A fusion protein and its application in the preparation of Brazil sweet

Abstract

This invention provides a fusion protein and its application in the preparation of Brazil sweetener. The fusion protein comprises a tag protein and a Brazil sweetener protein linked by a linker; wherein the linker comprises an amino acid sequence as shown in SEQ ID NO:1, and the amino acid sequence as shown in SEQ ID NO:1 is directly linked to the Brazil sweetener protein. This invention also provides a method for preparing the Brazil sweetener protein, comprising treating the fusion protein with a TEV protease and obtaining the Brazil sweetener protein. Large-scale expression of the fusion protein and the Brazil sweetener protein is achieved using an *E. coli* expression system; the resulting Brazil sweetener protein exhibits correct folding and a high sweetness.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, in particular to a fusion protein and its application in the preparation of brazzein. BACKGROUND

[0002] Brazzein protein is a natural sweet protein isolated from the red fresh fruit of the wild plant Pentadiplandra brazzeana Baillon in West Africa. The yield of brazzein protein accounts for 0.05-0.2% of the weight of the mature fruit. The isolated natural brazzein protein exists in two forms: the main form (accounting for 80%) is Glu-Brazzein with 54 amino acid residues containing pyroglutamic acid (pGlu) at the N-terminus, and the other form (accounting for 20%) is des-Glu Brazzein, which is the same as the former except that the N-terminus does not contain pyroglutamic acid. Brazzein can form natural dimers called Pentadin and also produce sweetness. Brazzein protein is currently the smallest single-chain sweet protein found, consisting of 54 amino acids and having a molecular weight of 6.5 kDa. Brazzein protein is a hydrophilic polypeptide with good solubility.

[0003] The practical application of natural sweet protein brazzein still has many limitations. The production of brazzein protein is limited by the growth conditions and growth cycle of the plant, and has the defects of low yield, low extraction rate and high cost. Therefore, obtaining a large amount of recombinant brazzein protein by genetic engineering is the main way to produce the protein. At present, the expression of recombinant brazzein protein mainly focuses on prokaryotic expression system based on Escherichia coli and eukaryotic expression system based on Pichia pastoris. Studies have shown that the heterologous expression of brazzein protein in Escherichia coli cannot be correctly folded due to the lack of post-processing, resulting in insoluble inclusion bodies. The expression in Pichia pastoris usually selects a methanol-inducible promoter, and the culture medium is complex, which brings safety problems to the later application in food.

[0004] The expression system of Escherichia coli may produce insoluble inclusion bodies. Usually, a solubility-promoting tag is added to make it soluble, and the tag is cut off after expression to obtain a soluble protein. The common enzyme cutting site between the common tag and the target protein is the TEV enzyme cutting site, i.e. ENLYFQ / G(S). The first amino acid of brazzein protein is Q, and if the common TEV enzyme cutting site is used, other amino acids will be introduced. If you want to get the correct brazzein protein sequence, you need to perform enzyme cutting at the ENLYFQ / Q site.

[0005] Advantages of E. coli expression system: (1) High expression level: with efficient protein synthesis mechanism, it can quickly produce a large amount of target protein; (2) Economical: E. coli is easy to culture, the culture medium is cheap, and the culture process is simple; (3) Scalability: E. coli expression system is suitable for small-scale and large-scale protein expression; (4) Rich genetic toolbox: E. coli has a rich genetic toolbox, such as various recombinant vectors, promoters, tags, etc., which can be used for customized protein expression experiments.

[0006] Isotope labeling of proteins includes the following processes: constructing a plasmid containing the target protein gene sequence, introducing it into a host organism, culturing in a medium containing an isotope label, expressing the isotope-labeled protein by the host organism, and separating and purifying it using biochemical techniques to obtain high-purity isotope-labeled protein. Common methods for isotope labeling of proteins include chemical labeling and metabolic labeling. Chemical labeling refers to introducing an isotope label at a specific site on the protein or peptide segment after collecting the protein sample using chemical reactions; metabolic labeling refers to adding N or H or other isotopes to the culture medium, and after several generations of culture, the protein will be completely labeled with isotopes. Among them, chemical labeling can only replace locally, and metabolic labeling can achieve uniform labeling of isotopes. 15 N or 2 H or other isotopes, and after several generations of culture, the protein will be completely labeled with isotopes. Among them, chemical labeling can only replace locally, and metabolic labeling can achieve uniform labeling of isotopes.

