Auto-transport protein secretion system, foreign protein and preparation method of foreign protein

Abstract

The invention relates to the technical field of biology, in particular to a self-transport protein secretion system, a foreign protein and a preparation method of the foreign protein. The self-transport protein secretion system comprises an expression vector and a plurality of groups of functional units on the expression vector, the multiple groups of functional units comprise a transporter signal peptide, a transporter beta-bucket structural domain as shown in SEQ ID NO.1, a transporter alpha hinge region protease hydrolysis mutation site as shown in SEQ ID NO.2 and a thrombin cutting site as shown in SEQ ID NO.3; wherein the transporter signal peptide comprises at least one of ESPp signal peptide, pet signal peptide, PIC signal peptide and Hbp signal peptide. The functional unit of the autotransporter secretion system can promote the secretion expression of the foreign protein, reduce the pollution of endogenous protein and reduce the residue of transporter in the foreign protein, so as to improve the purity of the foreign protein.

Description

Technical Field

[0001] The present application relates to the field of biotechnology, and in particular to an autotransporter secretion system, an exogenous protein and a preparation method thereof. Background Art

[0002] Prokaryotic protein secretion expression refers to a technology that secretes exogenous proteins outside the cell membrane of Escherichia coli. This technology can reduce the cleavage of exogenous proteins by proteases in Escherichia coli cells, making it easier to purify the target protein to reduce impurities in the target protein. However, at this stage, the expression of prokaryotic proteins is mainly based on expression in Escherichia coli cells, so how to use Escherichia coli to achieve secretory expression of prokaryotic proteins is currently a technical difficulty. In addition, Escherichia coli serine protease (EspP) is a member of the autotransporter secretion system, consisting of an N-terminal signal peptide, a middle serine protease domain (passenger domain), a self-chaperone domain that assists passenger folding, and a C-terminal β-barrel domain. EspP is mainly cut through the EVNNLN structure of the α-hinge region protease hydrolysis site, thereby releasing the serine protease domain.

[0003] Although the autotransporter secretion system can be used to secrete exogenous proteins at this stage, the autotransporter secretion system is only aimed at optimizing the secretion of individual proteins, which limits the applicability of the autotransporter secretion system. If other types of proteins are used in combination with the autotransporter secretion system, the yield of the corresponding protein product may be reduced. In addition, based on the expression characteristics of the autotransporter secretion system, these factors will cause the expressed protein to have some residual transporter structure, which will cause the exogenous protein to be contaminated by endogenous proteins, further reducing the purity of the corresponding protein product.

[0004] At present, the autotransporter secretion system is generally a type V secretion system of Gram-negative bacteria, which can transport the cargo domain of the autotransporter to the cell surface or culture medium of Gram-negative bacteria. The structural characteristics of these autotransporters include: ① N-terminal signal peptide; ② middle cargo domain; ③ C-terminal β-barrel domain. The working process of these autotransporters is: first, the "passenger" protein of the cargo domain, under the guidance of the signal peptide, passes through the inner membrane to the periplasm through the sec secretion pathway, and the signal peptide is cleaved in the periplasmic cavity to obtain the "passenger" protein; then the "passenger" protein passes through the outer membrane through the β domain. Summary of the invention

[0005] The present application provides an autotransporter secretion system, an exogenous protein and a preparation method thereof to solve the following technical problems: how to use the autotransporter secretion system to promote the secretory expression of the exogenous protein and to improve the purity of the secreted exogenous protein.

[0006] In a first aspect, the present application provides an autotransporter secretion system, the autotransporter secretion system comprising:

[0007] An expression vector and multiple groups of functional units on the expression vector; the multiple groups of functional units include a transporter signal peptide, a transporter β-barrel domain, a transporter α hinge region protease hydrolysis mutation site and a thrombin cleavage site;

[0008] Wherein, the transporter β-barrel domain has a nucleotide sequence as shown in SEQ ID NO.1, the transporter α hinge region protease hydrolysis mutation site has a nucleotide sequence as shown in SEQ ID NO.2, and the thrombin cleavage site has a nucleotide sequence as shown in SEQ ID NO.3;

[0009] The transporter signal peptide comprises at least one of the following:

[0010] ESPp signal peptide, pet signal peptide, PIC signal peptide and Hbp signal peptide.

[0011] Optionally, when the transporter signal peptide includes an ESPp signal peptide, the ESPp signal peptide has a nucleotide sequence as shown in SEQ ID NO.4.

[0012] Optionally, the functional unit has a nucleotide sequence as shown in SEQ ID NO.5.

[0013] Optionally, the ESPp signal peptide has an amino acid sequence as shown in SEQ ID NO.6, the transporter protein β-barrel domain has an amino acid sequence as shown in SEQ ID NO.7, the transporter protein α hinge region protease hydrolysis mutation site has an amino acid sequence as shown in SEQ ID NO.8, and the thrombin cleavage site has an amino acid sequence as shown in SEQ ID NO.9.

[0014] Optionally, the functional unit has an amino acid sequence as shown in SEQ ID NO.10.

