Sweet protein somatin secretion expression system and construction method and application thereof
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- SHENZHEN LIYING BIOTECHNOLOGY CO LTD
- Filing Date
- 2025-01-13
- Publication Date
- 2026-07-21
Smart Images
Figure CN122427263A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of genetic engineering technology, and in particular relates to a sweet protein semathys secretory expression system, its construction method and application. Background Technology
[0002] Thaumatin is a super-sweet protein derived from the fruit of the African arrowroot (Thaurnatocuccus danielli). Its sweetness is 2000-3000 times that of regular sucrose, while its caloric content per gram is comparable to sucrose. Its unique properties make thaumatin a common high-sweetness sweetener or flavor enhancer in the food industry. Compared to other sweeteners such as aspartame and saccharin, it is heat- and acid-resistant, structurally stable, and does not disrupt the balance of amino acids in the body after digestion. It also does not cause tooth decay or affect the health of diabetic patients. Furthermore, numerous toxicological studies have confirmed its safety as a food additive. With the increasing pursuit of healthy living, these sweet but calorie-free sugar alternatives are gaining popularity among consumers. However, naturally extracted thaumatin cannot meet market demand (Joseph JA, et al. Front Microbiol. 2019 Apr8; 10:695.).
[0003] The presence of eight disulfide bonds in semathy makes it difficult for the protein to fold correctly, hindering recombinant expression. Although the gene encoding semathy has been transferred into many microorganisms and plants under the transcriptional control of a heterologous promoter, the yield has remained low to date, failing to reach commercial production levels. Summary of the Invention
[0004] Based on this, in this invention, Pichia pastoris is used as the host strain to heterologously express semasin, and sweet-tasting semasin is expressed through genetic engineering technology. The expression level of semasin is also improved by optimizing fermentation conditions or co-expressing disulfide isomerase.
[0005] The first objective of this invention is to provide a recombinant expression system for semathy protein, including the target protein, tag protein, recombinant vector, engineered bacteria, codon optimization, and expression condition optimization.
[0006] Pichia pastoris (Komagataella phaffii) is a methanol-nutritive yeast first isolated from oak trees by Herman Phaff in 1956. It is one of the most commonly used protein expression systems, widely applied in laboratory-scale protein preparation, characterization, structural analysis, and functional validation. Pichia pastoris has been recognized as a GRAS (Generally Recognized as Safe) microorganism by the US FDA, expanding its applications in food and medicine. In 1995, P. pastoris was reclassified into the genus Komagataella, and subsequently subdivided into two species: Komagataella pastoris and Komagataella phaffii. Currently, the Pichia pastoris type strain CBS7435 and the widely used commercial chassis strains X-33 and GS115 all belong to the species K. phaffii.
[0007] In the Pichia pastoris expression system, ethanol oxidase is encoded by two genes, AOX1 and AOX2. The vast majority of ethanol oxidase activity in the cell is provided by AOX1, and the rate at which the strain utilizes methanol is mainly driven by the AOX1 protein expressed by the AOX1 gene. When AOX1 is absent and only AOX2 is present, most of the ethanol oxidase activity is lost. Such cells have low methanol utilization capacity, and strains that grow slowly on methanol-containing media exhibit the phenotype of slow methanol utilization (Mut). S For example, strain KM71. When AOX1 is present, cells utilize methanol for normal growth and grow rapidly on methanol-containing media; this strain exhibits a rapid methanol utilization phenotype. + ), including but not limited to X-33, SMD1168, and GS115, which are the two Mut expression species in methanol-nutritional Pichia pastoris. S and Mut + The principle behind its production. The optimal growth temperature for Pichia pastoris is 28-30℃. Temperatures exceeding 32℃ during induction are detrimental to protein expression and may lead to cell death. Mut S and Mut + The strain showed the same growth rate in the absence of methanol, but in the presence of methanol, the AOX1 promoter was strongly induced, and Mut... + Compared to Mut S It grows faster (4-5 times). There is also a methanol-independent type, such as MC100-3, in which both AOX1 and AOX2 genes are knocked out.
