WO2019019218A1

WO2019019218A1 - Protein regulatory system, preparation method therefor and use thereof

Info

Publication number: WO2019019218A1
Application number: PCT/CN2017/096346
Authority: WO
Inventors: 梁兴祥; 王美玲; 郭文中; 秦莉; 姚永超; 陈小平
Original assignee: 广州中科蓝华生物科技有限公司
Priority date: 2017-07-28
Filing date: 2017-08-08
Publication date: 2019-01-31
Also published as: CN107312797B; CN107312797A

Abstract

Disclosed are a protein regulatory system, a preparation method therefor and the use thereof. The dihydrofolate reductase in the protein regulatory system is linked to a protein of interest to be regulated using a rigid linker peptide. The rigid linker peptide is (EAAAK)n or A(EAAAK)nA, wherein n is any integer from 1-6, and preferably n is 6. The protein regulatory system has a higher expression amount of the protein of interest, a higher regulatory ratio and a higher regulating range, and has a very broad application value.

Description

Protein regulation system and preparation method and application thereof

Technical field

The invention belongs to the field of bioengineering, relates to a protein regulating system and a preparation method and application thereof, and particularly relates to a protein regulating system optimized by using a rigid connecting peptide and the application thereof.

Background technique

DDD (DHFR degradation domain) regulation system is a regulation system that regulates the target protein by using ubiquitin protease system. It uses Escherichia coli dihydrofolate reductase (ecDHFR) to fuse with target protein by controlling the addition of stabilizer. The target protein regulates protein levels.

ecDHFR can be stabilized by the DHFR inhibitor trimethoprim (TMP). When the stabilizer TMP is not added, ecDHFR and the protein fused thereto are labeled with ubiquitin, and then recognized and degraded by the proteasome, thereby achieving non-expression of the target protein. When the stabilizer TMP is added, TMP binds to ecDHFR and stabilizes ecDHFR, so that the ecDHFR and the protein fused thereto are kept in a stable state without being degraded by ubiquitination, and the target protein can be normally expressed. The combination of TMP and ecDHFR to stabilize the protein from degradation is reversible. The addition of TMP stabilizes ecDHFR, and the withdrawal of TMP leads to the degradation of ecDHFR and its fusion protein, thereby controlling the expression level of the target protein by controlling the amount of TMP.

TMP is a commonly used and inexpensive human medication that can be used directly on the human body. In addition, TMP has a strong stabilizing effect on DHFR, and TMP has a stronger stabilizing effect on ecDHFR than mammalian DHFR, so only a small amount of TMP is needed to stabilize ecDHFR, so the control cost of using DDD control system is relatively low. In addition, TMP can also pass the blood-brain barrier and the placental barrier, so DDD can be used to regulate protein expression in different periods and different ranges, and has a wide application space. In summary, the DDD control system is a convenient and widely used protein regulation system. System. However, the current DDD regulatory system still has defects: (1) the expression level of the target protein is lower than that when the regulatory sequence is removed; (2) the expression level of the regulatory protein is low.

A common method for constructing a fusion protein is to directly link two functional molecules, which usually include a protein, a globular domain of a protein, and some highly expressed protein sequences. Another method is to link functional molecules by linking peptide sequences. In general, the following factors may affect the biological activity of the fusion protein: 1. The spatial position between the fusion proteins; 2. The respective receptor structures and relationships of the fusion proteins; 3. The length of the fusion protein-linked peptide, folding, The amino acid composition, whether it is glycosylated, the adaptability between the linker peptide and the two linked protein molecules, and the flexibility and hydrophobicity of the linker peptide are important for not disturbing the functional domain of the protein.

