Pichia pastoris for highly expressing porcine epidermal growth factor fusion protein and application of pichia pastoris
By expressing the vector pPIC-fuEGF in Pichia schizophrenia strain RT01, the culture conditions are optimized, and the problem of difficulty in expression of epidermal growth factor in E. coli is solved, efficient and high expression and soluble improvement are achieved, and it is suitable for modern intensive aquaculture.
Patent Information
- Application Number
- CN202510693544.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-05-27
- Publication Date
- 2025-08-19
AI Technical Summary
In the prior art, epidermal growth factor exists in the form of inactive protein inclusion bodies when E. coli is expressed, the process is complex and costly, and the content of pig epidermal growth factor in breast milk is limited, making it difficult to obtain efficiently.
The pig epidermal growth factor gene was cloned on the Pichia cervical growth factor expression vector pPIC9K-fu using Pichia cervical strain RT01 through the EcoRI/NotI site, and methanol-induced culture was carried out to optimize the medium composition and induction conditions to achieve high expression of the pig epidermal growth factor fusion protein.
In the fermenter experiment, the expression level of pig epidermal growth factor reached 600mg/L, which increased the expression of soluble epidermal growth factor and had good application prospects.
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Figure CN120505215A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of fermentation engineering, and particularly relates to a Pichia pastoris strain highly expressing porcine epidermal growth factor fusion protein and uses thereof. Background Art
[0002] Epidermal growth factor (EGF) is a multifunctional growth factor with a strong mitogenic effect on a variety of tissue cells both in vitro and in vivo. EGF binds to the epidermal growth factor receptor (EGFR) on the cell surface, stimulating the activity of the receptor's intrinsic tyrosine kinase, thereby initiating a signaling cascade that leads to multiple biochemical changes. These include elevated intracellular calcium levels, increased glycolysis and protein synthesis, and increased expression of certain genes (including the EGFR), ultimately leading to DNA synthesis and cell proliferation. EGF is abundant in milk, and its receptors are widely distributed throughout the digestive tract, thus playing a crucial role in gastrointestinal development in newborn animals. In modern intensive farming, the use of EGF can enhance the health and overall production performance of young animals, representing a fundamental solution to the conflict between rapid animal growth and delayed organ development.
[0003] Epidermal growth factor exists in the form of a proprotein in animal breast milk, which is digested in the gastrointestinal tract to produce a mature polypeptide. Due to the high cost of obtaining epidermal growth factor from breast milk and the large molecular weight of the proprotein, direct exogenous expression of epidermal growth factor is difficult. Currently, expression in Escherichia coli is the primary method for producing epidermal growth factor, but this method often exists as inactive protein inclusion bodies, requiring subsequent separation, refolding, and purification. This process is complex and relatively costly.
[0004] A paper (Dowland F, Swayne A, Bhuta S, et al. Treatment of autoimmune encephalitis with immune-based therapies. Journal of Neurology, Neurosurgery & Psychiatry 2017;88:e1.) discloses a method for expressing and identifying porcine epidermal growth factor (PEF) in Pichia pastoris. Under optimal conditions, the engineered bacteria obtained in this study produced 82 mg / L of pEFG in the supernatant from shake flask-induced expression. However, the expression level of PEF still needs to be improved. Therefore, developing new methods for high EGF expression is of great research value. Summary of the Invention
[0005] In order to solve the above problems existing in the prior art, the object of the present invention is to provide a Pichia pastoris strain that highly expresses porcine epidermal growth factor fusion protein and its use.
[0006] In order to achieve the above object, the present invention adopts the following technical solutions:
[0007] The invention provides a Pichia yeast strain, and its preservation number is CCTCC NO: M 2025014.
[0008] The present invention also provides a method for producing epidermal growth factor by fermentation, which comprises inoculating the above-mentioned Pichia pastoris into a growth medium for cultivation, collecting the bacterial bodies by centrifugation, adding a methanol induction medium to suspend the bacterial bodies, adding methanol every day for induction cultivation, and collecting the supernatant after the induction cultivation is completed to obtain the epidermal growth factor.
