Expression and purification of trichocaulin hts in escherichia coli

Abstract

The application relates to the field of sweeteners, in particular to a method for expressing and purifying truffle sweet protein HTS in Escherichia coli. A recombinant expression vector coding truffle sweet protein HTS is introduced into a competent cell of Escherichia coli for protein induction expression; the recombinant expression vector coding truffle sweet protein HTS comprises a basic vector and a truffle sweet protein HTS coding gene inserted into the basic vector; the amino acid sequence of the truffle sweet protein HTS is shown in SEQ ID NO: 3. The application limits the conditions such as the concentration of an inducer, induction time and induction temperature, centrifugal collection of bacterial liquid after induction expression, ultrasonic crushing treatment and collection of supernatant, and through affinity chromatography and gel chromatography, the prokaryotic expression and purification of truffle sweet protein HTS can be realized, and a large amount of truffle sweet protein HTS with a purity of more than 95% can be obtained within two days, and the truffle sweet protein HTS is easy to mass-produce.

Description

Technical Field

[0001] This application relates to the field of sweeteners, and more specifically to a method for expressing and purifying truffle sweet protein HTS in Escherichia coli. Background Technology

[0002] Sweeteners are essential in daily life, and the demand for zero-calorie, natural, and safe sweetener alternatives continues to grow. However, excessive intake of nutritional sweeteners such as sucrose and high-fructose corn syrup is directly linked to diet-related health problems such as obesity, diabetes, cardiovascular disease, metabolic syndrome, and tooth decay. The food industry urgently needs to develop new sweeteners that can perfectly mimic the sweetness of sucrose without unpleasant aftertastes to replace traditional sugars and artificial sweeteners.

[0003] Truffles Truffle Truffles (Ascomycota) are underground fruiting bodies of fungi belonging to the class Discomycetes, and are a rare type of ectomycorrhizal fungus. In vitro digestibility, allergenicity, and toxicity were assessed through predictive testing; they were found to be non-allergenic, non-toxic, and easily digestible, meeting food-grade safety standards. Some scholars have studied Hungarian sweet truffles (… Mattirolomyces terfezioides Honey Truffle Sweetener (HTS), a sweet protein with a sweetness 1000-2000 times that of sucrose, was discovered and identified in [a study]. It consists of 121 amino acids and has a molecular weight of approximately 13 kDa. Due to the rarity of wild Hungarian sweet truffles, the extreme difficulty in artificial cultivation, the long growth cycle, and the extremely low yield, it is impossible to meet the needs of large-scale production. Furthermore, the complex purification process poses significant challenges to the industrial application of HTS. According to reports, although HTS has been expressed secreted in Pichia pastoris, the fermentation cycle is long, the induction conditions are harsh, and the cost of large-scale production is high.

[0004] Therefore, developing methods for the expression and homozygosity of HTS sweet proteins in Escherichia coli can overcome the bottleneck of sweet protein industrialization and fill the gap in industrial production technology of fungal sweet proteins, which has important technical and commercial value. Summary of the Invention

[0005] To address or partially address the technical shortcomings of existing technologies in expressing truffle sweet protein HTS in Escherichia coli, such as easy formation of inclusion bodies, low solubility, complex purification processes, and inability to achieve large-scale production, this study provides an efficient, low-cost, easily scalable, and high-purity method for expressing and purifying truffle sweet protein HTS in Escherichia coli. This method aims to achieve industrial-scale production of HTS and promote its widespread application in the food, health product, and pharmaceutical industries.

[0006] On the one hand, this application provides a recombinant Escherichia coli engineered strain containing pET-28a-HTS, wherein pET-28a-HTS is a gene encoding truffle sweet protein HTS inserted into the NcoI and HindIII sites of pET-28a(+); the nucleotide sequence of the HTS encoding gene is shown in SEQ ID NO.1.

[0007] Furthermore, the HTS encoding gene matches the restriction site of pET-28a(+), and the recombinant expression vector pET-28a-HTS is constructed by double restriction and ligation.