As a link between fusion proteins, the linker peptides generally require flexibility and hydrophilicity. Therefore, glycine is a small molecular weight, simple structure, and a hydrophilic amino acid is used in the design of the linker peptide. Since 1988 (GGGGS)n (n ≤ 6) was invented, it has been used as an excellent linker peptide until now, and is widely used in the construction of various fusion proteins. Because of the richness of glycine, the linker peptide of this structure is relatively soft and curved, and thus this linker peptide is called a flexible linker peptide. However, such flexible linker peptides also have disadvantages. Because of the softness, all the proteins linked at both ends of the linker peptide can be freely moved, and the hairpin structure is easily formed, so that the fusion protein is entangled to easily form a dimer, and at the same time, the active site of the protein may be masked. Affect the activity of the protein. In 2001, Arai et al. designed an α-helical-linked peptide (EAAAK) _n (n≤6), which has a rigid structure that can effectively segment the two proteins that are fused to each other. The smallest, its secondary structure is α-helix, which is not easy to bend, which ensures the relative stability of functional protein spacing.

The effect of different linker peptides is different. Some of the linker peptides can increase the expression level of the target protein regulated by the DDD regulatory system, but also increase the background level of the target protein without adding a stabilizer. Instead, the sensitivity of the DDD regulatory system is reduced, so it is necessary to select a suitable linker peptide to link the ecDHFR protein with the desired protein to be expressed.

Summary of the invention

In view of the technical defects existing in the prior art, the present invention provides a protein regulation system and a preparation method and application thereof, by optimizing DDDHFR (dihydrofolate reductase) in a DDD control system (dihydrofolate reductase degradation domain regulation system) The way in which the protein is fused to the protein of interest increases the level of expression of the protein of interest and the extent of regulation when TMP is added.

In order to achieve the above object, the present invention adopts the following technical solutions:

In a first aspect, the present invention provides a protein regulatory system in which a ligated linker is ligated between ecDHFR and a protein of interest to be regulated.

In the present invention, by connecting the regulatory system to the target protein by the rigid linker peptide, the background level of the protein can be reduced, the amplitude of the expression of the target protein can be effectively increased, and the regulatory effect can be improved. The inventors found the rigid connection. The peptide can be ligated to the C-terminus or N-terminus of ecDHFR. The attachment of the rigid linker peptide at either end does not affect the regulatory effect of the regulatory system, but the rigid linker peptide must be linked between the ecDHFR and the protein of interest.

In the present invention, the amino acid sequence of the ecDHFR is as shown in SEQ ID NO. 1, and the amino acid sequence of the SEQ ID NO. 1 is as follows: MISLIAALAVDYVIGMENAMPWNLPADLAWFKRNTLNKPVIMGRHTWESIGRPLPGRKNIILSSQPGTDDRVTWVKSVDEAIAACGDVPEIMVIGGGRVIEQFLPKAQKLYLTHIDAEVEGDTHFPDYEPDDWESVFSEFHDADAQNSHSYCFEILERR.

According to the invention, the rigid linker peptide is (EAAAK) _n or A(EAAAK) _n A, wherein n is independently selected from any integer from 1 to 6, for example 1, 2, 3, 4, 5 or 6 Preferably, n is 6.

According to the invention, the rigid linker peptide is A(EAAAK) _n A, wherein n is any integer from 1 to 6, for example 1, 2, 3, 4, 5 or 6, preferably n is 6.

In the present invention, the length of the linker peptide directly affects the distance of the functional protein, and the peptide which is too long or too short affects the stability and activity of the fusion protein, and the length is too short to cause a steric hindrance effect, and the protein at both ends is insufficient. Folding or unfolding, forming a wrong configuration or hindering the active site, resulting in inactive or low activity of the fusion protein, and the dimer is abundantly present. Too long will result in loose structure of the fusion protein and is easily cleaved by protease hydrolysis in the host cell. Therefore, the selected linker peptide should have sufficient length and good flexibility to ensure that the fusion-ligated protein can have sufficient freedom in space to exert its function, and the α-helix should be avoided as much as possible in the inter-chain linker peptide. And the effect of β-sheet folding on the stability of the fusion protein.