[0015] Optionally, when the transporter signal peptide includes a pet signal peptide, the pet signal peptide has an amino acid sequence as shown in SEQ ID NO.11;

[0016] In the case where the transporter signal peptide includes a PIC signal peptide, the PIC signal peptide has an amino acid sequence as shown in SEQ ID NO.12;

[0017] When the transporter signal peptide includes an Hbp signal peptide, the Hbp signal peptide has an amino acid sequence as shown in SEQ ID NO.13.

[0018] In a second aspect, the present application provides a method for preparing the autotransporter secretion system according to the first aspect, the method comprising:

[0019] The signal peptide region of the original expression vector is replaced with the functional unit to obtain a recombinant expression vector.

[0020] In a third aspect, the present application provides an exogenous protein, wherein the exogenous protein comprises the autotransporter secretion system described in the first aspect and a target gene carried on the autotransporter secretion system; wherein the target gene comprises at least one of the following:

[0021] Mouse Cd3e gene, mouse Prl gene, human WNT3A gene, human WNT5A gene, and rat Zg16 gene;

[0022] When the target gene is a mouse Cd3e gene, the mouse Cd3e gene has a nucleotide sequence shown in SEQ ID NO.14;

[0023] When the target gene is a mouse Prl gene, the mouse Prl gene has a nucleotide sequence shown in SEQ ID NO.15;

[0024] In the case where the target gene is a human WNT3A gene, the human WNT3A gene has a nucleotide sequence shown in SEQ ID NO.16;

[0025] In the case where the target gene is a human WNT5A gene, the human WNT5A gene has a nucleotide sequence shown in SEQ ID NO.17;

[0026] When the target gene is rat Zg16 gene, the rat Zg16 gene has a nucleotide sequence shown in SEQ ID NO.18.

[0027] In a fourth aspect, the present application provides a method for preparing the exogenous protein according to the third aspect, the method comprising:

[0028] The autotransporter secretion system is double-digested to obtain a digestion product;

[0029] The target gene is subjected to the double enzyme digestion to obtain a gene fragment;

[0030] Using a preset amplification primer set to perform PCR amplification on the gene fragment to obtain an amplified product;

[0031] Performing DNA ligation on the enzyme-cut product and the amplified product to obtain a recombinant expression vector;

[0032] Using host bacteria to transfect the recombinant expression vector to obtain recombinant cells; and

[0033] The recombinant cells are cultured, and then a large number of recombinant cells obtained by culture are subjected to enzyme digestion and purification to obtain foreign proteins.

[0034] Optionally, the preset amplification primer set includes a first primer set, a second primer set, a third primer set, a fourth primer set and a fifth primer set, the preset amplification primer set includes the first primer set, the second primer set, the third primer set, the fourth primer set and the fifth primer set, the upstream primer of the first primer set has a nucleotide sequence as shown in SEQ ID NO.19, and the downstream primer of the first primer set has a nucleotide sequence as shown in SEQ ID NO.20;

[0035] The upstream primer of the second primer set has a nucleotide sequence as shown in SEQ ID NO.21, and the downstream primer of the second primer set has a nucleotide sequence as shown in SEQ ID NO.22;

[0036] The upstream primer of the third primer set has a nucleotide sequence as shown in SEQ ID NO.23, and the downstream primer of the third primer set has a nucleotide sequence as shown in SEQ ID NO.24;

[0037] The upstream primer of the fourth primer set has a nucleotide sequence as shown in SEQ ID NO.25, and the downstream primer of the fourth primer set has a nucleotide sequence as shown in SEQ ID NO.26;

[0038] The upstream primer of the fifth primer set has a nucleotide sequence as shown in SEQ ID NO.27, and the downstream primer of the fifth primer set has a nucleotide sequence as shown in SEQ ID NO.28.

[0039] The above technical solution provided by the embodiment of the present application has the following advantages compared with the prior art:

[0040] The embodiment of the present application provides an autotransporter secretion system, which uses multiple groups of functional units including a transporter signal peptide, a transporter β-barrel domain, a transporter α hinge region protease hydrolysis mutation site and a thrombin cleavage site to replace part of the functional region of the expression vector, and the transporter signal peptide includes at least one of an ESPp signal peptide, a pet signal peptide, a PIC signal peptide and an Hbp signal peptide. The transporter signal peptide in the functional unit can transfer the exogenous protein translated from the target gene from the cell inner membrane to the periplasmic space of the cytoplasmic matrix, thereby facilitating the normal physiological activities of other subsequent functional units. In addition, the transporter β-barrel domain in the functional unit can The interaction with the protease hydrolysis mutation site in the α hinge region of the transport protein can avoid the shearing effect of the transport protease on the exogenous protein through the hydrolysis site when the exogenous protein is transported to the outside of the cell membrane for display, so as to promote the secretory expression of the exogenous protein; in addition, the use of the thrombin cleavage site can promote the enzymatic cleavage of the exogenous protein in the designated area outside the cell membrane, which can reduce the risk of the exogenous protein being enzymatically cleaved in the cell, thereby promoting the secretory expression of the exogenous protein. In addition, since the exogenous protein is enzymatically cleaved in the designated area outside the cell membrane, the functional unit can also reduce the contamination of endogenous proteins and reduce the residual transport protein in the exogenous protein, thereby improving the purity of the secreted exogenous protein. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments consistent with the present application and, together with the description, serve to explain the principles of the present application.