[0008] On the other hand, this application also provides a method for inducing truffle sweet protein HTS in Escherichia coli, the induction method comprising: 1) The above-mentioned recombinant Escherichia coli engineered strain was inoculated into LB liquid medium for seed culture, and then autoclaved to obtain seed culture; 2) The seed culture obtained in 1) is inoculated into LB liquid medium for scale-up culture; 3) Induction of expression: IPTG was added to the logarithmic phase bacterial culture to a final concentration of 0.1-1.0 mM and cultured for a period of time to obtain fermentation broth containing HTS protein; 4) Collection of bacterial culture: After centrifuging the fermentation broth containing HTS protein obtained in step 3) at low temperature, collect the precipitated bacterial cells, discard the supernatant, and obtain the recombinant Escherichia coli engineered strain containing pET-28a-HTS. The LB liquid culture medium is formulated as follows: 10 g / L tryptone, 5 g / L yeast extract, and 10 g / L NaCl.

[0009] Furthermore, the seed culture temperature is 37℃, the time is 10-16h, and the frequency is 180-220r / min.

[0010] Furthermore, the expanded culture involves inoculating the seed culture at a volume ratio of 1:10-100 into 500-1000 mL of LB liquid medium and incubating at 37°C with shaking at 180-220 r / min until the bacterial culture reaches its OD value. 600 The value reaches 0.4-1.0.

[0011] Furthermore, the continued culture temperature is 10-36℃, the frequency is 180-220 r / min, and the time is 12-30h.

[0012] On the other hand, this application also provides a method for isolating and purifying truffle sweet protein HTS in Escherichia coli. The method uses the above-mentioned recombinant Escherichia coli engineered strain containing pET-28a-HTS as raw material, and performs a combination of ultrasonic disruption, affinity chromatography and gel filtration chromatography in sequence to obtain a high-purity HTS protein solution, which is then concentrated until the protein concentration is 20-30 mg / mL.

[0013] Furthermore, the mass-to-volume ratio of the recombinant Escherichia coli engineered strain containing pET-28a-HTS to the lysis buffer in the ultrasonic disruption is 1:10-1:100 (g / mL), the power is 100-600W, the operation time is 1-5s with an interval of 1-5s, and the total disruption time is 10-30 min. After disruption, the mixture is centrifuged at 4℃ and 12000r / min for 30-60 min, and the supernatant containing soluble HTS protein is collected.

[0014] Furthermore, the affinity chromatography involves passing the supernatant containing soluble HTS protein through a Ni-NTA affinity chromatography column and collecting the corresponding components to obtain an HTS protein solution with a purity of over 90%.

[0015] Furthermore, after eluting the HTS protein solution obtained by affinity chromatography using gel filtration chromatography, a single elution peak component is collected at 10-100 mL to obtain the purified HTS protein.

[0016] Beneficial effects 1. This application constructs a recombinant expression vector for the truffle sweet protein HTS in Escherichia coli and common engineered strains. By combining His tag fusion expression and low-temperature induction, the technical problems of HTS easily forming inclusion bodies and low soluble expression level in Escherichia coli are solved. After optimizing the induction conditions, the soluble expression level of HTS can reach more than 35% of the total bacterial protein, which is significantly higher than the expression level of existing plant / yeast expression systems.

[0017] 2. The expression and purification process designed in this application adopts a combination of "ultrasonic disruption-affinity chromatography-gel filtration chromatography", which realizes two-step purification. The process is simple, easy to operate, and low in cost. Affinity chromatography can quickly remove most of the impurity proteins, and gel filtration chromatography can further remove trace impurity proteins. The purity of the HTS protein obtained can reach more than 95%, and the protein activity is stable.

[0018] 3. This application uses Escherichia coli as the expression host. Compared with plant expression systems, the culture cycle is short (only 2 days in total) and is not affected by environmental factors. Compared with eukaryotic expression systems such as Pichia pastoris, the fermentation cost is low, the operation is simple, and it is easy to scale up. It is suitable for industrial mass production, which greatly reduces the total production cost of HTS and breaks through the bottleneck of the scarcity of naturally obtained HTS and the inability of existing expression systems to produce it on a large scale.

[0019] 4. The HTS protein expressed and purified in this application retains its natural sweetness, is non-allergenic, non-toxic, and easily digestible, and has good thermal and acid-base stability. It is suitable for high-temperature processing in the food industry (such as baking and sterilization) and can be widely used in the fields of flavoring beverages, candies, baked goods, dairy products, health products, and pharmaceuticals. It has extremely high commercial value and application prospects.

[0020] 5. This application fills the technical gap in the efficient expression and purification of fungal sweet protein HTS in Escherichia coli. The constructed recombinant expression vector and engineered strain can be stably passaged, and the expression and purification process is highly reproducible, providing effective technical reference and theoretical support for the subsequent industrial development of HTS and the expression of related sweet proteins in Escherichia coli.

