CN117088946A - Total biosynthesis method of cyclic bacteriocin AS-48 - Google Patents

Total biosynthesis method of cyclic bacteriocin AS-48 Download PDF

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CN117088946A
CN117088946A CN202311055350.3A CN202311055350A CN117088946A CN 117088946 A CN117088946 A CN 117088946A CN 202311055350 A CN202311055350 A CN 202311055350A CN 117088946 A CN117088946 A CN 117088946A
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buffer
linear polypeptide
bacteriocin
polypeptide precursor
mbp
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胡松青
赵劲松
侯轶
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South China University of Technology SCUT
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Abstract

The invention discloses a biosynthesis method of a cyclic bacteriocin AS-48. The invention provides an AS-48 linear polypeptide precursor with an amino acid sequence shown AS SEQ ID NO. 1, which can obviously improve the expression level of the precursor in escherichia coli. The invention also provides a preparation method of the linear polypeptide precursor, which realizes the efficient and soluble expression of the AS-48 linear polypeptide precursor, is simple and efficient, has simple raw materials, high expression quantity and easy separation and purification, is convenient for mass expression, and is suitable for industrial production; the invention also provides a full biosynthesis method of the annular bacteriocin AS-48, which realizes the efficient synthesis of the annular AS-48 and mutants thereof; the structure and the antibacterial activity of the annular AS-48 and the mutant thereof are consistent with those of the natural AS-48.

Description

Total biosynthesis method of cyclic bacteriocin AS-48
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a full biosynthesis method of cyclic bacteriocin AS-48.
Background
The antibacterial peptide is used as an important representative of natural food preservatives, plays an important role in killing or inhibiting food-borne pathogenic bacteria and spoilage bacteria and guaranteeing food quality and safety, and has the advantages of environmental protection, safety, high efficiency and the like compared with chemical preservatives. The cyclic bacteriocin AS-48 is a cyclic antibacterial peptide with the carboxyl end and the amino end connected end to end, and has higher stability and wider antibacterial performance compared with the linear antibacterial peptide. The annular bacteriocin AS-48 is expected to develop into a novel polypeptide natural food preservative because of the advantages of good stability, strong antibacterial capability, high safety, no drug resistance and the like. At present, the food preservation scientific research which is carried out around the annular bacteriocin AS-48 has good effect for controlling the pathogenic bacteria or spoilage bacteria of the food, and the industrial application of the annular bacteriocin is beneficially explored. However, due to limitations of large-scale preparation technology, cyclic bacteriocins are not widely used in the food field at present.
One of the main reasons for slow application progress is the relative lag in the in vitro synthesis studies of the cyclic bacteriocin AS-48. The content of the natural cyclic bacteriocin is low, and the separation and purification are relatively complicated. By utilizing a high-density fermentation technology and on the basis of optimizing culture conditions, only 11.16mg of annular AS-48 can be separated from 1L of enterococcus faecalis fermentation liquor; the in vivo biosynthetic pathway of cyclic bacteriocins is complex, and synthetic gene clusters involve at least 10 or more genes, so that it is difficult to increase metabolic synthesis yield through gene regulation. The chemical synthesis of cyclic bacteriocins is difficult to realize at present because of more amino acid compositions and more than 50% of hydrophobic amino acids. In recent years, the research on recombinant expression of antibacterial peptides by taking escherichia coli, pichia pastoris and insect cells as main expression systems has achieved good results; however, due to the special cyclic framework structure, the high hydrophobic amino acid composition and the potential host toxicity, the heterologous expression of the cyclic bacteriocin is difficult at present. Although E.coli is used as a host for research, the cyclic bacteriocin linear polypeptide precursor is fusion expressed, the yield and antibacterial activity of the finally obtained linear polypeptide precursor are not ideal. Based on chemical solid phase synthesis of linear polypeptide precursors, the chemical-enzymatic synthesis of the cyclic bacteriocin AS-48 can be realized by utilizing enzymatic catalysis of the end-to-end cyclization of linear polypeptides. However, chemical-enzymatic synthesis of cyclic bacteriocins is also difficult to achieve in large scale production, limited by chemical synthesis of AS-48 linear polypeptide precursors.
Disclosure of Invention
In view of the drawbacks and deficiencies of the prior art, a first object of the present invention is to provide a method for total biosynthesis of the cyclic bacteriocin AS-48.
It is a second object of the present invention to provide a circular bacteriocin AS-48 linear polypeptide precursor fusion protein.
It is a third object of the present invention to provide a circular bacteriocin AS-48 linear polypeptide precursor.
A fourth object of the present invention is to provide a cyclic bacteriocin AS-48.
The aim of the invention is mainly realized by the following technical scheme:
a circular bacteriocin AS-48 linear polypeptide precursor has an amino acid sequence shown in SEQ ID NO. 1. Compared with the amino acid sequence SEQ ID NO. 2 of the cyclic AS-48, the amino acid sequence has the following structure and amino acid changes: losing the annular skeleton structure; cleavage of peptide bond between Asn17-Val 18; the carboxyl terminus of Asn17 was added with the HVKKK pentapeptide amino acid motif.
A DNA molecule encoding the linear polypeptide precursor has a nucleotide sequence shown in SEQ ID NO. 5. The nucleotide sequence optimizes codons based on an escherichia coli expression system, and can remarkably improve the expression efficiency of heterologous genes in host bacteria.
A circular bacteriocin AS-48 linear polypeptide precursor fusion protein has an amino acid sequence shown AS SEQ ID NO. 3 or SEQ ID NO. 4. Compared with the AS-48 linear polypeptide precursor, the amino acid sequence is fused with MBP-SUMO and MBP labels at the N terminal.
