Pichia pastoris capable of efficiently expressing TFF3 fusion protein as well as construction method and application of pichia pastoris

Abstract

The invention provides pichia pastoris capable of efficiently expressing TFF3 fusion protein as well as a construction method and application of the pichia pastoris, and belongs to the technical field of engineering bacteria. The preservation number of the pichia pastoris is CCTCC (China Center For Type Culture Collection) NO: M 2025013. The pichia pastoris constructed by the invention can obviously improve the expression quantity of the TFF3, the shake flask expression quantity is up to 391 mg / L, and the pichia pastoris has a wide application prospect in large-scale industrial production of the TFF3.

Description

Technical Field

[0001] This invention belongs to the field of engineered bacteria technology, specifically relating to a Pichia pastoris strain that efficiently expresses the TFF3 fusion protein, its construction method, and its application. Background Technology

[0002] Trefoil factor peptides (TFFs), with a 40-amino acid motif including six conserved cysteine ​​residues forming intramolecular disulfide bonds, are a family of mucus-associated secretory molecules that mediate numerous physiological functions to maintain and restore gastrointestinal (GI) mucosal homeostasis. There are three mammalian TFF peptides: TFF1 (formerly pS2), TFF2 (formerly PSP), and TFF3 (formerly intestinal trefoil factor (ITF)). TFF peptides play a crucial role in the response to gastrointestinal mucosal injury and inflammation. In the response to acute gastrointestinal mucosal injury, TFF peptides accelerate cell migration to seal the damaged area, protecting it from luminal contents, while chronic inflammation leads to increased TFF expression to prevent further disease progression. TFF peptides have clear benefits for both healthy and damaged gastrointestinal tissues. Since Playford proposed using TFF peptides as luminal surveillance peptides in 1995, TFF peptides have been shown to physiologically contribute to epithelial repair, synergistically reinforce the mucus layer with mucins, and prepare for small-scale or large-scale epithelial emergencies. TFF peptides also play an important role in epithelial regeneration and in the regulation of inflammation and the immune system. In modern intensive farming, the application of trifoliate peptides can enhance the intestinal health of young animals and improve their overall production performance, which is one of the fundamental means to solve the contradiction between rapid growth and lagging organ development in animals.

[0003] TFF3 was initially isolated from the gastrointestinal tract of animals. However, traditional methods for extracting TFF3 from the animal gastrointestinal tract are characterized by low yields and high costs. In contrast, fermentation with genetically engineered bacteria enables large-scale industrial production, significantly increasing protein yield and meeting the needs of scientific research and clinical applications. Therefore, developing engineered bacteria capable of efficiently expressing the TFF3 protein is of great significance. Summary of the Invention

[0004] The purpose of this invention is to provide a Pichia pastoris strain that efficiently expresses the TFF3 fusion protein, its construction method, and its application.

[0005] This invention provides a Pichia pastoris strain, with the preservation number CCTCC NO: M 2025013.

[0006] This invention also provides the application of the aforementioned Pichia pastoris R5312 strain for the fermentation preparation of TFF3 fusion protein.

[0007] This invention also provides a method for preparing TFF3 fusion protein by fermentation, comprising the following steps: The Pichia pastoris strain described above was inoculated into growth medium and cultured. The cells were collected by centrifugation, and the cells were resuspended in methanol induction medium. Methanol was added daily for induction culture, and the supernatant was collected after the culture was completed to obtain the TFF3 fusion protein.

[0008] Furthermore, the growth medium is mBSM medium, containing the following components: glucose 48-52 g / L, KH2PO4 3-7 g / L, CaSO4 0.80-1.20 g / L, K2SO4 16.0-20.0 g / L, MgSO4 13.0-17.0 g / L, NH4H2PO4 38-42 g / L, KOH 1.3-1.7 g / L; pH value 5.5-6.5.

[0009] Furthermore, the growth medium contains the following components: glucose 50 g / L, KH2PO4 5 g / L, CaSO4 0.93 g / L, K2SO4 18.2 g / L, MgSO4 14.9 g / L, NH4H2PO4 40 g / L, KOH 1.5 g / L; pH 6.0.

