Method for improving expression of recombinant human epidermal growth factor by pichia pastoris

Abstract

The invention relates to the technical field of bioengineering and fermentation engineering, and particularly discloses a method for improving expression of recombinant human epidermal growth factors by pichia pastoris, which comprises the step of adding carboxymethylation modified passion fruit peel polysaccharide derivatives into a culture medium in a pichia pastoris fermentation culture process. According to the method for improving the expression of the recombinant human epidermal growth factor by the pichia pastoris, a proper amount of carboxymethylation-modified passion fruit peel polysaccharide derivative (C-WPEP) is added into a pichia pastoris expression system, so that the expression quantity, stability and activity of the rhEGF can be remarkably improved, the operation is simple and convenient, the cost is low, and the method is suitable for industrial large-scale production of the rhEGF.

Description

Technical Field

[0001] This invention relates to the fields of bioengineering and fermentation engineering, and in particular to a method for enhancing the expression of recombinant human epidermal growth factor in Pichia pastoris. Background Technology

[0002] Recombinant human epidermal growth factor (rhEGF) is a small-molecule active protein and a highly efficient multifunctional cytokine that can promote the proliferation and differentiation of epithelial cells and fibroblasts. It has wide applications in wound repair, burn treatment, diabetic ulcer healing, and skin care. Currently, rhEGF is in high demand in the market as an important active ingredient in biopharmaceuticals and cosmetics. However, due to its small molecular weight, numerous disulfide bonds, and structural sensitivity to the external environment, large-scale production and improved stability remain bottlenecks for its industrialization.

[0003] Pichia pastoris has become a common host for the industrial production of rhEGF due to its advantages such as high-density culture, secretory expression, eukaryotic modification capabilities, and low cost. However, during fermentation, rhEGF yield is limited by the secretory pathway load and protein folding efficiency, and the expression level is usually difficult to further increase. Existing methods mainly involve modifying the host through molecular biology techniques, such as co-expressing molecular chaperones or optimizing signal peptides to improve secretion efficiency, but these methods generally suffer from problems such as complex processes, limited effectiveness, or high costs. Meanwhile, rhEGF is easily inactivated in fermentation broth and purified products due to temperature, pH, and environmental factors, resulting in insufficient stability and biological activity, thus affecting the preservation, transportation, and application of the product. Currently, in industrial production, there is still a lack of ideal solutions for simultaneously improving rhEGF yield and thermal stability with simple operation and low cost. Summary of the Invention

[0004] To address the above shortcomings, this invention provides a method for enhancing the expression of recombinant human epidermal growth factor (rhEGF) in Pichia pastoris, which can significantly improve the expression level and stability of rhEGF. The method is simple to operate and low in cost. The specific technical solution is as follows: A method for enhancing the expression of recombinant human epidermal growth factor in Pichia pastoris includes: adding a carboxymethylated passion fruit peel polysaccharide derivative (C-WPEP) to the culture medium during the fermentation culture of Pichia pastoris.

[0005] Preferably, in the above-mentioned method for enhancing the expression of recombinant human epidermal growth factor in Pichia pastoris, the degree of carboxymethylation (DS) of the passion fruit peel polysaccharide derivative is 0.2~1.2, more preferably 0.4~0.8.

[0006] Preferably, in the above-mentioned method for enhancing the expression of recombinant human epidermal growth factor by Pichia pastoris, the amount of passion fruit peel polysaccharide derivative added is 1.0 g / L to 5.0 g / L, and more preferably 2.0 g / L to 3.0 g / L.

[0007] Preferably, in the above-mentioned method for enhancing the expression of recombinant human epidermal growth factor in Pichia pastoris, the preparation method of the passion fruit peel polysaccharide derivative includes the following steps: (1) Take passion fruit peel and decolorize it by reflux with ethanol solution; (2) Crude polysaccharide was extracted by microwave-assisted water extraction and purified by precipitation with anhydrous ethanol to obtain crude polysaccharide; (3) Add the crude polysaccharide from step (2) to an alkaline solution, add a carboxymethylation reagent under stirring, react at 50~80℃ for 2~3h, and obtain C-WPEP by neutralization, dialysis and freeze drying.

