Method for high-efficiency recombinant expression of sweet protein brazzein and application of sweet protein brazzein

Abstract

The invention discloses a method for efficient recombinant expression of Brazzein sweet protein and application thereof, and belongs to the technical field of genetic engineering and recombinant microorganisms. The preparation method comprises the following steps: by taking pET-28a or pMAL-c6t as a carrier and Escherichia coli SHuffle T7 as a host, carrying out induced expression on an encoding gene of a sweet protein Brazzzein, and purifying, so as to obtain a fusion protein Brazzzein-His or His-MBP-Brazzzein; and the His-MBP-Brazzein is subjected to TEV protease cutting, and the cut His-MBP-Brazzein is purified to obtain the sweet protein mutant G-Brazzein. The sweetness of the obtained sweet protein mutant G-Bragg zein exceeds 1500 times of that of cane sugar, the sweet protein mutant G-Bragg zein can still taste obvious sweetness under the concentration of 6.6 mu g / mL, and the sweetness is pure; the sweetness of the Bragg zein-His is 467 times that of cane sugar, the lasting time of the sweetness is as long as 12s, and additional enzyme digestion is not needed in the preparation process. The obtained brazzein sweet protein can be used for improving coffee fragrance and reducing the sugar consumption.

Description

Technical Field

[0001] This invention relates to a method for efficient recombinant expression of Brazzein sweet protein and its application, belonging to the fields of genetic engineering and recombinant microbial technology. Background Technology

[0002] Excessive sugar intake has become one of the major challenges to human health worldwide, leading to increasingly prominent problems such as obesity, overweight, and diabetes. With rising health awareness, low-sugar, reduced-sugar, and sugar substitutes have become common goals for consumers and industry. Against this backdrop, plant-based sweet proteins, as protein-based sweeteners derived from natural plants, possess extremely high sweetness and good safety profiles, are receiving increasing attention. These sweet proteins produce a sweet sensation by activating the human sweet taste receptors T1R2 / T1R3, and their sweetness can typically be hundreds to thousands of times greater than that of sucrose. Compared to traditional small-molecule artificial sweeteners, sweet proteins are characterized by not participating in human metabolism, extremely low calories, and a lower risk of causing tooth decay, obesity, and diabetes, thus better meeting the current global demand for healthy, safe, and low-calorie natural sweeteners.

[0003] Brazzein is a typical example of plant-based sweet proteins, extracted from wild plants in West Africa. Pentadiplandra brazzeana Baillon The fruit of the plant. It is a single-chain polypeptide composed of 54 amino acid residues, containing 8 cysteine ​​residues forming 4 pairs of intramolecular disulfide bonds, with a relative molecular mass of approximately 6500 and an isoelectric point of 5. Brazil glycoprotein is approximately 2000 times sweeter than an equal mass of sucrose, and among many sweet proteins, it has the advantages of the smallest molecular weight and best water solubility. Furthermore, it retains its sweetness even after heating at 80°C for 4 hours, demonstrating good thermal and pH stability. As a natural sweetener, Brazil glycoprotein not only possesses the advantages of high sweetness, low calories, and high safety, but it can also improve bitterness, reduce astringency, and prolong the duration of sweetness. Therefore, it can be used as a high-quality raw material for food flavoring, and can be compounded with other sweeteners to enhance the overall flavor, showing broad application prospects, especially suitable for consumers who need to reduce or control sugar intake.

[0004] However, the production of brazzein currently faces numerous limitations. Traditional methods primarily rely on extraction from plant fruits, which suffers from low extraction efficiency, limited production areas, and significant environmental impact. In contrast, microbial fermentation is considered a greener, more economical, and sustainable production approach. However, this technology still faces challenges in practical application: for example, in prokaryotic expression systems (such as *E. coli*), due to the lack of eukaryotic post-translational modification capabilities, the multiple disulfide bonds of brazzein are difficult to fold correctly, easily forming inactive inclusion bodies. Even with in vitro refolding, there are still problems of low efficiency, high cost, and low sweetness (CN 119431606 A). Furthermore, in eukaryotic expression systems such as *Pichia pastoris*, although the secretory expression of brazzein can be achieved, the sweetness of the obtained product is still far from ideal (only about 40 times the sweetness of sucrose) due to incorrect protein folding. Therefore, developing an efficient, simple microbial fermentation process that can ensure the correct structural folding and sweetness of brazzein is a key research direction for realizing the large-scale production and commercial application of brazzein. Summary of the Invention

[0005] Technical issues The technical problem to be solved by this invention is that existing methods for preparing Brazilian sweet protein by microorganisms have problems such as complicated operation, easy formation of inclusion bodies, abnormal folding of sweet protein structure requiring in vitro refolding, and sweetness far lower than that of natural sweet protein.