[0021] It should be understood that the above general description and the following detailed description are exemplary and explanatory only, and do not limit this application. Attached Figure Description

[0022] The above and other objects, features and advantages of this application will become more apparent from the more detailed description of exemplary embodiments thereof in conjunction with the accompanying drawings.

[0023] Figure 1 The flowchart of this application scheme.

[0024] Figure 2 The chromatogram is for HTS protein affinity and gel chromatography purification.

[0025] Figure 3 This is a schematic diagram illustrating the construction of the recombinant expression vector pET-28a-HTS.

[0026] Figure 4 SDS-PAGE electrophoresis images for HTS protein expression and purification; where, 1: before induction; 2: after induction; 3: cell disruption supernatant (small-scale test); 4: cell disruption precipitate; 5: before disruption (large bottle); 6: after disruption; 7: cell disruption supernatant; 8: cell disruption precipitate; 9: flow through nickel column; 10: after 10% elution; 11: 10% elution result; 12: 60% elution result; 13: after 100% elution. Detailed Implementation

[0027] Embodiments of this application will now be described in more detail with reference to the accompanying drawings. While embodiments of this application are shown in the drawings, it should be understood that this application may be implemented in various forms and should not be limited to the embodiments set forth herein. Rather, these embodiments are provided to make this application more thorough and complete, and to fully convey the scope of this application to those skilled in the art.

[0028] Currently discovered sweet proteins are all plant-derived. HTS is the first high-sweetness sweet protein derived from fungi, filling a technological gap in fungal-derived sweet proteins. It provides a pure, natural, and aftertaste-free sweetness, and current research indicates it lacks bitterness and metallic taste, with a flavor close to sucrose, demonstrating significant industrial application potential. *E. coli*, as a prokaryotic expression host, offers advantages such as a clear genetic background, high expression levels, short culture cycles, simple protein purification processes, low production costs, ease of operation, and large-scale production, making it the preferred choice for industrial recombinant protein production. To date, there is no mature *E. coli* expression and purification protocol for HTS. Achieving rapid expression and purification in *E. coli* will accelerate the industrialization and application of HTS. In summary, HTS sweet protein, with its core advantages of natural high sweetness and safety, represents an important technological route in the field of sugar substitutes. Its development, moving from natural extraction to recombinant expression and molecular modification, is gradually overcoming cost and stability bottlenecks, becoming a crucial direction for future food sweeteners.

[0029] Unless otherwise specified, all reagents and materials used in the following examples were purchased from the market.

[0030] Example 1: Construction of the recombinant expression vector pET-28a-HTS; The expression vectors used are common pET series vectors, such as pET-21b(+), pET-28a(+), and pET-32a(+). Taking pET-28a(+) as an example, in... NcoI and HindⅢ The coding gene for the truffle sweet protein HTS was inserted at the site, and the nucleotide sequence of the HTS coding gene is shown in SEQ ID NO.1. The 3' end of the HTS coding gene is connected to a His tag coding sequence and a stop codon TAA. The HTS coding gene matches the restriction enzyme sites of the base vector. The recombinant expression vector pET-28a-HTS was constructed by double digestion and ligation. The His tag coding sequence is a six-histidine (His6) coding sequence, used for subsequent affinity purification of the HTS protein, and does not affect the sweetness activity or spatial conformation of the HTS protein.

[0031] SEQ ID NO:1 MPDLSSFITIKNNSNHVFTRTAIYSKYAAVQWSPEPQLSISPGKWDLFILKDILSIRGTSGYVQYRVGDGPGWVRVTFSSLVGADEVAEWSSGDLPDGFVLQKPVRTGSRPLQATFEATKQ Example 2: Induction and optimization of HTS protein expression conditions in Escherichia coli; 1. A recombinant Escherichia coli engineered strain containing the above expression vector Common expression strains of recombinant Escherichia coli (such as BL21 and Rosetta) were used as host strains. The recombinant expression vector obtained in Example 1 was introduced into competent cells by heat shock transformation. After resistance screening, PCR identification and sequencing verification, a recombinant Escherichia coli strain that could stably express truffle sweet protein HTS was obtained and named HTS-His.

