HK1072610B - High level constitutive production of anthrax protective antigen - Google Patents

Description

High level constitutive production of anthrax protective antigen

Technical Field

The present invention relates to high-level constitutive production of anthrax protective antigen in E.coli using fed Batch culture (fed Batch culture).

Background

Anthrax is an animal-derived disease caused by the gram-positive spore-forming bacterium bacillus anthracis (bacillus anthracaris). Protective antigen PA is the major component in all vaccines against anthrax. To date, culture supernatants of Bacillus anthracis have been the primary source of purified PA. However, the work of culturing Bacillus anthracis requires facilities of grade P3, which is cost prohibitive. In addition, PA prepared from B.anthracis often contaminates other anthrax toxic proteins. Researchers have attempted to express and purify PA in other microorganisms, such as Bacillus subtilis, baculovirus and E.coli. The yield of PA purified from Bacillus subtilis is low, requiring growth in rich media, and a large amount of PA is degraded due to protease secretion by the organism (L.W.J.Bailie et al, Lett.Appl.Microbial (1994)19, 225-. Baculovirus vectors express PA in insect cells; however, purification is not possible due to low yields. Although PA has been expressed in E.coli, attempts to overproduce proteins have not been successful (M.H. Vodkin et al, Cell, (1983)34, 693-697). Researchers also directed the protein to the periplasmic space, but the yield of purified PA was low. All known E.coli expression systems for protection of antigen expression are inducible systems requiring the use of IPTG, an expensive chemical.

U.S. Pat. No. 2,017,606 describes the preparation of anthrax antigen by culturing Bacillus anthracis on a suitable medium and separating the Bacillus from the medium.

U.S. Pat. No. 2,151,364 describes a method for producing an anthrax vaccine comprising preparing an anthrax spore suspension and adding the suspension to a sterile suspension solution containing alum.

The disadvantage of the above-mentioned us patents is that they all use bacillus anthracis cultures or spores. Bacillus anthracis is an infectious organism that cannot be handled without protective facilities. The expression level of protective antigen in B.anthracis is very low. Such vaccine preparations can also be contaminated with toxic and non-toxic proteins in bacillus anthracis, leading to a number of side effects and reactogenicity.

The object of the present invention is to establish a constitutive expression system for the rapid, efficient, cost-effective and high-level production of anthrax protective antigen in E.coli using fed-batch culture.

To achieve the above object of the present invention, there is provided a method for preparing anthrax Protective Antigen (PA) in Escherichia coli using fed-batch culture, comprising:

transforming E.coli DH 5. alpha. cells with a recombinant constitutive expression plasmid containing the PA gene to produce recombinant DH 5. alpha. cells expressing the PA protein,

-culturing the above recombinant DH5 α cells and testing the expression of PA by lysis of the above cells, followed by denaturing gel electrophoresis and Western Blotting (Western Blotting) technique using PA antibodies.

-fermenting the cells in a bioreactor using the following steps:

● adding polyhydroxy compound, carbohydrate or organic acid as main supplement into Luria Broth culture medium at 32-42 deg.C,

● fed batch culture technique, and

● nutrient loss was detected by a pH-DO static method to produce high cell density cultures expressing PA protein,

-collecting said cells by centrifugation, centrifuging said high density cell culture at 5000-,

-using 6-8 molar urea solution to lyse the high density cell culture and stirring for 1-2 hours at ambient temperature,

-centrifuging said high density cell culture debris at 10,000-15,000rpm at 32-42 ℃ for 30-60 minutes and collecting the supernatant comprising urea denatured PA,

-separating said urea denatured PA from said supernatant, purifying said PA by Ni-NTA chromatography and binding said PA to an affinity column by stepwise removal of urea,

-eluting said purified renatured PA and storing the Protected Antigen (PA) protein as a frozen aliquot at-20 to-70 ℃, depending on the point of use or long term use.

The recombinant constitutive expression plasmid for expression of the PA protein is insoluble inclusion bodies in E.coli strain DH5 alpha cells.