The preparation method of the annular bacteriocin AS-48 linear polypeptide precursor fusion protein comprises the following preparation steps: cloning the nucleotide sequence encoding the AS-48 linear precursor into a prokaryotic expression plasmid to construct a recombinant expression vector; then the recombinant expression vector is transformed into a prokaryotic expression system for induced expression, bacterial precipitation is collected, and the AS-48 linear polypeptide precursor recombinant fusion protein is obtained after crushing and purification; the prokaryotic expression plasmid is an expression vector containing MBP or MBP-SUMO label.
The prokaryotic expression plasmid is preferably pET-28a-MBP or pET-28a-MBP-SUMO, so that the expression yield and the solubility of the target protein can be increased.
The prokaryotic expression system is preferably an escherichia coli expression system; more preferably E.coli over express C43 (DE 3), host toxicity can be effectively avoided.
The specific operation steps of the induced expression are preferably as follows: inoculating positive transformant into LB culture solution for culturing, and culturing when bacterial solution OD 600 When reaching 0.4 to 0.6, adding IPTG with the final concentration of 0.2 to 0.4mmol/L, and inducing for 6 to 20 hours at the temperature of 18 to 37 ℃; the LB culture solution contains 50-100 mug/mL kanamycin.
The steps of crushing and purifying are preferably as follows: after the bacterial cells are broken, the supernatant component is collected by centrifugation, and after the purification by metal ion affinity chromatography, AS-48 linear precursor recombinant fusion protein is obtained by elution.
The method for breaking cells is preferably as follows: suspending the strain in buffer A, and performing cell lysis by ice bath ultrasonic disruption; the formula of the buffer A is 20+/-5 mM NaH 2 PO 4 -Na 2 HPO 4 、100±20mM NaCl，pH＝7。
The amount of the buffer A is preferably 10mL of the buffer A per gram of wet weight of the bacterial cells.
The conditions of the ultrasonic disruption method are preferably as follows: the power is 100-200W, the ultrasonic wave is 2-4 s, the interval is 2-4 s, and the duration is 10-20 min.
The operation of centrifugally collecting the supernatant is preferably as follows: centrifuging at 12000g for 40min at 4deg.C, discarding precipitate, and filtering with 0.22 μm microporous membrane to obtain supernatant.
The metal ion affinity chromatography purification is preferably Ni ion affinity chromatography.
The elution procedure is preferably to equilibrate with buffer A and then to elute with buffer B; the formula of the buffer A is 20mM NaH 2 PO 4 -Na 2 HPO 4 100mm nacl, ph=7; the formula of the buffer B is as follows: 20mM NaH 2 PO 4 -Na 2 HPO 4 100mM NaCl, 500mM imidazole, pH=7.
The preparation method of the annular bacteriocin AS-48 linear polypeptide precursor comprises all steps in the preparation method of the annular bacteriocin AS-48 linear polypeptide precursor fusion protein and the following preparation steps:
and (3) performing first dialysis on the linear polypeptide precursor recombinant fusion protein under a buffer system, then performing premixed enzyme digestion with protease, further performing second dialysis under the buffer system, separating enzyme digestion products by utilizing metal ion affinity chromatography and RP-HPLC, and collecting AS-48 linear polypeptide precursors after freeze drying.
The buffer system for the first dialysis is preferably buffer C, and the formula of the buffer C is as follows: 50+ -10 mM Tis-HCl, 50+ -10 mM NaCl, 0.5+ -0.1 mM EDTA, 1+ -0.5 mM DTT, pH 8.0.
The buffer system for the second dialysis is preferably buffer A, and the formula of the buffer A is as follows: 20+ -5 mM NaH 2 PO 4 -Na 2 HPO 4 、100±20mM NaCl，pH＝7。
When the N-terminal fusion of the annular bacteriocin AS-48 linear polypeptide precursor fusion protein is MBP label, the protease is TEV; when the N-terminal fusion of the linear polypeptide precursor fusion protein of the circular bacteriocin AS-48 is MBP-SUMO label, the protease is Ulp1.
In the enzyme digestion system, the equivalent ratio of the substrate to the protease is preferably 50:1-100:1.
The temperature of the enzyme digestion is preferably 4-30 ℃, and the time length is preferably 1-12 h; preferably under buffer A.
The metal ion affinity chromatography is preferably Ni ion affinity chromatography.
The buffer solution used for Ni ion affinity chromatography is preferably buffer solution A and buffer solution B.
The RP-HPLC separation procedure is preferably: 0-5min,5% acetonitrile; 5-35min,5-90% acetonitrile; 35-40min,90% acetonitrile; 40-45min,90-5% acetonitrile; 45-50min,5% acetonitrile.
A method for total biosynthesis of a cyclic bacteriocin AS-48, comprising all steps in the above method for preparing a linear polypeptide precursor of a cyclic bacteriocin AS-48 and the following steps:
and re-dissolving the collected AS-48 linear polypeptide precursor in a cyclization buffer solution, and adding cyclase to catalyze the end-to-end cyclization of the AS-48 linear polypeptide precursor, so that the cyclized product is the cyclic bacteriocin AS-48.
The cyclase is preferably the cyclase Butelase 1.
The cyclization buffer is preferably buffer D; the formula of the buffer solution D is as follows: 20+ -5 mM NaH 2 PO 4 -Na 2 HPO 4 、100±20mM NaCl、0.5±0.1mM EDTA、1±0.5mM DTT，pH 6.5。
The equivalent ratio of the AS-48 linear polypeptide precursor to the cyclase is preferably 50:1-100:1.
The cyclization reaction temperature is preferably 37-45 ℃, the reaction time is preferably 6-12 h, and the pH is preferably 6.0-6.5.
A cyclic bacteriocin AS-48 and its mutant are prepared through the above method, and have the amino acid sequence shown in SEQ ID NO. 2 or SEQ ID NO. 11, wherein the amino group of the N-terminal amino acid residue and the carboxyl group of the C-terminal amino acid residue are connected in the form of peptide bond to form a cyclic skeleton.
The invention analyzes the structure and function of the synthesized cyclic bacteriocin AS-48 and mutants thereof, including secondary structure and antibacterial activity analysis.