[0032] 2. Expression method of truffle sweet protein HTS in Escherichia coli 1) Seed culture: Inoculate recombinant Escherichia coli into 1-5 mL LB liquid medium and culture at 37℃ with shaking at 180-220 r / min for 10-16 h; the formula of the LB liquid medium is: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L NaCl, pH adjusted to 7.0-7.2, and autoclaved at 121℃ and 0.1 MPa for 20 min before use.

[0033] 2) Scale-up culture: Inoculate the seed culture at a volume ratio of 1:50 into 500-1000 mL of LB liquid medium and culture at 37℃ with shaking at 180-220 r / min until the bacterial OD of the culture is reached. 600 The value reaches 0.4-1.0; 3) Induction of expression: Add IPTG (isopropyl-β-D-thiogalactoside) to the logarithmic phase bacterial culture to a final concentration of 0.1-1.0 mM, adjust the culture temperature to 10-36℃, and induce expression by shaking at 180-220 r / min for 12-30 h to obtain fermentation broth containing HTS protein; 4) Collection of bacterial culture: Centrifuge the fermentation broth at 4℃ and 6000-8000 r / min for 10-20 min, collect the precipitated bacterial cells, and discard the supernatant.

[0034] Example 3: Purification and purity detection of HTS protein; 1. Purification methods for HTS protein The purification method uses bacterial cell precipitates containing HTS protein as raw materials and employs a combined process of "ultrasonic disruption-affinity chromatography-gel filtration chromatography," specifically including the following steps: 1) Ultrasonic disruption: The bacterial cells were resuspended in lysis buffer at a mass-to-volume ratio of 1:10 to 1:100 (g / mL) of lysis buffer (60mM phosphate buffer, 200mM NaCl, pH 7.0). The cells were ultrasonically disrupted under ice bath conditions. The ultrasonic parameters were: power 100-600W, working time 1-5s, interval 1-5s, total disruption time 10-30 min. After disruption, the cells were centrifuged at 4℃ and 12000r / min for 30-60 min, and the supernatant (containing soluble HTS protein) was collected. 2) Affinity chromatography: Pass the supernatant through a Ni-NTA affinity chromatography column. First, equilibrate the column with equilibration buffer (60mM phosphate buffer, 200mM NaCl, pH 7.0) until the baseline is stable. Load the sample at a rate of 1-3 mL / min. After loading, wash the column with washing buffer (60mM phosphate buffer, 200mM NaCl, 500mM imidazole, pH 7.0) to remove impurities. Then, elute the HTS protein with elution buffer (60mM phosphate buffer, 200mM NaCl, pH 7.0), collect the corresponding fractions, and obtain an HTS protein solution with a purity of over 90%. The elution conditions for affinity chromatography are as follows: elute buffer at a rate of 3 mL / min, and collect the eluted fraction with an absorbance value greater than 50 at a wavelength of 280 nm.

[0035] 3) Gel filtration chromatography: The HTS protein solution obtained by affinity chromatography was concentrated to 2-5 mL and loaded onto a HiLoad 16 / 600 Superdex 75 prep grade gel filtration chromatography column. Elution was performed with elution buffer (60 mM phosphate buffer, 200 mM NaCl, pH 7.0, the same as in step 2 above). A single elution peak was collected at 80 mL to obtain high-purity HTS protein. The elution rate of gel filtration chromatography was 1.0 mL / min. Finally, the HTS protein solution was detected and collected by SDS-PAGE electrophoresis.

[0036] 4) Protein concentration and preservation: Concentrate the high-purity HTS protein solution to a protein concentration of 20-30 mg / mL through an ultrafiltration centrifuge tube, and store at -80 ℃ for later use.

[0037] 2. Purity testing and result analysis Protein purity was determined by SDS-PAGE electrophoresis, and its absorbance was measured at 280 nm according to the formula. Where c is the concentration, A is the UV absorption at 280 nm, ε is equal to 27960, and L is 1, and then the HTS protein concentration is calculated.

[0038] Comparative Example 1 1. Effects of seed culture time on induction time and protein yield The seed culture was placed at 37℃ and shaken at 180-220 r / min for 10-16 h. If the culture time was too short (0-10 h), the protein expression cycle would be prolonged, affecting the experimental cycle. If the culture time was too long (more than 16 h), the protein yield would be affected, the HTS solubility would be reduced, and the inclusion body content would increase.

