Fusion protein and application thereof in preparation of Brazilian sweet

Abstract

The invention provides a fusion protein and application thereof in preparation of Brazilian sweet. The fusion protein comprises a tag protein and a Brazilian sweet protein which are connected 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 connected with the Brazilian sweet protein. The invention also provides a preparation method of the Brazilian sweet protein. The preparation method comprises the following steps: treating the fusion protein by using TEV protease and obtaining the Brazilian sweet protein. Large-scale expression of the fusion protein and the brazitame protein is realized by utilizing an escherichia coli expression system, and the obtained brazitame protein can be correctly folded and has high sweetness.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a fusion protein and application thereof in the preparation of Brazilian sweet. Background Art

[0002] Brazzein protein is a natural sweet protein isolated from the red fresh fruit of Pentadiplandra brazzeana Baillon, a wild plant in western Africa. Its yield accounts for 0.05-0.2% of the weight of 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 another structure (accounting for 20%) is called des-GluBrazzein, which is the same as the former except that the N-terminus does not contain pyroglutamic acid. Brazzein will form a natural dimer Pentadin, which can also produce sweetness. Brazzein is the single-chain sweet protein with the smallest molecular weight found so far, consisting of 54 amino acids and a molecular weight of 6.5kDa. Brazzein is a hydrophilic polypeptide with good solubility.

[0003] The practical application of the natural sweet protein Brazilian sweet still has many limitations. Brazilian sweet protein is limited by its plant growth conditions and growth cycle, and has defects such as low yield, low extraction rate and high cost. Therefore, the main way to produce this protein is to use genetic engineering to obtain a large amount of recombinant Brazilian sweet protein. At present, the expression research of recombinant Brazilian sweet protein mainly focuses on the prokaryotic expression system based on Escherichia coli and the eukaryotic expression system based on Pichia pastoris. At present, studies have shown that its heterologous expression in Escherichia coli cannot be folded correctly due to the lack of post-processing, and insoluble inclusion bodies will be produced. The expression in Pichia pastoris currently usually selects a promoter that requires methanol induction, and the culture medium conditions are complicated, which brings safety issues to the later application in food.

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

[0005] Advantages of the E. coli expression system: (1) High expression level: It has an efficient protein synthesis mechanism and can quickly produce a large amount of target protein; (2) Economical: E. coli is easy to culture, the culture medium is low-cost, and the culture process is simple; (3) Scalability: The 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 a variety of recombinant vectors, promoters, tags, etc. to choose from, which facilitates customized protein expression experiments.

[0006] Isotope labeling of proteins includes the following processes: constructing a plasmid containing the gene sequence of the target protein, introducing it into the host organism, culturing it in a culture medium containing an isotope marker, expressing the isotope-labeled protein through the host organism, and separating and purifying it using biochemical techniques to obtain high-purity isotope-labeled protein. Common protein isotope labeling methods include chemical labeling and metabolic labeling. Chemical labeling mainly refers to the use of chemical reactions to introduce isotope labels at specific sites of proteins or peptides after collecting protein samples; metabolic labeling refers to adding isotope labels to the culture medium. 15 N or 2 H or other isotopes, after several generations of culture, the protein will be completely labeled with isotopes. Chemical labeling can only replace part of the protein, while metabolic labeling can achieve uniform isotope labeling.

[0007] The MBP tag consists of 367 amino acids and has a molecular weight of 42.5KDa. It 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 technology has a disadvantage that cannot be ignored, that is, the affinity of the amylose matrix currently sold on the market for MBP is too weak, which often leads to too low a yield of purified protein. His tag is a technology commonly used in protein purification and biological research. It involves a string of short peptide sequences composed of histidine, making it easy to capture and identify during expression and purification. Since the His tag can specifically bind to affinity chromatography media chelated with metal ions (such as nickel or cobalt), this eliminates the need to use multiple purification steps to gradually separate proteins, achieves rapid purification, and reduces the complexity of the operation. At the same time, the His tag affinity chromatography method is easy to scale up and is suitable for small-scale laboratory purification to industrial-scale production. Summary of the invention