[0006] Technical solution This invention provides a method for efficiently expressing the sweet protein Brazzein, the method comprising the following steps: (1) The gene encoding the sweet protein Brazzein was cloned into the prokaryotic expression vector pET-28a or pMAL-c6t to obtain the recombinant expression vector pET-28a-Brazzein or pMAL-c6t-Brazzein. Then, it was transformed into Escherichia coli SHuffle T7 to induce expression. The bacterial cells were collected by centrifugation, and the bacterial cells were broken by ultrasonication and centrifuged to obtain the supernatant. The supernatant was purified by affinity chromatography to obtain the fusion protein Brazzein-His or His-MBP-Brazzein.

[0007] (2) The Brazzein-His obtained in step (1) was dialyzed in ultrapure water and freeze-dried to obtain the sweet protein Brazzein-His. The sweet protein Brazzein-His has a 6×His tag at the C-terminus and an MGSS sequence at the N-terminus of the sweet protein with an amino acid sequence as shown in SEQ ID NO:2. The fusion protein His-MBP-Brazzein obtained in step (1) was dialyzed at low temperature, and then TEV protease was added to remove the fusion tag His-MBP. After separation and purification by affinity chromatography, the Brazzein protein mutant was obtained. The impurities were removed by dialyzing in ultrapure water and freeze-dried to obtain the sweet protein mutant G-Brazzein. The sweet protein mutant G-Brazzein has an added glycine (Gly) residue at the N-terminus of the sweet protein with an amino acid sequence as shown in SEQ ID NO:2.

[0008] In one embodiment of the present invention, the coding gene of the sweet protein Brazzein in step (1) has been codon optimized according to the codon preference of Escherichia coli; the coding gene sequence of the optimized sweet protein Brazzein is shown in SEQ ID NO: 1.

[0009] In one embodiment of the present invention, the method for inducing expression in step (1) is as follows: recombinant Escherichia coli SHuffle T7 carrying the recombinant expression vector pET-28a-Brazzein or pMAL-c6t-Brazzein is cultured in LB medium at 37°C until OD. 600 The concentration was set at 0.4-0.6, and then the temperature was lowered to 16℃. IPTG was added at a final concentration of 0-0.8mM to induce expression for 16-20h.

[0010] In one embodiment of the present invention, the parameters for centrifuging and collecting bacterial cells in step (1) are: 8000~10000 r / min, time 5~10 min, and temperature 4~8℃; the collected bacterial cells are resuspended in buffer A (20±5 mmol / L Tris-HCl, pH=8.0), and the ratio of the amount of buffer A is 1g wet bacterial cells to 10~20 mL buffer A.

[0011] In one embodiment of the present invention, the parameters for ultrasonic crushing in step (1) are: power 100-200W, time 10-30min, ultrasonic 3s, stop 5s, and performed on an ice bath; the parameters for centrifugation after crushing are: 10000~12000 r / min, time 10~15 min, and temperature 4~8℃.

[0012] In one embodiment of the present invention, the affinity chromatography in step (1) is performed using nickel ion affinity chromatography.

[0013] In one embodiment of the present invention, the affinity chromatography purification in step (1) includes the following steps: the obtained supernatant is loaded into a nickel ion affinity chromatography column using a peristaltic pump; impurities are first washed with buffer A, and then the target protein is eluted with buffer B using a gradient elution program; the eluent is collected in separate tubes and analyzed by SDS-PAGE; and the eluent containing the fusion protein is collected. The composition of buffer A is: 20±5 mmol / L Tris-HCl, pH=8.0; and the composition of buffer B is: 20±5 mmol / L Tris-HCl, 400mM imidazole, pH=8.0.

[0014] In one embodiment of the present invention, the Brazzein-His dialysis in step (2) involves loading the fusion protein Brazzein-His obtained in step (1) into a dialysis bag with a molecular weight cutoff of 100-1000 Da, and then placing it in ultrapure water for dialysis at 4-8°C for 6-14 h.

[0015] In one embodiment of the present invention, the dialysis in step (2) involves loading the His-MBP-Brazzein fusion protein obtained in step (1) into a dialysis bag with a molecular weight cutoff of 100-1000 Da, and then placing it in buffer A for dialysis at 4-8°C for 6-14 h.

[0016] In one embodiment of the present invention, the dialysis bag in step (2) is preferably a dialysis bag with a molecular weight cutoff of 500 Da; the volume ratio of the fusion protein solution to the dialysis fluid is preferably 1:50.

[0017] In one embodiment of the present invention, the ratio of TEV protease used in step (2) is 50:1 molar concentration of fusion protein to protease. The TEV protease is digested at 4-30°C for 2-12 hours, and mixed every 30 minutes.

[0018] In one embodiment of the present invention, the affinity chromatography separation and purification in step (2) involves further separating and purifying the protein solution after TEV protease cleavage by nickel ion affinity chromatography to remove the cleaved protein tag, the uncleaved fusion protein, and the added TEV protease.