[0042] In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, for ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative labor.

[0043] Figure 1 A diagram of an autotransporter secretion system provided in an embodiment of the present application;

[0044] Figure 2 A schematic flow chart of a method for preparing an autotransporter secretion system provided in an embodiment of the present application;

[0045] Figure 3 A schematic diagram of a method for preparing exogenous protein provided in an embodiment of the present application;

[0046] Figure 4 This is a diagram showing the SDS-PAGE test results of a small test of the 004931-pET22b-ESPp vector provided in the examples of the present application;

[0047] Figure 5 This is a diagram showing the SDS-PAGE test results of a small-scale test of the 018724-pET22b-ESPp vector provided in the examples of the present application;

[0048] Figure 6 This is a diagram showing the SDS-PAGE test results of a small test of the 026136-pET22b-ESPp vector provided in the examples of the present application;

[0049] Figure 7 This is a diagram showing the SDS-PAGE test results of a small test of the 026138-pET22b-ESPp vector provided in the examples of the present application;

[0050] Figure 8 This is a diagram showing the SDS-PAGE test results of a small-scale test of the 807545-pET22b-ESPp vector provided in the examples of the present application;

[0051] Fig. 9 This is a diagram showing the results of SDS-PAGE detection of the purified 04931-pET22b-ESPp recombinant protein provided in the examples of the present application;

[0052] Fig.10 This is a diagram showing the results of SDS-PAGE detection of the purified 018724-pET22b-ESPp recombinant protein provided in the examples of the present application;

[0053] Fig.11 This is a diagram showing the results of SDS-PAGE detection of the purified 026136-pET22b-ESPp recombinant protein provided in the examples of the present application;

[0054] Fig.12 This is a diagram showing the results of SDS-PAGE detection of the purified 026138-pET22b-ESPp recombinant protein provided in the examples of the present application;

[0055] Fig.13 This is a diagram showing the results of SDS-PAGE detection of the purified 807545-pET22b-ESPp recombinant protein provided in the examples of the present application;

[0056] Fig.14 This is a diagram showing the results of SDS-PAGE detection of the purified 026136-pET22b-ESPp-t recombinant protein provided in the examples of the present application;

[0057] Fig.15 This is a diagram showing the results of SDS-PAGE detection of the purified 026136-pET22b-ESPp-C recombinant protein provided in the examples of the present application;

[0058] Fig.16This is a diagram of the SDS-PAGE detection results of the purified 026136-pET22b-ESPp-p recombinant protein provided in the examples of the present application. DETAILED DESCRIPTION

[0059] In order to make the purpose, technical solution and advantages of the embodiments of the present application clearer, the technical solution in the embodiments of the present application will be clearly and completely described below in conjunction with the drawings in the embodiments of the present application. Obviously, the described embodiments are part of the embodiments of the present application, not all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of this application.

[0060] Various embodiments of the present application may be presented in the form of a range; it should be understood that the description in the form of a range is only for convenience and brevity and should not be understood as a rigid limitation on the scope of the present application; therefore, it should be considered that the range description has specifically disclosed all possible sub-ranges and single numerical values ​​within the range; for example, it should be considered that the range description from 1 to 6 has specifically disclosed sub-ranges, such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., as well as single numbers within the range, such as 1, 2, 3, 4, 5 and 6, which applies regardless of the range; in addition, whenever a numerical range is indicated in this document, it is meant to include any cited numbers (fractions or integers) within the indicated range.

[0061] In this document, the terms including "comprising" and the like mean "including but not limited to". "At least one" means one or more, "plurality" means two or more; "at least one", "at least one of the following" or similar expressions refer to any combination of these items, including any combination of single items or plural items; for example, "at least one of a, b, or c", or "at least one of a, b, and c" can all represent: a, b, c, ab (i.e. a and b), ac, bc, or abc, where a, b, c can be single or multiple, respectively. Unless otherwise specified, the various raw materials, reagents, instruments and equipment used in this application can be purchased on the market or prepared by existing methods.

[0062] It should be noted that, regarding the autotransporter secretion system described in the background art, the inventors have found that, although the "passenger" protein can be replaced with the desired protein through the secretion principle of the autotransporter secretion system to achieve the secretory expression of exogenous protein, this type of method is mainly aimed at optimizing the secretion of individual proteins. Not only is its applicability narrow, but most of the expressed proteins contain structural residues of the autotransporter protein.

[0063] Figure 1 An atlas of an autotransporter secretion system provided in an embodiment of the present application is exemplarily shown;

[0064] like Figure 1 As shown, the present application embodiment provides an autotransporter secretion system, and the autotransporter secretion system comprises:

[0065] An expression vector and multiple groups of functional units on the expression vector; the multiple groups of functional units include a transporter signal peptide, a transporter β-barrel domain, a transporter α hinge region protease hydrolysis mutation site and a thrombin cleavage site;

[0066] Wherein, the transporter β-barrel domain has a nucleotide sequence as shown in SEQ ID NO.1, the transporter α hinge region protease hydrolysis mutation site has a nucleotide sequence as shown in SEQ ID NO.2, and the thrombin cleavage site has a nucleotide sequence as shown in SEQ ID NO.3;

[0067] The transporter signal peptide comprises at least one of the following:

[0068] ESPp signal peptide, pet signal peptide, PIC signal peptide and Hbp signal peptide.