[0008] Preferably, in the above-described method for enhancing the expression of recombinant human epidermal growth factor in Pichia pastoris, when the carboxymethylating agent is chloroacetic acid, its addition amount is preferably 2 to 3 times the mass of WPEP.

[0009] Preferably, in the above-mentioned method for enhancing the expression of recombinant human epidermal growth factor in Pichia pastoris, in step (1), the volume percentage concentration of ethanol in the ethanol solution is 80%.

[0010] Preferably, in the above-mentioned method for enhancing the expression of recombinant human epidermal growth factor in Pichia pastoris, in step (2), the microwave power is 500~700W and the microwave time is 3~5min.

[0011] Preferably, in the above-described method for enhancing the expression of recombinant human epidermal growth factor in Pichia pastoris, the alkaline solution is a mixed solution of NaOH aqueous solution and isopropanol; the mass concentration of the NaOH aqueous solution is 20%, and the volume ratio of NaOH aqueous solution to isopropanol is 1:2~3.

[0012] Preferably, in the above-mentioned method for enhancing the expression of recombinant human epidermal growth factor by Pichia pastoris, in step (3), dialysis is performed using a dialysis bag with a molecular weight cutoff of 3000 Da for 24 hours.

[0013] Preferably, in the above-described method for enhancing the expression of recombinant human epidermal growth factor in Pichia pastoris, the passion fruit peel polysaccharide derivative is added all at once at the beginning of Pichia pastoris fermentation induction or added in several installments during the induction phase.

[0014] Preferably, in the above-mentioned method for enhancing the expression of recombinant human epidermal growth factor in Pichia pastoris, the fermentation conditions are: temperature 25~30℃, pH maintained at 6.0~7.0, and dissolved oxygen not less than 20%.

[0015] Compared with the prior art, the beneficial effects of the present invention are: 1. In the method for enhancing the expression of recombinant human epidermal growth factor in Pichia pastoris of the present invention, adding an appropriate amount of carboxymethylated passion fruit peel polysaccharide derivative (C-WPEP) to the Pichia pastoris expression system can significantly improve the expression level, stability and bioactivity of rhEGF without changing the host strain and gene construction. The method is simple to operate, low in cost, and suitable for large-scale industrial production of rhEGF, showing good prospects for industrial application.

[0016] 2. In the method for enhancing the expression of recombinant human epidermal growth factor in Pichia pastoris according to the present invention, compared with the control group without C-WPEP, the expression yield of rhEGF increased by more than 30% with the addition of C-WPEP; after treatment at 55℃ for 60 minutes, the residual activity of rhEGF remained above 80%; NIH-3T3 cell proliferation assay showed that the cell proliferation promotion effect of rhEGF in the C-WPEP-added group reached 135% of that in the control group; the experimental results show that the method of the present invention can significantly enhance the expression level, stability and biological activity of rhEGF, which is beneficial for storage and application.

[0017] 3. This invention only requires the addition of C-WPEP to the fermentation system, making the process simple and easy to operate; the raw materials are abundant (passion fruit peel is a by-product of juice processing), and the cost is low; it can be directly integrated into existing industrial fermentation systems and has promotional value. Attached Figure Description

[0018] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0019] Figure 1 Fourier transform infrared (FT-IR) spectra of carboxymethylated passion fruit peel polysaccharide (C-WPEP) prepared according to this invention. The figure shows the wavelength of C-WPEP at 1611 cm⁻¹. -1 (Carboxyl C=O asymmetric stretching), 1422 cm -1 (CH2 bending vibration), 1328 cm -1 The presence of a characteristic absorption peak at the (C–O symmetric stretching) position serves as proof of successful carboxymethylation modification.

[0020] Figure 2The effect of C-WPEP with different degrees of carboxymethylation (DS) on rhEGF expression (72 h, n = 3). The figure compares the rhEGF expression levels of the DS = 0.2, 0.4, 0.6, 0.8, 1.0, 1.2 and the untreated group, showing that the promoting effect is most significant when DS = 0.4~0.8.