[0019] In one embodiment of the present invention, the affinity chromatography separation and purification in step (2) includes the following steps: loading the protein solution digested by TEV protease into a nickel ion affinity chromatography column using a peristaltic pump, and collecting the flow-through liquid, which is the obtained sweet protein G-Brazzein mutant.

[0020] In one embodiment of the present invention, the dialysis in step (2) involves loading the flow-through solution of nickel ion affinity chromatography into a dialysis bag with a molecular weight cutoff of 100-1000 Da, and then dialyzing it twice in ultrapure water at 4-8°C for 6-14 h. The dialysis bag is preferably a dialysis bag with a molecular weight cutoff of 100 Da; the volume ratio of the sweet protein solution to ultrapure water is preferably 1:50.

[0021] In one embodiment of the present invention, the freeze-drying in step (2) involves freezing the dialyzed Brazzein-His and G-Brazzein sweet protein solution to -80°C and then freeze-drying it to obtain the sweet protein Brazzein-His and its mutant G-Brazzein.

[0022] The present invention provides a sweet protein Brazzein obtained by the above method, which is Brazzein-His or a sweet protein mutant G-Brazzein. The amino acid sequence of Brazzein-His is shown in SEQ ID NO:3, which is a sweet protein with a 6×His tag at the C-terminus and an MGSS sequence at the N-terminus. The amino acid sequence of G-Brazzein is shown in SEQ ID NO:6, which is a sweet protein with an added glycine (Gly) residue at the N-terminus.

[0023] The present invention also provides the application of the sweet protein Brazzein-His or the sweet protein mutant G-Brazzein in increasing the aroma and improving the flavor of coffee, wherein the sweet protein Brazzein-His or the sweet protein mutant G-Brazzein partially or completely replaces the sugar used in coffee.

[0024] Beneficial effects: (1) The Shuffle T7 expression host of this invention can promote the correct folding of disulfide bond proteins without the need for in vitro refolding. Compared with the Brazzein expressed by ordinary E. coli and after denaturation and refolding, it has a more correct protein structure. It can not only effectively improve the sweetness of Brazzein, but also promote the protein expression yield. 235 mg of G-Brazzein mutant or 126 mg of Brazzein-His can be obtained per 1 L of fermentation broth.

[0025] (2) The sweet protein prepared in this invention has an extra Gly residue at the N-terminus of Brazzein, which can effectively increase the sweetness of Brazzein. The sweet protein mutant G-Brazzein has a high sweetness, which can exceed 1500 times that of sucrose. It can still taste obvious sweetness at a concentration of 6.6 μg / mL and has a good sensory flavor. Brazzein-His is 467 times sweeter than sucrose, and the sweetness lasts for up to 12 seconds. Moreover, the preparation process does not require additional enzymatic digestion.

[0026] (3) The sweet protein Brazzein-His or G-Brazzein prepared by this invention can enhance the aroma of coffee and improve the taste of coffee in coffee making, and has important application scenarios. Attached Figure Description

[0027] Figure 1 This is a schematic diagram illustrating the preparation steps of the sweet protein Brazzein of this invention.

[0028] Figure 2 This shows the expression of the Brazzein fusion protein in different hosts in Example 1.

[0029] Figure 3 Expression, purification, and SDS-PAGE analysis of the Brazzein fusion protein.

[0030] Figure 4 This is an SDS-PAGE analysis diagram of the Brazzein fusion protease digested in Example 1.

[0031] Figure 5 Secondary structure of the sweet protein G-Brazzein mutant. Detailed Implementation

[0032] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. The specific embodiments described below further illustrate the present invention.

[0033] The testing method involved in this invention: 1. Protein content detection methods Protein concentration was determined according to the instructions of the BCA protein assay kit. A standard curve for protein concentration determination was plotted using bovine serum albumin (BSA) as the standard protein.

[0034] The specific procedure for determining the protein concentration of the sample is as follows: Prepare an appropriate amount of BCA working solution at a volume ratio of solution A: solution B = 50:1 and mix thoroughly. Take 100 μL of the dialyzed protein sample, using 100 μL of dialysis buffer as a blank control. Add 2 mL of BCA working solution to both the protein sample and the control sample. Incubate at 37℃ for 30 min, then cool to room temperature and measure the absorbance at 562 nm. Substitute the measured absorbance value (y) into the BSA protein standard curve equation y = 0.0011x to obtain x, which is the concentration of the protein sample.

[0035] 2. Protein purity detection methods Protein samples were collected at different stages and prepared for electrophoresis at a sample volume to loading buffer ratio of 5:1. SDS-PAGE analysis was then performed using a protein gel containing 12% acrylamide. Electrophoresis conditions were: 60 V for 20-30 min, followed by 120 V for 60-80 min, stopping electrophoresis when the bromophenol blue indicator reached the protein gel substrate. Coomassie brilliant blue staining was applied, and the protein expression yield was determined using a BCA kit.