[0010] Furthermore, the methanol-inducing medium is mBSM-M methanol-inducing medium, containing the following components: KH2PO4 3-7 g / L, CaSO4 0.80-1.20 g / L, K2SO4 16.0-20.0 g / L, MgSO4 13.0-17.0 g / L, NH4H2PO4 38-42 g / L, KOH 1.3-1.7 g / L, methanol 8-12 g / L; pH value 5.5-6.5.

[0011] Furthermore, the methanol-induced culture medium contains the following components: KH2PO4 5 g / L, CaSO4 0.93 g / L, K2SO4 18.2 g / L, MgSO4 14.9 g / L, NH4H2PO4 40 g / L, KOH 1.5 g / L, methanol concentration 10 g / L; pH value 6.0.

[0012] Furthermore, during the induction culture, methanol is added daily until the final methanol concentration is 0.5%-2.0%, the induction culture temperature is 28-30℃, and the induction culture time is more than 90 hours.

[0013] Furthermore, during the induction culture, methanol is added daily until the final methanol concentration reaches 1.0%, the induction culture temperature is 30°C, and the induction culture time is 96-120 hours.

[0014] The present invention has achieved the following beneficial effects: the engineered Pichia pastoris strain constructed in this invention can significantly increase the expression level of TFF3, with the expression level in shake flasks reaching as high as 391 mg / L. It has broad application prospects in large-scale industrial production of TFF3.

[0015] Obviously, based on the above description of the present invention, and according to common technical knowledge and conventional methods in the field, various other modifications, substitutions or alterations can be made without departing from the basic technical concept of the present invention.

[0016] The following detailed embodiments further illustrate the above-described content of the present invention. However, this should not be construed as limiting the scope of the present invention to the following embodiments. All technologies implemented based on the above-described content of the present invention fall within the scope of the present invention. Attached Figure Description

[0017] Figure 1 pPIC9K-fu plasmid map.

[0018] Figure 2 TFF3 concentration standard curve.

[0019] Figure 3 SDS-PAGE analysis results of bacterial expression products. Lane 1: 0h; Lane 2: 34h starting with mixed feeding; Lane 3: 40h starting with induction; Lanes 4-9: 44h, 60h, 68h, 89h, 120h, and 132h samples, respectively. M: Molecular weight marker.

[0020] Figure 4 : Changes in fermentation levels of TFF3 fusion protein.

[0021] Figure 5 Results of pretreatment using G25 desalination column.

[0022] Figure 6 Results of anion exchange column separation and purification.

[0023] Figure 7 SDS-PAGE analysis results. M: Molecular weight marker; Lane 1: 10L fermentation broth sample; Lanes 2 and 3: Samples collected from segments 4 and 5 of the G25 desalting column; Lane 4: Mixed sample from segments 9-13 of the first stage of constant flow; Lanes 5, 6, 7, and 8: Samples collected from segments 14, 15, 16, and 17 of gradient elution, respectively; Lane 9: Samples collected from segments 18-21 of the 50% constant flow stage; Lane 10: Segment 22.

[0024] Figure 8Results of in vitro experiments promoting cell migration in each group. (A) Control group, with 1 μg / μl of fermentation broth without TFF3 gene purified sample; (B) with 1 μg / μl of purified TFF3; (C) with 1 μg / μl of TFF3 standard; (D) with combined application of 1 μg / μl of purified TFF3 and 1 μg / μl of EGF. Detailed Implementation

[0025] This invention's RT5312 strain has been deposited with the China Center for Type Culture Collection (CCTCC). The address of the collection institution is: Wuhan University, No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province. The accession number is CCTCC NO: M 2025013. The classification name is: Komagataella pastoris R5312, the deposit date is January 3, 2025, and the preservation period is 30 years.

[0026] The raw materials and equipment used in this invention are all known products, obtained by purchasing commercially available products.

[0027] The DNA sequence of TFF3 was synthesized by Invitrogen.