The inventors have found that using the rigid linker peptide selected by the present invention can effectively increase the amplitude of the target protein regulated by the protein regulatory system, improve the regulation effect, and the background level is also low, and the inventors have verified a large number of experiments, compared with (EAAAK) _n The regulation effect of other rigid linker peptides is obvious, especially the (EAAAK) _6- linked peptide has the most obvious effect, has the lowest background level, and can effectively improve the target protein after administration of TMP (trimethoprim). The expression amount greatly improved the expression level of the protein regulated by the protein regulatory system.

According to the invention, the protein of interest is an intracellular protein and/or a membrane protein.

According to the invention, the membrane protein is a chimeric antigen receptor.

In a second aspect, the present invention provides a method for preparing a protein regulatory system according to the first aspect, comprising the steps of:

The ecDHFR-rigid linker-target protein expression vector is constructed, and the constructed expression vector is transfected into an expression host to obtain the protein regulation system.

According to the invention, the rigid linker peptide is (EAAAK) _n , wherein n is independently selected from any integer from 1 to 6, for example 1, 2, 3, 4, 5 or 6, preferably n is 6.

According to the present invention, the expression vector and the expression host can be selected according to different proteins of interest, and are not particularly limited herein.

In a third aspect, the present invention provides a method for protein regulation using the protein regulatory system of the first aspect, comprising the steps of:

The constructed protein regulatory system was cultured, and TMP was added to regulate the expression of the protein.

According to the present invention, by externally adding TMP for controlling the expression of the protein of interest, controlling the amount of TMP added can control the expression level of the target protein, and the expression of the different proteins has different response values to the amount of TMP added, and those skilled in the art can The controlled protein of interest is adjusted to adjust the amount of TMP. The concentration of TMP according to the present invention is 5 to 250 μM, and may be, for example, 5 μM, 10 μM, 15 μM, 20 μM, 25 μM, 30 μM, 35 μM, 40 μM, 50 μM, 60 μM, 70 μM, 80 μM, 90 μM. 100 μM, 110 μM, 120 μM, 130 μM, 140 μM, 150 μM, 160 μM, 170 μM, 180 μM, 190 μM, 200 μM, 210 μM, 220 μM, 230 μM, 240 μM or 250 μM.

In a fourth aspect, the invention provides a protein regulatory system according to the first aspect for use in the regulation of protein expression of intracellular proteins and/or membrane proteins.

In a fifth aspect, the present invention provides a protein regulatory system according to the first aspect, for use in the preparation of a medicament for controlling the regulation of protein expression of an intracellular protein and/or a membrane protein.

According to the invention, the medicament further comprises TMP.

In a sixth aspect, the present invention provides a protein regulatory system according to the first aspect for use in the preparation of a medicament for alleviating the side effects of CAR-T cell therapy.

In the present invention, CAR-T cell therapy is effective in the treatment of leukemia and lymphoma, but it is accompanied by serious side effects, especially cytokine release syndrome (CRS), and highly proliferating CAR-T cells can cause CRS. The drug prepared by the protein regulatory system can selectively control the expression of CAR-T cells and control the expression of CAR-T cells, thereby ensuring the efficacy of CAR-T cells to reduce or even prevent side effects.

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

(1) The protein regulatory system of the present invention has a higher expression level of the target protein: the target protein has a higher expression level after the addition of the stabilizer TMP, and is even higher than the expression level of the directly expressed protein of interest;

(2) The protein regulatory system of the present invention has a higher regulatory ratio: when an equal amount of TMP is used to induce expression of a protein of interest, the protein regulatory system of the present invention can express more target protein than the original system, so control, etc. When the target protein is expressed, the protein regulatory system of the present invention requires less TMP and is more sensitive to regulation;

(3) The protein regulatory system of the present invention has a higher regulatory amplitude: an improved DDD regulatory system using (EAAAK) _6- linked peptide compared to other linker-optimized DDD regulatory systems and unoptimized DDD regulatory systems The background level is the lowest, the target protein expressed is the least when the stabilizer TMP is not added, and the regulation range is high because the amount of protein expressed is large.

