CN112458106A

CN112458106A - Preparation method of multi-copy golden pomfret delicious peptide, expression vector and recombinant bacteria

Info

Publication number: CN112458106A
Application number: CN202011406616.0A
Authority: CN
Inventors: 林洪; 邓小飞; 郭晓华; 隋建新; 董浩
Original assignee: Shandong Meijia Group Co ltd; Ocean University of China
Current assignee: Shandong Meijia Group Co ltd; Ocean University of China
Priority date: 2020-12-04
Filing date: 2020-12-04
Publication date: 2021-03-09

Abstract

The invention discloses a preparation method of multi-copy golden pomfret delicious peptide, which is characterized by comprising the following steps: 1) designing and synthesizing multi-copy series-connected umami peptide genome and amplifying; 2) connecting the amplified umami peptide genome to a plasmid with a His-sumo tag to obtain a recombinant expression vector; 3) transforming the expression vector into host bacteria, screening, and inducing expression; 4) obtaining the delicious peptide with His-sumo label after primary purification; 5) and (3) cracking the umami peptide with the His-sumo label to remove the His-sumo label, and carrying out secondary purification to obtain the umami peptide. The invention also provides an expression vector and a recombinant bacterium containing the umami peptide genome and application thereof. The umami peptide prepared by the method is quick, simple and convenient, has high cloning efficiency, and can quickly and effectively remove the influence of the tag sequence on the umami peptide.

Description

Preparation method of multi-copy golden pomfret delicious peptide, expression vector and recombinant bacteria

The technical field is as follows:

the invention relates to the technical field of food additives, and particularly relates to a preparation method of multi-copy golden pomfret delicious peptide, an expression vector and recombinant bacteria.

Background art:

umami is one of five basic tastes that can create a pleasant taste experience. The delicious peptide is a novel peptide flavor enhancer after flavor enhancers such as sodium glutamate, guanylic acid, inosinic acid and the like, not only has delicious taste, but also can provide amino acid nutrient substances, and the delicious peptide has great market potential in the field of seasonings.

The mass production of the delicious peptide is the basis of the wide application of the delicious peptide in the field of seasonings. In recent years, the preparation of small molecular umami peptide by adopting a recombinant cloning expression method is gradually developed, most of the research on the microbial expression of the umami peptide is based on BMP, the related reports of other umami peptide are very limited, the field is still in the starting stage, the technology mainly clones the gene of the foreign protein into a proper vector, and the foreign protein is transformed into a proper host bacterium, and the high-efficiency expression of the foreign protein is finally realized by utilizing the high-efficiency multiplication capacity of the host bacterium.

Wang Yan Liang Lian et al adopts secretory expression plasmid pPIC9 as a vector, and integrates multiple copies of BMP gene on Pichia pastoris GS115 host bacteria chromosome in series for fusion and secretion expression, firstly, obtains 4 copies of BMP gene by adopting a method of artificially synthesizing the gene, and connects 6 continuous histidines (6 × His) in series at the tail part of the 4 copies of BMP gene so as to separate and purify the expressed target protein by using metal chelate and chromatographic columns, and then constructs Pichia pastoris engineering strains expressing the 4 copies, 8 copies, 12 copies and 16 copies of BMP gene respectively on the basis, but 6 × His tag is introduced into C-end; according to the analysis of the structures of the flavor-developing peptides GD, AD and VD, specific sites of formic acid and trypsin are introduced, a monomer in series with the flavor-developing peptides is designed, 8 copies of genes of the three peptides are synthesized by a splicing method, the three peptides are cloned and expressed to an expression vector pET-30a and are transformed into a host bacterium BL21(DE3), a target gene is successfully expressed through IPTG induction, then enzyme digestion is carried out, and the flavor-developing peptide monomers are obtained through anion exchange chromatography and purification according to charge difference, so that the whole process is still complex and difficult to purify. At present, the technology has the defects of difficult expression, low efficiency, difficult recovery and the like, and often introduces a specific affinity tag at the C-terminal in the expression of the small molecule peptide, and the tag sequence such as 6 XHis tag and the like can not be effectively removed, thereby causing the inevitable negative influence on the taste of the delicious peptide.