[0007] MBP tag is composed of 367 amino acids, with a molecular weight of 42.5 KDa, and is a good protein tag for increasing the solubility of fusion proteins. MBP-tag can increase the solubility of fusion proteins overexpressed in prokaryotic expression systems, and improve their expression levels. However, this purification technique has an overlooked disadvantage, which is that the amylose matrix currently sold on the market has too weak affinity for MBP, often resulting in low protein yield after purification. His-tag is a technology commonly used for protein purification and biological research. It involves attaching a short peptide sequence composed of histidine (His) to the protein, making it easy to capture and identify during expression and purification. Because His-tag can specifically bind to metal ion (such as nickel or cobalt) chelated affinity chromatography media, it eliminates the need for multiple purification steps to gradually separate proteins, enabling rapid purification and reducing the complexity of operations. At the same time, His-tag affinity chromatography method is easy to scale up, suitable for purification from small-scale laboratory to industrial-scale production. SUMMARY

[0008] As described above, the traditional plant extraction method has the problems of harsh growth environment requirement, limited planting area and low yield, which limits the large-scale production and application of Brazzein. In order to meet the market demand, researchers use transgenic technology to introduce Brazzein into different hosts such as microorganisms and higher plants, in order to obtain a large amount of recombinant active Brazzein. However, the recombinant Brazzein protein is mostly expressed in inclusion bodies, and the purification is complex, and the conformation and sweet taste characteristics will be affected. The MBP tag can increase the solubility of the fusion protein overexpressed in the prokaryotic expression system, and improve the expression amount. The His tag can be specifically combined with the nickel column, so as to purify the protein. The nickel column has the advantages of good chemical stability, strong tolerance, not easy to be interfered by other components in the supernatant after cell disruption, and simple sample pretreatment. The Brazzein carrying the His-MBP tag can realize faster and more convenient purification. However, whether the MBP tag between the protein fragment and the target protein can be cut by TEV enzyme, and whether the conformation and sweetness of the protein during the purification process are affected still need to be solved.

[0009] In order to solve the above-mentioned prior art problems, the present application uses the E. coli expression system to recombinantly express Brazzein by genetic engineering technology, so that it has high sweetness, high purity and high yield, and simplifies the protein purification method. In addition, the present application develops a stable isotope labeling method for Brazzein, which lays a foundation for subsequent research on protein interaction and protein structure using nuclear magnetic resonance (NMR). Traditional E. coli expression may cause incorrect folding of the protein. By genetic engineering and stable isotope labeling technology, the E. coli expression system is used to realize large-scale expression of Brazzein, and ordinary NH4Cl in M9 culture medium is replaced with isotope 15 NH4Cl, to realize isotope labeling of Brazzein, and to use 1 H- 15 N HSQC spectrum, and compare with the standard spectrum to judge whether the protein is correctly folded. It is confirmed by nuclear magnetic resonance (NMR) that the conformation of Brazzein is correct. After sensory evaluation, it is confirmed that the Brazzein protein with correct conformation has high sweetness.

[0010] The first aspect of the present application provides a fusion protein comprising a tag protein and a brazzein protein connected by a linker; wherein the linker comprises a sequence as set forth in SEQ ID NO: 1 (ENLYFQ). The sequence as set forth in SEQ ID NO: 1 is directly connected to the brazzein protein. Thus, the sequence forms a special cleavage site (ENLYFQ / Q) with the first amino acid residue Q of the brazzein protein. The inventors have found that TEV enzyme can precisely cleave at this special site, thereby obtaining the intact brazzein protein with the tag protein and the linker precisely removed. In some embodiments, the linker can further comprise an additional sequence before the sequence ENLYFQ, which does not affect the expression and folding of the fusion protein, nor the precise cleavage of TEV enzyme at ENLYFQ / Q. For example, the amino acid sequence of the linker comprising the additional sequence can be as set forth in SEQ ID NO: 7.