[0009] Furthermore, the culture time is 10 to 100 hours, and the culture temperature is 10 to 60°C; the amount of methanol added daily is to add methanol every day to a final concentration of 0.1 to 10%, and the concentration is a volume concentration; the induction culture time is 50 to 200 hours, and the induction culture temperature is 10 to 60°C.
[0010] Furthermore, the culture time is 48 hours, and the culture temperature is 30°C; the amount of methanol added each day is to a final concentration of 1%; the induction culture time is 96 hours, and the induction culture temperature is 30°C.
[0011] Furthermore, the growth medium comprises glucose, KH2PO4, CaSO4, K2SO4, MgSO4, NH4H2PO4, KOH; the pH is 4.5 to 6.9;
[0012] The methanol induction culture medium contains KH2PO4, CaSO4, K2SO4, MgSO4, NH4H2PO4, KOH and methanol; and the pH value is 4.5-6.9.
[0013] Furthermore, in the growth medium, the glucose concentration is 10-100 g / L, the KH2PO4 concentration is 0.1-20 g / L, the CaSO4 concentration is 0.1-10 g / L, the K2SO4 concentration is 5-50 g / L, the MgSO4 concentration is 5-50 g / L, the NH4H2PO4 concentration is 10-100 g / L, and the KOH concentration is 0.1-10 g / L; the pH value is 4.5-6.9;
[0014] In the methanol induction culture medium, the concentration of KH2PO4 is 0.1-20 g / L, the concentration of CaSO4 is 0.1-10 g / L, the concentration of K2SO4 is 5-50 g / L, the concentration of MgSO4 is 5-50 g / L, the concentration of NH4H2PO4 is 10-100 g / L, the concentration of KOH is 0.1-10 g / L, the concentration of methanol is 1-50 g / L, and the pH value is 4.5-6.9.
[0015] Furthermore, in the growth medium, the concentration of glucose is 50 g / L, the concentration of KH2PO4 is 5 g / L, the concentration of CaSO4 is 0.93 g / L, the concentration of K2SO4 is 18.2 g / L, the concentration of MgSO4 is 14.9 g / L, the concentration of NH4H2PO4 is 40 g / L, and the concentration of KOH is 1.5 g / L; the pH value is 6.0;
[0016] In the methanol induction medium, the concentration of KH2PO4 is 5 g / L, the concentration of CaSO4 is 0.93 g / L, the concentration of K2SO4 is 18.2 g / L, the concentration of MgSO4 is 14.9 g / L, the concentration of NH4H2PO4 is 40 g / L, the concentration of KOH is 1.5 g / L, the concentration of methanol is 10 g / L, and the pH value is 6.0.
[0017] The present invention also provides use of the Pichia yeast in fermentation production of epidermal growth factor.
[0018] The present invention has achieved the following beneficial effects:
[0019] The present invention clones the epidermal growth factor (EGF) gene sequence into the Pichia pastoris expression vector pPIC9K-fu via the EcoRI / NotI sites to generate the recombinant vector pPIC-fuEGF. The recombinant vector pPIC-fuEGF is then transformed into Pichia pastoris GS115, and the RT01 strain, which can highly express a porcine epidermal growth factor fusion protein, is screened and obtained. The strain has a CCTCC deposit number of M2025014 and can express up to 600 mg / L of porcine epidermal growth factor in a fermenter experiment. The porcine epidermal growth factor fusion protein expressed by the present invention in Pichia pastoris can increase the expression of soluble epidermal growth factor and has good application prospects in modern intensive aquaculture.
[0020] Obviously, based on the above contents of the present invention, according to common technical knowledge and customary means in this field, without departing from the above basic technical ideas of the present invention, other various forms of modifications, replacements or changes can be made.