The invention content is as follows:

aiming at the defects in the prior art, the application provides a novel seamless cloning expression system of the delicious peptide, His-sumo is selected as a label, 16 copies of the delicious peptide WDDMEK are successfully expressed in a series connection mode, the label is efficiently cut off from the fusion protein by utilizing specific proteolytic enzyme, a target peptide segment can be obtained by removing a label protein part through affinity chromatography, and the fermentation control conditions of the target peptide segment are explored.

In order to achieve the purpose, the invention provides the following technical scheme:

a method for preparing multi-copy golden pomfret delicious peptide comprises the following steps:

1) designing and synthesizing multi-copy tandem golden pomfret delicious peptide genome and amplifying;

2) connecting the amplified umami peptide genome to a plasmid with a His-sumo tag to obtain a recombinant expression vector;

3) transforming the expression vector into host bacteria, screening, and inducing expression;

4) obtaining the delicious peptide with His-sumo label after primary purification;

5) and (3) cracking the umami peptide with the His-sumo label to remove the His-sumo label, and carrying out secondary purification to obtain the umami peptide.

In one embodiment according to the invention, the umami peptide gene copy number in the umami peptide genome is greater than 10; preferably 16.

In one embodiment according to the invention, the sequence of the umami peptide encoded by the umami peptide gene is SEQ ID NO 1MGHHHHHHMSDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKI KKTTPLRRLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIGGWDDMEKWDDMEKWDDMEKWDDMEKWDDMEKWDDMEKWDDMEKWDDMEKWDDMEKWDDMEKWDDMEKWDDMEKWDDMEKWDDMEKWDDMEKWDDMEK.

In one embodiment according to the invention, the umami peptide genomic sequence is SEQ ID NO 2 atgggtcaccatcaccatcaccatatgtcggactcagaagtcaatcaagaagctaagccagaggtcaagccagaagtcaagcctgagactcacatcaatttaaaggtgtccgatggatcttcagagatcttcttcaagatcaaaaagaccactcctttaagaaggctgatggaagcgttcgctaaaagacagggtaaggaaatggactccttaagattcttgtacgacggtattagaattcaagctgatcagacccctgaagatttggacatggaggataacgatattattgaggctcacagagaacagattggtggcTGGGACGATATGGAAAAATGGGATGACATGGAAAAGTGGGACGACATGGAAAAATGGGACGATATGGAAAAGTGGGATGATATGGAAAAATGGGACGATATGGAAAAATGGGACGACATGGAAAAATGGGATGATATGGAAAAATGGGATGACATGGAAAAATGGGACGACATGGAAAAGTGGGATGACATGGAAAAATGGGATGATATGGAAAAGTGGGACGATATGGAAAAGTGGGATGACATGGAAAAGTGGGACGATATGGAAAAATGGGATGATATGGAAAAATAACTCGAG.

In one embodiment according to the invention, the 5' end of the primer contains a plasmid homology arm sequence during amplification.

In one embodiment according to the invention, the plasmid is pATX-sumo; the host bacteria are selected from BL21(DE3) or BL21(DE3) pLys.

In one embodiment according to the invention, the lysis is followed by dialysis and then by removal of His-tag bearing proteins with Ni resin.

The invention also provides an expression plasmid for expressing the umami peptide, wherein the plasmid contains a His-sumo label and a multicopy serial umami peptide genome; preferably, the copy number of the umami peptide gene in the umami peptide genome is more than 10, preferably 16; preferably, the umami peptide genomic sequence is SEQ ID NO 2.