Natural cyclic AS-48 is composed of 5 alpha-helical structures, has two distinct negative peaks in the circular dichroism spectrum (CD) at 208nm and 220nm, has a typical alpha-helical structure characteristic spectrum, and can inhibit the growth and reproduction of gram-negative and gram-positive bacteria.
The synthesized AS-48 secondary structure was analyzed using far ultraviolet CD spectroscopy. The AS-48 linear precursor and the cyclic AS-48 were dissolved in buffer, respectively, and then subjected to circular dichroism spectroscopy.
The buffer system is preferably buffer E; the formulation of the buffer E is preferably 20mM NaH 2 PO 4 -Na 2 HPO 4 ，pH 7.2。
The final concentration of the dissolved polypeptide is preferably 10-50. Mu.M.
The CD test conditions are preferably: the scanning wavelength range is 190-260nm, the bandwidth is 1nm, the scanning step length is 1nm, the slit width is 0.02nm, the path is 1mm, and the scanning speed is 50nm/min. The polypeptide sample spectrum was baseline corrected with buffer E as a blank and by subtracting the blank spectrum.
Diluting the indicator bacteria cultured to logarithmic phase with fresh LB medium until the bacterial density is 10 7 CFU/mL. The AS-48 is diluted by the buffer E in a gradient way, 100 mu L of polypeptide sample and 100 mu L of bacterial liquid are taken and added into a 96-well plate after being evenly mixed, and are cultured for 24 hours at 37 ℃, and the OD is measured by an enzyme-labeled instrument 600 Absorbance under conditions. The concentration of bacteriocin added when no apparent cell growth was observed was defined as the minimum inhibitory concentration.
The indicator bacteria are preferably E.coli ATCC 43895, salmonella ATCC 14028, vibrio parahaemolyticus ATCC 17969, staphylococcus aureus ATCC 25923, listeria ATCC 19118, enterococcus faecalis ATCC 29212.
Compared with the prior art, the invention has the following advantages:
1. the invention adopts the prokaryotic expression system of the escherichia coli, efficiently expresses the recombinant fusion protein of the soluble AS-48 linear polypeptide precursor, has the target protein content of more than 200mg in 1L fermentation liquor, has high expression quantity, is convenient to separate and purify, is simple and efficient, is easy to express in large quantities and is suitable for industrial production compared with the existing recombinant expression technology of the AS-48 linear precursor.
2. The invention optimizes the codon aiming at a prokaryotic expression system, fuses MBP or MBP-SUMO dissolution promoting label at the N end of an AS-48 linear polypeptide precursor, and blends HVKKK pentapeptide motif at the C end, thereby not only being beneficial to increasing the expression quantity and the solubility of target protein, but also being capable of effectively shielding the antibacterial activity and the host toxicity of AS-48 and promoting the over-expression of the target protein in E.coli cells.
3. According to the invention, the AS-48 linear precursor fusion protein is digested by using TEV and Ulp1 protease, MBP or MBP-SUMO labels can be removed under mild conditions, the normal structure and function of the AS-48 linear precursor are not affected, and compared with chemical cleavage, the method is more efficient, economical and environment-friendly.
4. The invention adopts cyclase Butelase 1 to catalyze the cyclization of AS-48 linear polypeptide precursor, and the structure and function of the generated annular AS-48 and mutants thereof are consistent with those of the natural extracted AS-48. Moreover, compared with the existing chemical cyclization strategy, the method is more convenient, efficient and environment-friendly, the reaction system is suitable for amplification, and industrial production is easy. .
5. Compared with the existing annular bacteriocin synthesis technology, the biosynthesis method has the characteristics of high efficiency, convenience, rapidness, environment friendliness and the like, and the preparation method has sustainability and is beneficial to promoting the application of the annular bacteriocin in the fields of foods, medicines and the like.
Drawings
FIG. 1 is a SDS-PAGE map of recombinant fusion protein expression of AS-48 linear polypeptide precursors.
FIG. 2 is a SDS-PAGE map of AS-48 linear polypeptide precursor fusion proteins before and after cleavage.
FIG. 3 is a schematic diagram of the separation and purification of AS-48 linear polypeptide precursors.
FIG. 4 is a MALDI-TOF-MS mass spectrometry analysis of AS-48 linear polypeptide precursors.
FIG. 5 is a RP-HPLC analysis of Butelase 1 catalyzed cyclization of AS-48 linear polypeptide precursors.
FIG. 6 is a mass spectrometry chart of MALDI-TOF-MS for Butelase 1 to catalyze cyclization of AS-48 linear polypeptide precursors.
FIG. 7 is a circular dichroism spectrum of a biosynthetic AS-48 and its mutants.
FIG. 8 is a first order sequence diagram of a biosynthetic AS-48 and its mutants.
Detailed Description
The present invention will be described in further detail with reference to examples and drawings, but embodiments of the present invention are not limited thereto.
The various reagents and materials used in the present invention are commercially available or may be prepared by known methods unless otherwise specified.
EXAMPLE 1 construction of expression vectors for expression of AS-48 Linear polypeptide precursor recombinant fusion proteins
Constructing a recombinant vector containing a nucleotide sequence encoding an AS-48 linear polypeptide precursor, comprising the steps of:
(1) According to the amino acid sequence of the AS-48 linear polypeptide precursor (shown AS SEQ ID NO: 1), codon optimization based on an escherichia coli expression system is carried out to obtain a DNA molecule capable of being efficiently expressed in escherichia coli, and the DNA molecule for encoding the AS-48 linear polypeptide precursor is artificially synthesized by utilizing an overlap extension PCR technology, and is specifically shown AS SEQ ID NO: 5.
(2) The DNA molecule encoding the AS-48 linear polypeptide precursor is subjected to homologous recombination with the expression vector pET-28a-MBP or pET-28 a-MBP-SUMO.