[0021] The following further describes the above content of the present invention in detail through specific embodiments in the form of examples. However, this should not be construed as limiting the scope of the above subject matter of the present invention to the following examples. All technologies implemented based on the above content of the present invention fall within the scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS
[0022] Figure 1 It is the pPIC9K-fu vector structure.
[0023] Figure 2 is the EGF concentration standard curve.
[0024] Figure 3 The results of SDS-PAGE electrophoresis analysis of EGF fusion protein expressed by RT01 strain.
[0025] Figure 4 This is the EGF fusion protein production curve of RT01 strain in a 10L fermentation tank.
[0026] Figure 5 These are the electrophoresis analysis results of samples from a 30-ton fermentation tank of RT01 strain at different induction times.
[0027] Figure 6 This is the EGF fusion protein production curve of the RT01 strain in a 30-ton fermentation tank. DETAILED DESCRIPTION
[0028] The raw materials and equipment used in the present invention are all known products and are obtained by purchasing commercially available products.
[0029] The following experiments, where no temperature is specified, are reactions conducted at room temperature, which is 25±5°C.
[0030] The RT01 strain of the present invention is deposited with the China Center for Type Culture Collection as a patented strain. The depository address is Wuhan University, 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province. The deposit number is CCTCC NO: M 2025014. The deposit date is January 3, 2025, with a 30-year validity period. The strain is designated Komagataella pastoris RT001.
[0031] Synthesis of porcine epidermal growth factor gene fragment: The EGF DNA sequence was sent to Invitrogen for synthesis.
[0032] The DNA sequence of EGF (SEQ ID No: 1) is as follows:
[0033] AATTCTTACTCTGAATGTCCACCTTCCCACGACGGTTACTGTTTGCACGGTGGTGTTTGTATGTATATTGAAGCTGTTGATTCTTACGCTTGTAACTGTGTTTTTGGTTACGTTGGTGAAAGATGTCAGCATAGAGATTTGAAGTGGTGGGAACTGAGA
[0034] The protein sequence of EGF (SEQ ID No: 2) is as follows:
[0035] NSYSECPPSHDGYCLHGGVCMYIEAVDSYACNCVFGYVGERCQHRD LKWWELR
[0036] Example 1: Construction of Pichia pastoris RT01 that efficiently expresses porcine epidermal growth factor fusion protein
[0037] The multi-copy Pichia pastoris expression kit from Invitrogen was purchased and the full gene sequence EGF was cloned into the Pichia pastoris expression vector pPIC9K-fu through the EcoRI / NotI sites. The pPIC9K-fu vector (structure as shown in the figure) Figure 1 The figure shows the modified expression vector carrying the tagged protein fu, resulting in the recombinant vector pPIC-fuEGF. DNA recombination operations were performed primarily according to the Molecular Cloning Manual.
[0038] The recombinant vector pPIC-fuEGF was linearized with Sal I and transformed into Pichia pastoris GS115 using a Biorad electroporator at 1500V. Positive transformants were screened by PCR and shake flask screening. The resulting strain with high expression was named RT01. For electroporation and screening methods, please refer to the Invitrogen manual, "Multi-Copy Pichia Expression Kit," pages 34-56, https: / / assets.thermofisher.cn / TFS-Assets / LSG / manuals / pichmulti_man.pdf.
[0039] High-expressing positive clones were screened using shake flask screening medium. The cells were cultured in 25 mL of mBSM medium at 30°C for 48 h, and the cells were collected by centrifugation. 15 mL of mBSM-M methanol induction medium was added to suspend the cells to obtain a bacterial suspension. Methanol was added daily to a final methanol concentration of 1% (i.e., the volume ratio of added methanol to bacterial suspension was 1:99). The cells were induced at 30°C for 96 h. The EGF content in the supernatant of each strain was sampled and detected, and transformants expressing EGF were screened.
[0040] 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.