[0008] As mentioned above, the traditional plant extraction method has problems such as demanding raw material growth environment, limited planting area and low yield, which limits the large-scale production and application of Brazilian sweet. In order to meet market demand, researchers use transgenic technology to introduce Brazilian sweet into different hosts, such as microorganisms and higher plants, in order to obtain a large number of recombinant active Brazilian sweet proteins. However, recombinant Brazilian sweet proteins are mostly expressed in inclusion bodies, which is complicated to purify and its conformation and sweetness characteristics will be affected. The MBP tag can increase the solubility of the overexpressed fusion protein in the prokaryotic expression system and increase its expression level. The His tag can specifically bind to the nickel column to purify the protein. Purification with a nickel column has the advantages of good chemical stability, strong tolerance, not easily interfered by other components in the supernatant after bacterial cell crushing, and simple sample pretreatment. Brazilian sweet carrying a His-MBP tag can achieve faster and more convenient purification. However, whether the MBP tag and the target protein fragment can be cleaved by TEV enzymes and whether the conformation and sweetness of the protein during purification are affected remain to be solved.

[0009] In order to solve the above-mentioned problems of the prior art, the present invention uses genetic engineering technology to utilize an Escherichia coli expression system to recombinantly express brazilian sweet protein, so that it has high sweetness, high purity, and high yield, while simplifying the protein purification method. In addition, the present invention has developed a stable isotope labeling method for brazilian sweet, laying the foundation for the subsequent use of nuclear magnetic resonance (NMR) to study protein interactions and protein structure. Traditional Escherichia coli expression may lead to incorrect protein expression and folding. Through genetic engineering and stable isotope labeling technology, the large-scale expression of brazilian sweet is achieved using an Escherichia coli expression system, and the ordinary NH4Cl in the M9 culture medium is replaced with the isotope NH4Cl. 15 NH4Cl, isotope labeling of Brazilian sweet, using 1 H- 15 N HSQC spectrum, compared with the standard spectrum to determine whether the protein is correctly folded, nuclear magnetic resonance (NMR) confirmed that the Brazilian sweet conformation is correct. After sensory evaluation, it was confirmed that the Brazilian sweet protein with the correct conformation has a high sweetness.

[0010] The first aspect of the present invention provides a fusion protein comprising a tag protein and brazilian tamanin connected by a linker; wherein the linker comprises an amino acid sequence as shown in SEQ ID NO:1 (ENLYFQ). The amino acid sequence as shown in SEQ ID NO:1 is directly connected to the brazilian tamanin. Therefore, the sequence forms a special enzyme cleavage site (ENLYFQ / Q) with the first amino acid residue Q of brazilian tamanin. The inventors have made a groundbreaking discovery that the TEV enzyme can be precisely cut at this special site, thereby obtaining a complete brazilian tamanin with the tag protein and linker precisely removed. In some embodiments, the linker may also include an additional sequence before the sequence ENLYFQ, which does not affect the expression and folding of the fusion protein, nor does it affect the precise cutting of the TEV enzyme at ENLYFQ / Q. For example, the amino acid sequence of the linker comprising the additional sequence may be as shown in SEQ ID NO:7.

[0011] As used herein, a tag protein refers to a polypeptide or protein sequence fused and expressed with a target protein, which is usually covalently bound to the target protein to facilitate detection, purification, positioning or functional studies of the target protein. Tag proteins can be used for purification, and corresponding affinity chromatography techniques can be used to purify the target protein through specific affinity tags, such as His tags, GST tags, FLAG tags, etc.; tag proteins can also be used to increase solubility, and through some tag proteins, such as MBP (maltose binding protein) and GST (glutathione S-transferase), the soluble expression of the target protein in host cells can be improved; tag proteins can also improve the stability of the target protein and reduce aggregation or degradation. Tag proteins can also be used for detection and quantification, and the expression and positioning of the target protein can be observed in real time in living cells using fluorescent tag proteins (such as GFP and its derivatives) or enzyme tags (such as luciferase); tag proteins can also be used as reporter genes, 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 a His tag protein, an MBP tag protein, a linker, and Brazilian sweet protein from the N-terminus to the C-terminus. In some specific embodiments, the amino acid sequence of the His tag protein is shown in SEQ ID NO:5. In some specific embodiments, the amino acid sequence of the MBP tag protein is shown in SEQ ID NO:6. In some specific embodiments, the amino acid sequence of the linker comprising an additional sequence is shown in SEQ ID NO:7.

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

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

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

[0016] The fourth aspect of the present invention provides a host cell, which comprises the nucleic acid molecule as described in the present invention or the recombinant plasmid as described in the present invention; preferably, the host cell is Escherichia coli; more preferably, the Escherichia coli is selected from DH5α and BL21.