The sequence of the synthetic primer is as follows:
AS-48-FP1：5'-gggagaacctgtacttccagGTGGTAGAAGCAGGTGGTTGGG-3'；
AS-48-RP1：5'-tcggatccgtcgacgatatcTCACACGTGATTCAGTACAGTACCC-3'；
AS-48-FP2：5'-agatgctgatgggcggccgcGGGTCGGACTCAGAAGTCAATC-3'；
AS-48-RP2：5'-tcggatccgtcgacgatatcTCACACGTGATTCAGTACAGTACCC-3'；
pET-28a-MBP-FP1：5'-GATATCGTCGACGGATCCGA-3'；
pET-28a-MBP-RP1：5'-CTGGAAGTACAGGTTCTCCCCG-3'；
pET-28a-MBP-SUMO-FP1：5'-GATATCGTCGACGGATCCGA-3'；
pET-28a-MBP-SUMO-RP1：5'-GCGGCCGCCCATCAGCAT-3'；
under the action of high-fidelity 2× PfuMax HiFi PCR ProMix (manufactured by Inz Biotechnology Co., ltd., enzyValley, cat. No. P217) (12.5. Mu.L), an AS-48 linear precursor gene is used AS a template (0.5. Mu.L), AS-48-FP1 (1. Mu.L) is used AS an upstream primer, AS-48-RP1 (1. Mu.L) is used AS a downstream primer, and a proper amount of sterilized water (10. Mu.L) is supplemented to amplify the AS-48 linear precursor gene fragment carrying the homology arm; pET-28a-MBP plasmid is used AS a template (0.5 mu L), pET-28a-MBP-FP1 is used AS an upstream primer (1 mu L), pET-28a-MBP-RP1 (1 mu L) is used AS a downstream primer, a proper amount of sterilizing water (10 mu L) is supplemented, pET-28a-MBP is linearly amplified, a homologous recombination AS-48 linear precursor gene fragment and a linearized pET-28a-MBP vector gene are linearly amplified, and a pET-28a-MBP-AS-48 recombinant expression vector is constructed.
Under the action of high-fidelity 2× PfuMax HiFi PCR ProMix (manufactured by Inz Biotechnology Co., ltd., enzyValley, cat. No. P217) (12.5. Mu.L), an AS-48 linear precursor gene is used AS a template (0.5. Mu.L), AS-48-FP2 (1. Mu.L) is used AS an upstream primer, AS-48-RP2 (1. Mu.L) is used AS a downstream primer, and a proper amount of sterilized water (10. Mu.L) is supplemented to amplify the AS-48 linear precursor gene fragment carrying the homology arm; pET-28a-MBP-SUMO plasmid is used AS a template (0.5 mu L), pET-28a-MBP-SUMO-FP1 is used AS an upstream primer (1 mu L), pET-28a-MBP-SUMO-RP1 (1 mu L) is used AS a downstream primer, a proper amount of sterilized water (10 mu L) is supplemented, pET-28a-MBP-SUMO is linearly amplified, a homologous recombination AS-48 linear precursor gene fragment and a linearized pET-28a-MBP-SUMO vector gene are linearly amplified, and a pET-28a-MBP-SUMO-AS-48 recombinant expression vector is constructed. The recombinant ligation product was transformed into DH 5. Alpha. Competent cells (purchased from Shanghai Weidi Biotech Co., ltd.).
The PCR amplification conditions are specifically as follows: pre-denaturation at 98 ℃ for 30s, denaturation at 98 ℃ for 10s, annealing at 65-55 ℃ for 30s, extension at 72 ℃ for 2-4min, 10 cycles are performed, the annealing temperature is reduced by 1 ℃ in each cycle, then the annealing at 98 ℃ is continued for 10s, annealing at 55 ℃ for 30s, extension at 72 ℃ for 2-4min, the cycle is performed for 30 times, and finally extension at 72 ℃ for 5min is finished.
(3) And (3) picking single colonies for colony PCR identification, sending positive single clones to sequencing company for sequencing verification, and screening positive clone strains. And (3) picking the recombinant plasmid with correct sequence, and transferring the recombinant plasmid into escherichia coli over express C43 (DE 3) for induced expression.
EXAMPLE 2 expression, identification and purification of AS-48 Linear precursor recombinant fusion proteins in E.coli
The recombinant expression vector obtained in example 1 was transformed into host cell E.coli over express C43 (DE 3) (purchased from Shanghai Biotechnology Co., ltd.) to select E.coli positive transformant, inoculated into LB medium containing 100. Mu.g/mL kanamycin, shake-cultured overnight in a shaker at 37℃as seed solution, inoculated into LB medium containing 100. Mu.g/mL kanamycin at a volume ratio of 1:100, and cultured at 37℃under 180r/min to bacterial liquid OD 600 To reach 0.5, IPTG was added to each bottle of the bacterial liquid to a final concentration of 0.4mmol/L, and after induction expression at 37℃for 5 hours, bacterial pellet was collected by centrifugation.
Taking thallus obtained by fermenting the 1L fermentation liquid, adding 10mL buffer A (20 mM NaH) per gram thallus 2 PO 4 -Na 2 HPO 4 100mM NaCl,pH 7.0) re-suspending the cells, and lysing the cells by an ultrasonic cytoclasis machine in an ice bath under the following ultrasonic conditions: the power is 150W, the ultrasonic wave is 4s, the interval is 4s, and the duration is 15min. Centrifuging the crushed bacterial liquid at 12000g/min for 30min at 4 ℃, taking supernatant, and filtering with a 0.22 mu m microporous filter membrane for later use.
Ni ion affinity chromatography purification of AS-48 linear precursor recombinant fusion proteins. The chromatographic column is connected to a column position valve of a rapid protein purification instrument, the system and the column are washed by ultrapure water, then balanced by buffer A, then the sample is loaded by a sample pump, the hybrid protein is washed by the buffer A, and then the hybrid protein is washed by the buffer B (20 mM NaH 2 PO 4 -Na 2 HPO 4 100mM NaCl, 500mM imidazole, pH 7.0). And collecting bacterial liquid before and after IPTG induction, crushing supernatant after centrifugation and purifying target protein for SDS-PAGE analysis, and observing protein expression.