The high cell density culture was centrifuged at 5000rpm for 10 minutes to collect the cells.

The high cell density culture debris was centrifuged at 10,000rpm for 30 minutes to collect the cells to the maximum extent.

The polyol used as the main supplement during fermentation added to Luria Broth medium is glycerol.

The carbohydrate used as a primary supplement to Luria Broth medium is glucose, galactose, maltose, fructose or lactose.

The organic acid used as the main supplement to Luria Broth medium was malic acid.

By using a polyol, carbohydrate or organic acid as the main supplement to the Luria Broth medium, the maximum cell density in shake flask culture ranges from 10 to 14 optical density units.

By using a catalyst containing MgSO₄The fed-batch culture of (4) yields the maximum cell density of the recombinant cells.

The concentration of Luria Broth medium used for the culture was 5 to 25X.

The concentration of Luria Broth medium used for the cultivation was 25X in order to minimize the feed volume added at the time of fermentation.

The plasmid is pQE series vector containing phage promoter recognizable to Escherichia coli.

Anthrax antigens include purified structurally, biologically and functionally active recombinant Protective Antigen (PA) proteins of bacillus anthracis, which are expressed as 6X histidine fusion proteins in e.coli DH5 α cells free of polysaccharides, dead bacteria, culture medium, water-soluble and water-insoluble by-products, and suspended impurities.

Brief description of the drawings

The invention is described with reference to the following schemes and figures.

FIG. 1 shows that the maximum optical density was obtained by using glucose as an additional carbon source and the minimum optical density was obtained by using maltose in shake flask culture.

FIG. 2 shows batch culture and use of MgSO₄And no MgSO is used₄The optical density obtained in fed-batch culture of (1).

FIG. 3 shows that the recombinant PA produced is biologically and functionally as active as native PA from Bacillus anthracis.

Detailed description of the invention

Recombinant constitutive expression plasmids containing the PA gene cloned in the pQE series vector were used to transform E.coli DH 5. alpha. competent cells. The cells containing the recombinant plasmid were grown overnight at 37 ℃ and 250rpm in Luria Broth medium with 100. mu.g/ml ampicillin. Cells were collected by centrifugation at 5,000rpm for 20 minutes. Expression and localization of PA was determined by SDS-PAGE. More than 90% of the recombinant protein is found in inclusion bodies. The increase in protein expression was found to be directly proportional to the increase in cell density in the growing culture.

Glucose, fructose, galactose, lactose, maltose, malic acid and glycerol were prepared as seven different carbon sources in shake flasks each at 5X concentration of modified 100ml LB complex medium. Only LB (Medium) without any additional carbon source served as a control. Equimolar amounts of carbon atoms were used as carbon source and the concentration was controlled to be equal to 0.5% glucose. 10mM MgSO was also added₄100mM potassium phosphate and trace elements. All flasks were inoculated at the same time with an overnight grown inoculum of DH5 α cells containing the recombinant plasmid. Samples were aseptically collected every hour and OD determined₆₀₀. Culture aliquots were collected and their recombinant protein content was analyzed by SDS-PAGE. The highest OD obtained with glucose as the main carbon source₆₀₀(at an optical density of 600 nm), the lowest OD is seen in the case of using LB and maltose₆₀₀(FIG. 1). Maximum protein concentration was obtained using malic acid, maltose and glycerol as the main carbon sources. The lowest protein concentration was seen in the case of Lb and lactose.

A5L Biostat B (B Braun Biotech International) fermentor equipped with pH, temperature, dissolved oxygen and antifoam probes was used for fermentation. The fermenter was connected to a personal computer. MFCS/win 2.0 software was used to acquire the data and run the fermentor in both batch and fed-batch modes.

