阅读说明：本技术 一种大规模生产重组Exendin-4多肽的发酵方法 (Fermentation method for large-scale production of recombinant Exendin-4 polypeptide ) 是由 樊欣迎 李静 郭静雅 刘月峰 梁国龙 闻亚磊 于 2020-12-18 设计创作，主要内容包括：本发明提供一种大规模生产重组Exendin-4多肽的发酵方法,采用pET32a(+)-Exendin-4/BL21(DE3)发酵培养,在发酵中,以乳糖作为发酵培养的诱导剂。乳糖代替IPTG作为工程菌发酵培养的诱导剂,乳糖在发酵过程中会被菌体做为碳源分解代谢,不会产生毒性残留物质；此外,为使重组Exendin-4多肽的工艺更加适用于产业化生产,对发酵工艺参数进行了优化,发酵规模由30L扩大为500L,优化后的菌体湿重、蛋白表达量(高达14.1％～14.6％)优于前工艺。(The invention provides a fermentation method for large-scale production of recombinant Exendin-4 polypeptide, which adopts pET32a (+) -Exendin-4/BL21(DE3) for fermentation culture, and uses lactose as an inducer for the fermentation culture during the fermentation. Lactose replaces IPTG to be used as an inducer for fermentation culture of engineering bacteria, and can be catabolized by thalli as a carbon source in the fermentation process without generating toxic residual substances; in addition, in order to make the process of recombining the Exendin-4 polypeptide more suitable for industrialized production, fermentation process parameters are optimized, the fermentation scale is enlarged to 500L from 30L, and the optimized wet weight of thalli and the protein expression amount (up to 14.1-14.6%) are superior to those of the prior process.)

1. A fermentation method for large-scale production of recombinant Exendin-4 polypeptide adopts pET32a (+) -Exendin-4/BL21(DE3) for fermentation culture, and is characterized in that lactose is used as an inducer for fermentation culture in fermentation.

2. The fermentation method for producing recombinant Exendin-4 polypeptide of claim 1, wherein the induction conditions are lactose addition at a final concentration of 5g/L to 20 g/L; optionally 5 g/L.

3. The fermentation method for producing recombinant Exendin-4 polypeptide according to claim 1 or 2, wherein the induction conditions are that the induction temperature is 25-40 ℃ and the induction time is 4-6 hours;

optionally, the induction temperature is 25-30 ℃ or 30-40 ℃;

optionally, the induction time is 4-6 hours, optionally 6 hours.

4. The fermentation method for producing recombinant Exendin-4 polypeptide of any one of claims 1-3, wherein the fermentation process further comprises the steps of adding a feed medium;

optionally, a feed medium is added at the beginning of induction;

optionally, the fermentation medium used for fermentation is a TB medium;

optionally, the TB medium contains 8mL/L glycerol.

5. The fermentation method for producing recombinant Exendin-4 polypeptide according to any of claims 1-4, wherein the OD of the bacterial body₆₀₀When the value is 8-12, adding an inducer.

6. The fermentation method for producing the recombinant Exendin-4 polypeptide according to any of claims 1-5, wherein the volume of the fermentation medium is 50L-500L; and optionally 200L.

7. The fermentation method for producing recombinant Exendin-4 polypeptide according to any of claims 1 to 6,

adding the inducer for 1-6 times; optionally adding inducer for 2-6 times; alternatively, the time of initiation of induction was designated as 0 hour, and 1/5 volumes of inducer were added at 0, 1, 2, 3, and 4 hours, respectively.

8. The fermentation method for producing the recombinant Exendin-4 polypeptide according to any of claims 1-7, further comprising before fermentation: 1) primary seed preparation and 2) secondary seed preparation.

9. The fermentation method for producing recombinant Exendin-4 polypeptide according to any of claims 1-8, wherein the feed medium is MgSO₄·7H₂O, tryptone, yeast extractAnd glycerin, MgSO₄·7H₂The concentration of O is 11-12g/L g/L, the concentration of tryptone is 98g/L-102g/L, the concentration of yeast extract is 98g/L-102g/L, and the volume percentage of glycerol is 20%.