[0069] It should be noted that multiple groups of functional units can use Escherichia coli serine protease as a skeleton, which facilitates the transport protein signal peptide to transfer the exogenous protein translated from the target gene from the cell membrane to the periplasmic space of the cytoplasmic matrix to ensure the normal physiological activities of other subsequent functional units; in addition, the skeleton can avoid the shearing of the exogenous protein by the transport protease through the hydrolysis site when the exogenous protein is transported to the outside of the cell membrane for display through the interaction between the transport protein β-barrel domain and the transport protein α hinge region protease mutation site. In addition, the use of thrombin cleavage sites can promote the enzymatic cleavage of the exogenous protein in the designated area outside the cell membrane, which can promote the secretory expression of the exogenous protein. In addition, it can also reduce the contamination of endogenous proteins in Escherichia coli, and reduce the residual transport protein in the exogenous protein, which can improve the purity of the secreted exogenous protein.

[0070] The transporter protein signal peptide may be an ESPp signal peptide, a pet signal peptide, a PIC signal peptide, or an Hbp signal peptide.

[0071] In some optional embodiments, when the transporter signal peptide includes an ESPp signal peptide, the ESPp signal peptide has a nucleotide sequence as shown in SEQ ID NO.4;

[0072] In these embodiments, when the transporter signal peptide includes the ESPp signal peptide, the exogenous protein translated from the target gene can be transferred from the intracellular membrane to the periplasmic space of the cytoplasmic matrix through the ESPp signal peptide having a specific nucleotide sequence to ensure the normal physiological activities of other subsequent functional units.

[0073] In some optional embodiments, the functional unit has a nucleotide sequence as shown in SEQ ID NO.5;

[0074] In these embodiments, the functional unit may have a nucleotide sequence as shown in SEQ ID NO.5, and the transporter signal peptide, transporter β-barrel domain, transporter α hinge region protease mutation site and thrombin cleavage site in the functional unit may be effectively expressed in the process of expressing the transporter secretion system, thereby effectively promoting the secretory expression of exogenous proteins and improving the purity of the secreted exogenous proteins through the functional unit.

[0075] In some optional embodiments, the ESPp signal peptide has an amino acid sequence as shown in SEQ ID NO.6, the transporter β-barrel domain has an amino acid sequence as shown in SEQ ID NO.7, the transporter α hinge region protease hydrolysis mutation site has an amino acid sequence as shown in SEQ ID NO.8, and the thrombin cleavage site has an amino acid sequence as shown in SEQ ID NO.9;

[0076] In these embodiments, when the transporter signal peptide includes the ESPp signal peptide, the ESPp signal peptide may have an amino acid sequence as shown in SEQ ID NO.6, so that the transporter signal peptide can transfer the exogenous protein translated from the target gene from the cell inner membrane to the periplasmic space of the cytoplasmic matrix to ensure the normal physiological activities of other functional units in the future; in addition, the transporter β-barrel domain may have an amino acid sequence as shown in SEQ ID NO.7 and the transporter α hinge region protease hydrolysis mutation site may have an amino acid sequence as shown in SEQ ID NO.8, which can avoid the shearing effect of the transport protease on the exogenous protein through the hydrolysis site when the exogenous protein is transported to the outside of the cell membrane for display. In addition, the use of the thrombin cleavage site can promote the enzymatic cleavage of the exogenous protein in the designated area outside the cell membrane, which can promote the secretory expression of the exogenous protein. In addition, it can also reduce endogenous protein contamination, reduce the residual transporter protein in the exogenous protein, and improve the purity of the secreted exogenous protein.

[0077] In some optional embodiments, the functional unit has an amino acid sequence as shown in SEQ ID NO.10;

[0078] In these embodiments, the functional unit may have an amino acid sequence as shown in SEQ ID NO.10, and the transporter signal peptide, transporter β-barrel domain, transporter α hinge region protease hydrolysis mutation site and thrombin cleavage site in the functional unit may be effectively expressed in the process of expressing the transporter secretion system, thereby effectively promoting the secretory expression of exogenous proteins in Escherichia coli through the functional unit, and in addition, reducing the contamination of endogenous proteins in Escherichia coli, and at the same time reducing the residual transporter in the exogenous protein, thereby improving the purity of the secreted exogenous protein.