[0008] In this invention, the engineered strain expressing sematrandezvous acid is *Pichia pastoris* (Komagataella phaffii), whose expressed amino acid sequence is shown in SEQ ID NO. 1. Preferably, the starting strain is selected from one of GS115, KM71, X-33, KM71, SMD1168, Y11430, MG1003, SMD1165, SMD1163, MP-36, and MC100-3. More preferably, the starting strain is GS115.
[0009] The engineered bacteria contain a recombinant vector, or have an exogenous nucleic acid molecule integrated into their chromosome. The engineered bacteria contain at least one copy of the nucleotide sequence encoding recombinant semathyme. In one embodiment, the recombinant semathyme protein produced by high-copy recombinant semathyme bacteria exhibits significantly greater thermostability, being 2.5 times that of naturally extracted semathyme protein.
[0010] Yeast expression products can be achieved through two methods: intracellular expression and extracellular secretion. Intracellular expression vectors mainly include pPIC3, pPICZ, pPSC3K, and pHIL-D2. These vectors express the target gene intracellularly, avoiding glycosylation in yeast. They are suitable for proteins that are typically expressed in the cytoplasm or are non-glycosylated proteins without -SS-. While they offer higher expression levels than extracellular secretion, purification is relatively more complex. Extracellular secretion vectors include, but are not limited to, pPIC9, pHIL-S1, and pYAM75P. These vectors secrete exogenous proteins into the extracellular space, facilitating the purification and accumulation of the target protein.
[0011] Multiple copy insertion expression vectors: pPIC9K, pPIC3.5K. In some cases, the integration of multiple copies of recombinant genes can increase protein expression levels.
[0012] In this invention, the recombinant vector is selected from one or more of pPIC9, pPIC9K, pHIL-S1, pPICZαA, pYAM75P, pHIL-D2, pAO815, pPIC3K, pPICZ, pHWO10, pGAPZ, pGAPZa, and pPIC3.5K. Preferably, the recombinant vector is one or both of pPICZαA and pPIC9K.
[0013] The recombinant expression vector contains the aforementioned nucleic acid molecule. The expression vector comprises any nucleic acid molecule derived from any source and capable of genome integration or autonomous replication (e.g., plasmids, granules, viruses, autonomously replicating polynucleotide molecules, bacteriophages, or linear or circular single-stranded or double-stranded DNA or RNA nucleic acid molecules), containing one or more nucleic acid molecules that are operatively linked. The vector may include, for example, one or more selectable markers, one or more origins of replication (e.g., prokaryotic and eukaryotic origins), at least one multiple cloning site, and / or elements that facilitate stable integration of the construct into the host cell genome.
[0014] The nucleic acid molecule contains a nucleotide sequence encoding the semathy protein or its complementary sequence.
[0015] Preferably, the recombinant vector further includes a nucleic acid sequence encoding a tag protein.
[0016] Furthermore, the tagged proteins include, but are not limited to, one or more of the following: His tag, GST tag, MBP tag, NusA tag, Strep tag, and SUMO tag, used for protein purification and / or for enhancing protein solubility.
[0017] When the transcription level of a foreign gene in Pichia pastoris exceeds the folding capacity of Pichia pastoris itself, overexpression of folding-promoting molecular chaperones can effectively promote the correct folding of foreign proteins, reduce unfolded protein response (UPR) pressure, and thus increase the expression level of foreign proteins.
[0018] The molecular chaperones on the endoplasmic reticulum mainly include protein disulfide isomerase (PDI), human immunoglobulin binding protein (BIP), calreticulin, and calcium conjugate protein.