Technical Field

The invention relates to the field of product fermentation, in particular to a fermentation method for producing recombinant Exendin-4 polypeptide in a large scale.

Background

IPTG is used as an inducer for regulating and controlling the lactose operon by the genetic engineering bacteria, can activate the lactose operon, and leads the gene of the rExendin-4 fusion protein coded at the downstream of the operon to express, thereby obtaining the fusion protein containing the rExendin-4. IPTG can not be degraded in the culture process of engineering bacteria, and has potential toxicity, so that the safety of the final stock solution is influenced.

Disclosure of Invention

Therefore, the technical problem to be solved by the invention is to overcome the defect that the recombinant Exendin-4 polypeptide produced by fermentation in the prior art has toxic substance residues, so that a fermentation method for producing the recombinant Exendin-4 polypeptide is provided, and toxic residue substances are not generated by the method.

A fermentation method for large-scale production of recombinant Exendin-4 polypeptide adopts pET32a (+) -Exendin-4/BL21(DE3) for fermentation culture, and lactose is used as an inducer for the fermentation culture in the fermentation process.

Optionally, the induction condition is that the final concentration of the added lactose is 5g/L-20 g/L; optionally 5 g/L.

Optionally, the induction conditions are that the induction temperature is 25-40 ℃, and the induction time is 4-6 hours;

optionally, the induction temperature is 25-30 ℃ or 30-40 ℃;

optionally, the induction time is 4-6 hours, optionally 6 hours.

Optionally, the fermentation process further comprises the step of adding a feed medium;

optionally, a feed medium is added at the beginning of induction;

optionally, the fermentation medium used for fermentation is a TB medium;

optionally, the TB medium contains 8mL/L glycerol.

Optionally, the bacterial body OD₆₀₀When the value is 8-12, adding an inducer.

Optionally, the volume of the fermentation medium is 50L-500L; and optionally 200L.

Optionally, the inducer is added in 1-6 times; optionally adding for 2-6 times; alternatively, the time of initiation of induction was designated as 0 hour, and 1/5 volumes of inducer were added at 0, 1, 2, 3, and 4 hours, respectively.

Optionally, the method further comprises the following steps before fermentation: 1) primary seed preparation and 2) secondary seed preparation.

Optionally, the first-order seed preparation method comprises: inoculating original bacterial liquid of pET32a (+) -Exendin-4/BL21(DE3) prepared by application number 200410052039.4 into 1500mL of primary seed culture medium according to the inoculation amount of 1 per thousand (v/v), wherein the culture temperature is 36-38 ℃ (optionally 37 ℃), the rotation speed is 220-270 rpm (optionally 250rpm), and performing shaking culture for 15-17 h to obtain primary seed culture;

optionally, the preparation method of the second-level seeds comprises: transferring the first-class seed culture into a fermentation tank with a culture medium volume of 30L-50L, setting the temperature at 37 ℃, rotating at 250rpm, performing shake culture for 2.0-3.0 h, and measuring OD₆₀₀Obtaining a secondary seed culture after the value reaches 3.0-5.0;

alternatively, the feed medium is MgSO₄·7H₂O, tryptone, yeast extract and glycerol, MgSO₄·7H₂The concentration of O is 11-12g/L g/L, the concentration of tryptone is 98g/L-102g/L, the concentration of yeast extract is 98g/L-102g/L, and the volume percentage of glycerol is 20%.

Alternatively, the feed medium is MgSO₄·7H₂O, tryptone, yeast extract and glycerol, MgSO₄·7H₂The concentration of O was 11.4g/L, the concentration of tryptone was 100g/L, the concentration of yeast extract was 100g/L, and the volume percentage of glycerol was 20%.