[0079] In some optional embodiments, when the transporter signal peptide includes a pet signal peptide, the pet signal peptide has an amino acid sequence as shown in SEQ ID NO.11;

[0080] In the case where the transporter signal peptide includes a PIC signal peptide, the PIC signal peptide has an amino acid sequence as shown in SEQ ID NO.12;

[0081] In the case where the transporter signal peptide includes an Hbp signal peptide, the Hbp signal peptide has an amino acid sequence as shown in SEQ ID NO.13;

[0082] In these embodiments, when the transporter signal peptide includes a pet signal peptide, the pet signal peptide may have an amino acid sequence as shown in SEQ ID NO.11, and the expression yield of some types of exogenous proteins may be increased by the pet signal peptide; further, when the transporter signal peptide includes a PIC signal peptide, the PIC signal peptide may have an amino acid sequence as shown in SEQID NO.12, and the expression yield of some types of exogenous proteins may be increased by the PIC signal peptide; further, when the transporter signal peptide includes an Hbp signal peptide, the Hbp signal peptide may have an amino acid sequence as shown in SEQ ID NO.13, and the expression yield of some types of exogenous proteins may be increased by the Hbp signal peptide.

[0083] Figure 2 A schematic flow chart of a method for preparing an autotransporter secretion system provided in an embodiment of the present application is shown exemplarily;

[0084] Based on a general inventive concept, such as Figure 2 As shown, the present application embodiment provides a method for preparing the autotransporter secretion system, the method comprising:

[0085] S1. Use the functional unit to replace the signal peptide region of the original expression vector to obtain a recombinant expression vector.

[0086] The method is a method for preparing the above-mentioned autotransporter secretion system. The specific composition of the autotransporter secretion system can refer to the above-mentioned embodiment. Since the method adopts part or all of the technical solutions of the above-mentioned embodiment, it at least has all the beneficial effects brought by the technical solutions of the above-mentioned embodiment, which will not be described one by one here.

[0087] It should be noted that the replacement method may be enzyme cutting or the like.

[0088] Based on a general inventive concept, an embodiment of the present application provides an exogenous protein, wherein the exogenous protein includes the autotransporter secretion system and a target gene carried on the autotransporter secretion system; wherein the target gene includes at least one of the following:

[0089] Mouse Cd3e gene, mouse Prl gene, human WNT3A gene, human WNT5A gene, and rat Zg16 gene;

[0090] When the target gene is a mouse Cd3e gene, the mouse Cd3e gene has a nucleotide sequence shown in SEQ ID NO.14;

[0091] When the target gene is a mouse Prl gene, the mouse Prl gene has a nucleotide sequence shown in SEQ ID NO.15;

[0092] In the case where the target gene is a human WNT3A gene, the human WNT3A gene has a nucleotide sequence shown in SEQ ID NO.16;

[0093] In the case where the target gene is a human WNT5A gene, the human WNT5A gene has a nucleotide sequence shown in SEQ ID NO.17;

[0094] When the target gene is rat Zg16 gene, the rat Zg16 gene has a nucleotide sequence shown in SEQ ID NO.18.

[0095] The exogenous protein is realized based on the above-mentioned autotransporter secretion system. The specific composition of the autotransporter secretion system can be referred to the above-mentioned embodiment. Since the exogenous protein adopts part or all of the technical solutions of the above-mentioned embodiments, it at least has all the beneficial effects brought by the technical solutions of the above-mentioned embodiments, which will not be described one by one here.

[0096] It should be noted that the target gene may include at least one of the mouse Cd3e gene, mouse Prl gene, human WNT3A gene, human WNT5A gene and rat Zg16 gene. The optimized nucleotide sequences of these genes can cover most biological species, indicating that the autotransporter secretion system has good universality.

[0097] Figure 3 A schematic flow chart of a method for preparing exogenous protein provided in an embodiment of the present application is shown exemplarily;

[0098] Based on a general inventive concept, such as Figure 3 As shown, the present application embodiment provides a method for preparing the exogenous protein, the method comprising:

[0099] S1. double-digesting the autotransporter secretion system to obtain a digestion product;

[0100] S2. performing double enzyme digestion on the target gene to obtain gene fragments;

[0101] S3. using a preset amplification primer set to perform PCR amplification on the gene fragment to obtain an amplified product;

[0102] S4. DNA ligating the digested product with the amplified product to obtain a recombinant expression vector;

[0103] S5. transfecting the recombinant expression vector using a host bacterium to obtain a recombinant cell;

[0104] S6. Cultivating the recombinant cells, and then performing enzyme digestion and purification on a large number of recombinant cells obtained by culture to obtain exogenous proteins.

[0105] This method is a method for preparing the above-mentioned exogenous protein. The specific composition of the exogenous protein can refer to the above-mentioned embodiment. Since this method adopts part or all of the technical solutions of the above-mentioned embodiment, it at least has all the beneficial effects brought by the technical solutions of the above-mentioned embodiment, which will not be described one by one here.

[0106] It should be noted that the restriction sites of the double enzyme digestion are determined according to the functional units in the designed autotransporter secretion system, and the endonucleases used for the double enzyme digestion can be CpoI enzyme and NotI enzyme.