[0011] As used herein, a tag protein refers to a polypeptide or protein sequence that is expressed in fusion with a protein of interest, which is usually covalently bound to the target protein to facilitate detection, purification, localization or functional study of the target protein. The tag protein can be used for purification, through specific affinity tags such as His tag, GST tag, FLAG tag, etc., the target protein can be purified using the corresponding affinity chromatography technology; the tag protein can also be used to increase solubility, through some tag proteins such as MBP (maltose binding protein) and GST (glutathione S-transferase), the solubility expression of the target protein in host cells can be improved; the tag protein can also improve the stability of the target protein, reduce aggregation or degradation. The tag protein can also be used for detection and quantification, using fluorescent tag proteins (such as GFP and its derivatives) or enzyme tags (such as luciferase) can observe the expression and localization of the target protein in living cells in real time; the tag protein can also be used as a reporter gene, such as luciferase or GFP, to monitor gene expression or cell signaling.

[0012] In some embodiments, the tag protein comprises an MBP tag protein. In other embodiments, the tag protein comprises an MBP tag protein and a His tag protein, and the His tag protein is located at the N-terminus of the MBP tag protein. In some preferred embodiments, the fusion protein comprises, from N-terminus to C-terminus, a His tag protein, an MBP tag protein, a linker and a brazzein protein. In some specific embodiments, the amino acid sequence of the His tag protein is as set forth in SEQ ID NO: 5. In some specific embodiments, the amino acid sequence of the MBP tag protein is as set forth in SEQ ID NO: 6. In some specific embodiments, the amino acid sequence of the linker comprising an additional sequence is as set forth in SEQ ID NO: 7.

[0013] In some specific embodiments, the amino acid sequence of the fusion protein is as set forth in SEQ ID NO: 3.

[0014] The second aspect of the present application provides a nucleic acid molecule encoding the fusion protein as described in the present application. In some specific embodiments, the nucleotide sequence of the nucleic acid molecule is as set forth in SEQ ID NO: 4.

[0015] The third aspect of the present application provides a recombinant plasmid comprising the nucleic acid molecule as described in the present application.

[0016] The fourth aspect of the present application provides a host cell comprising the nucleic acid molecule as described in the present application or the recombinant plasmid as described in the present application; preferably, the host cell is Escherichia coli; more preferably, the Escherichia coli is selected from the group consisting of DH5a and BL21.

[0017] The fifth aspect of the present application provides a method for constructing an Escherichia coli engineering strain, comprising the step of transforming the recombinant plasmid as described in the present application into an Escherichia coli strain to obtain the Escherichia coli engineering strain.

[0018] The sixth aspect of the present application provides an Escherichia coli engineering strain obtained according to the method for constructing an Escherichia coli engineering strain as described in the fifth aspect of the present application.

[0019] The seventh aspect of the present application provides a method for preparing a fusion protein, the fusion protein being the fusion protein as described in the first aspect of the present application, the method comprising fermenting the Escherichia coli engineering strain as described in the sixth aspect of the present application and isolating and recovering the fusion protein from the fermentation broth.

[0020] In some embodiments, the fermentation comprises a bacterial enrichment phase and an expression induction phase, and the induction expression of the fusion protein is achieved by adding galactose to the culture medium. In some embodiments, the isolation and recovery comprises purifying the fusion protein using an affinity chromatography technique corresponding to the tag protein; for example, purifying the fusion protein using a Ni-NTA column corresponding to the His tag protein.

[0021] The eighth aspect of the present application provides a method for preparing Brazzein, comprising treating the fusion protein as described in the first aspect of the present application or the fusion protein obtained by the method as described in the seventh aspect of the present application using TEV protease, and obtaining Brazzein.