[0039] 2. Effect of different induction temperatures on the solubility of HTS The expression yield of truffle sweet protein HTS in Escherichia coli was compared at different temperatures. At 37°C, HTS was almost entirely expressed in the form of inclusion bodies, while at a specific temperature, HTS showed the highest solubility and the lowest inclusion body content.

[0040] 3. Effects of different IPTG concentrations on HTS protein expression levels By comparing the effects of different IPTG concentrations on HTS solubility and yield at different temperatures, it was found that at a certain temperature, the lower the IPTG concentration (greater than 0.1 mM), the stronger the HTS solubility.

[0041] The various embodiments of this application have been described above. These descriptions are exemplary and not exhaustive, nor are they limited to the disclosed embodiments. Many modifications and variations will be apparent to those skilled in the art without departing from the scope and spirit of the described embodiments. The terminology used herein is chosen to best explain the principles, practical application, or improvement of the technology in the market, or to enable others skilled in the art to understand the embodiments disclosed herein.

Claims

1. A recombinant Escherichia coli engineered strain containing pET-28a-HTS, characterized in that, The pET-28a-HTS is a gene encoding the truffle sweet protein HTS inserted into the NcoI and HindⅢ sites of pET-28a(+); the nucleotide sequence of the HTS encoding gene is shown in SEQ ID NO.

1.

2. The recombinant Escherichia coli engineered strain according to claim 1, characterized in that, The HTS encoding gene matches the restriction site of pET-28a(+), and the recombinant expression vector pET-28a-HTS is constructed by double restriction and ligation.

3. A method for inducing truffle sweet protein HTS in Escherichia coli, characterized in that, The induction method includes: 1) The recombinant Escherichia coli engineered strain as described in claim 1 is inoculated into LB liquid medium for seed culture, and then autoclaved to obtain seed culture; 2) The seed culture obtained in 1) is inoculated into LB liquid medium for scale-up culture; 3) Induction of expression: IPTG was added to the logarithmic phase bacterial culture to a final concentration of 0.1-1.0 mM and cultured for a period of time to obtain fermentation broth containing HTS protein; 4) Collection of bacterial culture: After centrifuging the fermentation broth containing HTS protein obtained in step 3) at low temperature, collect the precipitated bacterial cells, discard the supernatant, and obtain the recombinant Escherichia coli engineered strain containing pET-28a-HTS. The LB liquid culture medium is formulated as follows: 10 g / L tryptone, 5 g / L yeast extract, and 10 g / L NaCl.

4. The induction method according to claim 3, characterized in that, The seed culture was conducted at a temperature of 37℃ for 10-16 hours and at a frequency of 180-220 r / min.

5. The induction method according to claim 3, characterized in that, The expansion culture involves inoculating the seed culture into 500-1000 mL of LB liquid medium at a volume ratio of 1:10-100, and incubating at 37°C with shaking at 180-220 r / min until the bacterial culture reaches OD. 600 The value reaches 0.4-1.

0.

6. The induction method according to claim 3, characterized in that, The continued culture was carried out at a temperature of 10-36℃, a frequency of 180-220 r / min, and a time of 12-30h.

7. A method for isolating and purifying truffle sweet protein HTS in Escherichia coli, characterized in that, The separation and purification method uses the recombinant Escherichia coli engineered strain containing pET-28a-HTS as the raw material according to claim 1, and performs a combination of ultrasonic disruption, affinity chromatography and gel filtration chromatography in sequence to obtain a high-purity HTS protein solution, which is then concentrated until the protein concentration is 20-30 mg / mL.

8. The separation and purification method according to claim 7, characterized in that, The mass-to-volume ratio of the recombinant Escherichia coli engineered strain containing pET-28a-HTS to the lysis buffer in the ultrasonic disruption was 1:10-1:100 (g / mL), the power was 100-600W, the operation time was 1-5s with an interval of 1-5s, and the total disruption time was 10-30 min. After disruption, the mixture was centrifuged at 4℃ and 12000r / min for 30-60 min, and the supernatant containing soluble HTS protein was collected.

9. The separation and purification method according to claim 7, characterized in that, The affinity chromatography involves passing the supernatant containing soluble HTS protein through a Ni-NTA affinity chromatography column and collecting the corresponding components to obtain an HTS protein solution with a purity of over 90%.

10. The separation and purification method according to claim 7, characterized in that, The gel filtration chromatography elutes the HTS protein solution obtained by affinity chromatography, and collects a single elution peak fraction at 10-100 mL to obtain the purified HTS protein.

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