[0021] Figure 3 The following are the ELISA results for rhEGF expression in Example 5 of this invention. (a) Effect of different fermentation times (24, 48, 72, 96, 120 h) on rhEGF expression; (b) Effect of different C-WPEP addition amounts (0~5.0 g / L) on rhEGF expression. The results show that rhEGF expression was highest at 72 h of induction; and the best promoting effect was observed within the addition range of 2.0~3.0 g / L.

[0022] Figure 4 SDS-PAGE electrophoresis analysis of rhEGF in Example 5 of this invention: (a) Electrophoretic band display; (b) Comparison of protein secretion levels at 72 h between the control group and the C-WPEP treatment group (2.0 g / L). The rhEGF band was significantly enhanced in the C-WPEP treatment group, indicating that its expression-promoting effect was consistent with the ELISA results.

[0023] Figure 5 This figure shows the natural biological activity of rhEGF in Example 6 of the present invention (NIH-3T3 cell proliferation experiment). The figure compares the promoting effect of rhEGF on NIH-3T3 cell proliferation in the control group, WPEP group and C-WPEP group. The results show that the cell proliferation rate of C-WPEP group is the highest (approximately 135% of the control group).

[0024] Figure 6 This is a comparison of the residual bioactivity of rhEGF after heat treatment in Example 6 of the present invention. After treating each group of rhEGF at 55℃ for 60 min, their promoting effect on NIH-3T3 cells was detected. The results showed that the residual activities were: control group 52%, WPEP group 65%, and C-WPEP group 82%, indicating that C-WPEP significantly improved the heat resistance of rhEGF. Detailed Implementation

[0025] The specific embodiments of the present invention are described in detail below, but it should be understood that the scope of protection of the present invention is not limited to the specific embodiments. Unless otherwise defined, all technical terms used below have the same meaning as commonly understood by those skilled in the art. The technical terms used herein are for the purpose of describing specific embodiments only and are not intended to limit the scope of protection of the present invention. Unless otherwise specifically stated, all raw materials, reagents, instruments, and equipment used in the present invention are commercially available or can be prepared by existing methods.

[0026] Example 1 This example provides a method for preparing a carboxymethylated passion fruit peel polysaccharide derivative, including the following steps: (1) Select 250g of fresh passion fruit peel, wash and cut it into pieces, place it in 80% ethanol (material-liquid mass ratio 1:20), reflux in a water bath at 80℃ for 2h, repeat 3 times to remove pigments, polyphenols and small molecule impurities; filter the obtained peel and dry it for later use. (2) The pericarp obtained in step (1) was crushed, 5.0 g of powder was weighed and added to 135 mL of distilled water, and extracted by microwave-assisted water extraction at a microwave power of 600 W for 3.5 min. The extract was collected by suction filtration, concentrated at 95 °C, and precipitated by adding 4 times the volume of anhydrous ethanol. It was then refrigerated at −4 °C overnight. The supernatant was removed by centrifugation, the precipitate was redissolved, and the above operation was repeated twice. Finally, the crude polysaccharide powder (WPEP) was obtained by freeze drying, with a yield of about 6.2%.

[0027] (3) Weigh 120 mg WPEP and dissolve it in a mixture of 10 mL 20% NaOH and 25 mL isopropanol. Stir in an ice bath for 3 h to obtain a uniform suspension. Dissolve 300 mg chloroacetic acid in 25 mL isopropanol and mix it with 10 mL 20% NaOH. Slowly add the mixture to the reaction system and stir at 60 °C for 3 h. Adjust the pH of the reaction solution to 7, dialyze (molecular weight cutoff 3000 Da) for 24 h and freeze dry to obtain carboxymethylated passion fruit peel polysaccharide (C-WPEP).