[0041] mBSM-M methanol induction medium: KH2PO4 5g / L, CaSO4 0.93g / L, K2SO4 18.2g / L, MgSO414.9g / L, NH4H2PO4 40g / L, KOH 1.5g / L, methanol concentration 10g / L, pH 6.0.
[0042] Use the porcine EGF detection kit produced by Cloud Clone, USA, to detect the content of EGF in the culture medium (culture medium after 96 hours of induction expression). The operation steps are as follows: Figure 2 and as shown in Table 1.
[0043] Table 1 EGF concentration standard curve
[0044] EGF standard concentration (pg / mL) 250 125 62.5 31.75 15.875 OD450 1.3564 0.7235 0.4386 0.2958 0.1842
[0045] Using the standard curve as a reference, the culture fluid samples were tested by ELISA according to the dilution factor to determine the expression level.
[0046] The calculation formula is EGF content = (202.92 * (Sample A - AGS115) - 24.66) * dilution factor, where Sample A is the light absorption value of the fermentation broth sample and AGS115 is the light absorption value of the blank strain. The EGF content in the fermentation broth of the RT01 strain of the present invention after 96 hours of induction expression was 153 mg / L.
[0047] Example 2: Fermentation experiment of RT01 strain in 10L fermenter
[0048] According to 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 10 L fermentor. The fermentation process was recorded, samples were taken at appropriate times, and the expression of the fusion protein was analyzed by SDS-PAGE electrophoresis (e.g. Figure 3The porcine EGF detection kit produced by Cloud Clone Company of the United States was used to determine the content of EGF fusion protein and draw an expression curve, as shown in FIG. Figure 4 As shown, the final EGF content of the fermentation broth was 366 mg / L.
[0049] Example 3: Fermentation experiment of RT01 strain in 30-ton fermenter
[0050] The culture medium used in the 30-ton tank was consistent with the culture medium used in the Pichia Fermentation Process Guidelines provided by Invitrogen (version B053002, pages 1-7, https: / / tools.thermofisher.com / content / sfs / manuals / pichiaferm_prot.pdf). The process flow was as follows: after culturing for 29 hours, the biomass reached 100 g / L, the carbon source of the base feed was exhausted, and the dissolved oxygen increased rapidly. The glycerol feeding stage began at 100 L / h. After culturing for 38 hours, the biomass reached 222 g / L, and the glycerol feeding was stopped. The mixed feeding process was carried out with 75 L / h of glycerol and methanol. 10L / h, gradually increase the proportion of methanol, after 6 hours of mixed feeding, stop feeding, wait for the dissolved oxygen to rebound to the highest point, continue for 15 minutes, start methanol induction, the initial flow addition rate is 40L / h, gradually increase the methanol dosage, slowly reduce the dissolved oxygen, stabilize the flow addition after the methanol flow rate is stable at 75L / h, the dissolved oxygen in the fermentation process is maintained between 15-35%, stop methanol flow addition every 4 hours during the fermentation process, the dissolved oxygen can rise rapidly in the early 2 minutes, and the dissolved oxygen can rise in the later 10 minutes, the frequency of ammonia flow addition becomes longer 3-4 hours after fermentation for 150 hours, fermentation for 182 hours, OD390 of the tank is released, the wet weight is 427g / L, and the tank volume is 19m 3 , glycerol consumption 1.5m 3 , methanol consumption 7.5m 3 .
[0051] The electrophoresis analysis results of samples from the RT01 strain 30-ton fermenter fermentation experiment at different induction times are as follows: Figure 5 shown.
[0052] The porcine EGF detection kit produced by Cloud Clone Company of the United States was used to detect the content of EGF in the fermentation broth ( Figure 6 ), the final EGF expression level of the fermentation broth was 466 mg / L, but it can be seen that the EGF content can reach 600 mg / L between 110 and 140 hours, so the induction time can be set between 110 and 140 hours during production.