[0017] The fifth aspect of the present invention provides a method for constructing an engineered Escherichia coli bacterium, which comprises the step of transforming the recombinant plasmid of the present invention into an Escherichia coli strain to obtain the engineered Escherichia coli bacterium.

[0018] The sixth aspect of the present invention provides an engineered Escherichia coli obtained according to the construction method described in the fifth aspect of the present invention.

[0019] The seventh aspect of the present invention provides a method for preparing a fusion protein, wherein the fusion protein is the fusion protein described in the first aspect of the present invention, and the method comprises fermenting the engineered Escherichia coli bacteria described in the sixth aspect of the present invention and separating and recovering the fusion protein from the fermentation broth.

[0020] In some embodiments, the fermentation includes a bacterial enrichment stage and an induced expression stage, and the induced expression of the fusion protein is achieved by adding galactose to the culture medium. In some embodiments, the separation and recovery includes 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 invention provides a method for preparing brasilien, which comprises treating the fusion protein described in the first aspect of the present invention or the fusion protein obtained by the method described in the seventh aspect of the present invention with TEV protease, and obtaining brasilien.

[0022] In the present invention, the fusion protein is correctly folded. In some embodiments, the correct folding of the fusion protein is achieved by auxiliary folding. The auxiliary folding includes denaturation reduction and oxidation folding. The denaturation reduction includes mixing the fusion protein with a solution containing EDTA, DTT and guanidine hydrochloride and dialyzing with ultrapure water containing acetic acid. The oxidation folding includes mixing several times the volume of Tris solution with the dialyzed product. In some embodiments, the auxiliary folding occurs before TEV protease treatment.

[0023] Considering the safety of sweet protein consumption and the retention of sweetness, the linker and tag protein used in the present invention need to be completely removed during the preparation of the sweet protein. The ideal state is that when the fusion protein is subsequently truncated with a protease, the linker and the sweet protein can be separated, but in actual operation, it is usually difficult to ensure accurate excision. For example, the cleavage site of TEV protease is ENLYFQ / G(S), but the first amino acid residue of Brazilian sweet protein is not G or S, so in theory, it is impossible to use TEV protease to achieve accurate excision. Introducing the cleavage site of the complete TEV protease will introduce the terminal amino acid residue of the cleavage site into the resulting sweet protein.

[0024] Unexpectedly, the inventors found that TEV protease also has cleavage efficiency on the cleavage site of ENLYFQ / Q. The present invention connects the tag protein and brazilian tamanin through a linker (ENLYFQ, SEQ ID NO: 1), thereby forming the cleavage site of ENLYFQ / Q, achieving complete and accurate removal of the linker and tag protein, and retention of intact brazilian tamanin.

[0025] In some embodiments, the enzymatic cleavage product is separated by affinity chromatography corresponding to the tag protein to obtain brazilian tamanin. For example, a Ni-NTA column corresponding to the His tag is used, and the flow-through is brazilian tamanin. In some embodiments, the flow-through product is further purified using a molecular sieve (e.g., a Hiload column) to obtain high-purity brazilian tamanin.

[0026] A ninth aspect of the present invention provides a stable isotope labeling method, which comprises fermenting and culturing the Escherichia coli engineered bacteria according to the present invention 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 culture 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 with TEV protease and obtaining brazilian sweet protein.

[0027] The tenth aspect of the present invention provides a fusion protein with an isotope stable label, which is obtained according to the labeling method described in the ninth aspect of the present invention. The present invention also provides a brasil protein with an isotope stable label, which is obtained according to the labeling method described in the ninth aspect of the present invention.

[0028] The eleventh aspect of the present invention provides the use of fusion proteins and / or brazilian tamarind proteins having isotope stable labels in studying protein interactions and / or protein structures; preferably, the research is carried out using nuclear magnetic resonance.

[0029] The beneficial effects of the present invention are: constructing a fusion protein and realizing large-scale expression of the fusion protein and brazilian tamanin by using an E. coli expression system. The fusion protein comprises a tag protein and brazilian tamanin connected by a linker. The linker can be recognized and cleaved by TEV protease, realizing complete and accurate removal of the tag protein and the linker and complete retention of brazilian tamanin. 1 H- 15 N HSQC spectrum confirmed the correct folding of the fusion protein and brazilian thauma protein. Sensory evaluation confirmed that the brazilian thauma protein obtained had a high sweetness.