[0028] like Figure 1 As shown, Fourier transform infrared spectroscopy (FT-IR) analysis indicates that C-WPEP is at 1611 cm⁻¹. -1 1422 cm -1 1328 cm -1 New characteristic peaks appeared at locations such as 1611 cm⁻¹. -1 This is an asymmetric stretching vibration of the C=O group in the carboxylate (-COO-) group, a typical absorption introduced by the carboxymethyl structure; 1422 cm⁻¹ -1The absorption primarily corresponds to the symmetric stretching vibration of the carboxylate COO-, accompanied by the bending vibration absorption of –CH2–, indicating the formation of the carboxymethyl side chain (-CH2–COO-); 1328 cm⁻¹ -1 This is due to the enhanced symmetric stretching vibrations of C–O / C–O–C in the polysaccharide structure after carboxymethylation, reflecting the structural changes after the hydroxyl groups of the polysaccharide are replaced.

[0029] The aforementioned absorption peaks were not observed in unmodified WPEP, but were significantly enhanced in C-WPEP, fully demonstrating that the carboxymethylation reaction had occurred and successfully introduced the carboxymethyl group into the polysaccharide molecule. The degree of substitution was determined by the 2,7-dihydroxynaphthalene colorimetric method, with an average value of 0.65.

[0030] Example 2: A method for enhancing the expression of recombinant human epidermal growth factor in Pichia pastoris, comprising: Pichia pastoris strain: The recombinant Pichia pastoris expression strain GS115-rhEGF used in this invention is a commercially available strain purchased from Invitrogen. This engineered strain can secrete and express rhEGF, and its gene composition and expression mode were provided by the supplier. This invention does not involve the construction process of this engineered strain, but rather focuses on the optimization of the fermentation process and the technical research on adding C-WPEP to improve expression levels and protein stability based on this engineered strain.

[0031] Host strain Pichia pastoris GS115 (his4, Mut + Pichia pastoris is a widely used methanol-nutritional expression host internationally, and its application in recombinant protein secretory expression has been extensively reported in the literature. For example, Eissazadeh et al. reported in the Brazilian Journal of Microbiology their study on rhEGF expression in the Pichia pastoris GS115 background, demonstrating that this host strain is suitable for expression systems of exogenous proteins. The engineered strain used in this invention was obtained based on such a mature system.

[0032] Fermentation medium composition: YPD seed culture medium: 20 g glucose, 10 g yeast extract, 20 g tryptone, add water to 1 L, sterilize at 121℃ for 20 min.

[0033] BMGY glycerol growth medium: 10 g yeast extract, 20 g tryptone, 10 g glycerol, 1.34 g KH2PO4, 11.67 g K2HPO4, 0.5 g MgSO4·7H2O, 4.35 mL PTM1 trace elements, add water to 1 L.

[0034] BMMY methanol-induced medium: 10 g yeast extract, 20 g tryptone, 0.5% initial methanol (v / v), 1.34 g KH2PO4, 11.67 g K2HPO4, 0.5 g MgSO4·7H2O, 4.35 mL PTM1 trace elements, and water to 1 L.

[0035] PTM1 Trace Element Solution: CuSO4·5H2O 6.0 g, ZnCl2 20.0 g, NaI 0.08 g, MnSO4·H2O 3.0 g, Na2MoO4·2H2O 0.2 g, H3BO3 0.02 g, CoCl2·6H2O 0.5 g, FeSO4·7H2O 65.0 g, Biotin 0.2 g, concentrated sulfuric acid 5 mL, add water to 1 L.

[0036] Fermentation process: 1. Seed culture: The frozen recombinant Pichia pastoris was inoculated into 50 mL of YPD liquid medium and cultured at 28℃ with shaking at 220 rpm for 16-20 h, allowing the cells to grow to the OD level. 600 Version 2.0~4.0.

[0037] 2. Scale-up culture: Inoculate 10% (v / v) into 500 mL of BMGY medium and continue culturing at 28℃ and 220 rpm for 12-18 h until the bacterial density reaches OD500. 600 ≥6 is used for fermenter inoculation.