The invention also provides a recombinant bacterium for expressing the umami peptide, which comprises the expression plasmid; preferably, the host bacterium of the recombinant bacterium is BL21(DE 3).

The invention further provides application of the expression plasmid or the recombinant bacterium in preparation of the delicious peptide.

The preparation method of the delicious peptide provided by the invention has the following beneficial effects:

the preparation method of the delicious peptide provided by the invention is quick, simple and convenient, and has high cloning efficiency; the methods of the present application can be used for the high efficiency clonal expression of other novel taste-conferring peptides.

Description of the drawings:

FIG. 1 is a flow chart of recombinant expression of 16 copies of umami peptide WDDMEK;

FIG. 2 is a graph showing the results of detection of 16WDDMEK expressed by two different recombinant bacteria;

FIG. 3 is a graph showing the results of the target protein expression test;

FIG. 4 is a graph showing the results of the tag removal of a target protein;

FIG. 5 is a graph showing the results of tag-removal purification of a target protein;

FIG. 6(a) is a graph showing sensory evaluation results of 16WDDMEK purified peptides.

FIG. 6(b) is a graph showing the results of the analysis of the major components of the 16WDDMEK purified solution.

The specific implementation mode is as follows:

the following detailed description of the preferred embodiments of the present invention, taken in conjunction with the accompanying drawings, will make the advantages and features of the invention more readily understood by those skilled in the art, and thus will more clearly and distinctly define the scope of the invention.

Example 1: construction examples of vectors

1.1 amplification of the Gene sequence of interest

Obtaining the target gene sequence of the umami peptide through an NCBI database, designing and synthesizing enzyme cutting sites and primers by using Primer Premier 5.0 software according to the sequences of a carrier and the target gene, and cloning the target gene by using the obtained cDNA or a plasmid containing a target fragment as a template.

The gene sequence SEQ ID NO: 2:

atgggtcaccatcaccatcaccatatgtcggactcagaagtcaatcaagaagctaagccagaggtcaagccagaagtcaagcctgagactcacatcaatttaaaggtgtccgatggatcttcagagatcttcttcaagatcaaaaagaccactcctttaagaaggctgatggaagcgttcgctaaaagacagggtaaggaaatggactccttaagattcttgtacgacggtattagaattcaagctgatcagacccctgaagatttggacatggaggataacgatattattgaggctcacagagaacagattggtggcTGGGACGATATGGAAAAATGGGATGACATGGAAAAGTGGGACGACATGGAAAAATGGGACGATATGGAAAAGTGGGATGATATGGAAAAATGGGACGATATGGAAAAATGGGACGACATGGAAAAATGGGATGATATGGAAAAATGGGATGACATGGAAAAATGGGACGACATGGAAAAGTGGGATGACATGGAAAAATGGGATGATATGGAAAAGTGGGACGATATGGAAAAGTGGGATGACATGGAAAAGTGGGACGATATGGAAAAATGGGATGATATGGAAAAATAACTCGAG

primer:

the PCR reaction system is as follows:

the PCR reaction procedure was as follows:

1.2 cleavage, ligation, transformation

The vector and the target fragment are subjected to double enzyme digestion and reacted for 1h at 37 ℃. And (4) recovering the enzyme digestion product, then performing recombination connection, and reacting at 25 ℃.

The corresponding reaction system is as follows:

the ligation product was converted to DH5 α, plated and incubated overnight at 37 ℃. The method comprises the following specific steps:

(1) adding 10 μ L of ligation product into 50 μ L of Escherichia coli competence, gently mixing, and ice-cooling for 30 min;

(2) placing the transformed bacteria liquid in a water bath kettle at 42 ℃, thermally shocking for 90s, quickly taking out and placing on ice for 3-5 min;

(3) adding 800 μ L liquid LB culture medium, and recovering by shaking table at 37 deg.C and 150rpm for 40-50 min;

(4) centrifuging at 3000rpm for 5min at room temperature, leaving 100 μ L of supernatant, and resuspending the bacteria;

(5) transferring the thallus to an LB solid plate containing 50mg/L Kan, uniformly coating the thallus by using a sterilized coating rod, and inversely placing the thallus in a thermostat at 37 ℃ for overnight culture after the thallus is sucked dry.

