Universal in-situ renaturation method for thallus outer membrane protein

Abstract

The invention provides a universal in-situ renaturation method for thallus outer membrane protein, which is suitable for various thalli including acinetobacter baumannii, the method uses a nickel ion affinity chromatography column to separate and purify target outer membrane protein, and a denaturant is replaced on the nickel ion affinity chromatography column at an artificially controllable extremely low rate, so that the target outer membrane protein is separated and purified. The removal rate of the denaturant is controlled, so that the protein can be slowly renatured in situ, the success rate of protein renaturation is remarkably improved, and large-scale amplification preparation is easy.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and particularly relates to a universal in-situ renaturation method of bacterial outer membrane proteins. Background Art

[0002] Acinetobacter baumannii is a Gram-negative bacterium and an important opportunistic pathogen in hospitals. Due to the increase in the number of multidrug-resistant strains and their global prevalence, Acinetobacter baumannii infection has become a challenging problem in clinical practice. Vaccines are an effective tool for preventing and controlling Acinetobacter baumannii infections. Many researchers are studying subunit vaccines against Acinetobacter baumannii. Subunit vaccines have the advantages of high purity, safety and stability, easy production, and strong targeted induction of immune responses. So far, no Acinetobacter baumannii subunit vaccine candidate has entered clinical trials. This may be related to the fact that subunit vaccines are easily degraded in the body and have weak immunogenicity.

[0003] Many subunit vaccine antigens of Acinetobacter baumannii have been explored with varying degrees of success. The immunogenicity and protective effects of approximately twenty subunit vaccines of Acinetobacter baumannii have been evaluated in vivo. The most studied antigens in Acinetobacter baumannii are outer membrane proteins OmpA, Omp33-36, Omp22, OmpW, and Ata, etc. However, since membrane proteins are usually transmembrane proteins, in prokaryotic expression systems, due to factors such as excessive protein expression, too fast expression, or amino acid composition, protein solubility, etc., outer membrane proteins basically form inclusion bodies after expression. Inclusion bodies are inactive solid particles formed by intracellular agglutination, which are huge challenges to protein purification and activity.

[0004] At present, the commonly used inclusion body purification method is basically in vitro denaturation and renaturation. Inclusion body denaturation is the process of converting inclusion bodies from an inactive state to a natural conformation with biological activity. For example, denaturants (urea, guanidine hydrochloride, etc.) are used to break the chemical bonds between inclusion body protein molecules through ionic interactions to stretch the polypeptide chain. Renaturation refers to the removal of denaturants to restore the target protein from a fully stretched denatured state to a normal folded structure. Commonly used renaturation methods mainly use dilution or dialysis to reduce the concentration of denaturants, and finally completely replace the denaturant with a buffer. However, in the renaturation process after inclusion body denaturation, the setting of renaturation conditions not only determines whether the protein can be successfully renatured, but also plays a vital role in the recovery of protein activity. In the conventional renaturation process, it is often difficult for the protein to fold into the correct conformation and form an inactive white precipitate due to the high protein concentration during renaturation or the too fast removal of the denaturant during the renaturation process. Even if the renatured protein can be obtained, its proportion is very low, making it difficult to achieve large-scale preparation. Therefore, the conditions for protein renaturation are particularly important.

[0005] How to achieve efficient and large-scale production of bacterial outer membrane proteins while ensuring protein activity is a problem that needs to be solved. Summary of the invention

[0006] In view of the problems existing in the prior art, the purpose of the present invention is to provide a universal in situ renaturation method for bacterial outer membrane proteins. This method replaces the denaturant at an extremely low rate that can be artificially controlled on a nickel ion affinity chromatography column, controls the removal rate of the denaturant, and allows the protein to slowly renature in situ, significantly improves the success rate of protein renaturation, and is easy to scale up for preparation.

[0007] The objective of the present invention is achieved through the following technical solutions:

[0008] The first aspect of the present invention provides a general method for in situ renaturation of bacterial outer membrane proteins, comprising the following steps:

[0009] (1) Obtaining bacterial solution expressing the target outer membrane protein and collecting bacterial cells;

[0010] (2) washing the cells with the detergent TritonX-100 to obtain inclusion bodies;

[0011] (3) The inclusion bodies are sonicated under urea denaturation conditions to obtain denatured proteins;

[0012] (4) Using a nickel ion affinity chromatography column to separate and purify the target outer membrane protein, the specific steps are as follows:

[0013] S41, the chromatography column is balanced in buffer 3, and the denatured protein dissolved in urea is loaded; the buffer 3 is 8M urea, 0.5M NaCl, 20mM Na n H2PO4, pH 8.0;

[0014] S42, using buffer 4 containing imidazole to wash the weakly bound proteins from the chromatography column; the buffer 4 is 0.5M imidazole, 8M urea, 0.5M NaCl, 20mM Na n H2PO4, pH 8.0;