[0038] 3. Glycerol growth stage (fermentation and proliferation period): Introduce the expansion culture medium into a 5 L fermenter at a ratio of 5~10% (v / v) and control the following conditions: temperature: 28℃, pH: 6.0 (adjusted with 28% ammonia), aeration rate: 1.0~1.5 vvm, stirring: 400~700 rpm, dissolved oxygen: ≥20%.

[0039] After glycerol is depleted (dissolved oxygen levels rise), the induction phase begins.

[0040] Methanol-induced culture: After glycerol depletion (significant increase in dissolved oxygen), the methanol-induced culture stage was initiated. During the methanol-induced culture, C-WPEP prepared in Example 1 was added to the fermentation system at different amounts, setting up C-WPEP treatment groups (0, 1.0, 2.0, 3.0, 4.0, 5.0 g / L), a control group (no C-WPEP added), and a WPEP group (added with an equal amount of unmodified polysaccharide WPEP).

[0041] To improve feasibility, this invention further provides preferred induction process conditions for a 5 L fermenter. The total volume of the fermenter is 5 L, with a working volume of approximately 3 L.

[0042] Methanol induction can be divided into two stages: the induction initiation stage and the steady-state induction stage, as detailed below: (1) Induction initiation stage (0~12 h) At the start of induction, methanol is added all at once to bring the methanol volume fraction in the tank to 0.5% (v / v).

[0043] Subsequently, based on changes in dissolved oxygen, methanol was added at an average flow rate of approximately 0.4 mL / (L·h) to maintain the methanol content in the tank at approximately 0.5% (v / v) to avoid cytotoxicity caused by a sudden increase in methanol.

[0044] (2) Steady-state induction phase (12~72 h) The steady-state induction phase is the main expression period of rhEGF.

[0045] At this stage: The methanol flow acceleration was controlled at 0.4~0.8 mL / (L·h); For a 3 L working volume, add approximately 1.2~2.4 mL / h or 15~30 mL of methanol every 12 h. Maintain the methanol volume fraction in the tank at 0.5~1.0% (v / v).

[0046] Fermentation conditions: temperature 28℃, pH 6.0 (adjusted with 28% ammonia), dissolved oxygen control ≥20%, aeration rate 1.0~1.5vvm (approximately 3.0~4.5 L / min air), stirring speed automatically adjusted to 400~700 rpm according to dissolved oxygen.

[0047] Under the above conditions, rhEGF expression levels typically reach their peak around 72 hours.

[0048] (3) Late induction stage (72~96 h) To reduce metabolic stress in the later stages, the methanol load can be appropriately reduced: Reduce the methanol flow rate to 0.2-0.4 mL / (L·h) or add a small dose every 24 h. Terminate fermentation when the expression level no longer increases.

[0049] The above-mentioned stage divisions and parameters are not limiting conditions of the present invention, but are used to illustrate preferred embodiments that can achieve good expression results in a typical 5 L fermenter.

[0050] Example 3: Effect of C-WPEP with different degrees of carboxymethylation (DS) on rhEGF expression. To investigate the effect of C-WPEP carboxymethylation degree on enhancing rhEGF expression, C-WPEP with DS values ​​of approximately 0.2, 0.4, 0.6, 0.8, 1.0, and 1.2 were prepared according to the method described in Example 1. Each group was added to a Pichia pastoris fermentation system under the same fermentation conditions at a concentration of 2.0 g / L, with the fermentation system without added polysaccharides serving as a control group. Fermentation supernatant was collected after 72 h of methanol induction, and the concentration of rhEGF was detected using ELISA. Each condition was repeated in triplicate. The results are shown below. Figure 2 As shown (mean ± SD, n = 3).

[0051] like Figure 2 As shown, C-WPEP with different degrees of carboxymethylation significantly affected rhEGF expression. Expression levels were slightly increased when DS = 0.2; the promoting effect was most significant when DS = 0.4–0.8, with the highest expression level (approximately 270 mg / L) at DS = 0.6, representing an increase of approximately 35% compared to the control group (approximately 200 mg / L). Expression levels decreased when DS increased to 1.0 and 1.2, indicating that excessive carboxymethylation may weaken its ability to promote fermentation expression.