[0019] PDI is a multifunctional molecular chaperone protein located in the endoplasmic reticulum. It catalyzes the formation of disulfide bonds and helps proteins fold correctly. The promoting effect of PDI co-expression on the expression of exogenous proteins may be related to the disulfide bonds within the proteins themselves. Li et al. increased the expression level of recombinant silkworm acetylcholinesterase 2 (rBmAChE2) by 5-fold through PDI co-expression. Since there are four disulfide bonds in the rBmAChE2 protein, it is inferred that PDI co-expression facilitates the formation of disulfide bonds during rBmAChE2 folding, which is an important reason for its increased expression. Furthermore, to improve the thermostability of AppA phytase, Navone et al. successfully obtained a thermostable mutant ApV1 by introducing disulfide bonds, and PDI co-expression increased the expression level of ApV1 protein by 12-fold. Additionally, the copy number of the target gene also affects the effectiveness of this strategy. Huang et al. found that Pichia pastoris exhibited endoplasmic reticulum stress when expressing 4 copies of the lipase gene, but did not when expressing 2 copies of the RML gene; co-expression of PDI increased the expression level of 4-copy RML strains, but had no effect on 2-copy RML strains (Huang, et al. Bioengineered. 2020 Dec; 11(1):375-385.).
[0020] BIPs mainly stabilize immature proteins by binding to the elongation ends of hydrophobic amino acids. Since molecular chaperones play an important role in protein folding, molecular chaperones on the endoplasmic reticulum improve protein secretion in Pichia pastoris by manipulating the levels of transcription factors. Some studies have also reported that overexpression of protein chaperone genes such as CUP5, SSA4, BMH2, and KIN2 promotes the secretion of target proteins in Pichia pastoris (Zhu Taicheng, et al. Overview and trend of Pichia pastoris expression system development [J]. Chinese Journal of Biotechnology, 2015, 31(6):929-938).
[0021] The Ssa1 protein derived from Pichia pastoris belongs to the HSP40 family and helps nascent peptide chains maintain the correct conformation. Molecular chaperones and their co-chaperones of the HSP family can promote the expression of exogenous proteins (Prabhu, AA, et al. (2018). Chemical Engineering Science, 181, 58–67.).
[0022] In this invention, the target protein is co-expressed with a molecular chaperone. Preferably, the target protein is co-expressed with PDI, which can be expressed in hosts with different copy numbers and produce correctly folded semathy protein. The amino acid sequence of the PDI is shown in SEQ ID NO.3. In one embodiment, the thermostability of recombinant semathy protein produced in high-copy-count recombinant semathy bacteria is increased to twice that of naturally extracted protein; in another embodiment, the recombinant protein exhibits a higher yield in low-copy-count recombinant semathy bacteria.
[0023] Compared with the prior art, the present invention has the following beneficial effects:
[0024] The semathy protein expressed by the engineered bacteria constructed in this invention has higher purity and thermal stability compared to semathy naturally extracted from African arrowroot. This biosynthesis method is milder and yields higher output than chemical extraction, making it suitable for industrialization. Attached Figure Description
[0025] Figure 1 The image shows the SDS-PAGE results of the target protein obtained from the expression and purification of recombinant X-33. Lane D5 represents the supernatant after 5 days of fermentation, FT represents the supernatant after the flow line, and E2, E4, and E6 represent the supernatants after the second, fourth, and sixth elutions, respectively.
[0026] Figure 2 The image shows the SDS-PAGE results of the target protein obtained from the expression and purification of recombinant GS115. Lanes D3 and D5 represent the supernatant after 3 and 5 days of fermentation, respectively. FT represents the supernatant after 5 days of fermentation, and E represents the elution supernatant.
[0027] Figure 3 The image shows the DNA agarose gel electrophoresis results of the positive transformant GS115-thaumatin-PDI after co-expression of thaumatin at high and low copies and PDI. The target band PDI is located at 1757 bp and thaumatin is located at 1168 bp.
[0028] Figure 4 The SDS-PAGE results of semastem protein after co-expression with high and low copy numbers and PDI are shown in the figure. Lanes D3 and D5 represent the supernatant after 3 and 5 days of fermentation, respectively. FT represents the supernatant after 5 days of fermentation, and E represents the elution supernatant.
[0029] Figure 5 Thermostability test results of semastem protein were obtained by expressing and purifying high-copy GS115 for semastem.
[0030] Figure 6 Thermostability test results of semastem protein were obtained by expressing and purifying GS115 low-copy semastem.