1.3 colony PCR identification and sequencing

3-5 clones are selected for colony PCR identification, and two positive clones are selected for sequencing.

The PCR reaction system is as follows:

the PCR reaction procedure was as follows:

and (3) carrying out agarose gel electrophoresis on the PCR product, carrying out gel cutting recovery on the obtained positive band containing the size of the target fragment, and carrying out sequencing detection.

Example 2: bacterial activation and induced expression, expression test

1. Single colonies containing the recombinant plasmid were picked from the plate, inoculated into 5mL of LB medium containing kanamycin antibiotic (50ug/mL), and cultured overnight at 37 ℃.

2. 200. mu.L of the suspension was inoculated into 20mL of LB medium containing resistance and cultured at 37 ℃ until OD600 became about 0.6.

3. Expression testing was performed for both strains: BL21(DE3), BL21(DE3) pLys. Two strains of 1mL uninduced bacteria were aspirated into sterilized 2mL centrifuge tubes to serve as uninduced controls. Culturing the residual bacterial liquid until OD600 is 0.5-0.6, adding IPTG (isopropyl-beta-D-thiogalactoside) to a final concentration of 1mM, and culturing the bacterial liquid added with IPTG and an uninduced control according to the following different conditions: the cells were incubated at 16 ℃ overnight and at 37 ℃ for 4 hours.

4. Samples of different expression conditions were collected, centrifuged at 12,000rpm for 1min to collect the cells. The cells were suspended in 200ul PBS and disrupted by sonication for 2s at 2s intervals for a total of 4 min. The supernatant (native) and pellet (denated) were centrifuged at 12,000rpm for 2min and the pellet was dissolved in 8Murea + PBS. Expression was verified by running 12ul samples on SDS-PAGE (12%, 80v gel concentrate, 20min, 120v gel isolate, 45 min.) separately. Marker (brand: SMOBIO, PM2500), loading buffer (self-contained, formulation as follows)

The results of the 16WDDMEK assay are shown in fig. 2, a correctly verified recombinant strain (e.coli bl21(DE3)/(DE3) pLys) pATX-sumo-16WDDMEK strain was inoculated into 20mL LB broth containing Amp resistance selection at 200 μ L inoculum size, two strain expression tests were performed when OD600 was 0.6, the fermentation broth was subjected to centrifugation, ultrasonication, centrifugation at 12,000rpm to separate supernatant (native) and precipitate (denatured), NPE and DPE obtained from the control and different strains under two different culture conditions without induction were subjected to SDS-PAGE protein electrophoresis to detect the expression of 16WDDMEK, and as shown in fig. 2, the non-induced bacterial broth was used as a control and had no band at the target molecular weight, and the supernatant had a band at the theoretical molecular weight 35 of the target umami polypeptide 16 kDa under two different fermentation conditions of BL21(DE3), sequencing results prove that the umami peptide is successfully expressed, and the band in the sediment is shallow; in addition, under two different fermentation conditions of BL21(DE3) pLys, no band is generated at the target molecular weight in both supernatant and precipitate, and the expression effect is not ideal; further, Western blot verification analysis is carried out by using an anti-His-tag antibody, and the blank takes histidine as a control, and the supernatant and the precipitate of BL21(DE3) have binding effects at the target molecular weight, and the supernatant has the best effect, so that the target peptide segment is proved to be expressed, and the result of BL21(DE3) pLys has no any binding mark at the target band, has some hybrid bands and fails to express; combining the above experimental results, the optimal induced expression conditions of 16WDDMEK were determined as follows: BL21,1mM IPTG, treated at 37 ℃ for 4 h.