As a result, as shown in FIG. 1, the arrow indicates the target protein expressed, and the protein expressed content is high and all exist in a soluble form. Analyzing the content of the collected target protein, and purifying 1L fermentation liquid to obtain 202.59mg MBP-AS-48 and 175.32mg MBP-SUMO-AS-48 recombinant fusion proteins.
EXAMPLE 3 cleavage, purification and identification of AS-48 Linear precursor recombinant fusion proteins
The recombinant expression vector obtained in example 1 was transformed into host cell E.coli over express C43 (DE 3), E.coli positive transformants were selected, inoculated into LB medium containing 100. Mu.g/mL kanamycin, shake-cultured overnight in a shaker at 37℃as seed solution, inoculated into fresh LB medium containing 75. Mu.g/mL kanamycin in a volume ratio of 1:50, and cultured at 37℃under 200r/min to a bacterial liquid OD 600 When the concentration reaches 0.6, IPTG is added into the bacterial liquid to reach the final concentration of 0.3mmol/L, and after induced expression is carried out for 6 hours at 37 ℃, bacterial precipitate is collected by centrifugation. Adding 10mL of buffer solution A to each gram of thalli to resuspend the thalli, and using an ultrasonic cell disruption instrument to crack the thalli in an ice bath, wherein the ultrasonic conditions are as follows: the power is 120W, the ultrasonic wave is 5s, the interval is 5s, and the duration is 20min. The crushed bacterial liquid is centrifuged for 30min at 12000g/min at 4 ℃, and the supernatant is filtered by a microporous filter membrane of 0.22 mu m. Loading the supernatant into a Ni ion affinity chromatography column which is balanced by buffer A in advance, washing the hybrid protein by the buffer A, and eluting the target protein by the buffer B to prepare the AS-48 linear precursor recombinant fusion protein.
The AS-48 linear precursor recombinant fusion protein in the embodiment 2 is dialyzed overnight in buffer C (50 mM Tis-HCl, 50mM NaCl, 0.5mM EDTA, 1mM DTT, pH 8.0), and then TEV protease or Ulp1 protease is added according to the substrate to enzyme ratio of 50:1 equivalent, and the AS-48 linear polypeptide precursor is obtained after enzyme digestion for 2 hours at 30 ℃, and the enzyme digestion effect is detected by SDS-PAGE protein electrophoresis. As a result, AS shown in FIG. 2, the TEV and Ulp1 proteases can remove the MBP tag and MBP-SUMO tag, respectively, to give an AS-48 linear polypeptide precursor.
The AS-48 linear polypeptide precursor was isolated and purified using the schematic diagram shown in FIG. 3. The digested product was dialyzed into buffer a, then loaded into a Ni ion affinity chromatography column previously equilibrated with buffer a, the permeate was collected and further loaded on to semi-preparative RP-HPLC, at the elution procedure: 0-5min,5% acetonitrile; 5-35min,5-90% acetonitrile; 35-40min,90% acetonitrile; 40-45min,90-5% acetonitrile; and collecting a target product peak under the action of 45-50min and 5% acetonitrile to obtain the AS-48 linear polypeptide precursor with the purity of 95%.
MALDI-TOF-MS was further used to identify the desired product. The results are shown in FIG. 4. The molecular weights of the collected target products are 7786.52Da and 7846.51Da respectively, which are consistent with the theoretical molecular weights 7788.35Da and 7845.40Da, thus indicating that the AS-48 linear polypeptide precursor is successfully prepared. The 1L broth was purified to obtain 19.48mg of AS-48 linear polypeptide precursor (shown AS SEQ ID NO: 1) and 29.97mg of G-AS-48 linear polypeptide precursor mutant (shown AS SEQ ID NO: 10), respectively.
EXAMPLE 4Butelase 1 catalysis of cyclization of AS-48 Linear polypeptide precursor to Cyclic AS-48 and mutants thereof
The recombinant expression vector obtained in example 1 was transformed into host cell E.coli over express C43 (DE 3), E.coli positive transformants were selected, inoculated into LB medium containing 50. Mu.g/mL kanamycin, shake-cultured overnight in a shaker at 37℃as seed solution, inoculated into fresh LB medium containing 50. Mu.g/mL kanamycin at a volume ratio of 1:75, and cultured at 37℃under 180r/min to a bacterial liquid OD 600 When the concentration reaches 0.4, IPTG is added into the bacterial liquid to reach the final concentration of 0.5mmol/L, and after induced expression is carried out for 6 hours at 37 ℃, bacterial precipitate is collected by centrifugation. Adding 10mL of buffer solution A to each gram of thalli to resuspend the thalli, and using an ultrasonic cell disruption instrument to crack the thalli in an ice bath, wherein the ultrasonic conditions are as follows: the power is 180W, the ultrasonic wave is 3s, the interval is 3s, and the duration is 12min. The crushed bacterial liquid is centrifuged for 30min at 12000g/min at 4 ℃, and the supernatant is filtered by a microporous filter membrane of 0.22 mu m. Loading the supernatant into a Ni ion affinity chromatography column which is balanced by buffer A in advance, washing the hybrid protein by the buffer A, and eluting the target protein by the buffer B to prepare the AS-48 linear precursor recombinant fusion protein.
After the recombinant fusion protein of the AS-48 linear precursor in example 2 was dialyzed overnight in buffer C, TEV protease and Ulp1 protease were added in a substrate to enzyme ratio of 100:1 equivalent, and cleaved for 4 hours at 25℃to obtain the AS-48 linear polypeptide precursor. The digested product was dialyzed overnight in buffer a and loaded onto a Ni ion affinity column pre-equilibrated with buffer a, the permeate was collected and loaded onto semi-preparative RP-HPLC, following the elution procedure: 0-5min,5% acetonitrile; 5-35min,5-90% acetonitrile; 35-40min,90% acetonitrile; 40-45min,90-5% acetonitrile; under the action of 45-50min and 5% acetonitrile, AS-48 linear polypeptide precursor with purity up to 95% is obtained, and freeze-dried for later use.