[0036] Example 1: Preparation of the sweet protein Brazzein 1. Construction of recombinant expression plasmid for the sweet protein Brazzein (1) Based on the amino acid sequence of the sweet protein Brazzein (SEQ ID NO:2), codon optimization was performed on the E. coli expression system to obtain a DNA molecule that can be efficiently expressed in E. coli. The DNA molecule encoding the sweet protein Brazzein was artificially synthesized using the overlap extension PCR method, as shown in SEQ ID NO:1.

[0037] (2) DNA molecules encoding the sweet protein Brazzein were amplified by PCR. Simultaneously, pET-28a, pET-28a-SUMO, and pMAL-c6t expression vectors were linearized by PCR. After digestion with DPN1, they were homologously recombined with the DNA molecules encoding Brazzein to construct pET-28a-Brazzein, pET-28a-SUMO-Brazzein, and pMAL-c6t-Brazzein recombinant expression vectors. These vectors were used to prepare fusion proteins Brazzein-His (SEQ ID NO:3) with a His tag at the C-terminus and an MGSS sequence at the N-terminus, His-SUMO-Brazzein (SEQ ID NO:4) with a His tag and a SUMO tag at the N-terminus, and His-MBP-Brazzein (SEQ ID NO:5) with a His tag and an MBP tag at the N-terminus, respectively. The above recombinant ligation products were transformed into DH5α competent cells.

[0038] The primer sequences used for PCR are as follows: Amplify the Brazzein forward primer (FP1) to construct the pET-28a-Brazzein recombinant expression vector: gatataccatgggcagcagcCAAGACAAATGCAAGAAAGTGTACG; Amplify the Brazzein reverse primer (RP1) to construct the pET-28a-Brazzein recombinant expression vector: tggtggtggtggtgctcgagGTATTCGCAGTAGTCACAGATGCAC; Forward primer (FP2) for linearizing the pET-28a expression vector: CTCGAGCACCACCACCACC; Reverse primer (RP2) for linearizing the pET-28a expression vector: GCTGCTGCCCATGGTATATCTC; Amplify the Brazzein forward primer (FP3) to construct the pET-28a-SUMO-Brazzein recombinant expression vector: acagagaacagattggtggaCAAGACAAATGCAAGAAAGTGTACG; Amplify the Brazzein reverse primer (RP3) to construct the pET-28a-SUMO-Brazzein recombinant expression vector: cggagctcgaattcggatcaGTATTCGCAGTAGTCACAGATGCAC; Forward primer (FP4) for linearizing the pET-28a-SUMO expression vector: TGATCCGAATTCGAGCTCCG; Reverse primer (RP4) for linearizing the pET-28a-SUMO expression vector: TCCACCAATCTGTTCTCTGTGAGC; Amplify the Brazzein forward primer (FP5) to construct the pMAL-c6t-Brazzein recombinant expression vector: agaacctgtacttccagggtCAAGACAAATGCAAGAAAGTGTACG; Amplify the Brazzein reverse primer (RP5) to construct the pMAL-c6t-Brazzein recombinant expression vector: gatccgtcgacgatatctcaGTATTCGCAGTAGTCACAGATGCAC; Forward primer (FP6) for linearizing the pMAL-c6t expression vector: TGAGATATCGTCGACGGATCCG; The reverse primer (RP6) for linearizing the pMAL-c6t expression vector is: ACCCTGGAAGTACAGGTTCTCCC.

[0039] The PCR process is as follows: Under the action of a high-fidelity 2×PfuMax HiFi PCR ProMix, using SEQ ID... Using the Brazzein gene shown in NO:1 as a template, with FP1 / 3 / 5 as the upstream primer and RP1 / 3 / 5 as the downstream primer, and with the addition of an appropriate amount of sterile water, the Brazzein gene fragment carrying the homologous arm was amplified. Using pET-28a, pET-28a-SUMO, and pMAL-c6t plasmids as templates, with FP2 / 4 / 6 as the upstream primer and RP2 / 4 / 6 as the downstream primer, and with the addition of an appropriate amount of sterile water, the linearized pET-28a, pET-28a-SUMO, and pMAL-c6t vectors were amplified. The PCR amplification products were digested with DPN1, and then the Brazzein gene fragment and the linearized expression vector were recovered using a PCR product recovery kit. Using seamless cloning technology, the Brazzein gene fragment and the linearized vector were homologously recombined to construct the pET-28a-Brazzein, pET-28a-SUMO-Brazzein, and pMAL-c6t-Brazzein recombinant expression plasmids.

[0040] (3) Select single colonies for colony PCR identification, and send positive single clones to sequencing companies for sequencing verification to screen positive clone strains.