The fermentation specifically comprises the following steps:

transferring 30L of the secondary seed culture into a 500L fermentation tank (the volume of the culture medium is 200L) through a seed transfer pipeline; the temperature is 37 ℃ plus or minus 2 ℃, the dissolved oxygen is 30 percent (the ventilation and the stirring are coupled), and the pH fluctuation range is 7.00 plus or minus 0.20; OD₆₀₀Starting induction when 8-12 ℃, namely supplementing 460mL of supplemented culture medium and 400mL of lactose solution, reducing the culture temperature to 30 +/-2 ℃, supplementing 5 times of 400mL of lactose, supplementing 80mL of lactose each time,from 0h of induction, the first 5h is completely supplemented to make the final lactose concentration in the tank be 5 g/L.

The technical scheme of the invention has the following advantages:

1. the invention provides a fermentation method for large-scale production of recombinant Exendin-4 polypeptide, which adopts pET32a (+) -Exendin-4/BL21(DE3) for fermentation culture, and uses lactose as an inducer for the fermentation culture during the fermentation. Lactose replaces IPTG to be used as an inducer for fermentation culture of engineering bacteria, and can be catabolized by thalli as a carbon source in the fermentation process, so that toxic residual substances can not be generated.

2. In order to make the process of recombining the Exendin-4 polypeptide more suitable for industrialized production, fermentation process parameters are optimized, the fermentation scale is enlarged to 500L from 30L, the optimized wet weight of thalli and the protein expression amount (up to 14.1-14.6%) are superior to those of the prior process, and potential toxic residues are not generated.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.

FIG. 1 comparison of the results induced by different lactose concentrations;

FIG. 2 shows 4-hour electrophoretograms induced at different temperatures; in the figure, S1 represents an experiment number, which has no practical significance, and 20080417 and 20080422 are experiment times without other meanings;

FIG. 3 shows an electropherogram at different temperatures for 6 hours; in the figure, S1 represents an experiment number, which has no practical significance, and 20080417 and 20080422 are experiment times without other meanings;

FIG. 430 ℃ is an electrophoretogram showing the expression level at different times; in the figure, S1 represents an experiment number and has no practical significance;

FIG. 5 electrophoresis chart of different media induced at 30 ℃ for 6 hours; in the figure, S1 represents an experiment number and has no practical significance

FIG. 6 electrophoretogram of lactose in different modes of addition;

FIG. 7 is a flow diagram of a fermentation process.

Detailed Description

The invention uses the following authorization notice numbers: CN100535111C recombinant Exendin-4 polypeptide was prepared from the engineered bacterium disclosed in example 1 (pET32a (+) -Exendin-4/BL21(DE 3)).

EXAMPLE 1 preparation of recombinant Exendin-4 polypeptide

The general process flow is shown in fig. 7, and the induction concentration, induction temperature and induction time of lactose are determined by experiment exploration in the example. The expression levels of TB, LB and LM9 in the three media were compared with each other using lactose as an inducer. After the selection of TB medium was determined, the effect of lactose addition on fermentation and expression was compared. According to the experimental data, the amplification and the research are carried out in the fermentation tank.

1. Determination of lactose Induction concentration

Inoculating the strain into 250mL shake flask (containing 50mL LB culture medium), and culturing at 37 deg.C to OD₆₀₀Values 2, lactose addition at 5, 10, 20, 30, 40g/L (final concentration), induction culture for 4 hours sampling test, comparison of the effect of different lactose concentrations on expression, results show: when lactose is added above 30g/L, the expression level is reduced (FIG. 1 a); lactose is added according to 5, 10, 15 and 20g/L respectively for induction, sampling detection is carried out after 4 hours of induction culture, and the result shows that the expression amount of the added lactose is approximate to that of 5-20g/L (figure 1b), and the result of 5g/L lactose is slightly higher, so that the induction concentration of lactose is determined to be 5 g/L.