The AS-48 linear polypeptide precursor of example 3 was reconstituted in cyclization buffer D (20 mM NaH 2 PO 4 -Na 2 HPO 4 100mM NaCl, 0.5mM EDTA, 1mM DTT, pH 6.5) and then the cyclase Butelase 1 was added in a substrate to enzyme ratio of 50:1 equivalent and reacted at 37℃for 12 hours. 20. Mu.L of the reaction mixture was analyzed by RP-HPLC. As a result, AS shown in FIG. 5, the AS-48 linear polypeptide precursor chromatographic peak was decreased by the catalytic action of Butelase 1, and a new chromatographic peak appeared after about 4min from the peak position. The yield of cyclized product was calculated by the area of the liquid phase peaks and the yield of the reaction product was calculated to be about 50-60%. To further determine the reaction products, the effluent of the product peak liquid phase was taken and identified by MALDI-TOF-MS analysis. As shown in FIG. 6, the molecular weights of the products are 7205.92Da and 7148.71Da respectively, which are consistent with the theoretical molecular weights 7206.61Da and 7149.56Da, and the peaks of the products are determined to be cyclized products, and the reaction yield is the cyclized yield. By calculation, about 10.28mg of cyclic AS-48 and 19.22mg of cyclic G-AS-48 mutant were prepared from 1L of fermentation broth (FIG. 7).
EXAMPLE 5 structural and functional analysis of biosynthesis of AS-48 and G-AS-48
The recombinant expression vector obtained in example 1 was transformed into host cell E.coli over express C43 (DE 3), E.coli positive transformants were selected, inoculated into LB medium containing 50. Mu.g/mL kanamycin, shake-cultured overnight in a shaker at 37℃as seed solution, inoculated into fresh LB medium containing 50. Mu.g/mL kanamycin at a volume ratio of 1:100, and cultured at 37℃under 200r/min to a bacterial liquid OD 600 When the concentration reaches 0.6, IPTG is added into the bacterial liquid to reach the final concentration of 0.4mmol/L, and after induced expression is carried out for 5 hours at 37 ℃, bacterial precipitate is collected by centrifugation. Adding 10mL of buffer solution A to each gram of thalli to resuspend the thalli, and using an ultrasonic cell disruption instrument to crack the thalli in an ice bath, wherein the ultrasonic conditions are as follows: the power is 160W, the ultrasonic wave is 4s, the interval is 6s, and the duration is 20min. The crushed bacterial liquid is centrifuged for 30min at 12000g/min at 4 ℃, and the supernatant is filtered by a microporous filter membrane of 0.22 mu m. Loading the supernatant to a pre-useIn the Ni ion affinity chromatographic column balanced by the buffer solution A, firstly, washing the hybrid protein by the buffer solution A, and then eluting the target protein by the buffer solution B to prepare the AS-48 linear precursor recombinant fusion protein.
After the recombinant fusion protein of the AS-48 linear precursor in example 2 was dialyzed overnight in buffer C, TEV protease and Ulp1 protease were added in a substrate to enzyme ratio of 75:1 equivalent, and cleaved for 12h at 4℃to obtain the AS-48 linear polypeptide precursor. After dialysis of the cleaved product overnight in buffer a, it was again loaded into a Ni ion affinity column pre-equilibrated with buffer a, the permeate was collected and loaded onto semi-preparative RP-HPLC, following the elution procedure: 0-5min,5% acetonitrile; 5-35min,5-90% acetonitrile; 35-40min,90% acetonitrile; 40-45min,90-5% acetonitrile; under the action of 45-50min and 5% acetonitrile, AS-48 linear polypeptide precursor with purity up to 97% is obtained, and freeze-dried for later use.
The AS-48 linear polypeptide precursor in example 3 is redissolved in cyclization reaction buffer D, and then Butelase 1 is added according to the ratio of substrate to cyclase Butelase 1 of 100:1, and the mixture is reacted for 8 hours at 40 ℃ to prepare the cyclic AS-48 and cyclic G-AS-48 mutant.
After freeze-drying the cyclic AS-48 and G-AS-48 of example 4, a portion of the sample was dissolved in buffer E (20 mM NaH, respectively 2 PO 4 -Na 2 HPO 4 pH 7.2) to a final concentration of 25. Mu.M, followed by CD analysis. The CD test conditions were set as follows: the scanning wavelength range is 190-260nm, the bandwidth is 1nm, the scanning step length is 1nm, the slit width is 0.02nm, the path is 1mm, and the scanning speed is 50nm/min. The polypeptide sample spectrum was baseline corrected with buffer E as a blank and by subtracting the blank spectrum. As a result, AS shown in FIG. 8, both the synthesized cyclic AS-48 and G-AS-48 had typical alpha-helical characteristic spectra, forming distinct negative peaks at 208 and 220nm, indicating that the correct secondary structure had been formed.
After freeze-drying the cyclic AS-48 and G-AS-48 of example 4, a portion of the sample was dissolved in buffer E (20 mM NaH, respectively 2 PO 4 -Na 2 HPO 4 pH 7.2) to a final concentration of 24. Mu.M, and then subjected to an antibacterial activity assay. The results are shown in Table 1, the present inventionThe synthesized AS-48 and G-AS-48 have different degrees of antibacterial activity on escherichia coli ATCC 43895, salmonella ATCC 14028, vibrio parahaemolyticus ATCC 17969, staphylococcus aureus ATCC 25923, listeria ATCC 19118 and enterococcus faecalis ATCC 29212, and the minimum antibacterial concentration is similar to that of the natural extracted AS-48, so that the synthesized cyclic bacteriocin has correct functional activity.
Table 1 bacteriostasis spectra and MIC values of biosynthetic AS-48 and mutants thereof.