Example 3: 1000ml amplification and purification

1. Sample treatment: selecting the optimal induction expression conditions: the 16WDDMEK protein was treated with expression strain BL21,1mM IPTG for 4h at 37 ℃. Culturing 1000ml of thallus, culturing for a certain time, adding IPTG (isopropyl-beta-D-thiogalactoside) to a concentration of 1mM for induction expression when OD600 is detected to be 0.8, performing induction expression on the 16WDDMEK protein at 37 ℃ for 4 hours, centrifugally collecting the thallus after induction expression (when the thallus is collected), adding 10ml of PBS buffer solution, performing ultrasonic treatment for 2s, separating for 2s, and totally 30 min. The supernatant was collected after centrifugation.

2. Preparing a chromatography column

The resin was packed into a suitable column, with an upper gasket over the resin.

3. Resin cleaning: according to the resin dosage, 10 times of resin volume binding buffer is selected for cleaning.

Binding buffer:PBS pH 7.5,10％Glycerol

4. Loading: the collected supernatant sample was passed through the column, and this process was not accelerated. The speed must be controlled if acceleration is required: one drop for 1-2 seconds.

5. Leaching: the binding solution must be allowed to drop down by gravity itself, and the speed (one drop in 1-2 seconds) must be controlled to strictly prevent the drop from forming a line.

W1: washing 10ml with binding buffer (pH:7.5)

W2: binding buffer (pH:7.5) containing 10mM imidazole 5ml was rinsed

W3: binding buffer (pH:7.5) containing 30mM imidazole 5ml was rinsed

6. And (3) elution: inhibiting acceleration

Binding buffer (pH:7.5) containing 300mM imidazole 0.5ml × 9

Each eluted sample was run on SDS-PAGE gels (10 ul for IN and FT and 20ul for the other). The results are shown IN FIG. 3, IN: an infusion solution; FT: flowing through the liquid; W1-W3: a washing section; E1-E9: an elution portion; after repeated impurity washing, obvious target band expression is observed at the 35kDa molecular weight in the eluent, and the eluent of each part is collected and mixed for subsequent Western blot, sensory evaluation and electronic tongue verification.

7. Delabelling lysis assay

Lysis buffer: 50mM Tris, pH 8.0150mM NaCl;

and (3) cracking conditions: incubation at 4 ℃ overnight with gentle rotation;

after lysis and dialysis overnight, the protein with His-tag was removed by Ni resin, and as shown in FIG. 4, the fusion protein was subjected to ultrasonic lysis, and the target protein appeared untagged at about 15 kDa.

8. Endotoxin removal test experiment

Endotoxin removal was performed by a standard Triton X114 phase distribution procedure as follows:

add Triton X114 to a final concentration of 1%;

continuously and gently stirring at 4 ℃ for 30 min;

incubating at 37 ℃ for 10 min;

centrifuging at 2000g for 10min at 25 deg.C;

recovering the upper aqueous phase (containing the target protein);

determining endotoxin levels according to IFU using a chromogenic LAL endotoxin assay kit;

9. desalting and replacing buffer

The purified sample was placed in dialysis bags and filled into displacement buffer (pbs) for 4 degrees dialysis overnight to substantially remove imidazole.

10. Concentration:

the dialyzed protein sample was loaded into a 10kDa ultrafiltration tube (Millipore) and centrifuged at 4,000rpm for 30 min. The filtrate was discarded and the appropriate volume of TBS was added to continue the concentration of protein by centrifugation. After three repetitions, the protein was concentrated to the appropriate concentration.

The retained final protein samples were aliquoted and the retained samples (2ug) were run on SDS-PAGE for detection. The detection result is shown in FIG. 5, after the target protein without the label appears at about 15kDa after the target protein is cracked, purified and concentrated.