[0031] Figure 7 Thermostability test results of semastem protein were obtained by co-expressing high-copy semastem with PDI and GS115 expression and purification.
[0032] Figure 8 Thermostability test results of semastem protein were obtained by co-expressing semastem low copy with PDI using GS115 expression and purification.
[0033] Figure 9 The image shows the SDS-PAGE results of the thermal stability test of naturally extracted semacin protein. Detailed Implementation
[0034] This invention does not impose any particular limitation on the preparation method of the recombinant vector; conventional recombinant vector preparation methods in the art can be used. In this invention, the gene can be synthesized by a biotechnology company. This invention does not impose any particular limitation on the separation and purification method; conventional protein separation and purification methods in the art can be used; preferred technical solutions are described in the examples. Unless otherwise specified, the linearization system, amplification reaction system, and ligation reaction system in this invention are all commercially available, along with the relevant enzymes, buffers, and kits.
[0035] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.
[0036] Example 1: Construction of recombinant Pichia pastoris X-33 and expression of the target protein
[0037] 1. Linearization of recombinant plasmid pPICZαA-Thaumatin
[0038] Linear plasmids have higher transformation efficiency than circular plasmids; therefore, before integrating the target gene into the Pichia pastoris genome, the plasmid needs to be linearized by single-enzyme digestion. The plasmid pPICZαA-Thaumatin, synthesized from the whole genome by Anhui General Biotechnology, was linearized using the restriction endonuclease Pme I. The linearization system is shown in Table 1. After preparation, the system was incubated in a 37℃ metal bath for 20 min, and then purified. A small amount of the purified product was taken and subjected to DNA agarose gel electrophoresis to verify whether the plasmid was completely linearized.
[0039] Table 1 Linearization System
[0040]
[0041] 2. Preparation of Pichia pastoris X-33 competent cells
[0042] Using a sterile toothpick, pick a single colony of Pichia pastoris X-33 from a YPD plate and inoculate it into 10 mL of LYPD medium. Incubate overnight on a shaker at 30°C and 250 rpm. Transfer an appropriate amount of the overnight culture to a 250 mL Erlenmeyer flask containing 100 mL of LYPD, and control the initial OD of the culture. 600 The value was 0.5, and the culture was carried out at 30℃ and 250 rpm for 3.0-4 hours to adjust the OD value of the bacterial suspension. 600 The value should be within the range of 1.3-1.5. In a laminar flow hood, evenly distribute the bacterial culture into two 50mL sterile centrifuge tubes and centrifuge at 6000rpm and 4℃ for 5min. Discard the supernatant, add 40mL of freshly prepared LDST solution to each centrifuge tube, mix gently, and incubate at 30℃ for 30min. Centrifuge at 6000rpm and 4℃ for 5min, discard the supernatant, add 1mL of pre-chilled 1M sorbitol to resuspend the bacteria, and transfer to a new 1.5mL sterile EP tube. Wash the bacteria three times with pre-chilled sorbitol, and finally resuspend in 400μL of pre-chilled 1M sorbitol. Aliquot 100μL into sterile 1.5mL EP tubes and immediately store at -80℃.
[0043] 3 Recombinant plasmid pPICZαA-Thaumatin transfected into Pichia pastoris X-33 competent cells
[0044] The linearized recombinant plasmid was transformed into Pichia pastoris X-33 competent cells using electroporation. An appropriate amount of linearized plasmid (1-5 μg) was added to the aliquoted Pichia pastoris X-33 competent cells, gently mixed with a sterile pipette tip, and transferred to a pre-ice-warmed 0.2 cm electroporation cuvette. The cells were incubated on ice for 5 min. Electroporation was then performed using an electroporator with the following conditions: voltage 1.5 kV, resistance 200 Ω, capacitance 25 mF, and electroporation time approximately 5 ms. After electroporation, 1 mL of pre-ice-warmed 1 M sorbitol was added and quickly transferred to a 1.5 mL centrifuge tube. The cells were incubated at 30°C for 90 min. Remove the bacterial suspension, pipette 100 μL of the bacterial suspension and spread it on a YPDZ resistant plate (Zeocin final concentration 100 μg / mL). Centrifuge the remaining bacterial suspension at room temperature, 8000 rpm for 1 min, remove part of the supernatant, and retain about 150 μL. Resuspend the bacterial suspension and spread it on another YPDZ resistant plate. Incubate at 30°C upside down for 36-72 h until single yeast colonies grow.