[0107] In some optional embodiments, the preset amplification primer set includes a first primer set, a second primer set, a third primer set, a fourth primer set and a fifth primer set, the preset amplification primer set includes a first primer set, a second primer set, a third primer set, a fourth primer set and a fifth primer set, the upstream primer of the first primer set has a nucleotide sequence as shown in SEQ ID NO.19, and the downstream primer of the first primer set has a nucleotide sequence as shown in SEQ ID NO.20;

[0108] The upstream primer of the second primer set has a nucleotide sequence as shown in SEQ ID NO.21, and the downstream primer of the second primer set has a nucleotide sequence as shown in SEQ ID NO.22;

[0109] The upstream primer of the third primer set has a nucleotide sequence as shown in SEQ ID NO.23, and the downstream primer of the third primer set has a nucleotide sequence as shown in SEQ ID NO.24;

[0110] The upstream primer of the fourth primer set has a nucleotide sequence as shown in SEQ ID NO.25, and the downstream primer of the fourth primer set has a nucleotide sequence as shown in SEQ ID NO.26;

[0111] The upstream primer of the fifth primer set has a nucleotide sequence as shown in SEQ ID NO.27, and the downstream primer of the fifth primer set has a nucleotide sequence as shown in SEQ ID NO.28.

[0112] In these embodiments, by using a preset amplification primer set including a first primer set, a second primer set, a third primer set, a fourth primer set and a fifth primer set, the gene fragment can be amplified into a sufficient number of amplification products, and the sufficient number of amplification products can be effectively connected to the enzyme cleavage products by DNA to obtain a sufficient number of recombinant expression vectors, so as to facilitate the subsequent secretion of sufficient exocrine proteins by the recombinant cells.

[0113] The present application is further described below in conjunction with specific examples. The experimental methods in the following examples that do not specify specific conditions are usually measured according to industry standards; if there are no corresponding industry standards, they are carried out according to common international standards, conventional conditions, or conditions recommended by the manufacturer.

[0114] Example 1

[0115] 1. Experimental materials:

[0116] 1. Experimental instruments:

[0117] UV instrument (WD-9403F, Beijing Liuyi);

[0118] Desktop high-speed centrifuge (H1650-W, Xiangyi);

[0119] Constant temperature water bath (DK-S22, Shanghai Jinghong);

[0120] Constant temperature incubator (GNP-P270, Shanghai Jinghong);

[0121] Clean bench (SW-CJ-1FD, Suzhou Antai);

[0122] PCR amplifier (DL9700, Beijing Donglin Changsheng);

[0123] Constant temperature shaker (TS-100C, Shanghai Tiancheng);

[0124] -80℃ refrigerator (DW-86L628, Qingdao Haier Special Electric Appliance Co., Ltd.);

[0125] Restriction endonucleases CpoI and NotI were purchased from Fermentas.

[0126] 2. Experimental reagents:

[0127] Methanol, agar powder, sodium chloride, imidazole, EDTA, etc. were purchased from Sinopharm Chemical Reagent Co., Ltd.;

[0128] Thrombin (20402ES05, ​​Yisheng Biotechnology).

[0129] 2. Experimental steps:

[0130] 1. Design of recombinant expression vector:

[0131] The pET22b(+) vector is selected, and the original signal peptide region is removed. The sequence shown at the position of the signal peptide 1aa to 55aa of the ESPp transporter protein itself is used, and then the 908aa to 1300aa of the ESPp transporter protein beta-barrel domain is added, and then the protease hydrolysis site of the ESPp transporter α hinge region is mutated to promote the 1023rd amino acid Asn of the protease hydrolysis site of the ESPp transporter α hinge region to mutate to Ala, and obtain the transporter protein α hinge region protease hydrolysis mutation site, and then the thrombin cleavage site and linker are used to replace the 56aa to 907aa of the passenger domain of the ESPp transporter protein to obtain a recombinant expression vector. The amino acid sequence of the specifically changed functional unit region is shown in SEQ ID NO.10:

[0132] MNKIYSLKYSHITGGLIAVSELSGRVSSRATGKKKHKRILALCFLGLLQSSYSFA(SEQ ID NO.6)RTVHGYRN *FGSEFELRRQACGR(SEQ ID NO.8)LVPRGS(SEQ ID NO.9)+GGGGS+YAMRTNLSESDKLEVKKHLSGENNILLVDFLQKPTPEKQLNIELVSAPKDTN ENVFKASKQTIGFSDVTPVITTRETDDKITWSLTGYNTVANKEATRNAAALFSVDYKAFLNEVANLNKRMGDLRDINGEAGAWARIMSGTGSASGGFSDNYTHVQVGVDKKHELDGLDLFTGFTVTHTDSSASADVFSGKTKSVGAGLYASAMFDSGAYIDLIGKYVHHDNE YTATFAGLGTRDYSTHSWYAGEAAGYRYHVTEDAWIEPQAELVYGSVSGKQFAWKDQGMHLSMKDKDYNPLIGRTGVDVGKSFSGKDWKVTARAGLGYQFDLLANGETVLRDASGEKRIKGEKDSRMLMSVGLNAEIRDNVRFGLEFEKSAFGKYNVDNAVNANFRYSF(SEQ ID NO.7).