[0052] These results indicate that the degree of substitution of C-WPEP is a crucial factor in regulating its expression-promoting effect, with a preferred substitution range of DS = 0.4–0.8, where DS = 0.6 is optimal. This result provides experimental support for the preferred substitution degree described in this invention.

[0053] Example 4: Effects of fermentation time and C-WPEP addition on rhEGF expression levels After determining the optimal degree of substitution (DS) of 0.6 in Example 3 (Substitution Degree Screening), a C-WPEP addition amount of 2.0 g / L was selected, and the expression level was detected at different time points during methanol induction. Fermentation supernatant was collected at 24, 48, 72, 96, and 120 h of induction, and the rhEGF concentration was determined using ELISA. The results are shown below. Figure 3 (a).

[0054] The results showed that rhEGF expression gradually increased with increasing induction time, reaching its peak at 72 h, an increase of over 250% compared to 24 h. After 72 h, the expression level no longer increased significantly, and showed a decreasing trend at 96 h and 120 h. These results indicate that 72 h is the optimal induction time for the system of this invention.

[0055] Based on the optimal degree of substitution (DS = 0.6), the C-WPEP addition amounts were set to 0, 1.0, 2.0, 3.0, 4.0, and 5.0 g / L. After induction for 72 h under the same fermentation conditions, rhEGF expression was measured using ELISA. The results are shown below. Figure 3 (b)

[0056] like Figure 3 As shown in (b), when the amount of C-WPEP added was 2.0~3.0 g / L, the expression level was significantly increased by 30%~38%; the promoting effect was not obvious when the amount added was ~1.0 g / L, while the promoting effect decreased when it exceeded 3.0 g / L.

[0057] The overall results show that the optimal addition amount of C-WPEP is 2.0~3.0 g / L.

[0058] Example 5: SDS-PAGE analysis of rhEGF secretion levels To further validate the ELISA results, SDS-PAGE was used to analyze the protein secretion levels in the fermentation supernatant. The treatment group with 2.0 g / L C-WPEP was compared with the control group and the WPEP group; the results are shown in Figure 4.

[0059] like Figure 4 As shown, the rhEGF band in the C-WPEP treatment group was significantly enhanced, increasing by approximately 31% compared to the control group, consistent with the ELISA results, further demonstrating that C-WPEP can increase the secretory expression level of rhEGF.

[0060] Example 6: Biological activity and thermal stability tests of rhEGF To systematically evaluate the biological performance of rhEGF under different treatment conditions (control group, WPEP group and C-WPEP group), the natural activity of unheated samples was first determined by NIH-3T3 cell proliferation assay, and then the same batch of samples were heat-treated at 55℃ and their residual biological activity was determined.

[0061] (1) Natural biological activity of rhEGF The NIH-3T3 cell proliferation assay was used to evaluate the biological activity of rhEGF. Unheated purified protein was used in all three rhEGF groups (control group, WPEP group, and C-WPEP group) to maintain a consistent final concentration. The experimental procedures are as follows: 1) NIH-3T3 cells were seeded in 96-well plates and cultured until adherent; 2) The medium was replaced with serum-free medium; 3) The same final concentration of rhEGF protein sample was added to each well; 4) CCK-8 reagent was added after 24 h of culture; 5) The absorbance at 450 nm was measured, and the relative proliferation rate was compared with the control group as 100%.

[0062] Experimental results are as follows Figure 5 As shown: Control group: 100%; WPEP group: approximately 115%; C-WPEP group: approximately 135%.

[0063] The results showed that rhEGF obtained by fermentation after C-WPEP treatment had higher natural biological activity.

[0064] (2) Calculation method of thermal stability and residual activity of rhEGF To evaluate the thermal stability of rhEGF, samples of the same batch of rhEGF were placed in a 55°C water bath for 60 min, and then immediately cooled to 4°C in an ice bath. The residual biological activity was then determined according to the following method.