2. Determination of Induction temperature and Induction time

Inoculating the strain into 250mL shake flask (containing 50mL LB culture medium), and culturing at 37 deg.C to OD₆₀₀Value 2, lactose was added at a final concentration of 5g/L, the cells were incubated at 25 ℃, 28 ℃, 30 ℃, 32 ℃, 37 ℃ and 40 ℃ respectively, and samples were taken 4 and 6 hours after induction for SDS-PAGE analysis (see FIGS. 2-4), and the semi-quantitative results of SDS-PAGE were statistically analyzed to obtain tables 1 and 2. The results showed that the expression level was highest after 6 hours of induction at 30 ℃ (tables 1 and 2), and therefore, induction culture at 30 ℃ was confirmed. By comparison at 30 ℃ for 4 to 8 hours, at 6 hoursThe expression decreased after the incubation (Table 3, FIG. 4), and the induction temperature and time were determined to be 30 ℃ for 6 hours.

TABLE 1 lactose Induction of 4 hours expression at different temperatures

The expression amount is expressed as the mass of the fusion protein/the mass of the total bacterial protein × 100%.

TABLE 2-1 lactose induction for 6 hours of expression at different temperatures

TABLE 2 Induction of expression at 230 ℃ for various periods of time

The expression amount is expressed as the mass of the fusion protein/the mass of the total bacterial protein × 100%.

3. Determination of the culture Medium

Inoculating the strain into 250mL shake flasks containing 50mL TB, LB, LM9 culture medium, respectively, and culturing at 37 deg.C to OD₆₀₀The value 2, adding lactose according to 5g/L, sampling after induction culture for 6 hours at 30 ℃ for SDS-PAGE analysis and biomass determination, repeating the experiment for 4 times, and the result shows that the expression level and biomass of the TB culture medium are higher than those of the other two culture media (table 3 and figure 5), and the TB culture medium is determined to be the TB culture medium because the TB culture medium has a more complete nutritional structure and is suitable for scale-up production.

TABLE 3-1 LM9 Medium

TABLE 3-2 TB Medium

TABLE 3-3 LB Medium

TABLE 3-4 comparison of expression levels in different media

The expression amount is expressed as the mass of the fusion protein/the mass of the total bacterial protein × 100%.

4. Effect of lactose addition on results

Lactose is used as an inducer and also as a carbon source, and when the concentration is excessively high, the growth of thalli and the expression of products are influenced. Therefore, under the condition that the total amount of lactose is not changed, the lactose is added in a plurality of times for expression comparison.

Transferring the strain to TB culture medium, and culturing at 37 deg.C to OD₆₀₀The culture temperature is 2, the culture is transferred to 30 ℃ for induction culture, lactose is added in 1, 2, 4, 5 and 6 times on average (1 time is all added at the beginning of induction, 2 times are half added after the beginning of induction and three hours after induction, 4 times are 1/4 added every other hour from the beginning of induction and are supplemented in the first 4 hours, 5 times are 1/5 added every other hour from the beginning of induction and are supplemented in the first 5 hours, 6 times are 1/6 added every other hour from the beginning of induction), and the total addition amount of lactose is 5g of lactose added in each liter of TB culture medium. A final concentration of 0.5mM IPTG was added as a parallel control. The above experiment was repeated 2 times, i.e. experiment 1 and experiment 2, with 4 trials of the lactose addition regime, and 4 replicates were performed (see fig. 6a and b). The test results were averaged. The results of the experiment showed that the expression level and biomass were slightly higher in 5-fold average additions (Table 4, FIG. 6). Thus, the total addition in lactose was determinedUnder the condition of constant input, the induction is carried out in a mode of supplementing 1/5 volumes of lactose solution every hour, namely, the induction is completed at the 4 th hour from the beginning of the induction.

TABLE 4 comparison of expression levels for different lactose addition modes

Note: the expression amount is the mass of the fusion protein/the mass of the total bacterial protein multiplied by 100 percent;

of these, 4 experiments with lactose addition were performed in 4 replicates (see fig. 6a and b), with a mean of 13.3; 2 of these are listed in the table.