[0015] S43, after returning to buffer 3 to allow the chromatography column to reach equilibrium again, the pump B of the FPLC is replaced with a pump containing buffer 1, and the buffer is replaced by running a linear gradient from 8 M urea to 0 M urea at a speed of 1 ml / min, while the target outer membrane protein is bound to the column;

[0016] The buffer 1 is 5mM imidazole, 0.5M NaCl, 20mM Na n H2PO4, pH 8.0;

[0017] S44, after the buffer exchange is completed, pump A of the FPLC is changed to contain buffer 1, and pump B is changed to contain buffer 2;

[0018] Buffer 2 is 0.5 M imidazole, 0.5 M NaCl, 20 mM Na n H2PO4, pH 7.4;

[0019] S45, using a 5-500 mM imidazole gradient to elute the bound protein, thereby obtaining the target outer membrane protein.

[0020] Furthermore, the bacteria include Acinetobacter baumannii.

[0021] Furthermore, the bacterial outer membrane proteins include but are not limited to the Acinetobacter baumannii outer membrane proteins OmpA, Omp22 and OmpW.

[0022] Furthermore, in step (3), the ultrasonic conditions are: ultrasonic time: 10 minutes, working time: 10 seconds, interval time: 10 seconds, alarm temperature 4°C, power: 25%.

[0023] Furthermore, the target outer membrane protein is eluted at 300-500 mM imidazole.

[0024] The second aspect of the present invention provides a bacterial outer membrane protein obtained by the method described in the first aspect.

[0025] The third aspect of the present invention provides the use of the bacterial outer membrane protein described in the first aspect in the preparation of a vaccine composition.

[0026] Compared with the prior art, the present invention has the following beneficial effects:

[0027] 1. In the prior art, the inclusion bodies are generally denatured and then the denaturing agent is removed by dialysis bag dialyzation for renaturation. However, protein aggregation and inactivation often occur, and the denatured protein is extremely difficult to renature, and the renaturation efficiency is very low. The present invention uses a nickel ion affinity chromatography column to separate and purify the target outer membrane protein. The denaturing agent is replaced at an extremely low rate that can be artificially controlled on the nickel ion affinity chromatography column, and the removal rate of the denaturing agent is controlled, so that the protein can be slowly renatured in situ, which significantly improves the success rate of protein renaturation and is easy to scale up for preparation.

[0028] 2. In the in situ renaturation method of bacterial outer membrane protein described in the present invention, the denatured protein is renatured and folded at the same time, and the impurity protein that has not been successfully renatured is removed; at the same time, the denatured protein is automatically gradient dialyzed without manual solution replacement. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] The present invention will be further described below in conjunction with the accompanying drawings and embodiments:

[0030] Figure 1This is a diagram showing the purification effect of the outer membrane protein of Acinetobacter baumannii described in the test example;

[0031] Figure 2 The figure shows the purification effect of the outer membrane protein of Acinetobacter baumannii expressed by different expression vectors;

[0032] Figure 3 The results showed that the purified Acinetobacter baumannii outer membrane protein had good immunogenicity. DETAILED DESCRIPTION

[0033] In order to better understand the present invention, the present invention will be further described in detail below in conjunction with the accompanying drawings so that those skilled in the art can implement the present invention with reference to the description text.

[0034] It should be noted that the experimental methods described in the following experiments, unless otherwise specified, are all conventional methods, and the reagents and materials, unless otherwise specified, can be obtained from commercial channels.

[0035] Experimental reagents:

[0036] In the following examples, the nickel ion affinity chromatography filler was purchased from Cytiva, product model: NI SEPHAROSE 6FF.

[0037] Example 1

[0038] This embodiment provides a method for in situ renaturation of an expressed Acinetobacter baumannii outer membrane protein, comprising the following steps:

[0039] (1) Using genetic engineering methods, the outer membrane proteins OmpA, OmpW, and Omp22 were expressed in Acinetobacter baumannii using different expression vectors, and bacterial liquid expressing the target outer membrane protein was obtained and the bacterial cells were collected.

[0040] (2) Wash the cells with the detergent Triton X-100 to remove related impurities and obtain inclusion bodies.

[0041] (3) The inclusion bodies were sonicated under urea denaturation conditions to obtain denatured proteins; the sonication time was 10 minutes, the working time was 10 seconds, the interval time was 10 seconds, the alarm temperature was 4° C., and the power was 25%.

[0042] (4) Using a nickel ion affinity chromatography column to separate and purify the target outer membrane protein, the specific steps are as follows:

[0043] S41, the chromatography column is balanced in buffer 3, and the denatured protein dissolved in urea is loaded; the buffer 3 is 8M urea, 0.5M NaCl, 20mM Na n H2PO4, pH 8.0.

[0044] S42, using buffer 4 with a concentration of 20 mM imidazole to wash the weakly bound proteins from the chromatography column; the buffer 4 is 0.5 M imidazole, 8 M urea, 0.5 M NaCl, 20 mM Na n H2PO4, pH 8.0.