The medium used for the fermentation is composed of MgSO₄.7H₂Potassium phosphate O (10mM) (5g/L) and glycerol (1%), pH7.4 Luria Broth medium. For fed batch cultures, MgSO is added before autoclaving₄. Glycerol was also added prior to autoclaving. 25 Xfeeds were prepared with 25% glycerol (w/v) and 25 XLB and autoclaved separately. Potassium phosphate was also autoclaved separately and, after reaching room temperature, added aseptically before starting the run. The pH probe was calibrated with standard buffers at pH7.0 and 9.2 prior to autoclaving. After autoclaving, the fermenter medium was automatically adjusted to pH7.4 by adding 1N NaOH/1M HCl, the temperature was set at 37 deg.C. The DO (dissolved oxygen) probe was calibrated by setting the electronic zero value of dissolved oxygen in the range zero to +15 nanoamperes (nAmp), the 100% DO value was set at a stirring speed of 250rpm and the oxygen tension of the pump air was 2 vvm. The fermenter started in batch phase with a working volume of 2L. Cells containing the recombinant plasmid were grown overnight at 37 ℃ and 250rpm in LB under 100. mu.g/ml ampicillin at selective pressure. The fermentor was inoculated with 1% of overnight grown culture. The DO value was set at 40% and the agitator was set to cascade mode. In this mode, after inoculation, the DO value begins to drop below 40% and the agitator speed is automatically increased to maintain the DO value at 40%. Samples were collected every 1 hour. Culture aliquots were diluted to Optical Density (OD)₆₀₀) About 0.5 units or less. When the growing culture reached the mid-log phase, the feed was started by feeding 25 Xfeed using a peristaltic pump (Pharmacia). The feed rate was monitored and manually controlled to maintain the pH between 7.2 and 7.4. Oxygen supplementation was required after the OD reached 70. Oxygen was supplied to the culture using the Gasmix function of the fermentor. Growth to OD in culture₆₀₀Cells were collected after 120 units. The antifoam is added to the medium initially before autoclaving and later, if necessary.

To optimize the media composition and other conditions for continuous operation of the fermentation, MgSO was added or not added to the media of modified LB with glycerol₄To perform a series of batch cultures. In the absence of MgSO₄The maximum OD of the modified LB (with glycerol as a carbon source) is 14, and the modified LB is in the presence of MgSO₄Improved LB in which the OD of more than 18 can be obtained by batch operation₆₀₀The value is obtained. It can be concluded that MgSO₄Is essential for high density cell growth. Adding MgSO into growth medium₄Is necessary to obtain a higher biomass yield in the fermentation (FIG. 2).

5ml of high density cell culture was centrifuged at 10,000g for half an hour in a pre-weighed tube. After the supernatant was drawn, the tube with centrifuged cells was weighed on a balance to determine the wet cell weight. To determine the dry cell weight, the same tubes were placed in an incubator at 70 ℃ overnight and weighed the following day.

To check the stability of the plasmid pMW1 in the fermentor, culture samples were aseptically collected every hour during the fermentation. The samples were diluted to the OD of each sample₆₀₀5.0, centrifuge at 10,000g for one minute. After the supernatant was completely extracted, the sediment was suspended in 100. mu.l of lysis buffer containing 100mM potassium phosphate buffer, 8M urea, pH 8.0. These samples were subjected to SDS-PAGE to examine protein expression of the recombinant plasmids. Colony preparation and miniprep of plasmid DNA was also used to directly check for the presence of recombinant plasmid within the expressing cells. No obvious plasmid-free cells were found to be produced.

The protein is purified by metal chelate affinity chromatography under denaturing conditions. Briefly, 100ml of the pellet of the culture was resuspended in 100ml of denaturing buffer containing 100mM sodium phosphate buffer, 300mM sodium chloride and 8M urea (pH 8.0). The resuspended pellet was incubated on a rotary shaker at 37 ℃ for 2 hours. The lysate was centrifuged twice at room temperature for 60 minutes each and the supernatant was mixed with 50% Ni-NTA slurry. The slurry was poured onto the column and allowed to settle. The flow-through was then applied to the column. The Ni-NTA matrix was washed with 500ml of denaturing buffer containing 8M urea, followed by renaturation of the protein on the column with an 8M-0M urea gradient. The protein was eluted with 250mM imidazolium chloride in elution buffer, 100mM sodium phosphate pH8.0 and 300mM sodium chloride with 250mM imidazolium. Each fraction, 10. mu.l, was analyzed on SDS-PAGE. The protein containing fractions were collected, pooled and dialyzed against 10mM HEPES buffer containing 50mM NaCl and stored frozen at-70 ℃ in aliquots.