[0041] The specific PCR amplification conditions were as follows: 98 °C pre-denaturation for 30 s, 98 °C denaturation for 10 s, 65 °C-55 °C annealing for 30 s, 72 °C extension for 1-4 min, for a total of 10 cycles. In each cycle, the annealing temperature was decreased by 1 °C, followed by 98 °C denaturation for 10 s, 55 °C annealing for 30 s, 72 °C extension for 2-4 min, for a total of 30 cycles, and finally 72 °C extension for 5 min to finish.

[0042] 2. Expression optimization and purification of Brazzein fusion protein (1) The three recombinant expression plasmids obtained in step 1 were transformed into three host cells of Escherichia coli BL21(DE3), Rosetta(DE3) and Shuffle T7, respectively. Positive transformants were selected, resulting in a total of 9 expression strains. These strains were then inoculated into LB medium containing 100 μg / mL kanamycin or 50 μg / mL ampicillin and cultured overnight at 37 °C in a shaker as seed culture. The seed culture was then inoculated into LB medium containing the corresponding antibiotic at a volume ratio of 1:100 and cultured at 37 °C and 180 r / min until the bacterial culture reached OD. 600 Once the concentration reaches 0.5, add IPTG to each bottle of bacterial culture to a final concentration of 0.1 mmol / L. After inducing expression at 16 ℃ for 20 h, centrifuge the fermentation broth and collect the bacterial cell precipitate.

[0043] (2) Take 1 L of the bacterial cells prepared in step (1), resuspend each gram of bacterial cells in 10 mL of buffer A (50 mM Tris-HCl, pH 8.0), and lyse the bacterial cells using an ultrasonic cell disruptor in an ice bath. The ultrasonic conditions are: power 150 W, ultrasonic for 3 s, interval 5 s, for a total of 20 min. Centrifuge the disrupted bacterial solution at 12000 g / min at 6 ℃ for 20 min, and collect the supernatant for later use.

[0044] (3) The whole bacterial culture induced by IPTG prepared in step (1) and the supernatant obtained in step (2) were subjected to SDS-PAGE analysis to compare the expression effects of the three fusion proteins in different expression hosts. The results are as follows: Figure 2As shown, pET-28a-Brazzein produced the most Brazzein-His protein in the Shuffle T7 expression host. Although Rosetta(DE3) could also express Brazzein-His, the yield was not as high as that of Shuffle T7, while BL21(DE3) hardly expressed Brazzein-His protein. The expression results of pET-28a-SUMO-Brazzein and pMA-c6t-Brazzein in the three strains were similar to those of pET-28a-Brazzein. The expression yields of His-SUMO-Brazzein and His-MBP-Brazzein fusion proteins were Shuffle T7 > Rosetta(DE3) > BL21(DE3).

[0045] The host optimization analysis above showed that, compared with BL21(DE3), Escherichia coli Shuffle T7 had better expression performance, thereby effectively improving the expression yield of the sweet protein Brazzein in Escherichia coli.

[0046] (4) Nickel affinity chromatography purification of Brazzein protein. The chromatography column was connected to the column position valve of a rapid protein purification instrument. The system and column were washed with ultrapure water, and then equilibrated with buffer A. The supernatant from (2) was then loaded into the purification column. Impurities were washed with buffer A, and then washed with 5% buffer B (50 mM Tris-HCl, 20 mM imidazole, pH 8.0) and 10% buffer B (50 mM Tris-HCl, 40 mM imidazole, pH 8.0). Finally, the fusion proteins were obtained by elution with buffer B (50 mM Tris-HCl, 400 mM imidazole, pH 8.0): Brazzein-His, His-SUMO-Brazzein, and His-MBP-Brazzein. The washings after washing with 5% buffer B and 10% buffer B, as well as the fusion proteins purified by nickel affinity chromatography, were analyzed by SDS-PAGE to observe protein expression. The results are as follows: Figure 3 As shown, the purified target protein bands are uniform with no obvious impurities. The molecular weights of the proteins shown are approximately 7kD, 20kD, and 45kD, which are consistent with the theoretical molecular weights of Brazzein-His, His-SUMO-Brazzein, and His-MBP-Brazzein, indicating that the purification effect is good.

[0047] Among them, under the condition of selecting the optimal expression host Shuffle T7, the expression yields of the fusion proteins were His-MBP-Brazzein > His-SUMO-Brazzein > Brazzein-His. It was calculated that under optimal expression conditions, approximately 126 mg of Brazzein-His, 326 mg of His-SUMO-Brazzein, and 1453 mg of His-MBP-Brazzein protein could be isolated and purified from 1 L of fermentation broth.