[0065] Methods for determining bioactivity after heat treatment Except for the heat treatment step, the other NIH-3T3 proliferation experimental methods are consistent with the above-described natural bioactivity analysis. Let: OD blank : Absorbance of blank well; OD unheated : Absorbance of cells after stimulation with unheated rhEGF; OD heated : Cell absorbance after heat treatment and rhEGF stimulation.

[0066] Using the proliferation effect of unheated rhEGF as 100% activity, its proliferation effect is defined as: Proliferation effect unheated = OD unheated - OD blank The multiplication effect after heat treatment: Proliferation effect heated = OD heated - OD blank Formula for calculating residual bioactivity (%): Residual activity (%) = (proliferative effect) heated / proliferative effect unheated ) × 100% Experimental results are as follows Figure 6 Residual activity in the control group: 52%, WPEP group: 65%, C-WPEP group: 82%.

[0067] The results showed that C-WPEP treatment significantly improved the stability of rhEGF at 55℃ compared with unmodified polysaccharides.

[0068] The foregoing description of specific exemplary embodiments of the invention is for illustrative and explanatory purposes. These descriptions are not intended to limit the invention to the precise forms disclosed, and it will be apparent that many changes and variations can be made in accordance with the foregoing teachings. The exemplary embodiments were chosen and described in order to explain the specific principles of the invention and its practical application, thereby enabling those skilled in the art to implement and utilize various different exemplary embodiments of the invention, as well as various different choices and variations. The scope of the invention is intended to be defined by the claims and their equivalents.

Claims

1. A method for enhancing the expression of recombinant human epidermal growth factor in Pichia pastoris, characterized in that, include: During the fermentation and culture of Pichia pastoris, a carboxymethylated passion fruit peel polysaccharide derivative was added to the culture medium.

2. The method for enhancing the expression of recombinant human epidermal growth factor in Pichia pastoris according to claim 1, characterized in that, The amount of passion fruit peel polysaccharide derivative added is 1.0 g / L to 5.0 g / L.

3. The method for enhancing the expression of recombinant human epidermal growth factor in Pichia pastoris according to claim 2, characterized in that, The amount of passion fruit peel polysaccharide derivative added is 2.0 g / L to 3.0 g / L.

4. The method for enhancing the expression of recombinant human epidermal growth factor in Pichia pastoris according to claim 1, characterized in that, The degree of carboxymethylation substitution of the passion fruit peel polysaccharide derivative is 0.2~1.

2.

5. The method for enhancing the expression of recombinant human epidermal growth factor in Pichia pastoris according to claim 1, characterized in that, The degree of carboxymethylation substitution of the passion fruit peel polysaccharide derivative is 0.4~0.

8.

6. The method for enhancing the expression of recombinant human epidermal growth factor in Pichia pastoris according to claim 1, characterized in that, The preparation method of the passion fruit peel polysaccharide derivative includes the following steps: (1) Take passion fruit peel and decolorize it by reflux with ethanol solution; (2) Crude polysaccharide was extracted by microwave-assisted water extraction and purified by precipitation with anhydrous ethanol to obtain crude polysaccharide; (3) Add the crude polysaccharide from step (2) to an alkaline solution, add a carboxymethylation reagent under stirring, react at 50~80℃ for 2~3h, and obtain C-WPEP by neutralization, dialysis and freeze drying.

7. The method for enhancing the expression of recombinant human epidermal growth factor in Pichia pastoris according to claim 6, characterized in that, The carboxymethylating agent is sodium chloroacetate or chloroacetic acid.

8. The method for enhancing the expression of recombinant human epidermal growth factor in Pichia pastoris according to claim 6, characterized in that, The alkaline solution is a mixture of NaOH solution and isopropanol.

9. The method for enhancing the expression of recombinant human epidermal growth factor in Pichia pastoris according to claim 1, characterized in that, The passion fruit peel polysaccharide derivative is added all at once at the start of Pichia pastoris fermentation induction or added in several stages during the induction phase.

10. The method for enhancing the expression of recombinant human epidermal growth factor in Pichia pastoris according to claim 1, characterized in that, The fermentation conditions are: temperature 25~30℃, pH maintained at 6.0-7.0, and dissolved oxygen not less than 20%.

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