Specific proteins can be determined in a number of ways. Optical density measurements were performed using the BioRad gel file management system and QuantityOne software. 50% J774A.1 macrophage-like cells (EC) were killed by calculation at 3 hours in combination with LF (1. mu.g/ml), 37 ℃₅₀) The amount of protein required determines the fold purification of the PA at different stages. The purified protein was measured by the Bradford method and determining the OD of the preparation at 280 nm. PA accounts for more than 30% of the total cell protein and exists in the form of inclusion bodies in the cells. 5-8g/L PA was produced in the cells.

The biological activity of rPA (recombinant PA) is also usefulCytotoxicity assays on j774a.1 macrophage-like cell line. All experiments were repeated three times. Briefly, different concentrations of rPA protein were added to the cells along with LF (1. mu.g/ml). PA native to B.anthracis was used as a positive control along with LF. After 3 hours, cell viability was determined using MTT dye and the resulting pellet was dissolved in a buffer containing 0.5% (w/v) sodium dodecyl sulfate, 25mM HCl in 90% isopropanol. The percent viability was determined by reading the absorbance at 540nm using a microplate reader (BioRad). rPA was found to lyse macrophages completely along with LF, and its biological activity was similar to that of PA produced by Bacillus anthracis. EC for rPA and nPA was found₅₀Each at-50 ng/ml (FIG. 3). Fold purification of the protein was determined at each stage of purification by the cytotoxicity assay described above (table 1).

TABLE 1

Purification of PA from E.coli

In part	Volume (ml)	Protein (mg/ml)	Activity (EC)50)a	Purification (multiple)b
					Affinity purification of cell lysate	1025	2911.8	57.30.0241	11965

^aEC₅₀Defined as the concentration of PA (. mu.g/ml) required to kill 50% of J774A.1 cells along with LF (1. mu.g/ml). Viability was determined by MTT dye after 3 hours incubation. Results represent the average of three experiments.

^bFold purification from EC of cell lysate₅₀EC divided by fractions obtained from different columns₅₀And (4) determining.

Discussion of the related Art

Overexpression of any recombinant protein depends on the optimal configuration of the various components of the expression system. Many factors strongly influence recombinant protein expression such as promoter strength, plasmid stability, plasmid copy number, transcription terminator, transcription and translation efficiency ultimately enhancing mRNA stability, translation terminator, tight regulation of gene transcription, availability of ribosomes, post-translational modifications, stability and solubility of the recombinant protein itself, and host cell and culture conditions. The high level steps towards heterologous protein expression use strong promoters such as T5, T7, PL, PR, Ptrc, Ptac to create an efficient expression system. Most of these systems carry inducible promoters and have a low or no product formation stage prior to induction. Upon induction, the specific yield reaches a maximum in a short time and the protein is produced continuously for 4-5 hours. It has been reported that there are many changes such as changes in carbon metabolism after induction leading to acetate accumulation, increase in respiration, decrease in synthesis of housekeeping proteins (housekeeping proteins), and occurrence of heat shock-like reaction and SOS reaction. All these reactions lead to degradation of the recombinant product. The formation of inclusion bodies protects the recombinant protein from the impact of cellular proteases which otherwise could lead to extensive degradation of the protein leading to low product recovery. SDS-PAGE and Western blot (Western Blotting) analysis at different time points confirmed the fact that PA expression of the recombinant plasmid pMW1 was not leaky and that expression was proportional to the OD of the culture. Without significant degradation, most proteins within the cell are in an inclusion body form.