[0022] In the present application, the fusion protein is correctly folded. In some embodiments, the correct folding of the fusion protein is achieved by assisted folding. The assisted folding includes denaturing reduction and oxidative folding. The denaturing reduction includes mixing the fusion protein with a solution containing EDTA, DTT and guanidine hydrochloride and dialysis using ultrapure water containing acetic acid. The oxidative folding includes mixing several volumes of Tris solution with the dialysis product. In some embodiments, the assisted folding occurs before the TEV protease treatment.

[0023] For the safety of brazzein consumption and the retention of sweetness, the linker and tag protein used in the present application need to be completely removed during the preparation of brazzein. Ideally, the linker and brazzein are separated exactly when the fusion protein is truncated using protease in subsequent use. However, it is usually difficult to ensure accurate removal in actual operation. For example, the enzyme cleavage site of TEV protease is ENLYFQ / G(S), but the first amino acid residue of brazzein is not G or S, so theoretically it is impossible to achieve precise removal using TEV protease. The introduction of the complete enzyme cleavage site of TEV protease will introduce the terminal amino acid residues of the enzyme cleavage site into the obtained brazzein.

[0024] Unexpectedly, the present inventors found that TEV protease also has cleavage efficiency for the enzyme cleavage site of ENLYFQ / Q. The present application connects the tag protein and brazzein through the linker (ENLYFQ, SEQ ID NO: 1), thereby forming the enzyme cleavage site of ENLYFQ / Q, achieving complete and precise removal of the linker and tag protein, and retaining the complete brazzein.

[0025] In some embodiments, the brazzein is separated from the enzyme cleavage product by using the corresponding affinity chromatography technology of the tag protein. For example, using the Ni-NTA column corresponding to the His tag, and the flow-through is the brazzein. In some embodiments, the flow-through product is further purified using a molecular sieve (such as a Hiload column) to obtain high-purity brazzein.

[0026] The ninth aspect of the present application provides a stable isotope labeling method, which comprises fermenting and culturing the engineered E. coli bacteria as described in the present application in a culture medium containing an isotope-labeled raw material, wherein the isotope-labeled raw material comprises 15 N-labeled ammonium chloride; preferably, the culture medium is M9 medium. In some embodiments, the method further comprises separating and recovering the fusion protein from the fermentation broth. In some embodiments, the method further comprises treating the fusion protein using TEV protease and obtaining brazzein.

[0027] The tenth aspect of the present application provides a fusion protein with isotopically stable label, which is obtained according to the labeling method of the ninth aspect of the present application. The present application also provides a brazzein with isotopically stable label, which is obtained according to the labeling method of the ninth aspect of the present application.

[0028] The eleventh aspect of the present application provides the use of the fusion protein and / or brazzein with isotopically stable label in the study of protein interaction and / or protein structure; preferably, the study is performed by nuclear magnetic resonance.

[0029] The present application has the advantages of constructing a fusion protein and realizing large-scale expression of the fusion protein and brazzein by using the E. coli expression system. The fusion protein comprises a tag protein and brazzein connected by a linker. The linker can be recognized and cut by TEV protease, realizing complete and accurate removal of the tag protein and linker and complete retention of the brazzein. The brazzein obtained by the method of the present application has high sweetness. 1 H- 15 N HSQC spectrum, which confirms the correct folding of the fusion protein and brazzein. The obtained brazzein is confirmed to have high sweetness by sensory evaluation.

[0030] The sequences used in the present application are as follows:

[0031]

[0032]

[0033] BRIEF DESCRIPTION OF DRAWINGS

[0034] Figure 1 The flowchart for preparing brazzein.

[0035] Figure 2 The map of the expression vector pETM-44.

[0036] Figure 3 The fusion protein with incorrect folding 1 H- 15 N HSQC spectrum.

[0037] Figure 4 The SDS-PAGE silver staining diagram of the fusion protein after direct assisted folding after protease digestion.

[0038] Figure 5 The SDS-PAGE experimental results of the fusion protein after protease digestion and passing through Ni-NTA column.

[0039] Figure 6 The brazzein passing through Hiload column for purification.