DRAWINGS

1 is a schematic diagram of a plasmid of pGPC3-EGFP-(EAAAK) ₆ -DHFR-CAR3(ecDHFR-(EAAAK) ₆ );

2 is a schematic diagram of a pGPC3-EGFP-(EAAAK) ₃ -DHFR-CAR3 (ecDHFR-(EAAAK) ₃ ) plasmid according to Example 2 of the present invention;

Figure 3 is a schematic diagram showing the plasmid of pGPC3-EGFP-A(EAAAK) ₅ A-DHFR-CAR3 (ecDHFR-A(EAAAK) ₅ A);

Figure 4 is a schematic diagram showing the plasmid of Comparative Example 1pGPC3-EGFP-DHFR-CAR3(ecDHFR);

Figure 5 is a schematic diagram showing the plasmid of Comparative Example 2pGPC3-EGFP-flag-DHFR-CAR3(ecDHFR-Flag);

Figure 6 is a schematic diagram of the plasmid of Comparative Example 3pGPC3-EGFP-(GGGGS) ₃ -DHFR-CAR3(ecDHFR-(GGGGS) ₃ );

7 is a flow analysis of the present invention comparing the average values of GFP fluorescence of each group, wherein FIG. 7(a) is a comparison of no TMP, 10 μm TMP, and 100 μm TMP; FIG. 7(b) changes with time. Comparison of adding 10 μm TMP and adding 100 μm TMP, blank is 293T cells without transfection, GPC3 control is pGPC3-EGFP-Flag, ecDHFR-(GGGGS) ₃ is Comparative Example 3, and ecDHFR-(EAAAK) ₃ is Example 2. ecDHFR-(EAAAK) ₆ is Example 1, ecDHFR-A(EAAAK) ₅ A is Example 3, ecDHFR is Comparative Example 1;

Figure 8 is a flow chart analysis comparing the average results of GFP fluorescence of each group in the flow analysis, wherein Figure 8 (a) compares the addition of TMP with the addition of 100 μm TMP; Figure 8 (b) compares the change of time with the addition of 100 μm TMP , blank is 293T cells not transfected, GPC3 control is pGPC3-EGFP-Flag, ecDHFR is comparative example 1; ecDHFR-A (EAAAK) ₅ A is Example 3, and ecDHFR-Flag is Comparative Example 2.

Detailed ways

In order to further illustrate the technical means and effects of the present invention, the following embodiments and drawings are combined. The invention is further illustrated. It is understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

The specific techniques or conditions are not indicated in the examples, according to the techniques or conditions described in the literature in the art, or in accordance with the product specifications. The reagents or instruments used are not specified by the manufacturer, and are conventional products that are commercially available through formal channels.

Example 1: Construction of a protein regulatory system

The 293T vector was constructed, and the DDD regulatory fragment of the GPC3 band (EAAAK) ₆ ligation peptide was inserted into the pEGFP-C1 vector by the AgeI and EcoRI cleavage sites to obtain the pGPC3-EGFP-(EAAAK) ₆ -DHFR-CAR3 vector. The carrier is shown in Figure 1.

Example 2: Construction of a protein regulatory system

The 293T vector was constructed, and the DDD regulatory fragment of the GPC3 band (EAAAK) ₃ ligated peptide was inserted into the pEGFP-C1 vector by the AgeI and EcoRI cleavage sites to obtain the pGPC3-EGFP-(EAAAK) ₃ -DHFR-CAR3 vector. The carrier is shown in Figure 2.

Example 3 is a peptide substituted with A(EAAAK) ₅ A

The 293T vector was constructed, and the DDD regulatory fragment of the GPC3 band A (EAAAK) ₅ A ligation peptide was inserted into the pEGFP-C1 vector by the AgeI and EcoRI cleavage sites to obtain the pGPC3-EGFP-AEAAAK5A-DHFR-CAR3 vector, and the obtained vector was obtained. As shown in Figure 3.

Comparative Example 1 does not contain a linker peptide

The 293T vector was constructed, and the DPC regulatory fragment of GPC3 without Flag tag was inserted into the pEGFP-C1 vector by the AgeI and EcoRI cleavage sites to obtain the pGPC3-EGFP-DHFR-CAR3 vector, and the obtained vector was shown in FIG.