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

[0031]

[0032]

[0033] BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 Schematic diagram of the process for preparing Brazilian sweet protein.

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

[0036] Figure 3 Misfolded fusion protein 1 H- 15 N HSQC spectrum.

[0037] Figure 4 This is the SDS-PAGE silver staining image after direct assisted folding after fusion proteinase cleavage.

[0038] Figure 5 The results of SDS-PAGE experiments after fusion protein cleavage and passage through a Ni-NTA column.

[0039] Figure 6 Brazilian sweet protein was purified by Hiload column.

[0040] Figure 7 These are the results of SDS-PAGE experiments on the collected solution after Brazilian sweet protein passed through molecular sieves.

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

[0042] Exemplary embodiments of the present invention will now be described in detail, which should not be considered as limiting the present invention but rather should be understood as a more detailed description of certain aspects, features, and embodiments 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 the present disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, preferred materials and methods are described below.

[0044] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0045] Example 1: Construction of recombinant expression vector

[0046] The expression vector pETM-44 (with a solubility tag MBP tag and a purification tag His tag) is as follows: Figure 2 As shown, it was purchased from the European Molecular Biology Laboratory EMBL (detailed information can be found on the website https: / / grp-pepcore.embl-community.io / vectors / ecoli.html). According to the conventional molecular cloning process, the target sequence was inserted into the NcoI and TliI restriction sites, the codons were optimized, suitable for the expression of Escherichia coli, and the recombinant expression vector of the fusion protein was obtained. The inserted target sequence includes the linker sequence and the brazilian sweet protein sequence in turn, and the amino acid sequences are ENLYFQ (SEQ ID NO: 1) and QDKCKKVYENYPVSKCQLANQCNYDCKLDKHARSGECFYDEKR NLQCICDYCEY (SEQ ID NO: 2), respectively.

[0047] Example 2: Construction of engineered Escherichia coli

[0048] E. coli DH5α and E. coli BL21 were purchased and stored by the experimenters. According to the conventional molecular cloning process, the recombinant expression vector of the fusion protein in Example 1 was transformed into E. coli DH5α. The plasmid was prepared by GenScript 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 competent cells (BL21), and the successfully transformed strains were screened on plates containing kanamycin sulfate. The strains with good growth were selected and inoculated into 3 mL LB medium. The culture was shaken at 37°C and 220 rpm for 12 h. 600 μL of the bacterial solution was injected into a cryopreservation tube, and 50% glycerol was added according to the volume ratio of bacterial solution to glycerol of 3:2 to obtain the recombinant expression strain, which was named MBP-Bra and stored in a -80°C refrigerator.

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

[0051] 1. Inducible expression

[0052] Add the recombinant expression strain MBP-Bra obtained in the previous step to 200 mL of LB medium and culture overnight in a shaker at 37°C and 220 rpm. 600 After 3-5 min, take part of the bacterial solution and centrifuge at 4000 rpm, 4 ° C, 30 min, pour out the supernatant to obtain the bacterial precipitate, resuspend it in M9 medium, and transfer it to 1L M9 medium (raw material is phosphate buffer, 15 NH4Cl, MgSO4, CaCl2, glucose, kanamycin sulfate), at OD 600 When the OD of the bacteria reached 0.1, the culture was carried out in a shaking incubator at 37°C and 220 rpm. 600 When the cell number reaches 0.6, 0.3 mM IPTG is added and induced at 37°C, 220 rpm for 5 h. After induction, centrifuge at 4200 rpm, 4°C, 30 min, discard the supernatant, obtain the bacterial pellet, and store it at -20°C.

[0053] 2. Purification of MBP-Bra

[0054] The bacterial cells obtained in step 1 were treated with buffer 1 (25mM phosphate, 100mM NaCl, 20mM imidazole, pH = 7.4), and an appropriate amount of protease inhibitors were added, the bacterial cells were resuspended, and the cells were broken with an ultrasonic disruptor, centrifuged at 14000rpm, 4°C, 30min, filtered with a 0.45μm filter membrane, and the supernatant was retained. The Ni-NTA column was balanced with buffer 1 (column volume was 2mL), the supernatant was added to the Ni-NTA column, placed in a low temperature oscillation combination for 1.5h, and after the combination, 5 column volumes and 5 column volumes of buffer 2 (25mM phosphate, 100mM NaCl, 40mM imidazole, pH = 7.4) were used to wash the impurities to obtain cleaning solution 1 and cleaning solution 2. The impurities were 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 solutions 3, 4, and 5. The impurities were washed 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 eluent 1, 2, and 3. All collected samples were subjected to 12% SDS-PAGE detection, and the relatively pure target protein was collected from washing solutions 4, 5, and 1 and then combined.