[0028] TFF3 DNA sequence (SEQ ID NO.1): ggggagtatgtgggcctgtcggcgaaccagtgtgccgtccctgccaaggacagggtggactgcggctacccccaggtcaccccccgagcagtgcaacaaccggggctgctgcttcgactccagcatccccggggtgccctggtgcttcaagcccctgcaggaaacagaatgcaccttctga TFF3 protein sequence (SEQ ID NO.2): geyvglsanqcavpakdrvdcgypqvtpeqcnnrgccfdssipgvpwcfkplqetectf* Example 1: Construction of Pichia pastoris R5312 for efficient expression of porcine TFF3 fusion protein Purchased the Invitrogen Pichia pastoris expression kit. The full-length TFF3 gene sequence was cloned into the Pichia pastoris expression vector pPIC9K-fu via the EcoRI / NotI site. The pPIC9K-fu vector (structure shown) Figure 1 (As shown) is the modified expression vector carrying the tagged protein fu, resulting in the recombinant vector pPIC-fuTFF3. The DNA recombination procedures were primarily performed according to the "Molecular Cloning Laboratory Manual".

[0029] The recombinant vector pPIC-fuTFF3 was linearized by digestion with Sal I, and then transformed into Pichia pastoris GS115 using a Biorad electroporator at 1500V. Positive transformants were screened by PCR. Figure 4 The strain was selected through shake-flask screening and named RT5312. Electroconversion and screening methods are detailed in the Invitrogen manual "Multi-Copy Pichia Expression Kit," pages 34-56, https: / / assets.thermofisher.cn / TFS-Assets / LSG / manuals / pichmulti_man.pdf.

[0030] The method for screening high-expression positive clones using shake-flask screening medium is as follows: Positive transformants are cultured in 25 mL mBSM medium at 30℃ on a shaker for 48 h. The bacterial cells are collected by centrifugation, and 15 mL mBSM-M methanol induction medium is added to suspend the bacterial cells to obtain a bacterial suspension. Methanol is added daily at a final concentration of 1% (i.e., the volume ratio of added methanol to bacterial suspension is 1:99). The cells are induced and cultured at 30℃ for 96 h. The EGF content in the supernatant of each strain is measured, and transformants that highly express EGF are screened out.

[0031] mBSM medium: glucose 50 g / L, KH2PO4 5 g / L, CaSO4 0.93 g / L, K2SO4 18.2 g / L, MgSO4 14.9 g / L, NH4H2PO4 40 g / L, KOH 1.5 g / L, pH 6.0.

[0032] mBSM-M methanol-induced medium: KH2PO4 5 g / L, CaSO4 0.93 g / L, K2SO4 18.2 g / L, MgSO4 14.9 g / L, NH4H2PO4 40 g / L, KOH 1.5 g / L, methanol concentration 10 g / L, pH 6.0.

[0033] The TFF3 content in the culture medium 96 hours after induction was detected using a porcine TFF3 detection kit purchased from Shanghai Jianglai Biotechnology Co., Ltd. The operating procedures were described in the kit instructions. A concentration standard curve was plotted using standards, as shown below. Figure 2 As shown in Table 1.

[0034] Table 1. Standard curve of TFF3 concentration Using the standard curve as a reference, the expression level of the culture medium samples was determined by ELISA according to the dilution factor.

[0035] The calculation formula is: TFF3 content = (2.492 * (A sample - AGS115) - 0.1067) * dilution factor, where A sample is the light absorbance value of the fermentation broth sample and AGS115 is the light absorbance value of the blank strain.

[0036] A strain with a TFF3 content of 330 mg / L in the fermentation broth after 96 h of shake-flask induction expression was obtained and named RT5312.

[0037] Example 2: 10L fermentation production of TFF3 fusion protein and SDS-PAGE analysis of expression products 1. Fermentation production of TFF3 fusion protein Following the instructions provided by Invitrogen, "Pichia Fermentation Process Guidelines," version B053002, pages 1-7, https: / / tools.thermofisher.com / content / sfs / manuals / pichiaferm_prot.pdf, horizontal fermentation was carried out in a 10L fermenter, going through four stages: cultivation, glycerol feeding, mixing with feed, and induction.