EXAMPLE 6 AS-48 Synthesis method according to the present invention compared with the existing Synthesis method
By using the high-density fermentation technology and on the basis of optimizing the culture conditions, 11.16mg of cyclic AS-48 can be separated from 1L of metabolic products of enterococcus faecalis, and the preparation yield is relatively low. The heterologous expression of AS-48 linear mutant by E.coli has also been studied, but the expression yield and antibacterial activity of the finally obtained linear mutant are not ideal. The cyclic AS-48 can be chemically synthesized in a solid phase by a special chemical crosslinking means, but the synthetic route is extremely complex, the synthetic yield is low, and in addition, the high economic cost also makes the large-scale preparation of the chemically synthesized AS-48 difficult.
The invention realizes the full biosynthesis of the annular bacteriocin AS-48 for the first time, and has higher synthesis yield compared with natural extraction and chemical synthesis; meanwhile, compared with the prior escherichia coli recombinant expression, the synthesized annular AS-48 has a more correct secondary structure and stronger antibacterial activity.
EXAMPLE 7 selection of expression vectors
In the course of the research of the present invention, it was attempted to recombinantly express AS-48 linear polypeptide precursors and fusion proteins thereof using 4 expression vectors, such AS pET-28a-SUMO, pET-28a-Trx, pET-31b-SUMO, and pET-28 a.
The constructed pET-28a-AS-48 recombinant vector expresses methionine M+AS-48 linear polypeptide precursor, and the amino acid sequence is shown in SEQ ID NO. 6. The pET-28a-AS-48 recombinant expression vector is transferred into escherichia coli over express C43 (DE 3) for induced expression, and no obvious target protein is produced.
The constructed pET-28a-SUMO-AS-48 and pET-28a-Trx-AS-48 recombinant vectors express SUMO or Trx tag +AS-48 linear polypeptide precursors, and the amino acid sequences are shown AS SEQ ID NO. 7 and SEQ ID NO. 8. The pET-28a-SUMO-AS-48 and pET-28a-Trx-AS-48 recombinant expression vectors are transferred into E.coli over express C43 (DE 3) for expression, and AS a result, only a very small amount of fusion protein is expressed.
The constructed pET-31b-SUMO-AS-48 recombinant expression vector expresses a KSI-SUMO label+AS-48 linear polypeptide precursor, and the amino acid sequence is shown AS SEQ ID NO. 9. The pET-31b-SUMO-AS-48 recombinant expression vector is transferred into E.coli over express C43 (DE 3) for expression. The results show that the target protein is expressed at high level, but exists in the form of inclusion bodies, and the soluble AS-48 linear polypeptide precursor fusion protein can be obtained after complex denaturation and renaturation operation.
The constructed pET-28a-MBP-AS-48 and pET-28a-MBP-SUMO-AS-48 recombinant expression vectors, the expressed recombinant protein is MBP or MBP-SUMO label +AS-48 linear polypeptide precursor, and the amino acid sequences are shown AS SEQ ID NO. 3 and SEQ ID NO. 4. The pET-28a-MBP-AS-48 or pET-28a-MBP-SUMO-AS-48 recombinant expression vector is transferred into E.coli over express C43 (DE 3) to induce expression. The result shows that the target protein has high expression yield and is soluble expression, and the cyclic AS-48 and the mutant thereof can be obtained after purification, enzyme digestion and cyclization.
In summary, through screening studies of various expression vectors, the present invention preferably employs an expression vector containing MBP or MBP-SUMO tag.
EXAMPLE 8 screening of expression Strain
During the course of the present invention, attempts were made to express AS-48 linear polypeptide precursor recombinant fusion proteins using E.coli BL21 (DE 3), E.coli ROSETTA (DE 3), E.coli Shuffle T7 and E.coli Overexpress C43 (DE 3) expression strains.
The recombinant vector obtained in example 1 was transformed into host cells such as E.coli BL21 (DE 3), ROSETTA (DE 3) and SHuffle T7, and E.coli positive transformants were selected for induction expression, but the cells were often unable to grow normally, even were lysed, etc. during the seed liquid expansion culture and induction expression. After the escherichia coli expression host is replaced by escherichia coli over express C43 (DE 3), the growth and the expression of host cells are normal, and abnormal conditions such as obvious cell death and the like are avoided.
In conclusion, the preferred expression strain was E.coli over express C43 (DE 3) after screening studies of the expression strain.
Example 9 molecular engineering optimization
In the course of the present invention, 5 mutation schemes have been tried.
Initial attempts to express MBP/MBP-SUMO-AS-48 fusion proteins with the C-terminal HV dipeptide have failed to achieve the desired expression levels. For this purpose, the present invention attempted to incorporate further 5 different amino acid motifs, K, KK, KKK, HHHHHH and KKKHHH, etc., at the C-terminal end of HV. The results of the expression induced according to the expression method in example 2 show that the MBP/MBP-SUMO-AS-48 fusion protein with KKKK at the C-terminus has a higher expression level. While HVKKK is preferred AS the C-terminal amino acid motif of MBP/MBP-SUMO-AS-48 fusion proteins in the present invention based on the beneficial effect of the C-terminal KKK tripeptide motif on Butelase 1 catalytic cyclization.
In summary, the C-terminal of the molecular engineered AS-48 is the HVKKK motif, AS a preferred embodiment.
The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.

Claims (10)

1. A circular bacteriocin AS-48 linear polypeptide precursor characterized by: has the amino acid sequence shown in SEQ ID NO. 1.
2. A DNA molecule encoding the linear polypeptide precursor of claim 1, characterized in that: has the nucleotide sequence shown in SEQ ID NO. 5.
3. A circular bacteriocin AS-48 linear polypeptide precursor fusion protein, characterized in that: has the amino acid sequence shown in SEQ ID NO. 3 or SEQ ID NO. 4.