Example 4: sensory evaluation and electronic tongue analysis of 16WDDMEK

(1) Preparing a standard solution:

0.08% of citric acid solution, 1% of sucrose solution, 0.08% of quinine solution, 0.35% of salt solution and 0.35% of monosodium glutamate solution are prepared respectively, and the five solutions are used as evaluation standards of sour taste, sweet taste, bitter taste, salty taste and delicate flavor respectively.

(2) Sensory evaluation:

8 panelists (4 for each of men and women) were recruited to form a sensory panel, and were trained with 5 taste standard solutions in order to ensure that these panelists could recognize the respective sensory characteristics. The sensory evaluation adopts a scoring inspection method, the taste development intensity is evaluated according to the standard of 0-10 points, wherein standard liquids prepared according to the specification are uniformly divided into 5 points, and five taste development attributes (sour, sweet, bitter, salty and delicate) of the sample are scored;

electronic tongue principal component analysis: the main component analysis experiment of the sample was also performed with the above-mentioned standard solution.

As shown in fig. 6(a) and fig. 6(b), it can be seen from fig. 6 that the umami attribute score of the 16WDDMEK dialysate is slightly higher than the standard solution taste intensity, indicating that the 16WDDMEK has a distinct umami taste (score 5.313); the dialysate has a slightly lower salty taste attribute score than that of the salt standard solution, but can also have a relatively obvious feeling (score is 4.25 points). In addition, the 16WDDMEK dialysate was substantially non-bitter and sweet, slightly sour. The successful analysis of the principal components of the electronic tongue in conjunction with fig. 6(b) revealed the taste characteristics of the purified liquid, with a Discrimination (DI) value of 99.86%, the closer the distance between the two samples in the PCA plan, the closer their taste, and it can be readily seen that the purified liquid was closest to the monosodium glutamate standard, indicating a predominantly umami taste. In conclusion, sensory evaluation and electronic tongue main component analysis verify that the recombinant bacterium E.coli BL21/pATX-sumo-16WDDMEK expressed serial sixteen peptide has obvious delicate flavor, does not have obvious bad peculiar smell, has the potential of further developing into healthy and safe delicate flavor seasoning products, and can neutralize and harmonize comprehensive flavor in subsequent delicate flavor peptide seasoning compounding for obvious salty taste.

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention.

Sequence listing

<110> Shandong Meijia group Co., Ltd, China university of oceans

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Claims

1. A preparation method of multi-copy golden pomfret delicious peptide is characterized by comprising the following steps:

1) designing and synthesizing multi-copy series-connected umami peptide genome and amplifying;

2. The method of claim 1, wherein the umami peptide gene copy number in the umami peptide genome is greater than 10; preferably 16.

3. The method of claim 1, wherein the sequence of the umami peptide encoded by the umami peptide gene is SEQ ID NO. 1.

4. The method of claim 3, wherein the umami peptide genomic sequence is SEQ ID NO 2.

5. The method of claim 1, wherein the 5' end of the primer contains a sequence of a plasmid homology arm during amplification.

6. The method according to claim 1, wherein the plasmid is pATX-sumo; the host bacteria are selected from BL21(DE3) or BL21(DE3) pLys.

7. The method according to claim 1, wherein the cleavage is followed by dialysis and then by removal of His-tag-bearing proteins with Ni resin.

8. An expression plasmid for expressing an umami peptide, which is characterized by comprising a His-sumo label and a multicopy tandem umami peptide genome; preferably, the copy number of the umami peptide gene in the umami peptide genome is more than 10, preferably 16; preferably, the umami peptide genomic sequence is SEQ ID NO 2.

9. A recombinant bacterium for expressing umami peptide, wherein the recombinant bacterium comprises the expression plasmid of claim 8; preferably, the host bacterium of the recombinant bacterium is BL21(DE 3).

10. Use of the expression plasmid of claim 8 or the recombinant bacterium of claim 9 for the preparation of an umami peptide.