[0045] S43, after returning to buffer 3 to allow the chromatography column to reach equilibrium again, the pump B of the FPLC is replaced with a pump containing buffer 1, and a 20 mL linear gradient is run from 8 M urea to 0 M urea at a speed of 1 ml / min to replace the buffer, while the target outer membrane protein is bound to the column;

[0046] The buffer 1 is 5mM imidazole, 0.5M NaCl, 20mM Na n H2PO4, pH 8.0.

[0047] S44, after the buffer exchange is completed, pump A of the FPLC is changed to contain buffer 1, and pump B is changed to contain buffer 2;

[0048] Buffer 2 is 0.5 M imidazole, 0.5 M NaCl, 20 mM Na n H2PO4, pH 7.4.

[0049] S45, bound proteins were eluted using a 5–500 mM imidazole gradient to obtain the target outer membrane protein, and His-tagged proteins were eluted at 300–500 mM imidazole.

[0050] Test example: protein purity and immunogenicity testing

[0051] In order to detect the purity of the target outer membrane protein obtained by the method described in Example 1, the total protein staining of the eluted fractions using Coomassie Brilliant Blue showed that Figure 1 As shown, the recovery purity of the target protein is about 98%. Figure 1 , Figure 2 As shown, the outer membrane proteins OmpA, OmpW, and Omp22 expressed in different expression vectors can be purified by this method ( Figure 2 ), further indicating that this method is generally applicable to the purification of bacterial outer membrane proteins.

[0052] The immunogenicity of the three purified Acinetobacter baumannii outer membrane proteins was further tested, and the test method was as follows:

[0053] (1) Coating: The bacterial membrane vesicles of the standard strain of Acinetobacter baumannii were coated overnight;

[0054] (2) 2% BSA blocking for 2 hours;

[0055] (3) Primary antibody: 1000-fold diluted mouse serum;

[0056] (4) Secondary antibody: HRP-labeled goat anti-mouse IgG antibody;

[0057] (5) TMB substrate color development;

[0058] (6) Detection by ELISA instrument.

[0059] Test results such as Figure 3 As shown, it was confirmed that the three Acinetobacter baumannii outer membrane proteins not only had high purity but also had good immunogenicity.

[0060] Finally, it should be noted that the above is only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred arrangement scheme, a person skilled in the art should understand that the technical solution of the present invention may be modified or replaced by equivalents without departing from the spirit and scope of the technical solution of the present invention.

Claims

1. A universal in situ renaturation method for bacterial outer membrane proteins, characterized in that: The method comprises the following steps: (1) Obtaining bacterial solution expressing the target outer membrane protein and collecting bacterial cells; (2) washing the cells with the detergent TritonX-100 to obtain inclusion bodies; (3) The inclusion bodies are sonicated under urea denaturation conditions to obtain denatured proteins; (4) Using a nickel ion affinity chromatography column to separate and purify the target outer membrane protein, the specific steps are as follows: S41, the chromatography column is balanced in buffer 3, and the denatured protein dissolved in urea is loaded; the buffer 3 is 8M urea, 0.5M NaCl, 20mM Na n H2PO4, pH 8.0; S42, using buffer 4 containing imidazole to wash the weakly bound proteins from the chromatography column; the buffer 4 is 0.5M imidazole, 8M urea, 0.5M NaCl, 20mM Na n H2PO4, pH 8.0; S43, after returning to buffer 3 to allow the chromatography column to reach equilibrium again, the pump B of the FPLC is replaced with a pump containing buffer 1, and the buffer is replaced by running a linear gradient from 8 M urea to 0 M urea at a speed of 1 ml / min, while the target outer membrane protein is bound to the column; The buffer 1 is 5mM imidazole, 0.5M NaCl, 20mM Na n H2PO4, pH 8.0; S44, after the buffer exchange is completed, pump A of the FPLC is changed to contain buffer 1, and pump B is changed to contain buffer 2; Buffer 2 is 0.5 M imidazole, 0.5 M NaCl, 20 mM Na n H2PO4, pH 7.4; S45, using a 5-500 mM imidazole gradient to elute the bound protein, thereby obtaining the target outer membrane protein.

2. The method according to claim 1, characterized in that The bacteria include Acinetobacter baumannii.

3. The method according to claim 2, characterized in that The bacterial outer membrane proteins include but are not limited to Acinetobacter baumannii outer membrane proteins OmpA, Omp22 and OmpW.

4. The method according to claim 1, characterized in that: In step (3), the ultrasonic conditions are: ultrasonic time: 10 minutes, working time: 10 seconds, interval time: 10 seconds, alarm temperature 4°C, power: 25%.

5. The method according to claim 1, characterized in that The target outer membrane protein is eluted at 300-500 mM imidazole.

6. A bacterial outer membrane protein obtained by the method according to any one of claims 1 to 5.

7. Use of the bacterial outer membrane protein according to claim 6 in preparing a vaccine composition.

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