[0048] 3. Enzymatic digestion of Brazzein fusion protein (1) The recombinant expression vectors pET-28a-SUMO-Brazzein and pMAL-c6t-Brazzein obtained in step 1 were transformed into Escherichia coli Shuffle T7, respectively. Positive E. coli transformants were selected and inoculated into LB medium containing 100 μg / mL kanamycin or 50 μg / mL ampicillin. The mixture was shaken and cultured overnight at 37 °C to obtain the seed culture. The seed culture was then inoculated into fresh LB medium containing the corresponding antibiotic at a volume ratio of 1:50 and cultured at 37 °C and 200 r / min until the bacterial culture reached OD. 600 To achieve a final concentration of 0.6, IPTG was added to the bacterial culture to a final concentration of 0.2 mmol / L. Expression was induced at 18 ℃ for 16 h, followed by centrifugation to collect the bacterial cell pellet. The bacterial cells were resuspended in 10 mL of buffer A per gram of bacterial cells and lysed using an ultrasonic cell disruptor on ice. The sonication conditions were: 120 W power, 3 s sonication, 5 s interval, for a total of 20 min. The disrupted bacterial culture was centrifuged at 12000 g / min for 30 min at 4 ℃. The supernatant was loaded into a Ni ion affinity chromatography column pre-equilibrated with buffer A. Impurities were washed with buffer A, followed by elution with buffer B to prepare the Brazzein fusion proteins: His-SUMO-Brazzein and His-MBP-Brazzein.

[0049] (2) After dialyzing the Brazzein fusion protein from step (1) overnight in buffer A at a volume ratio of 1:100, add TEV protease or Ulp1 protease at a substrate-to-enzyme molar ratio of 50:1, and digest at 25 °C for 5 h. His-SUMO-Brazzein, after being digested by Ulp1 protease, yields a sweet protein Brazzein without any extra amino acids, while His-MBP-Brazzein, after being digested by TEV protease, yields a Brazzein mutant (G-Brazzein) with an extra glycine residue (Gly) at the N-terminus. The digestion effect was detected by SDS-PAGE protein electrophoresis. The results are as follows:Figure 4 As shown, TEV and Ulp1 proteases can remove the His-MBP tag and His-SUMO tag from the fusion proteins His-MBP-Brazzein and His-SUMO-Brazzein, respectively, to obtain mutants G-Brazzein and Brazzein.

[0050] (3) Brazzein and its mutant G-Brazzein were isolated and purified. The enzyme-digested product was dialyzed into buffer A and then loaded into a nickel ion affinity chromatography column pre-equilibrated with buffer A. The permeate was collected to obtain Brazzein and its mutant G-Brazzein. It was calculated that 1 L of fermentation broth yielded 105 mg of Brazzein and 235 mg of G-Brazzein mutant, respectively. It can be seen that His-MBP tag fusion expression can obtain more Brazzein mutant protein compared with His tag only or His-SUMO fusion.

[0051] The Brazzein-His obtained in step 2, the Brazzein obtained in this step, and the G-Brazzein mutant were dialyzed in ultrapure water at a volume ratio to remove salt ions, and then freeze-dried for later use. In summary, using protein tag fusion to express Brazzein, and selecting the ShuffleT7 expression host which can promote protein disulfide bond formation and protein folding, effectively expresses Brazzein with good solubility. Compared with tagless auxiliary expression or the ordinary BL21(DE3) expression host, it has a higher protein expression yield. Moreover, the Brazzein mutant obtained after protease digestion does not require in vitro folding and can be directly obtained as shown in the image. Figure 5 The correct folding structure shown is easy to operate, and the final protein yield is higher than that of previous E. coli expression, showing better prospects for practical application.

[0052] Example 2: Brazzein sweetness assessment and threshold determination (1) Since Brazzein is not a glycosyl compound, its sweetness cannot be directly measured by a saccharimeter. Therefore, the human sensory evaluation method was used to determine its sweetness threshold. For most people, the sweetness threshold of sucrose in beverages is usually between 0.32% and 1.0%. To evaluate the sweetness threshold of each solution, a 1% sucrose solution was used as the lowest concentration standard for perceiving sweetness, that is, its sweetness threshold was set at 10000 μg / mL.

[0053] Samples used for sweetness determination included Brazzein-His, Brazzein, G-Brazzein, sucrose, and mineral water. Literature reports that the sweetness of the brazzein protein is approximately 500-3000 times that of sucrose; therefore, the brazzein powder was diluted with mineral water to create gradient concentration solutions ranging from 2 to 100 μg / mL for sensory evaluation. A total of 300 health assessors participated in this evaluation, including 150 men and 150 women aged 20 to 40 years. Sensory evaluation started at the lowest concentration (2 μg / mL), increasing by 2 μg / mL each time until the assessor perceived sweetness; then, the concentration was gradually decreased in increments of 0.1 μg / mL until the sweetness disappeared, thus determining the sweetness threshold of the sample. During testing, 20 mL of the sample was held in the mouth for at least 10 seconds, and its sweetness was evaluated on a scale of 0 to 10 (0 representing no sweetness, 10 representing extremely sweet). The final sweetness threshold was the average of the results from 30 evaluators. The sweetness calculation method, specifically the formula for calculating the sweetness multiple of Brazilin and its variants relative to 1% sucrose, is as follows: Sweetness multiple = 10000 / sample sweetness threshold (μg / mL) The results are shown in Table 1. The sweetness threshold concentrations of the three recombinant Brazzein sweet proteins and their mutants were 21.4, 26.3, and 6.6 μg / mL, respectively, with sweetness multiples relative to 1% sucrose of 467, 380, and 1515 times, respectively. This indicates that the recombinant Brazzein sweet proteins and their mutants prepared in this invention possess good sweetness. Previously, the sweetness of recombinant Brazzein sweet proteins expressed in microorganisms was often inferior to that of naturally extracted proteins, mainly due to misfolding of the protein structure leading to a reduction or loss of sweetness. The Shuffle T7 expression host used in this invention effectively overcomes this problem, producing Brazzein with the correct structure, resulting in a high sweetness.