Once a strong expression system is established, high cell density culture using different techniques can significantly increase the production of recombinant proteins in E.coli. Cell concentrations in excess of 50g/l dry weight can be routinely obtained to provide cost-effective recombinant protein production. However, high cell density cultures also have some disadvantages such as substrate inhibition, limited oxygen transport capacity, formation of growth inhibitory byproducts and limited heat dissipation leading to a decrease in the mixing efficiency of the fermentor. One major problem with recombinant protein production in High Cell Density Culture (HCDC) is the accumulation of acetate, a lipophilic agent that is detrimental to cell growth. Acetate accumulation in the growth medium reportedly reduces recombinant protein production. In fed-batch culture, many strategies have been developed to reduce acetate formation, such as controlling specific growth rates by limiting essential nutrients of the medium, such as carbon or nitrogen sources, changing growth conditions and strains of E.coli. Since the main goal of fermentation research is to produce recombinant products cost-effectively, it is important to develop culture methods that maximize the yield of the desired product. The composition of the growth medium is critical to increase product formation and reduce acetate. Acetate is produced when E.coli is overproduced in oxygen-limited or aerobic conditions when carbon flow in the central metabolic pathway exceeds its biosynthetic requirements and the capacity for intracellular energy production.

Glycerol was selected as the main carbon source for HCDC from 7 carbon sources, i.e., glucose, fructose, galactose, lactose, maltose, malic acid and glycerol, based on growth kinetics and recombinant protein production. The use of glycerol as a carbon source does not produce acetate and the use of glycerol relatively easily results in high cell densities. The lower rate of glycerol transport into the cell compared to glucose apparently results in reduced carbon flow through glycolysis; acetate formation is greatly reduced and cells grow slower in glycerol. Glycerol also has an antifoaming effect, resulting in less foaming during the fermentation run.

The method of using nutrient feeding is also critical to the success of HCDC because it affects cell density and cell productivity. Constant or intermittent feeding is carried out under conditions of nutrient limitation. Although other feeding methods have been successfully applied to HCDCs of E.coli, more complex feeding methods with feedback control schemes have recently been developed. The feed rate is combined with other physical parameters such as DO (dissolved oxygen), pH, microbial heat and CO₂Rate of evolution (CER). The DO stat (DO-stat) method of feeding is based on the fact that DO in the culture increases dramatically when the substrate is depleted. When the nutrient level drops and there is less oxygen available to the cell, the cell cannot grow rapidly. Thus, in the DO stat method, the substrate concentration is maintained within the desired range by automatically adding nutrients when the DO rises above a preset value. Alternatively, the pH static (pH-stat) method is based on the fact that the pH of the medium changes when the main carbon source is limited. When the carbon source is depleted, the pH begins to rise primarily as a result of the increased concentration of ammonium ions excreted/secreted by the cells.

The analysis of the feeding system starts from the identification of the probing and detecting method. Saturation in respiration was detected by examining the response of DO and pH to fluctuations in feed rate. Once feeding was started, E.coli entered the log phase and the consumption of the feed was more or less exponential. However, the feed rate must be controlled so as not to exceed the nutrient requirement or feed consumption rate. This is accomplished by maintaining the pH and DO close to their set points. The decrease in pH and DO is indicative of substrate excess. The increase in pH and DO values indicates a limitation of the carbon source or of a certain substrate, so that feeding is required. If the feed fluctuations/rate increase DO not produce any significant reaction, i.e. a decrease in DO or an increase in stirrer speed, an ideal indication is that some other factor, such as MgSO₄，KH₂ PO₄Or trace elements become limited, in which case the addition must be intermittent. The above variables change rapidly after addition. Coli can use acetate as a carbon source when there is no glucose or any other major carbon source. Acetate consumption is characterized by a deviation from the preset value to a lower pH and by the onset of a cyclic pattern of oxygen consumption until a gradual recovery in the cultureA preset pH value. At this point feeding was resumed.

