行培养，当菌液OD 600达到0.5～0.9时，加入终浓度为0.1～0.6mmol/L的IPTG，15～30℃诱导12～28h；所述的LB培养液中含有卡那霉素30～50μg/mL和氨苄青霉素50～100μg/mL。
  6. 一种重组多肽连接酶原的激活方法，其特征在于，包括如下步骤：
    将权利要求1所述的重组多肽连接酶原先在pH为3.5～6的环境下进行第一次透析，再于pH为4.5～7的环境下进行第二次透析，固液分离去除沉淀，得到重组多肽连接酶原激活产物。
  7. 根据权利要求6所述的重组多肽连接酶原的激活方法，其特征在于：
    所述的激活方法在25～45℃下进行；
    所述的第一次透析在包含如下组分的缓冲液Ⅰ中进行：20～50mM乙酸-乙酸钠、50～200mM NaCl、1～5mM EDTA、2～5mM DTT；
    所述的第一次透析的时间为8～24h；
    所述的第二次透析在包含如下组分的缓冲液Ⅱ中进行：20～50mM Na 2HPO 4-NaH 2PO 4或Na 2HPO 4-柠檬酸、50～200mM NaCl、1～5mM EDTA、0.5～1mM DTT；
    所述的第二次透析的时间为2～8h。
  8. 一种重组多肽连接酶原激活产物，其特征在于：
    通过权利要求6所述的重组多肽连接酶原的激活方法得到。
  9. 权利要求1所述的重组多肽连接酶原或权利要求8所述的重组多肽连接酶原激活产物的应用，其特征在于：
    所述的应用包括用于多肽或蛋白质的环化、多肽与多肽或蛋白质之间的连接、蛋白质与蛋白质之间的连接、蛋白质或多肽与其它化合物的连接中；
    当用于多肽或蛋白质的环化时，所述的多肽或蛋白质的C端含有天冬酰胺-组氨酸-缬氨酸序列，N端第一位氨基酸是除了脯氨酸以外的19种常见氨基酸，N端第二位氨基酸残基为异亮氨酸、亮氨酸、缬氨酸或者半胱氨酸；
    当用于多肽与多肽或蛋白质之间的连接、蛋白质与蛋白质之间的连接时，至少有一个多肽或蛋白质的C端含有天冬酰胺-组氨酸-缬氨酸序列，另一个多肽或蛋白质的N端第一位氨基酸是除了脯氨酸以外的19种常见氨基酸，N端第二位氨基酸残基为异亮氨酸、亮氨酸、缬氨酸或者半胱氨酸。
  10. 根据权利要求9所述的重组多肽连接酶原或所述的重组多肽连接酶原激活产物的应用，其特征在于：
    所述的应用的条件为温度为25～60℃，pH为4.5～7；
    所述的应用在含有如下成分的缓冲液中进行：
    (1)20～50mM Na 2HPO 4-NaH 2PO 4、Na 2HPO 4-柠檬酸或2-吗啉乙磺酸，50～200mM NaCl，1～5mM EDTA，0.5～1mM DTT；
    或
    (2)20～50mM Na 2HPO 4-NaH 2PO 4、Na 2HPO 4-柠檬酸或2-吗啉乙磺酸，50～200mM NaCl，1～5mM EDTA，0.5～1mM DTT，0.05％～0.3％的Triton X-100。
PCT/CN2020/120817 2020-01-06 2020-10-14 一种重组多肽连接酶原及其制备、激活方法与应用 WO2021139295A1 (zh)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202010010362.4A CN111117977B (zh) 2020-01-06 2020-01-06 一种重组多肽连接酶原及其制备、激活方法与应用
CN202010010362.4 2020-01-06

Publications (1)

Publication Number Publication Date
WO2021139295A1 true WO2021139295A1 (zh) 2021-07-15

Family

ID=70486915

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2020/120817 WO2021139295A1 (zh) 2020-01-06 2020-10-14 一种重组多肽连接酶原及其制备、激活方法与应用

Country Status (2)

Country Link
CN (1) CN111117977B (zh)
WO (1) WO2021139295A1 (zh)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114854782A (zh) * 2022-05-12 2022-08-05 华南理工大学 一种高效表达具有高活性的重组多肽连接酶原的方法

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111117977B (zh) * 2020-01-06 2021-09-10 广州英赞生物科技有限公司 一种重组多肽连接酶原及其制备、激活方法与应用
CN112010947B (zh) * 2020-09-10 2022-05-24 中新国际联合研究院 一种具有血管紧张素转换酶抑制活性的环十六肽及其制备方法与应用
CN114874299B (zh) * 2022-03-17 2023-04-28 温州大学 用于生成二氧化硅的多肽及其应用、组合物

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106459160A (zh) * 2014-04-24 2017-02-22 南洋理工大学 Asx特异性蛋白质连接酶
WO2017054044A1 (en) * 2015-09-30 2017-04-06 Hexima Limited A method
CN109790205A (zh) * 2016-09-23 2019-05-21 南洋理工大学 酶促肽连接的方法
CN111117977A (zh) * 2020-01-06 2020-05-08 华南理工大学 一种重组多肽连接酶原及其制备、激活方法与应用

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11739128B2 (en) * 2018-03-07 2023-08-29 Epicypher, Inc. Peptide ligase-mediated engineering of recombinant nucleosomes
CN110331157B (zh) * 2019-08-02 2021-06-08 湖北大学 一种aep环化酶在大肠杆菌中的融合表达方法、aep环化酶环化能力鉴定方法及其应用