[0040] Figure 7 The results of SDS-PAGE experiments on the collected solution of Brazilian sweet protein after passing it through a molecular sieve.

[0041] Figure 8 Brazilian sweet protein 1 H- 15 N HSQC spectrum. Detailed Implementation

[0042] Exemplary embodiments of the present invention will now be described in detail, which should not be considered as limitations on the present invention, but rather as a more detailed description of certain aspects, features, and implementations of the present invention.

[0043] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. While any methods and materials similar to or equivalent to those described herein may be used in practice or experimentation with this disclosure, preferred materials and methods are described below.

[0044] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be readily apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0045] Example 1: Construction of recombinant expression vector

[0046] Expression vector pETM-44 (with a solubility tag MBP and a purification tag His tag), such as Figure 2 As shown, the protein was purchased from the European Molecular Biology Laboratory (EMBL) (details can be found on the website https: / / grp-pepcore.embl-community.io / vectors / ecoli.html). Following standard molecular cloning procedures, the target sequence was inserted into the NcoI and TliI restriction sites, and the codons were optimized for expression in *E. coli*, resulting in a recombinant expression vector for the fusion protein. The inserted target sequence consisted of a linker sequence and a Brazil glycoprotein sequence, with amino acid sequences of ENLYFQ (SEQ ID NO:1) and QDKCKKVYENYPVSKCQLANQCNYDCKLDKHARSGECFYDEKR NLQCICDYCEY (SEQ ID NO:2), respectively.

[0047] Example 2: Construction of engineered Escherichia coli

[0048] E. coli DH5a and E. coli BL21 were routinely purchased and preserved by the experimenter. The recombinant expression vector of the fusion protein in Example 1 was transformed into E. coli DH5a according to the routine molecular cloning procedure. The plasmid preparation was completed by Kingsway Biotech Co., Ltd. The obtained plasmid dry powder was dissolved in TE buffer at 100 ng / μL and placed in a -20°C refrigerator.

[0049] The obtained plasmid was added to the competent cells (BL21), coated on a plate containing kanamycin sulfate to screen the successfully transformed strains, and well-grown ones were inoculated into 3 mL LB medium, cultured at 37°C with 220 rpm shaking for 12 h, 600 μL of the bacterial solution was injected into a cryotube, 50% glycerol was added according to the volume ratio of bacterial solution to glycerol 3:2, and the recombinant expression strain was obtained and named MBP-Bra and placed in a -80°C refrigerator for preservation.

[0050] Example 3: Expression and preparation of the fusion protein

[0051] 1. Inducing expression

[0052] The recombinant expression strain MBP-Bra glycerol bacteria obtained in the above step was added to 200 mL LB medium, cultured overnight at 37°C with 220 rpm shaking. When the OD 600 was 3-5, part of the bacterial solution was centrifuged at 4000 rpm, 4°C for 30 min, the supernatant was discarded, and the bacterial pellet was resuspended with M9 medium and transferred to 1 L of M9 medium (raw materials were phosphate buffer, NH4Cl, MgSO4, CaCl2, glucose, kanamycin sulfate) and cultured at 37°C with 220 rpm shaking when the OD 15 was 0.1. When the OD 600 of the bacteria grew to 0.6, 0.3 mM IPTG was added and induced at 37°C with 220 rpm for 5 h. After induction, the bacteria were centrifuged at 4200 rpm, 4°C for 30 min, the supernatant was discarded, and the bacterial pellet was stored at -20°C. 600