[0054] (2) To further evaluate the sweetness characteristics of Brazilin and its mutants, this invention diluted them to a concentration with the same sweetness as sucrose aqueous solution according to their sweetness multiples, and evaluated their sensory performance based on this. The evaluation indicators mainly include: sweetness delay (the time required from tasting to perceiving the sweetness) and sweetness duration (the duration of the sweetness in the mouth). The results are shown in Table 1. The sweetness delay time of sucrose solution is relatively short, only 0.5s, while the sweetness perception time of Brazilin is slightly longer, between 1.5s and 3s. However, the sweetness delay time of G-Brazzein recombinantly expressed in this invention is shorter than that of Brazzein and Brazzein-His, and is closer to the sweetness perception of sucrose. In addition, the sweetness duration of Brazzein recombinantly prepared in this invention is generally higher than that of sucrose, but the sweetness duration of G-Brazzein is 7s, which is close to the sweetness duration of sucrose, indicating that the sweetness and flavor of G-Brazzein are more suitable for application in the food field.

[0055] Table 1. Yield, sweetness threshold concentration, and sensory flavor evaluation of Brazzein

[0056] In addition, some literature has used the His-MBP-Brazzein method to recombinantly express Brazzein, but the resulting Brazzein, after in vitro refolding, is only 200 times sweeter than sucrose. This invention innovatively discovers that the G-Brazzein mutant produced by protease cleavage of the recombinant His-MBP-Brazzein can be more than 1500 times sweeter than sucrose. The main reason for this may be: I. The Shuffle T7 expression host used in this invention can promote the correct folding of the protein structure, resulting in a more correct protein structure compared to Brazzein obtained after denaturation and renaturation, which can enhance the sweetness of Brazzein. II. In addition to the good folding ability of the Shuffle T7 expression host, which can enhance the sweetness of Brazzein, the sweet protein prepared in this invention has an additional Gly residue at the N-terminus of Brazzein, which also increases the sweetness of Brazzein.

[0057] Example 3: Application of sugar as a substitute in coffee Sensory evaluation of coffee taste preferences was conducted using six participants as a test unit, based on the addition of equal amounts of glycoprotein (G-Brazzein) and sucrose solutions of equal sweetness. The specific evaluation was as follows: Three classic unsweetened lattes were prepared, containing 36g of coffee extract, 50g of milk base, and 225g of whole milk, labeled A (unsweetened), B (glycoprotein), and C (sucrose). Then, equal amounts of glycoprotein and sucrose solutions of equal sweetness were added to lattes B and C, respectively. The effects of no addition, glycoprotein, sucrose, and mixed additions on coffee flavor were compared, primarily evaluated from two aspects: coffee aroma (0-10 points) and bitterness (-10-0 points). The highest overall score was considered the optimal addition ratio, and the results are shown in Table 2. In a blind test, approximately two-thirds of the participants could clearly perceive a more pronounced nutty and smoky aroma in group B latte, with a slightly bitter aftertaste; group C had a fuller flavor and better masked the coffee bitterness. In this test, the sweetened protein solution exhibited a certain aroma-enhancing effect on coffee, but at the same time, it amplified the bitterness of the coffee itself. After trying a latte blend ratio of B:C=1:1, comparing groups A, B, and C, it was found that the blend ratio neutralized some of the coffee bitterness while still enhancing the aroma. Therefore, under the existing conditions (36g coffee extract, 50g milk base, 225g pure milk), adding a small amount of diluted sweetened protein and sucrose can significantly improve the sensory flavor of classic sugar-free latte, enhance the coffee aroma, and reduce the bitterness.