[0133] The codons of the amino acid sequence after overall optimization are shown in SEQ ID NO.5:

[0134]

[0135]

[0136] The optimized nucleotide sequence shown in SEQ ID NO.5 was synthesized by Wuhan Jinkairui Biotechnology Co., Ltd. and connected to the pET22b(+) vector without the signal peptide. The recombinant vector obtained is as follows: Figure 1 As shown, the recombinant vector was named: pET22b-ESPp.

[0137] 3. Preparation of exogenous protein:

[0138] (1) Acquisition of foreign protein gene fragments

[0139] The exogenous protein gene to be optimized was selected, and its corresponding Uniprot number, project number, tag, expression range and Escherichia coli preferred codon optimization sequence were shown in Table 1. The optimized gene fragment was synthesized by Wuhan Jinkairui Bioengineering Co., Ltd., and the gene synthesized fragment was amplified by PCR.

[0140] Table 1 Sequence information of exogenously expressed proteins

[0141]

[0142]

[0143]

[0144] (2) Obtaining foreign protein expression vector

[0145] Amplification primers were designed according to the optimized nucleic acid sequence. The amplification primers are shown in Table 2.

[0146] Table 2 Amplification primer sets corresponding to exogenous protein genes

[0147]

[0148]

[0149] The pET22b-ESPp vector was double-digested with CpoI and NotI to obtain the digestion product, and then the exogenous protein gene fragment in Table 1 was connected with the digestion product and the corresponding amplification primer set in Table 2 to obtain a recombinant expression vector, as shown in Table 3. The recombinant expression vector was transformed and sequenced, and the bacterial solution with correct sequencing was selected to extract the plasmid.

[0150] Table 3 Various recombinant expression vectors obtained

[0151] Gene name serial number Recombinant expression vector Mouse Cd3e 004931 004931-pET22b-ESPp Mouse Prl 018724 018724-pET22b-ESPp Human WNT3A 026136 026136-pET22b-ESPp Human WNT5A 026138 026138-pET22b-ESPp Rat Zg16 807545 807545-pET22b-ESPp

[0152] (3) Expression of exogenous recombinant proteins:

[0153] Prepare the E. coli expression host Rosetta (DE3) to melt on ice, then add the exogenous recombinant expression vector obtained in the above steps to obtain a mixture; the mixture is first placed on ice for 30 minutes, then heat-shocked at 42°C for 90 seconds, placed on ice for 1 minute, and then 800 μL of preheated LB medium is added, and placed in a 37°C shaker at a speed of 158r for 120 minutes. Then centrifuge at a speed of 6000r for 4 minutes, and then remove 800 μL of the supernatant in the clean bench. After the remaining bacterial solution is mixed, it is coated on the K + The plates were then inverted and incubated overnight in a 37°C incubator.

[0154] Small test: Pick a single colony and add it to the corresponding resistance LB (3mL) test tube. After culturing in a shaker for 3 hours, take 700μL of the suspension and add it to 100μL (C=50%) of seed preservation glycerol, shake it evenly, and freeze it in a refrigerator (-20℃). Then add IPTG to the remaining bacterial solution for induction for 4 hours. Collect the bacterial solution and directly add it to the loading buffer and boil it. SDS-PAGE is used to detect the expression of the exogenous protein. The results are as follows: Figures 4 to 8 shown.

[0155] Large-scale amplification: Add the strains with the expression results of exogenous proteins detected by SDS-PAGE to 300mL of LB medium, supplement with 300μL of antibiotics, grow at 37℃ and 220rpm for about 3h, then cool to 18℃, and finally add IPTG inducer with a final concentration of 500μM. Centrifuge after 4h to collect the bacteria.

[0156] (4) Recombinant protein purification:

[0157] The collected bacteria were washed three times with NTA buffer (20mM Tris-HCl, 150mM NaCl, pH 8.0); resuspended with 50mL TE buffer (10mM Tris-HCl, 1mM EDTA, pH 8.0), stirred at 4°C for 5min, centrifuged, and the bacteria were collected; the bacteria were washed three times with NTA buffer, and then resuspended with 50mL NTA buffer, and 5U / mL bovine thrombin was added, and incubated at 37°C for 30min; the supernatant was purified with a nickel column, and eluted with NTA buffer containing 20mM imidazole, 60mM imidazole, 200mM imidazole, and 500mM imidazole, respectively. The eluted proteins were combined and concentrated using an ultrafiltration tube. The concentrated protein was analyzed for purity and concentration, and the results are as follows: Figures 9 to 13 The output is calculated and the results are shown in Table 4.

[0158] Table 4 Expression of exogenous proteins in each recombinant vector

[0159]

[0160] (5) Expression vector optimization:

[0161] Considering that the yield of secretory proteins is relatively low, the signal peptides of other molecules in the transporter family were selected to replace the signal peptide of ESPp on the 026136-pET22b-ESPp vector to compare the yield of secretory proteins. The selected signal peptides are as follows:

[0162] pet: MNKIYSIKYSAATGGLIAVSELAKKVICKTNRKISAALLSLAVISYTNIIYA(SEQ IDNO.11);

[0163] PIC:

[0164] MNKVYSLKYCPVTGGLIAVSELARRVIKKTCRRLTHILLAGIPAICLCYSQISQA(SEQ IDNO.12)

[0165] Hbp: MNRIYSLRYSAVARGFIAVSEFARKCVHKSVRRLCFPVLLLIPVLFSAGSLA(SEQ IDNO.13)

[0166] The replaced vectors were named as 026136-pET22b-ESPp-t, 026136-pET22b-ESPp-C, and 026136-pET22b-ESPp-p. The optimized vectors were amplified and purified for comparison. The results are as follows: Figures 14 to 16 And as shown in Table 5.