[0038] The process flow is as follows: RT5312 bacteria are cultured until the biomass reaches 103 g / L after 20 hours. The carbon source in the substrate is depleted, and dissolved oxygen rises rapidly. Glycerol supplementation is then initiated at 33 mL / h. After 37 hours of culture, the biomass reaches 298 g / L. Glycerol feeding is then stopped, and mixed feeding begins. During this process, glycerol is added at 25 mL / h, and methanol at 3 mL / h, gradually increasing the methanol ratio. After 6 hours of mixed feeding, supplementation is stopped, and the dissolved oxygen is allowed to rebound to its peak for 15 minutes before methanol feeding begins. Induction was initiated with a methanol feed rate of 5 mL / h. The methanol feed rate was gradually increased, and the dissolved oxygen was slowly reduced. After the feed rate stabilized, the methanol feed rate was stabilized at 10 mL / h. The dissolved oxygen was maintained between 5-35% during the fermentation process. Methanol feed was stopped every 4 hours during the fermentation process. The dissolved oxygen could rise rapidly in the first 2 minutes and in the later 10 minutes. At 110 hours of fermentation, the frequency of ammonia feed increased to once every 3-4 hours. After 132 hours of fermentation, the OD of the tank was 158, the wet weight was 295 g / L, and the tank volume was 7 L.

[0039] Record the fermentation process, take samples at appropriate times, and analyze protein expression using SDS-PAGE electrophoresis.

[0040] 2. SDS-PAGE analysis of bacterial expression products Take samples of the culture medium, centrifuge, and collect 20 μL of the supernatant for SDS-PAGE analysis.

[0041] Experimental results showed that the expression of the TFF3 fusion protein was identified by SDS-PAGE. SDS-PAGE electrophoresis revealed a single protein band for the TFF3 fusion protein expressed in Pichia pastoris, with an apparent molecular weight of approximately 32 kDa, which is comparable to the theoretical molecular weight (kDa) inferred from the amino acid sequence. Figure 3 ).

[0042] 3. Expression level analysis The content of TFF3 fusion protein was determined using a porcine TFF3 detection kit produced by Jianglai Biotechnology Co., Ltd., and an expression curve was plotted. Figure 3 and Figure 4 As shown, the highest fermentation level of TFF3 was around 120 hours, with a content of 391 mg / L.

[0043] Example 3: Purification of TFF3 fusion protein The purification method for TFF3 fusion protein is as follows: Take the supernatant of the culture medium from Example 2, use 85% ammonium sulfate solution for salting out at 4°C, and centrifuge at 12000 rpm for 15 min to obtain protein precipitate.

[0044] The precipitate was dissolved in 20 mM pH 8.0 Tris buffer. The sample was pretreated using a G25 desalting column in the AKTA protein purification system at a flow rate of 1 mL / min, collected in fractions, with the target protein obtained in the 4th and 5th fractions. Figure 5 .

[0045] Take 5 mL of the G25 purified fraction 5 sample solution and purify it using a 1 mL anion exchange column in the AKTA protein purification system. Use 20 mM pH 8.0 Tris buffer as mobile phase A and 20 mM pH 8.0 Tris buffer containing 1 M sodium chloride as mobile phase B. Set the flow rate to 1 mL / min constant flow for 5 column volumes of mobile phase B, then elute 15 column volumes using a gradient of 1%–50% mobile phase B, and finally elute 5 column volumes using 1 mL / min constant flow for 50% mobile phase B. Figure 6 .

[0046] The separation of different sections of the anion exchange column was analyzed by protein electrophoresis, such as... Figure 7 In section 14, a single protein sample was obtained. The purified TFF3 fusion protein sample was then subjected to centrifugation and freeze-drying.

[0047] Example 4: In vitro experiment on TFF3 promoting cell migration 1. Experimental Methods The experimental method was improved based on that of Ciacci, C., SE Lind, and DK Podolsky. 1993. Transforming growth factor P regulation of migration in wounded rat intestinal epithelial monolayers. and Gastroenterology. 105:93-101. and McCormack, SA, MJ Viar, and LR Johnson. 1992. Migration of IEC-6 cells: a model for mucosal healing. Am. J. Physiol. 263G:426-435. The confluent monolayers of IEC-6 cells in 24-well multi-well plates were damaged using a razor blade, with each well measuring 4-5 mm × 1 cm. The cells were washed with fresh serum-free medium to remove any residual cell debris, and then, with or without protein, the damaged monolayers were cultured again in fresh plasma-free medium for 24 hours. The experiment was divided into four groups based on the substances applied: (A) control group, which received 1 μg / μl of fermentation broth purified without the TFF3 gene; (B) group receiving 1 μg / μl of purified TFF3; (C) group receiving 1 μg / μl of TFF3 standard; and (D) group receiving a combination of 1 μg / μl of purified TFF3 and 1 μg / μl of EGF. The purified TFF3 was prepared according to Example 3. The fermentation broth purified without the TFF3 gene was prepared as follows: Pichia pastoris without the TFF3 gene was fermented using the same method as in Example 2, and the resulting fermentation broth was purified using the same method as in Example 3 to obtain the TFF3 gene purified fermentation broth purified sample.