[0045] 4. Induced expression of various recombinant yeasts
[0046] Positive transformants selected from YPDZ plates (Zeocin final concentration 100 μg / mL) were inoculated into 10 mL of liquid YPD medium and cultured at 30℃ and 250 rpm for 12-16 h. Then, the yeast cells from the liquid YPD medium were transferred at a 1% inoculation rate to 100 mL of liquid BMGY medium and cultured at 30℃ and 250 rpm for 24-48 h. After culture, the bacterial suspension was centrifuged at 6000 rpm for 10 min to remove the BMGY medium. The cells were then resuspended in the same volume of BMMY medium and cultured at 30℃ and 250 rpm. 2 mL of methanol (2%) was added every 24 h, and the culture was continued until 168 h (the culture time can be extended or shortened as needed). The supernatant was collected by centrifugation and filtered through a 0.45 μM filter membrane. The filtrate was used directly for affinity chromatography. SDS-PAGE electrophoresis was used to verify the molecular weight and purity of the protein. The protein sample was mixed with protein loading buffer (DTT), placed in a metal bath, and boiled at 95°C for 10 minutes before gel loading. The results are as follows: Figure 1 As shown.
[0047] Example 2: Construction of recombinant Pichia pastoris GS115 and expression of the target protein
[0048] 1. Construction of recombinant plasmid pPIC9K-Thaumatin
[0049] Table 2 Primers for constructing the recombinant plasmid pPIC9K-Thaumatin
[0050]
[0051] Table 3. PCR amplification reaction system for the target gene semathy.
[0052]
[0053]
[0054] Table 4. PCR amplification procedure for the target gene semathy.
[0055]
[0056] Table 5. PCR amplification reaction system for vector pPIC9K
[0057]
[0058] Table 6. Reaction Procedure for pPIC9KPCR Amplification of Vector
[0059]
[0060] Table 7. Ligation system between target gene and vector
[0061]
[0062]
[0063] Thaw the cloned competent cells on ice. Add 10 μL of the ligation product to 100 μL of *E. coli* DH5α competent cells in a clean bench. Mix gently by pipetting and incubate on ice for 30 min. Place the transformation product in a 42°C water bath for 90 s heat shock, then immediately transfer to ice and incubate for 5 min. Add 400 μL of antibiotic-free SOC liquid medium and incubate at 37°C and 200 rpm for 1 h. Briefly centrifuge the transformation product at low speed to remove some of the medium, leaving 100 μL. Resuspend the bacterial cells at the bottom of the centrifuge tube and spread them onto LB agar plates (ampicillin final concentration 100 μg / mL). Invert the plates in a 37°C incubator and incubate for approximately 12-16 h. Observe the growth of colonies on the plates.
[0064] 2. Screening of positive transformants by pPIC9K-Thaumatin Escherichia coli DH5α colony PCR
[0065] Ten single colonies were randomly selected from the plate as templates for colony PCR amplification using universal primers AOX. The colony PCR amplification system is shown in Table 8, and the colony PCR amplification reaction procedure is shown in Table 9. After the PCR reaction, DNA agarose gel electrophoresis was performed, and the band size was observed using a DL5000 DNA Marker as a reference.
[0066] Table 8 Colony PCR Reaction System
[0067]
[0068] Table 9 Colony PCR Reaction Procedure
[0069]
[0070] 3. Preparation of Pichia pastoris GS115 competent cells
[0071] The preparation of Pichia pastoris GS115 competent cells is carried out according to step 2 of Example 1.