4. A method for preparing a circular bacteriocin AS-48 linear polypeptide precursor fusion protein according to claim 3, wherein: the preparation method comprises the following preparation steps: cloning the nucleotide sequence encoding the AS-48 linear precursor into a prokaryotic expression plasmid to construct a recombinant expression vector; then the recombinant expression vector is transformed into a prokaryotic expression system for induced expression, bacterial precipitation is collected, and the AS-48 linear polypeptide precursor recombinant fusion protein is obtained after crushing and purification; the prokaryotic expression plasmid is an expression vector containing an MBP label or an MBP-SUMO label.
5. The method for preparing a circular bacteriocin AS-48 linear polypeptide precursor fusion protein according to claim 4, wherein:
the expression vector containing the MBP tag is pET-28a-MBP; the expression vector containing the MBP-SUMO tag is pET-28a-MBP-SUMO;
the prokaryotic expression system is an escherichia coli expression system;
the specific operation steps of the induced expression are as follows: inoculating positive transformant into LB culture solution for culturing, and culturing when bacterial solution OD 600 When reaching 0.4 to 0.6, adding IPTG with the final concentration of 0.2 to 0.4mmol/L, and inducing for 6 to 20 hours at the temperature of 18 to 37 ℃; the LB culture solution contains 50-100 mug/mL kanamycin;
the steps of crushing and purifying are as follows: after the bacterial cells are crushed, centrifugally collecting supernatant components, purifying by metal ion affinity chromatography, and eluting to obtain AS-48 linear precursor recombinant fusion protein;
the method for crushing the bacterial cells comprises the following steps: suspending the strain in buffer A, and performing cell lysis by ice bath ultrasonic disruption; the formula of the buffer A is 20+/-5 mM NaH 2 PO 4 -Na 2 HPO 4 、100±20mM NaCl，pH＝7；
The metal ion affinity chromatography is purified into Ni ion affinity chromatography;
the elution process is to equilibrate with buffer A and then to elute with buffer B; the formula of the buffer A is 20mM NaH 2 PO 4 -Na 2 HPO 4 100mm nacl, ph=7; the formula of the buffer B is as follows: 20mM NaH 2 PO 4 -Na 2 HPO 4 100mM NaCl, 500mM imidazole, pH=7.
6. A method for preparing a precursor cyclic bacteriocin AS-48 linear polypeptide AS defined in claim 1, wherein: all steps in the preparation method of the circular bacteriocin AS-48 linear polypeptide precursor fusion protein AS claimed in claim 4 or 5 and the following preparation steps are included: and (3) performing first dialysis on the linear polypeptide precursor recombinant fusion protein under a buffer system, then performing premixed enzyme digestion with protease, further performing second dialysis under the buffer system, separating enzyme digestion products by utilizing metal ion affinity chromatography and RP-HPLC, and collecting AS-48 linear polypeptide precursors after freeze drying.
7. The method for preparing a circular bacteriocin AS-48 linear polypeptide precursor according to claim 6, wherein:
the buffer system for the first dialysis is buffer C, and the formula of the buffer C is as follows: 50+ -10 mM Tis-HCl, 50+ -10 mM NaCl, 0.5+ -0.1 mM EDTA, 1+ -0.5 mM DTT, pH 8.0;
the buffer system for the second dialysis is buffer A, and the formula of the buffer A is as follows: 20+ -5 mM NaH 2 PO 4 -Na 2 HPO 4 、100±20mM NaCl，pH＝7；
When the N-terminal fusion of the annular bacteriocin AS-48 linear polypeptide precursor fusion protein is MBP label, the protease is TEV; when the N-terminal fusion of the annular bacteriocin AS-48 linear polypeptide precursor fusion protein is MBP-SUMO label, the protease is Ulp1;
in the enzyme digestion system, the equivalent ratio of the substrate to the protease is 50:1-100:1;
the enzyme digestion is carried out under buffer solution A at the temperature of 4-30 ℃ for 1-12 hours;
the metal ion affinity chromatography is preferably Ni ion affinity chromatography;
the buffer solution adopted by the Ni ion affinity chromatography is a buffer solution A and a buffer solution B;
the RP-HPLC separation procedure was: 0-5min,5% acetonitrile; 5-35min,5-90% acetonitrile; 35-40min,90% acetonitrile; 40-45min,90-5% acetonitrile; 45-50min,5% acetonitrile.
8. A full biosynthesis method of a cyclic bacteriocin AS-48, which is characterized by comprising the following steps: all steps in the preparation method of the circular bacteriocin AS-48 linear polypeptide precursor AS claimed in claim 6 or 7 and the following preparation steps: and re-dissolving the collected AS-48 linear polypeptide precursor in a cyclization buffer solution, and adding cyclase to catalyze the end-to-end cyclization of the AS-48 linear polypeptide precursor, so that the cyclized product is the cyclic bacteriocin AS-48.
9. The method of total biosynthesis of cyclic bacteriocin AS-48 according to claim 6 wherein:
the cyclase is cyclase Butelase 1;
the cyclization buffer solution is buffer solution D; the formula of the buffer solution D is as follows: 20+ -5 mM NaH 2 PO 4 -Na 2 HPO 4 、100±20mM NaCl、0.5±0.1mM EDTA、1±0.5mM DTT，pH 6.5；
The equivalent ratio of the AS-48 linear polypeptide precursor to the cyclase is 50:1-100:1;
the cyclization reaction temperature is 37-45 ℃, the reaction time is 6-12 h, and the pH is 6.0-6.5.
10. A cyclic bacteriocin AS-48 and mutants thereof, characterized in that: has an amino acid sequence shown in SEQ ID NO. 2 or SEQ ID NO. 11, wherein the amino group of the N-terminal amino acid residue and the carboxyl group of the C-terminal amino acid residue are connected in a peptide bond form to form a cyclic skeleton.
CN202311055350.3A 2023-08-21 2023-08-21 Total biosynthesis method of cyclic bacteriocin AS-48 Pending CN117088946A (en)

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