[0053] 2. Purification of MBP-Bra

[0054] ​The bacteria obtained in step 1 were resuspended in buffer 1 (25 mM phosphate, 100 mM NaCl, 20 mM imidazole, pH = 7.4) with appropriate amount of protease inhibitors, broken by ultrasonic homogenizer, centrifuged at 14000 rpm, 4°C for 30 min, filtered by 0.45 μm filter, and the supernatant was reserved. The Ni-NTA column was equilibrated with buffer 1 (2 mL column volume), and the supernatant was added to the column, and combined at low temperature for 1.5 h. After combination, the column was washed with 5 column volumes of buffer 2 (25 mM phosphate, 100 mM NaCl, 40 mM imidazole, pH = 7.4) to obtain washing solution 1 and washing solution 2. The column was washed with 5 column volumes, 5 column volumes and 5 column volumes of buffer 3 (25 mM phosphate, 100 mM NaCl, 50 mM imidazole, pH = 7.4) to obtain washing solution 3, washing solution 4 and washing solution 5. The column was eluted with 5 column volumes, 5 column volumes and 5 column volumes of buffer 4 (25 mM phosphate, 100 mM NaCl, 500 mM imidazole, pH = 7.4) to obtain elution solution 1, elution solution 2 and elution solution 3. All the collected samples were detected by 12% SDS-PAGE, and the relatively pure target protein was collected from washing solution 4, washing solution 5 and elution solution 1.

[0055] 3. MBP-Bra assisted folding

[0056] (1) Denaturation and reduction

[0057] A solution containing 10 mM EDTA, 50 mM DTT and 6 M guanidine hydrochloride was prepared. The last collected sample in step 2 was mixed with the above prepared solution at room temperature for 2-3 hours to completely denature the protein. The sample was dialyzed against ultrapure water containing acetic acid at pH 4.0 for 4 times for 48 h to obtain a sample free of denaturant and reducing agent.

[0058] (2) Oxidative folding

[0059] 4-5 times the volume of 200 mM Tris, pH 8.0 solution was added to the above sample to assist the correct folding of the protein, thereby obtaining the correctly folded fusion protein.

[0060] The nuclear magnetic resonance spectrum of the product without the assisted folding step is shown in Figure 3 , and the conformation of the fusion protein is inconsistent with the standard product. It can be seen that without the assisted folding step, the fusion protein cannot be correctly folded. In addition, since the molecular weight of the target protein is small, if the assisted folding is performed after the enzyme digestion, it will lead to direct oligomerization, as shown in Figure 4 . It can be seen that the presence of the MBP tag assists the correct folding of the protein.

[0061] Example 4: Preparation of brazzein expression

[0062] 1. MBP-Bra cleavage

[0063] (1) Dialysis

[0064] The correctly folded fusion protein obtained in Example 3 was dialyzed against buffer 5 (50 mM tris, 0.5 mM EDTA, 1 mM DTT, pH = 8.0), the first exchange was performed after 1.5 h, the second exchange was performed after 3 h, and the sample was collected after overnight dialysis and the sample concentration was determined using a NanoDrop.

[0065] (2) Cleavage

[0066] The dialyzed sample was calculated for concentration, and TEV protease was added according to the enzyme concentration: protein concentration = 1:3, and the sample was incubated at room temperature for 3 h. The Ni-NTA column was equilibrated with buffer 6 (25 mM phosphate, 100 mM NaCl) (the column volume was 2 mL), and the cleaved sample was added to the Ni-NTA column and combined at low temperature for 1.5 h. After binding, the flow-through was collected and subjected to 12% SDS-PAGE detection ( Figure 5 ), and the protein purity was calculated to be about 50% according to the results.

[0067] 2. Hiload column purification

[0068] After obtaining the protein collection solution with high purity in Step 1, the solution was concentrated by ultrafiltration with a 3 kDa ultrafiltration tube and then subjected to molecular sieving with buffer 7 (25 mM CH3COOH / CH3COONa, pH = 5.2). The different peak samples were subjected to 12% SDS-PAGE detection ( Figure 5 ), and the peak time corresponding to the target protein was obtained ( Figure 6 ). The purity of the target protein, Brazzein, was about 99%.

[0069] 3. Freeze-drying

[0070] The sample was dialyzed against ultrapure water to remove salt, and the protein collection solution was obtained after 1 day. The protein solution was quantified by measuring the absorbance at 280 nm using a NanoDrop, and freeze-drying was performed to obtain powdered Brazzein. The final yield of Brazzein was 9.89 mg / L of fermentation broth precipitate.