[0072] 4. pPIC9K-Thaumatin recombinant plasmid was transformed into Pichia pastoris GS115 competent cells
[0073] Add an appropriate amount of linearized recombinant plasmid to GS115 competent cells, mix well, and transfer to a pre-sterilized and ice-bathed 0.2 cm electroporation cuvette. Incubate on ice for 5 min. Perform electroporation using an electroporator with a voltage of 1.5 kV and a duration of approximately 5 ms. Immediately after electroporation, add 1 mL of ice-cold sorbitol and mix well. Gently pipette the mixture to a 1.5 mL sterile EP tube and incubate at 30°C for 1.5 h. Pipette 50 μL of the bacterial suspension onto an antibiotic-free MD plate. Centrifuge the remaining bacterial suspension at 6000 rpm for 1 min at room temperature, remove some of the supernatant, and retain approximately 150 μL. Resuspend the suspension and spread it onto another antibiotic-free MD plate. Incubate upside down at 30°C for 36-72 h until single yeast colonies appear. Single colonies from MD plates were picked and transferred to 4 mg / mL G418 high-anti-YPD plates, and incubated upside down at 30°C for 36-72 h.
[0074] 5. Inducible expression of recombinant GS115-Thaumatin
[0075] Transformants with higher copy numbers grew more rapidly and produced larger volumes on high-concentration G418 (4 mg / mL) YPD plates. Two transformants with the largest difference in colony size were selected for fermentation culture; larger colonies were identified as high-copy-count recombinant semaphore, and smaller colonies as low-copy-count recombinant semaphore. Positive transformants were inoculated into 10 mL of liquid YPD medium and cultured at 30°C and 250 rpm for 12-16 h. The yeast cells from the liquid YPD medium were then transferred at a 1% inoculation rate to 100 mL of liquid BMGY medium and cultured at 30°C and 250 rpm for 24-48 h. After culture, the bacterial culture was centrifuged at 6000 rpm for 10 min, the BMGY medium was removed, and the cells were resuspended in the same volume of BMMY medium and cultured at 30°C and 250 rpm. 2 mL of methanol (2%) was added every 24 h, and the culture was continued until 168 h (the culture time can be extended or shortened as needed). Samples were collected every 24 h to monitor changes in expression levels. Centrifuge and collect the supernatant. SDS-PAGE electrophoresis results are as follows: Figure 2 As shown.
[0076] Example 3: Co-expression of the target protein and protein disulfide isomerase (PDI)
[0077] 1. Construct a plasmid containing the PDI encoding gene.
[0078] The sequence of the protein disulfide isomerase PDI from Pichia pastoris GS115 was obtained from NCBI, and primers were designed using Snapgene. Pichia pastoris GS115 cells were resuspended in PBS and lysed by repeated freeze-thaw cycles. The cells were centrifuged at 12000 rpm for 2 min at room temperature, and the supernatant was used as a template for amplification of the full-length target gene, constructing the recombinant plasmid pPICZαA-PDI. The primers for the target gene PDI, the full-gene amplification reaction system, and the PCR reaction program are shown in Tables 10, 11, and 12, respectively. The PCR amplification reaction system and reaction program for the vector pPICZαA are shown in Tables 13 and 14. The ligation system of the target gene PDI and the vector pPICZαA is shown in Table 15.
[0079] Table 10 Primers for constructing the recombinant plasmid pPICZαA-PDI
[0080]
[0081]
[0082] Table 11. Target gene PDIPCR amplification reaction system
[0083]
[0084] Table 12. PDIPCR amplification reaction procedure for the target gene.
[0085]
[0086] Table 13 pPICZα APCR amplification reaction system
[0087]
[0088] Table 14 PCR amplification reaction procedure for vector pPICZαA
[0089]
[0090]
[0091] Table 15. Ligation system between target gene and vector
[0092]
[0093] 2. PCR product transcloning strain DH5α
[0094] Thaw the cloned competent cells on ice. Add 10 μL of the ligation product to 100 μL of *E. coli* DH5α competent cells in a clean bench. Mix gently by pipetting and incubate on ice for 30 min. Place the transformation product in a 42°C water bath for 90 s heat shock, then immediately transfer to ice and incubate for 5 min. Add 400 μL of antibiotic-free SOC liquid medium and incubate at 37°C and 200 rpm for 1 h. Briefly centrifuge the transformation product at low speed to remove some of the medium, leaving 100 μL. Resuspend the bacterial cells at the bottom of the centrifuge tube and spread DH5α-pPICZαA-PDI onto LB agar plates (Zeocin final concentration 25 μg / mL). Invert the plates in a 37°C incubator and incubate for approximately 12-16 h. Observe the growth of colonies on the plates.