The DO static method reacts faster to nutrient loss than the pH static method. When a complex substrate is used with a carbohydrate substrate, the change in DO is not more pronounced than in cells that continuously use the complex substrate. The feeding method used in this experiment was a combination of the pH stat method and the DO stat method. Monitoring both parameters simultaneously better controls the growth conditions of the growth culture. Using this substrate feeding method we were able to obtain an OD of 120, which is 6 times higher than the OD obtained in batch culture and 23 times higher than in shake flask culture.

This is the first report of the optimized conditions for fed-batch HCDC to obtain high PA yields in e. This work attempts to obtain large amounts of non-reactive PA as a candidate vaccine for the future. The process for producing PA reported herein uses novel and advantageous substrates and a simple and easy controlled feeding method, which can also be successfully applied to other recombinant protein expression systems to obtain high yields of product.

Claims (12)

1. A method for preparing anthrax protective antigen in e.coli using fed-batch culture comprising the steps of:

(a) transforming E.coli DH5 alpha cells with a recombinant constitutive expression plasmid containing a protective antigen gene to produce recombinant DH5 alpha cells expressing a protective antigen protein,

(b) culturing the recombinant DH5 alpha cells and testing the expression of protective antigen by lysing the cells, denaturing gel electrophoresis and Western blotting using antibodies to the protective antigen,

(c) fermenting the cells in a bioreactor using the following steps:

(i) adding polyhydroxy compound, carbohydrate or organic acid as main supplement into Luria Broth culture medium at 32-42 deg.C,

(ii) fed-batch culture technique, and

(iii) nutrient loss was detected using a pH-DO static method to produce high cell density cultures expressing protective antigen proteins,

(d) the high density cell culture was centrifuged at 5000-,

(e) the high density cell culture is lysed using 6-8 molar urea solution and stirred at ambient temperature for 1-2 hours,

(f) centrifuging the high density cell culture debris at 10,000-15,000rpm at 32-42 ℃ for 30-60 minutes and collecting the supernatant containing the urea denaturation-protected antigen,

(g) separating said urea denatured protective antigen from said supernatant, purifying said protective antigen by Ni-NTA chromatography and binding said protective antigen to an affinity column by stepwise removal of urea,

(h) eluting the purified renatured protective antigen and storing the protective antigen protein in frozen aliquots at-20 to-70 ℃.

2. The method of claim 1, wherein the recombinant constitutive expression plasmid for expressing a protective antigen protein is insoluble inclusion bodies in E.coli strain DH5 a cells.

3. The method of claim 1, wherein said high density cell culture is centrifuged at 5000rpm for 10 minutes to collect said cells.

4. The method of claim 1, wherein said high density cell culture debris is centrifuged at 10,000rpm for 30 minutes to maximize collection of said cells.

5. The process according to claim 1, characterized in that the polyol used as a major supplement in the fermentation to which the Luria Broth medium is added at 37 ℃ is glycerol.

6. The method according to claim 1, characterized in that the carbohydrate used as a main supplement to the Luria Broth medium at 37 ℃ is glucose, galactose, maltose, fructose or lactose.

7. The method according to claim 1, characterized in that the organic acid used as a primary supplement to the Luria Broth medium at 37 ℃ is malic acid.

8. The method according to claim 1, characterized in that the maximum cell density in shake flask culture is in the range of 10-13 optical density units by using a polyol, carbohydrate or organic acid as the main supplement to the Luria Broth medium.

9. The method of claim 1, characterized in that the maximum cell density of the recombinant cells is obtained by using fed-batch culture containing MgSO 4.

10. The method according to claim 1, characterized in that the concentration of Luria Broth medium used for feeding is 5-25X.

11. The method as claimed in claim 1, wherein the concentration of Luria Broth medium used for feeding is 25x in order to minimize the feeding volume added during fermentation.

12. The method of claim 1, wherein the plasmid is a pQE series vector containing a phage promoter recognizable to E.coli.