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106459160A (zh) * 2014-04-24 2017-02-22 南洋理工大学 Asx特异性蛋白质连接酶
WO2017054044A1 (en) * 2015-09-30 2017-04-06 Hexima Limited A method
CN109790205A (zh) * 2016-09-23 2019-05-21 南洋理工大学 酶促肽连接的方法
CN111117977A (zh) * 2020-01-06 2020-05-08 华南理工大学 一种重组多肽连接酶原及其制备、激活方法与应用

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
JAMES AMY M., HAYWOOD JOEL, LEROUX JULIE, IGNASIAK KATARZYNA, ELLIOTT ALYSHA G., SCHMIDBERGER JASON W., FISHER MARK F., NONIS SAMU: "The macrocyclizing protease butelase 1 remains autocatalytic and reveals the structural basis for ligase activity", THE PLANT JOURNAL, BLACKWELL SCIENTIFIC PUBLICATIONS, OXFORD., GB, GB, XP055826973, ISSN: 0960-7412, DOI: 10.1111/tpj.14293 *
PI NI, GAO MENG, CHENG XIYAO, LIU HUILI, KUANG ZHENGKUN, YANG ZIXIN, YANG JING, ZHANG BAILING, CHEN YAO, LIU SEN, HUANG YONGQI, SU: "Recombinant Butelase-Mediated Cyclization of the p53-Binding Domain of the Oncoprotein MdmX-Stabilized Protein Conformation as a Promising Model for Structural Investigation", BIOCHEMISTRY, vol. 58, no. 27, 9 July 2019 (2019-07-09), pages 3005 - 3015, XP055826972, ISSN: 0006-2960, DOI: 10.1021/acs.biochem.9b00263 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114854782A (zh) * 2022-05-12 2022-08-05 华南理工大学 一种高效表达具有高活性的重组多肽连接酶原的方法
CN114854782B (zh) * 2022-05-12 2023-06-20 华南理工大学 一种高效表达具有高活性的重组多肽连接酶原的方法

Also Published As

Publication number Publication date
CN111117977A (zh) 2020-05-08
CN111117977B (zh) 2021-09-10

Similar Documents

Publication Publication Date Title
WO2021139295A1 (zh) 一种重组多肽连接酶原及其制备、激活方法与应用
JP4504014B2 (ja) インスリン分泌性ｇｌｐ−１（７−３６）ポリペプチドおよび／またはｇｌｐ−１類似体を生成する方法
CN102839182B (zh) 利用大肠杆菌表达系统制备重组人神经生长因子的方法
CN110343689A (zh) 一种新型链霉菌胰蛋白酶gm2938及其异源表达
Pozidis et al. Protein secretion biotechnology using Streptomyces lividans: Large‐scale production of functional trimeric tumor necrosis factor α
CN106939315B (zh) 一种草酸脱羧酶的制备方法及应用
CN113801240B (zh) 一种d-阿洛酮糖-3-差向异构酶活性聚集体及其制备方法与应用
CN112175971A (zh) 密码子优化的krd基因和gdh基因及其应用
CN101413009B (zh) 一种改构的人淀粉样多肽突变体－普兰林肽(pramlintide)的制备方法
US20160145303A1 (en) Strong Secretory Signal Peptide Enhancing Small Peptide Motifs and the Use Thereof
CN110776569B (zh) 一种具有粘附-抗冻双功能的二嵌段融合蛋白及合成方法和应用
CN112824527B (zh) 人工设计的赖氨酰内切酶及编码序列和发酵方法
CN112500495A (zh) 一种elp-ⅲ型胶原蛋白的纯化方法及应用
CN112143688A (zh) 一种重组大肠杆菌的构建及其应用
RU2453604C1 (ru) Гибридный белок (варианты), штамм бактерий escherichia coli - продуцент гибридного белка (варианты) и способ получения безметионинового интерферона альфа-2 человека
CN114410558A (zh) 具有降血压功能的ace抑制肽-aceip及其基因工程制备方法
RU2441072C1 (ru) ГИБРИДНЫЙ БЕЛОК, ШТАММ БАКТЕРИЙ ESCHERICHIA COLI - ПРОДУЦЕНТ ГИБРИДНОГО БЕЛКА И СПОСОБ ПОЛУЧЕНИЯ БЕЗМЕТИОНИНОВОГО ИНТЕРФЕРОНА АЛЬФА-2b ЧЕЛОВЕКА ИЗ ЭТОГО ГИБРИДНОГО БЕЛКА
CN113754726B (zh) 一种含多肽标签的重组酶及其在医药化学品合成中的应用
CN103146631A (zh) 一种表达可溶性猪γ-干扰素PoIFN-γ的基因工程菌及其构建方法和应用
CN113862290A (zh) 一种来源于甘草的异黄酮4′-o-甲基转移酶及其应用
CN113980880A (zh) 基因工程菌及其应用和以葡萄糖为原料生产阿洛酮糖的方法
CN114854782B (zh) 一种高效表达具有高活性的重组多肽连接酶原的方法
CN113699091B (zh) 一种重组枯草芽孢杆菌及其构建方法和应用
CN117466992B (zh) 一种纤连蛋白突变体及其制备和应用
CN114350643B (zh) 一种产氨肽酶的重组菌株及其在高效蛋白水解中的应用

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: 20913045

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

32PN Ep: public notification in the ep bulletin as address of the adressee cannot be established

Free format text: NOTING OF LOSS OF RIGHTS PURSUANT TO RULE 112(1) EPC (EPO FORM 1205A DATED 21/11/2022)

122 Ep: pct application non-entry in european phase

Ref document number: 20913045

Country of ref document: EP

Kind code of ref document: A1