[0095] 3. Screening for positive transformants by colony PCR
[0096] Ten single colonies were randomly selected from the plate as templates for colony PCR amplification using universal primers AOX. The colony PCR amplification system and reaction procedure are shown in the table below. After the PCR reaction, DNA agarose gel electrophoresis was performed, and the band size was observed using a DL5000 DNA Marker as a reference.
[0097] Table 16 Colony PCR Reaction System
[0098]
[0099] Table 17 Colony PCR Reaction Procedure
[0100]
[0101] 4. Preparation of GS115-pPIC9K-Thaumatin competent cells
[0102] Competent cells were prepared using the semaphore high-copy GS115 recombinant bacteria and the semaphore low-copy GS115 recombinant bacteria obtained in Example 2. The specific preparation operation is the same as step 2 in Example 1.
[0103] 5. pPICZαA-PDI recombinant plasmid transfected Pichia pastoris GS115-pPIC9K-Thaumatin competent cells
[0104] The construction process for co-expressing the PDI strain followed step 4 of Example 2, screening for a final concentration of 100 μg / mL of Zeocin (bleomycin) in YPD. The recombinant bacteria contained two exogenous proteins, thaumatin and PDI, at different AOX sites. After PCR with universal primers, two target bands should appear at 1757 bp and 1168 bp, as shown in the results. Figure 3As shown in the gel image, transformants 3, 6, and 8 of the semaphore high-copy GS115 recombinant bacterium are positive transformants, while transformants 1, 6, 8, 9, and 11 of the semaphore low-copy GS115 recombinant bacterium are positive transformants.
[0105] 6. Inducible expression of recombinant GS115-PDI-Thaumatin
[0106] The induction expression procedure is the same as step 5 in Example 2. The supernatant was collected by centrifugation. The SDS-PAGE electrophoresis results are as follows: Figure 4 As shown in the figure. The results showed that co-expression of the high-copy semaphore transformant and PDI on day 5 did not increase semaphore expression and even had an inhibitory effect. However, co-expression of the low-copy semaphore transformant and PDI significantly increased semaphore expression, and its expression level was higher than that of the high-copy semaphore transformant, increasing by 37.9%.
[0107] Example 4: Semamethin-induced expression, purification, and sweetness analysis before and after PDI co-expression
[0108] After centrifuging the collected fermentation broth, collect the supernatant, filter it through a 0.45 μm microporous membrane, and aliquot it. Add 1 ml of NI-Beads to every 100 mL of fermentation supernatant and incubate on a shaker at 4°C for 2 hours. Wash with wash buffer, washing away impurities at approximately 90 packing volume volumes. Elute the target protein with Elution buffer, adding Elution buffer each time and incubating for 10 min. Collect the eluted protein solution using an EP tube under a gravity column. Elute 10 ml at a time. Eluting 1 ml at a time reduces dilution of uncertain protein amounts.
[0109] Experiments showed that the maximum solubility of wild-type semaphore in pure water is 0.01 mg / mL. Precipitation will occur when the protein concentration exceeds 0.01 mg / mL. The protein was diluted to 0.01 mg / mL with Elution buffer and then dialyzed into Wahaha pure water.
[0110] In the sweetness test, the researchers first rinsed their mouths with pure water to clean their oral cavity, and then tasted the samples to determine the differences in sweetness. The test results showed that the recombinant protein prepared by the X-33 expression system described in Example 1 had no obvious sweetness, while the recombinant proteins prepared by the expression systems described in Examples 2 and 3 all had obvious sweetness, among which the semathy protein produced by the low copy number recombinant GS115 strain (Example 3) had the most significant sweetness.