[0053] In summary, the present invention clones the epidermal growth factor (EGF) gene sequence into the Pichia pastoris expression vector pPIC9K-fu via the EcoRI / NotI sites to generate the recombinant vector pPIC-fuEGF. The recombinant vector pPIC-fuEGF is then transformed into Pichia pastoris GS115, and the RT01 strain, which can highly express a porcine epidermal growth factor fusion protein, was screened. This strain expressed porcine epidermal growth factor at a level of 600 mg / L in a fermenter experiment. The porcine epidermal growth factor fusion protein expressed in Pichia pastoris can increase the expression of soluble epidermal growth factor and has promising application prospects in modern intensive aquaculture.
Claims
1. A Pichia yeast strain characterized by: Its accession number is CCTCC NO: M 2025014.
2. A method for producing epidermal growth factor by fermentation, characterized in that: The method comprises inoculating the Pichia yeast of claim 1 into a growth medium for cultivation, collecting the bacterial bodies by centrifugation, adding a methanol induction medium to suspend the bacterial bodies, adding methanol every day for induction cultivation, and collecting the supernatant after the induction cultivation is completed to obtain epidermal growth factor.
3. The method according to claim 2, wherein: The culture time is 10 to 100 hours, and the culture temperature is 10 to 60° C.; the amount of methanol added each day is to add methanol to a final concentration of 0.1 to 10%, and the concentration is a volume concentration; the induction culture time is 50 to 200 hours, and the induction culture temperature is 10 to 60° C.
4. The method according to claim 3, wherein: The culture time is 48 hours, and the culture temperature is 30°C; the amount of methanol added every day is such that the final concentration is 1%; the induction culture time is 96 hours, and the induction culture temperature is 30°C.
5. The method according to claim 2, wherein: The growth medium comprises glucose, KH2PO4, CaSO4, K2SO4, MgSO4, NH4H2PO4, and KOH; and has a pH of 4.5 to 6.9; The methanol induction culture medium contains KH2PO4, CaSO4, K2SO4, MgSO4, NH4H2PO4, KOH and methanol; and the pH value is 4.5-6.
9.
6. The method according to claim 5, characterized in that: The growth medium has a glucose concentration of 10-100 g / L, a KH2PO4 concentration of 0.1-20 g / L, a CaSO4 concentration of 0.1-10 g / L, a K2SO4 concentration of 5-50 g / L, a MgSO4 concentration of 5-50 g / L, a NH4H2PO4 concentration of 10-100 g / L, and a KOH concentration of 0.1-10 g / L; and a pH value of 4.5-6.9; In the methanol induction culture medium, the concentration of KH2PO4 is 0.1-20 g / L, the concentration of CaSO4 is 0.1-10 g / L, the concentration of K2SO4 is 5-50 g / L, the concentration of MgSO4 is 5-50 g / L, the concentration of NH4H2PO4 is 10-100 g / L, the concentration of KOH is 0.1-10 g / L, the concentration of methanol is 1-50 g / L, and the pH value is 4.5-6.
9.
7. The method according to claim 6, characterized in that: The growth medium has a glucose concentration of 50 g / L, a KH2PO4 concentration of 5 g / L, a CaSO4 concentration of 0.93 g / L, a K2SO4 concentration of 18.2 g / L, a MgSO4 concentration of 14.9 g / L, a NH4H2PO4 concentration of 40 g / L, and a KOH concentration of 1.5 g / L; and a pH value of 6.0; In the methanol induction medium, the concentration of KH2PO4 is 5 g / L, the concentration of CaSO4 is 0.93 g / L, the concentration of K2SO4 is 18.2 g / L, the concentration of MgSO4 is 14.9 g / L, the concentration of NH4H2PO4 is 40 g / L, the concentration of KOH is 1.5 g / L, the concentration of methanol is 10 g / L, and the pH value is 6.
0.
8. Use of the Pichia pastoris according to claim 1 in the fermentation production of epidermal growth factor.