[0048] Experimental results were recorded using an inverted microscope. IEC-6 cell migration was assessed by blind counting of IEC-6 cells observed at the wound edge, expressed as the average number of cells present at the wound edge within a standard length (2 mm). Two wells were used in each experiment, with three wound regions per well for quantifying migration; data from at least three separate experiments were used to determine the effect of various substances on recovery (batch-to-batch variation <10%), by fixing cells and taking photomicrographs (×100) after 24 hours of culture. Scratches represent the original edge of the wound. Data are expressed as the average number of cells per standard wound edge unit ± SD.

[0049] 2. Experimental Results The results are as follows Figure 8As shown. As previously described, standard wounds were created in confluent monolayers of IEC-6 cells using a razor blade. After washing with fresh culture medium, the wounded monolayers were cultured with four different substances for 24 hours. No enhanced migration of IEC-6 cells to the wounds was observed in the control group treated with fermentation broth purified without TFF3. Figure 8 A), while the application of purified TFF3 enhanced the migration of IEC-6 cells to the wound by approximately 4 times ( Figure 8 B), and. Figure 8 D showed that the combined administration of purified TFF3 and EGF led to a significant increase in the recovery rate, which was 6-15 times higher than that of the control group.

[0050] The above results indicate that the TFF3 fusion protein produced by fermentation in this invention can effectively promote IEC-6 cell migration and intestinal wound healing.

Claims

1. A Pichia pastoris strain, characterized in that, Its accession number is CCTCC NO: M 2025013.

2. The application of the Pichia pastoris R5312 strain according to claim 1 for the fermentation preparation of TFF3 fusion protein.

3. A method for preparing TFF3 fusion protein by fermentation, characterized in that, Includes the following steps: The Pichia pastoris strain described in claim 1 was inoculated into a growth medium and cultured. The cells were collected by centrifugation, and the cells were resuspended in methanol induction medium. Methanol was added daily for induction culture, and the supernatant was collected after the culture was completed to obtain the TFF3 fusion protein.

4. The method according to claim 3, characterized in that, The growth medium is mBSM medium, containing the following components: glucose 48-52 g / L, KH2PO4 3-7 g / L, CaSO4 0.80-1.20 g / L, K2SO4 16.0-20.0 g / L, MgSO4 13.0-17.0 g / L, NH4H2PO4 38-42 g / L, KOH 1.3-1.7 g / L; pH value 5.5-6.

5.

5. The method according to claim 4, characterized in that, The growth medium contains the following components: glucose 50 g / L, KH2PO4 5 g / L, CaSO4 0.93 g / L, K2SO4 18.2 g / L, MgSO4 14.9 g / L, NH4H2PO4 40 g / L, KOH 1.5 g / L; pH 6.

0.

6. The method according to claim 3, characterized in that, The methanol-inducing medium is mBSM-M methanol-inducing medium, containing the following components: KH2PO4 3-7 g / L, CaSO4 0.80-1.20 g / L, K2SO4 16.0-20.0 g / L, MgSO4 13.0-17.0 g / L, NH4H2PO4 38-42 g / L, KOH 1.3-1.7 g / L, methanol 8-12 g / L; pH value 5.5-6.

5.

7. The method according to claim 3, characterized in that, The methanol-induced medium contains the following components: 5 g / L KH2PO4, 0.93 g / L CaSO4, 18.2 g / L K2SO4, 14.9 g / L MgSO4, 40 g / L NH4H2PO4, 1.5 g / L KOH, and 10 g / L methanol; pH 6.

0.

8. The method according to any one of claims 3-7, characterized in that, During the induction culture, methanol is added daily until the final methanol concentration is 0.5%-2.0%, the induction culture temperature is 28-30℃, and the induction culture time is more than 90 hours.

9. The method according to claim 8, characterized in that, During the induction culture, methanol is added daily until the final methanol concentration reaches 1.0%, the induction culture temperature is 30℃, and the induction culture time is 96-120 hours.

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