Comparative Example 2 Ligation peptides were replaced with flag tags

The 293T vector was constructed, and the GPC3 flag-tagged DDD regulatory fragment was inserted into the pEGFP-C1 vector by the AgeI and EcoRI cleavage sites to obtain the pGPC3-EGFP-Flag-DHFR-CAR3 vector, and the obtained vector is shown in FIG.

Comparative Example 3 Ligation peptide was replaced with (GGGGS)3

The 293T vector was constructed, and the DDD regulatory fragment of the GPC3 band (GGGGS) ₃ ligated peptide was inserted into the pEGFP-C1 vector by the AgeI and EcoRI cleavage sites to obtain the pGPC3-EGFP-GGGGS3-DHFR-CAR3 vector. 6 is shown.

Example 4 Validation of the effect of rigid linker peptides and other linker peptides

Example 1-3 and Comparative Examples 1 and 3 were administered to TMP (10 μM, 100 μM) for 48 hours, and then GFP fluorescence was detected by flow cytometry. The fluorescence average data of each group were analyzed as shown in Fig. 7 (a). ) - Figure 7 (b), where blank is 293T cells that are not transfected and GPC3 control is pGPC3-EGFP-Flag.

As can be seen from Fig. 7(a) - Fig. 7(b), it can be seen from the fluorescence analyzed by flow cytometry that the rigid linker peptide (EAAAK) ₆ (Example 1), (EAAAK) ₃ (Example) 2), A(EAAAK) ₅ A (Comparative Example 3) The TMP-free experimental group had a lower background level of target protein expression, while the flexible linker (GGGGS) ₃ (Comparative Example 4) was not added. The background level of the target protein expression of the TMP experimental group was relatively high, even higher than the background level of pGPC3-EGFP-DHFR-CAR3 (Comparative Example 1) without the addition of the linker peptide, and at the same time, with a rigid connection Peptide (EAAAK) ₆ (Example 1), (EAAAK) ₃ (Example 2), A (EAAAK) ₅ A (Comparative Example 3) and (GGGGS) ₃ (Comparative Example 4) Administration of 10 μM, 100 μM TMP The expression level of the target protein was higher than that of pGPC3-EGFP-DHFR-CAR3 without unligated peptide (Comparative Example 1) and pGPC3-EGFP-Flag with pure expression of GPC3 without DDD regulatory system. Peptides can increase the regulation of DDD regulatory systems. Compared with Example 1-2 and Comparative Example 3-4, the rigid linker peptide (EAAAK) ₆ (Example 1) was found to have the most superior effect, low background, high regulation range, and far more control ratio than other examples and In the comparative example, there was a 5-fold regulatory ratio in the 10 μM group, and a nearly 7-fold regulatory ratio in the 10 μM group, and the regulatory ratio became apparent as the amount of TMP administered increased.

Example 5 Validation of the effect of a linker peptide and no linker peptide

Example 3 and Comparative Example 1-2 were administered to TMP (100 μM) for 48 hours, and then the GFP fluorescence of each group was detected by flow cytometry. The fluorescence average data of each group were analyzed as shown in Fig. 8(a)-Fig. (b) shows that blank is a non-transfected 293T cell and the GPC3 control is pGPC3-EGFP-Flag.

It can be seen from Fig. 8(a)-Fig. 8(b) that the control peptide without the linker peptide has the lowest amplitude, and has the A(EAAAK) ₅ A (Comparative Example 3) linker peptide and the Flag tag (Comparative Example 2). The experimental group as a linker peptide control was more regulated than the group without the linker peptide (Comparative Example 1), but the background with the Flag tag (Comparative Example 2) had a very high background level when no TMP was added. Therefore, the regulation ratio is lower than that of the group without the addition of the linker peptide. Therefore, although the inappropriate linker peptide can increase the regulation range of the target protein, the sensitivity of the regulation is decreased. The regulation ratio of the group with A(EAAAK) ₅ A (Comparative Example 3)-linked peptide was 6 times higher than that of the group without the addition of the linker peptide (Comparative Example 1), so the regulation range was still The group with A(EAAAK) ₅ A (Comparative Example 3) linked peptide was the highest. It can be seen that the DDD regulatory system has a linker peptide or a peptide of a certain length in the middle of the ecDHFR and the target protein to improve the regulation effect of the DDD regulatory system, but a suitable linker peptide can increase the sensitivity of the regulation while reducing the background level.