[0055] 3. MBP-Bra assisted folding

[0056] (1) Denaturation reduction

[0057] Prepare a solution containing 10mM EDTA, 50mM DTT and 6M guanidine hydrochloride. Take the last sample collected in step 2 and mix it with the above solution at room temperature for 2-3 hours to completely denature the protein. Dialyze it 4 times in ultrapure water with a pH of 4.0 containing acetic acid for 48 hours to obtain a sample free of denaturants and reducing agents.

[0058] (2) Oxidative folding

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

[0060] The NMR spectrum of the product without auxiliary folding step is as follows Figure 3 As shown in the figure, the conformation of the fusion protein is inconsistent with that of the standard. It can be seen that without the auxiliary folding of this step, the fusion protein cannot be folded correctly. In addition, since the molecular weight of the target protein is small, if the auxiliary folding is performed after enzyme cleavage, it will directly oligomerize, such as Figure 4 As shown, it can be seen that the presence of the MBP tag helps the protein to fold correctly.

[0061] Example 4: Expression and preparation of Brazilian sweet

[0062] 1. MBP-Bra digestion

[0063] (1) Dialysis

[0064] The correctly folded fusion protein obtained in Example 3 was dialyzed with buffer 5 (50 mM tris, 0.5 mM EDTA, 1 mM DTT, pH = 8.0). The first solution was changed after 1.5 hours and the second solution was changed after 3 hours. After overnight dialysis, the samples were collected and the sample concentration was determined using NanoDrop.

[0065] (2) Enzyme digestion

[0066] After dialyzed samples, the concentration was calculated, and TEV protease was added according to the condition of enzyme concentration: protein concentration = 1:3. The enzyme digestion was carried out at room temperature for 3 hours. The Ni-NTA column was balanced with buffer 6 (25mM phosphate, 100mM NaCl) (column volume was 2mL), and the digested samples were added to the Ni-NTA column and placed in low temperature shaking for 1.5 hours. After binding, the flow-through was collected for 12% SDS-PAGE detection ( Figure 5 ), based on which the protein purity was calculated to be approximately 50%.

[0067] 2. Hiload column purification

[0068] After obtaining a high purity protein collection solution in step 1, the protein was concentrated by ultrafiltration using a 3 kDa ultrafiltration tube and then passed through a molecular sieve. The molecular sieve was buffer 7 (25 mM CH3COOH / CH3COONa, pH = 5.2). Samples of different peaks were subjected to 12% SDS-PAGE detection ( Figure 5 ), and obtain the peak time corresponding to the target protein ( Figure 6 ) to obtain the target protein with a purity of about 99%, namely Brazilian sweet protein.

[0069] 3. Freeze Drying

[0070] The solution was dialyzed with ultrapure water to remove salt, and the protein solution was taken out after 1 day. The protein solution was quantified by measuring the absorbance of the solution at 280 nm using NanoDrop, and freeze-dried to obtain powdered brazilian sweet protein. The final yield of brazilian sweet was 9.89 mg / L fermentation liquid precipitate.

[0071] Example 5: Liquid NMR Experiment

[0072] Take 300 μL of the purified sample of brasilien obtained in Example 4, adjust the sample concentration to 500 μM, the background buffer is buffer 7, pH 5.2, and add 30 μL of D2O at a ratio of 1 / 10 (v / v) for field locking, field balancing and reducing solvent interference. After mixing, transfer to a 5 mm ordinary NMR tube, and avoid generating bubbles during the transfer process. Agilent 800 MHz NMR nuclear magnetic resonance spectrometer and low temperature probe ( 1 H( 13 C / 15 N) 5mm PFG Triple Resonance Probe, VT, 800NB), protein samples were collected at 37°C 1 H- 15 NHSQC 2D spectrum. 15 N and 1 The H dimension collected 80 and 1024 data points respectively, and 8 scans were performed at each time increment. The spectral width of the HSQC spectrum on the 1H axis is 11ppm, centered at 4.64ppm; the spectral width on the 15N axis is 30ppm, centered at 120ppm. The data were processed using NMR Pipe and Sparky software, and the results are shown below. Figure 8 As shown. 1 H- 15 NHSQC spectra were used to analyze the folding accuracy of brazilian sweet protein. Figure 8 All the resonance cross peaks in the sample are evenly dispersed, without overlapping areas, and the peak shapes are regular, indicating that the sample 15 The N atoms are spread out evenly and the molecular spatial structure is well folded.