[0058] Table 2. Effects of sweet protein on coffee flavor improvement

[0059] sequence SEQ ID NO:1 (Gene sequence of the sweet protein Brazzein) CAAGACAAATGCAAGAAAGTGTACGAGAACTATCCGGTGTCCAAATGCCAACTGGCGAACCAGTGCAACTACGACTGCAAACTGGATAAACACGCTCGTTCCGGTGAATGCTTCTACGATGAAAAACGTAACCTGCAGTGCATCTGTGACTACTGCGAATAC SEQ ID NO:2 (Amino acid sequence of the sweet protein Brazzein) QDKCKKVYENYPVSKCQLANQCNYDCKLDKHARSGECFYDEKRNLQCICDYCEY SEQ ID NO:3 (Amino acid sequence of the sweet protein Brazzein-His) MGSS QDKCKKVYENYPVSKCQLANQCNYDCKLDKHARSGECFYDEKRNLQCICDYCEY HHHHHH SEQ ID NO:4 (Amino acid sequence of His-SUMO-Brazzein) MGSSHHHHHHSSGLVPRGSHMASMSDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPLRRLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIGG QDKCKKVYENYPVSKCQLANQCNYD CKLDKHARSGECFYDEKRNLQCICDYCEY SEQ ID NO:5 (Amino acid sequence of His-MBP-Brazzein) MKIHHHHHHEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEEKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPF TWDAVRYNGKLIAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAADGGYAFKYENGKYDIKDVGVDNAGAKAG LTFLVDLIKNKHMNADTDYSIAEAAFNKGETAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLENYLLTDEGLEAVNKDKPLGAVALKSYEEELVKDPRIAATMENAQKGEIMPNIPQMSAFWYAVRTAVINAASGRQTVDEALKDAQTNSSSNNNNNNNNLGENLYFQG QDKCKKVYENYPVSKCQLANQCNYDCKLDKHARSGECFYDEKRNLQCICDYCEY SEQ ID NO:6 (Amino acid sequence of the G-Brazzein mutant) G QDKCKKVYENYPVSKCQLANQCNYDCKLDKHARSGECFYDEKRNLQCICDYCEY Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A method for efficiently expressing the sweet protein Brazzein, characterized in that, The method includes the following steps: (1) The gene encoding the sweet protein Brazzein was cloned into the prokaryotic expression vector pET-28a or pMAL-c6t to obtain the recombinant expression vector pET-28a-Brazzein or pMAL-c6t-Brazzein. Then, it was transformed into Escherichia coli SHuffle T7 to induce expression. The bacterial cells were collected by centrifugation, and the bacterial cells were broken by ultrasonication and centrifuged to obtain the supernatant. The supernatant was purified by affinity chromatography to obtain the fusion protein Brazzein-His or His-MBP-Brazzein. (2) The fusion protein Brazzein-His obtained in step (1) was dialyzed and then freeze-dried to obtain the sweet protein Brazzein-His. The amino acid sequence of the sweet protein Brazzein-His is shown in SEQ ID NO:

3. His-MBP-Brazzein obtained in step (1) was dialyzed at low temperature, and then TEV protease was added to remove the fusion tag His-MBP. After separation and purification by affinity chromatography, G-Brazzein protein mutant was obtained. After dialyzed and freeze-dried, the sweet protein mutant G-Brazzein was obtained. The amino acid sequence of the sweet protein mutant G-Brazzein is shown in SEQ ID NO:

6.

2. The method for efficiently expressing the sweet protein Brazzein according to claim 1, characterized in that, The gene sequence encoding Brazzein, the sweet protein mentioned in step (1), is shown in SEQ ID NO:

1.

3. The method for efficiently expressing the sweet protein Brazzein according to claim 1, characterized in that, The affinity chromatography described in step (1) uses nickel ion affinity chromatography.

4. The method for efficiently expressing the sweet protein Brazzein according to claim 1, characterized in that, The dialysis described in step (2) involves placing the fusion protein Brazzein-His obtained in step (1) into a dialysis bag with a molecular weight cutoff of 100-1000 Da, then placing it in ultrapure water and dialyzing it at 4-8°C for 6-14 hours. His-MBP-Brazzein is placed into a dialysis bag with a molecular weight cutoff of 100-1000 Da, then placed in buffer A and dialyzed at 4-8°C for 6-14 hours. The buffer A is 20±5 mmol / L Tris-HCl with pH=8.

0.

5. The method for efficiently expressing the sweet protein Brazzein according to claim 1, characterized in that, Step (2) involves separating and purifying the protein solution after TEV protease cleavage by nickel ion affinity chromatography to remove the cleaved protein tag, uncleaved fusion protein, and added TEV protease.

6. Brazzein, a sweet protein, characterized by, The amino acid sequence is shown in SEQ ID NO:3 or SEQ ID NO:

6.

7. The use of the sweet protein of claim 6 in the food, pharmaceutical or cosmetic fields.

8. The application according to claim 7, characterized in that, It is used as a sweetener in beverages, confectionery, dairy products, and baked goods, including coffee.

9. The application according to claim 7, characterized in that, It is used as a flavoring agent to improve the taste of oral medications.

10. The application according to claim 7, characterized in that, It is used as a sweetener in oral care products.

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