5. Determination of lactose Induction timing

The induction time and the regulation and control of the feeding are further optimized to improve the density and the expression quantity of the bacteria and increase the yield of unit fermentation volume. The main optimization conditions are as follows:

1) according to the growth curve of the strain cultured in 5L fermenter, OD was taken₆₀₀Inducing the engineering bacteria when the value is 3-5 and 8-12;

2) the content of glycerol in the TB culture medium is increased from 4mL/L to 8 mL/L;

3) feed medium (11.4g/L MgSO₄·7H₂O、100g/LTrytone100g/L Yeast extract (Yeast extract) and 20% (v/v) glycerol) the effects of 10% (v/v) and 20% (v/v) glycerol on expression and biomass were compared using glycerol as a carbon source.

The results show that: after the content of glycerol is increased in the initial TB culture medium, the early growth speed is accelerated; when inducing the timing OD₆₀₀The value is 8-12, 20% of glycerol is added into a feed culture medium, and under the condition, the biomass and the expression quantity are relatively high (Table 5).

Table 5: comparison of the results of the fermentations in 5L fermentors under different conditions

Note: the expression amount is expressed as the mass of the fusion protein/the mass of the total bacterial protein × 100%.

6. Optimized result of fermentation process

And (3) combining the experimental results to preliminarily determine the fermentation process conditions: the fermentation medium is TB medium (glycerol amount 8mL/L), the culture temperature is 37 deg.C, pH value is 7.0, Dissolved Oxygen (DO) value is 30%, and induction time OD₆₀₀The value is 8-12, the induction temperature is 30 ℃, the induction time is 6 hours, the lactose induction concentration is 5g/L (1/5 is added in each of 0, 1, 2, 3 and 4 hours of induction), and the culture medium is fed (11.4g/L MgSO)₄·7H₂O, 100g/L tryptone, 100g/L yeast extract, 20% glycerol (v/v)) were supplemented according to the dissolved oxygen and pH changes.

7. Fermentation process

The developed recombinant insulinotropic hormone secretagogue (rExendin-4) fermentation process was subjected to commercial scale studies.

1) First order seed liquid preparation

Taking one working seed, inoculating to 0.75L of first-class seed culture medium (TB culture medium) according to 1 ‰ (v/v) inoculum size, placing in a shaking table, culturing at 37 deg.C and 250rpm for 15-17 h, and measuring OD₆₀₀Values (see table below).

2) Second stage seed liquid preparation

Transferring the first-class seed culture to a 50L fermentation tank, culturing with a culture medium (TB culture medium) volume of 30L at 37 deg.C under dissolved oxygen of 30% and rotation speed of 250rpm for 2-3 h, and measuring OD₆₀₀And obtaining a secondary seed culture after the value reaches 3-5.

3) Fermentation of

30L of the secondary seed culture was transferred into a 500L fermenter (medium volume 200L) via a transfer line. The temperature is 37 ℃ plus or minus 2 ℃, the dissolved oxygen is 30 percent (the ventilation and the stirring are coupled), and the pH fluctuation range is 7.00 plus or minus 0.20. OD₆₀₀Starting induction when the temperature reaches 8-12, namely supplementing 460mL of supplemented medium and 400mL of lactose solution, and simultaneously reducing the culture temperature to 30 +/-2 ℃, wherein the temperature is 400 DEG CAnd (3) adding 80mL of lactose into the mL of lactose for 5 times, wherein the lactose adding time is completed within 4-5min each time, and the final concentration of the lactose added into the tank is 5g/L after the previous 5 hours from the induction time of 0 hour. When the pH value is higher than 7.0 and the rising is continued, the feed liquid is supplemented until the fermentation is finished. Inducing for 5-6 h, OD₆₀₀When the trend of the value changes to be flat and slow, the fermentation is stopped.

TABLE 6 results of fermentation Process study

The above experimental results show that: the protein expression amount is 14.1-14.6%, and the stable level can be ensured after the process is amplified.

It should be understood that the above examples are only for clarity of illustration and are not intended to limit the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. And obvious variations or modifications therefrom are within the scope of the invention.