[0073] Example 6: Sweetness Detection of Brazilian Thaumatin

[0074] 1. Sweetness detection principles

[0075] The sweetness of sweet protein is identified by blind test. The standard sample of the blind test is 2% (2g / 100mL) sucrose aqueous solution. The samples of sweet protein to be tested are configured into different concentration gradients and randomly numbered. An evaluation team of 8 people tastes the samples and judges the samples with the same or similar sweetness as the 2% sucrose aqueous solution in the samples with different concentration gradients. When more than 5 (60%) of the 8 sensory evaluators believe that the sweetness intensity and taste of the sample are no different from those of the standard sample, the ratio of the concentration of the standard solution to the concentration of the sample is the sweetness multiple of the sample.

[0076] 2. Preparation of different concentration gradient samples of sweet protein: The concentration of the sample mother solution is 1 / 10 of the standard sucrose aqueous solution, and it is further diluted with distilled water to form test solutions with different dilution multiples such as 2 times, 4 times, 6 times, 8 times, 10 times, 20 times, 50 times, 100 times, 200 times, and 300 times.

[0077] 3. The sensory evaluation results showed that the sweetness of the Brazilian tamarind obtained in Example 4 was 200 times that of standard sucrose.

Claims

1. A fusion protein, characterized in that The fusion protein comprises a tag protein and brazilian tamanin connected 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 connected to the brazilian tamanin.

2. The fusion protein according to claim 1, characterized in that The tag protein comprises an MBP tag protein.

3. The fusion protein according to claim 1, characterized in that 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.

4. The fusion protein according to claim 1, characterized in that The amino acid sequence of the fusion protein is shown in SEQ ID NO:

3.

5. A nucleic acid molecule, characterized in that The nucleic acid molecule encodes the fusion protein according to any one of claims 1 to 4.

6. The nucleic acid molecule according to claim 5, characterized in that The nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO:

4.

7. A recombinant plasmid, characterized in that: The recombinant plasmid comprises the nucleic acid molecule according to claim 5 or 6.

8. A host cell, characterized in that The host cell comprises the nucleic acid molecule according to claim 5 or 6 or the recombinant plasmid according to claim 8; Preferably, the host cell is Escherichia coli.

9. A method for constructing an engineered Escherichia coli, characterized in that: The method comprises the step of transforming the recombinant plasmid as claimed in claim 7 into an Escherichia coli strain to obtain the engineered Escherichia coli bacteria.

10. An engineered Escherichia coli obtained according to the construction method according to claim 9.

11. A method for preparing a fusion protein, wherein the fusion protein is the fusion protein according to any one of claims 1 to 4, characterized in that: The method comprises fermenting the engineered Escherichia coli bacteria as claimed in claim 10 and separating and recovering the fusion protein from the fermentation liquid.

12. A method for preparing Brazilian tamarind, characterized in that: The method comprises treating the fusion protein of any one of claims 1 to 4 or the fusion protein obtained by the method of claim 11 with TEV protease, and obtaining brazilian tamarind.

13. A stable isotope labeling method, characterized in that: The method comprises fermenting and culturing the Escherichia coli engineered bacteria according to claim 10 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.

14. The marking method according to claim 13, characterized in that: The method also includes separating and recovering the fusion protein from the fermentation broth.

15. The marking method according to claim 14, characterized in that: The method further comprises treating the fusion protein with TEV protease and obtaining brasilin.

16. A fusion protein with an isotope stable label, obtained according to the labeling method of claim 14.

17. Brazilian thauma protein with isotope stable labeling, obtained according to the labeling method according to claim 15.

18. Use of the isotope-stable labeled fusion protein according to claim 16 and / or the isotope-stable labeled brazilian tamarind protein according to claim 17 in studying protein interactions and / or protein structures; preferably, the study is performed using nuclear magnetic resonance.

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