In summary, the discovery of the rigid linker peptide (EAAAK) ₆ (Example 1) of Example 1-2 has the most superior effect, the background is low, the control range is high, and the regulation ratio is far superior to other examples and comparative examples. In the 10μM group, there is a 5-fold regulation ratio, while in the 10μM group, there is a nearly 7-fold regulation ratio. The regulation ratio becomes more apparent with the increase in the amount of TMP administered. The effect of the rigid linker peptide (EAAAK) _n is superior to that of the rigid junction. Peptide A (EAAAK) _n A, a rigid linker peptide is superior to other linker peptides in that it has a better effect than a linker peptide.

The ecDHFR of the DDD regulatory system is fused with the rigid linker peptide (EAAAK) _n- ligation peptide to obtain improved DDD regulatory system components and then regulate the target protein. The improved DDD regulatory system has a greatly improved regulation level, and the sensitivity is also improved. A great improvement, and the effect of the linker peptide (EAAAK) ₆ is most pronounced. The improved DDD control system is more suitable for regulating the expression of the target protein and has high use value.

The Applicant declares that the present invention is described by the above-described embodiments, but the present invention is not limited to the above detailed methods, that is, it does not mean that the present invention must be implemented by the above detailed methods. It should be apparent to those skilled in the art that any modifications of the present invention, equivalent substitution of the various materials of the products of the present invention, addition of auxiliary components, selection of specific means, and the like, are all within the scope of the present invention.

Claims

A protein regulatory system characterized in that a dihydrofolate reductase in the protein regulatory system is linked to a protein of interest to be regulated by a rigid linker peptide.
The protein regulatory system according to claim 1, wherein the rigid linker peptide is (EAAAK) n or A(EAAAK) n A, wherein n is independently selected from any integer from 1 to 6, preferably n is 6;

Preferably, the rigid linker peptide is A(EAAAK) n A, wherein n is any integer from 1 to 6, preferably n is 6.
The protein regulatory system according to claim 1 or 2, wherein the protein of interest is an intracellular protein and/or a membrane protein;

Preferably, the membrane protein is a chimeric antigen receptor.
A method for preparing a protein regulation system according to any one of claims 1 to 3, comprising the steps of:

A dihydrofolate reductase-rigid linker-target protein expression vector is constructed, and the constructed expression vector is transfected into an expression host to obtain the protein regulation system.
The method according to claim 4, wherein the rigid linker peptide is (EAAAK) n or A(EAAAK) n A, wherein n is independently selected from any integer of 1-6, preferably n is 6 ;

Preferably, the rigid linker peptide is A(EAAAK) n A, wherein n is any integer from 1 to 6, preferably n is 6.
A method for protein regulation using the protein regulatory system according to any one of claims 1 to 3, comprising the steps of:

The constructed protein regulatory system was cultured, and trimethoprim was added to regulate the expression of the protein.
The method of protein regulation according to claim 6, wherein the concentration of the trimethoprim is from 5 to 250 μM.
A protein regulatory system according to any one of claims 1 to 3 for protein expression regulation of intracellular proteins and/or membrane proteins;

Preferably, the membrane protein is a chimeric antigen receptor.
A protein regulatory system according to any one of claims 1 to 3 for use in the preparation of a medicament for controlling the regulation of protein expression of intracellular proteins and/or membrane proteins;

Preferably, the membrane protein is a chimeric antigen receptor;

Preferably, the medicament further comprises trimethoprim.
A protein regulatory system according to any one of claims 1 to 3 for use in the preparation of a medicament for alleviating the side effects of chimeric antigen receptor T cell therapy.