[0071] Example 5: Liquid nuclear magnetic resonance experiment

[0072] Take the purified sample of Brazzein 300 μL obtained in Example 4, adjust the sample concentration to 500 μM, the background buffer is buffer 7, pH 5.2, add 30 μL D2O for field locking, field homogenization and solvent interference reduction at a ratio of 1 / 10 (v / v). After mixing, transfer to a 5 mm normal NMR tube, avoid generating bubbles during the transfer process. Use Agilent 800 MHz NMR spectrometer and low temperature probe (<5mm PFG Triple Resonance Probe, VT, 800NB) to collect the two-dimensional spectrum of protein sample at 37℃. 1 H( 13 C / 15 N)5mm PFG Triple Resonance Probe, VT, 800NB), at 37℃. Collect the two-dimensional spectrum of protein sample at 37℃. 1 H- 15 NHSQC. 15 Nand 1 Hrespectively. Collect 80 and 1024 data points respectively, and perform 8 scans for each time increment. The spectrum width of HSQC spectrum is 11 ppm in 1H axis, and the center is at 4.64 ppm; the spectrum width of 15N axis is 30 ppm, and the center is at 120 ppm. Data processing uses NMR Pipe and Sparky software, and the results are shown in Figure 8 1 H- 15 NHSQC spectrum to analyze the folding accuracy of Brazzein, Figure 8 all resonance cross peaks are uniformly dispersed without overlapping regions, and the peak shape is regular, indicating that the sample 15 Natom is uniformly expanded, and the molecular spatial structure folding is good.

[0073] Example 6: Sweetness detection of Brazzein

[0074] 1. Sweetness detection principle

[0075] The sweetness identification of sweet protein uses blind test experiment, and the blind test sample uses 2% (2g / 100mL) sucrose aqueous solution. The sample to be detected is configured into different concentration gradients and randomly numbered. An evaluation team consisting of 8 people tastes the sample to judge the sample with the same or similar sweetness as 2% sucrose aqueous solution in different concentration gradients. When more than 5 people (60%) in the 8 sensory evaluators think that the sample has no difference in sweetness intensity and taste from the sample, the ratio of sample concentration value to sample concentration value is the sweetness multiple of the sample

[0076] 2. Configuration of different concentration gradient samples of sweet protein: the sample mother liquor concentration is 1 / 10 of the sample sucrose aqueous solution, and is further diluted into 2 times, 4 times, 6 times, 8 times, 10 times, 20 times, 50 times, 100 times, 200 times, 300 times and other different dilution times of the sample to be detected. ​

[0077] 3. Sensory evaluation results show that the sweetness of the Brazilian sweet protein obtained in Example 4 is 200 times that of standard sucrose.

Claims

1. A method for the preparation of Brazzein characterized in that, The method comprises the following steps: a) fermenting an engineered E. coli bacterium comprising a nucleic acid molecule encoding a fusion protein of an amino acid sequence as set forth in SEQ ID NO: 3; b) isolating and recovering the fusion protein from the fermentation broth; c) assisting folding of the fusion protein, wherein the assisting folding comprises denaturing reduction in a solution of 10 mM EDTA, 50 mM DTT and 6 M guanidine hydrochloride, and oxidative folding in a solution of 200 mM Tris, pH 8.0; d) treating the fusion protein obtained in step c) using TEV protease, and e) obtaining brazzein.

2. A stable isotope labeling method characterized by, The method comprises the following steps: a) fermenting an engineered E. coli bacterium comprising a nucleic acid molecule encoding an amino acid sequence as set forth in SEQ ID NO: 3 in a culture medium containing an isotopically labeled feedstock, wherein the isotopically labeled feedstock comprises 15 N-labeled ammonium chloride; b) isolating and recovering the fusion protein from the fermentation broth; c) assisting folding of the fusion protein, the assisting folding comprising denaturing reduction and oxidative folding; d) treating the fusion protein obtained in step c) using a TEV protease, and e) obtaining brazzein.

3. The marking method according to claim 2, characterized in that, The culture medium is M9 medium.

Images