[0111] Example 5: Thermostability Test of Recombinantly Expressed Semamate Protein
[0112] The purified thauplatin from Examples 1, 2, and 3, and the thauplatin extracted from natural plants (Cat: T93001), were aliquoted into 1.5 mL centrifuge tubes, 50 μL per tube. A water bath was set to 95 °C, and samples were boiled for 0 min, 5 min, 10 min, 15 min, 20 min, 30 min, 45 min, 60 min, and 75 min, respectively. After boiling, the samples were centrifuged at 4 °C and 14800 rpm for 10 min (no obvious precipitation was observed). 40 μL of the supernatant was collected for sample preparation, and the remaining supernatant was completely removed. 40 μL of PBS was added to the original centrifuge tube for sample preparation, and SDS-PAGE was used to observe the protein composition.
[0113] Figure 5 The results showed that the semaphore protein expressed by the high-copy recombinant semaphore strain in Example 2 of this invention did not show significant differences before and after treatment at 95°C for 30 minutes. After 75 minutes of treatment at 95°C, approximately 50% of the target protein remained in the supernatant.
[0114] Figure 6 The results show that after 30 minutes of treatment at 95°C, approximately 50% of the target protein remained in the supernatant of the semastem protein expressed by the low-copy recombinant strain of semastem in Example 2 of this invention.
[0115] Figure 7 After co-expression with PDI, the samaste protein produced by the recombinant GS115 strain with a high copy number of samaste (Example 3) had 50% protein remaining in the supernatant after heating at 95°C for 60 min; and the recombinant protein expressed in this system had a large amount of protein remaining in the supernatant after high-temperature treatment for 3 hours.
[0116] Figure 8 The recombinant GS115 strain (Example 3) co-expressing low-copy semaphore with PDI also produced 50% semaphore protein in the supernatant after heating at 95°C for 60 min, but the yield was 37.9% higher than that of the high-copy-number transformant.
[0117] Figure 9 The protein standard showed that degradation began after heating for 5 minutes, and 50% precipitation occurred after heating for 30 minutes.
Claims
1. An engineered bacterium expressing semaside protein, characterized in that, The starting strain of the engineered bacteria is Pichia pastoris (Komagataella phaffii), which expresses the target protein with the amino acid sequence shown in SEQ ID NO.
1.
2. The engineered bacteria according to claim 1, characterized in that, The engineered bacteria are selected from one of GS115, KM71, X-33, KM71, SMD1168, Y11430, MG1003, SMD1165, SMD1163, MP-36 and MC100-3.
3. The engineered bacteria according to claim 1, characterized in that, It contains recombinant vectors selected from one or more of the following: pPIC9, pPIC9K, pHIL-S1, pPICZαA, pYAM75P, pHIL-D2, pAO815, pPIC3K, pPICZ, pHWO10, pGAPZ, pGAPZa and pPIC3.5K.
4. The engineered bacteria according to claim 3, characterized in that, The recombinant vector contains a nucleic acid sequence encoding semathy protein, as shown in SEQ ID NO.
2.
5. The engineered bacteria according to claim 1, characterized in that, The target protein is linked to a tagged protein.
6. The engineered bacteria according to claim 1, characterized in that, It also expresses the molecular chaperone PDI.
7. The engineered bacteria according to claim 1, characterized in that, It contains high-copy plasmids.
8. The engineered bacteria according to claim 6, characterized in that, It contains low-copy plasmids.
9. A method for expressing semaside in Pichia pastoris, characterized in that, Includes the following steps: (1) Codon optimization and gene synthesis of the nucleotide sequence encoding recombinant semathy protein; (2) Connect the semathy protein encoding gene to construct the first expression vector, and connect the PDI encoding gene to construct the second expression vector; (3) The first expression vector and the second expression vector are co-transformed into the host cell, or successively transformed into the host cell, the host cell is cultured, and the expression of the target protein is induced; (4) Recombinant semastem protein was obtained by separation and purification.