[0167] Table 5 Expression of optimized vectors of 026136-pET22b-ESPp-t, 026136-pET22b-ESPp-C and 026136-pET22b-ESPp-p

[0168] Recombinant expression vector purity,% Concentration, mg / mL Volume, mL Yield, mg / L 026136-pET22b-ESPp-t 94.6 2.31 2 15.4 026136-pET22b-ESPp-C 82.3 0.14 2 0.9 026136-pET22b-ESPp-p 98.2 0.83 2 5.5

[0169] In summary, the autotransporter secretion system provided in the embodiments of the present application is based on the autotransporter secretion system protein Escherichia coli serine protease (ESPp) as the skeleton, and the α-hinge region protease hydrolysis site of ESPp is mutated to obtain the α-hinge region protease hydrolysis mutation site. The α-hinge region protease hydrolysis mutation site and the β-barrel domain of the transporter protein are used to prevent the transporter protease from shearing the exogenous protein through the hydrolysis site when the exogenous protein is transported to the outside of the cell membrane for display, so that the exogenous protein can be displayed by using the carrier domain of ESPp, and then the introduction of the thrombin cleavage site can enable the exogenous protein to be artificially intervened by enzyme cleavage at the designated site, which can promote the secretory expression of the exogenous protein, and can also reduce the contamination of endogenous proteins in Escherichia coli, and reduce the residual transporter protein in the exogenous protein, which can improve the purity of the secreted exogenous protein.

[0170] In addition, an autotransporter secretion system provided in an embodiment of the present application can selectively select ESPp signal peptide, pet signal peptide, PIC signal peptide and Hbp signal peptide based on the transporter signal peptide site. These signal peptides can, on the one hand, improve the expression process of exogenous proteins to increase the yield of exogenous proteins, and on the other hand, can secrete exogenous proteins into the extracellular matrix of the host bacteria under the premise of adding thrombin to further promote the secretory expression of exogenous proteins.

[0171] In addition, the autotransporter secretion system provided in the embodiments of the present application can reduce the content of endogenous proteins through its purification method. Therefore, the autotransporter secretion system not only improves the yield and purity of exogenous secretory proteins, but also improves the purification efficiency of exogenous proteins.

[0172] The above is only a specific implementation of the present application, so that those skilled in the art can understand or implement the present application. It will be apparent to those skilled in the art that various modifications to these embodiments are possible, and the general principles defined in the present application can be implemented in other embodiments without departing from the spirit or scope of the present application. Therefore, the present application will not be limited to these embodiments shown in the present application, but will conform to the widest scope consistent with the principles and novel features applied for by the present application.

Claims

1. An autotransporter secretion system, characterized in that The autotransporter secretion system comprises: An expression vector and multiple groups of functional units on the expression vector; the multiple groups of functional units include a transporter signal peptide, a transporter β-barrel domain, a transporter α hinge region protease hydrolysis mutation site and a thrombin cleavage site; Wherein, the transporter β-barrel domain has a nucleotide sequence as shown in SEQ ID NO.1, the transporter α hinge region protease hydrolysis mutation site has a nucleotide sequence as shown in SEQ ID NO.2, and the thrombin cleavage site has a nucleotide sequence as shown in SEQ ID NO.3; The transporter signal peptide comprises at least one of the following: ESPp signal peptide, pet signal peptide, PIC signal peptide and Hbp signal peptide.

2. The autotransporter secretion system according to claim 1, characterized in that When the transporter signal peptide includes an ESPp signal peptide, the ESPp signal peptide has a nucleotide sequence as shown in SEQ ID NO.

4.

3. The autotransporter secretion system according to claim 2, characterized in that The functional unit has a nucleotide sequence as shown in SEQ ID NO.

5.

4. The autotransporter secretion system according to claim 2, characterized in that The ESPp signal peptide has an amino acid sequence as shown in SEQ ID NO.6, the transporter protein β-barrel domain has an amino acid sequence as shown in SEQ ID NO.7, the transporter protein α hinge region protease hydrolysis mutation site has an amino acid sequence as shown in SEQ ID NO.8, and the thrombin cleavage site has an amino acid sequence as shown in SEQ ID NO.

9.

5. The autotransporter secretion system according to claim 4, characterized in that The functional unit has an amino acid sequence as shown in SEQ ID NO.

10.

6. The autotransporter secretion system according to claim 1, characterized in that In the case where the transporter signal peptide includes a pet signal peptide, the pet signal peptide has an amino acid sequence as shown in SEQ ID NO.11; In the case where the transporter signal peptide includes a PIC signal peptide, the PIC signal peptide has an amino acid sequence as shown in SEQ ID NO.12; When the transporter signal peptide includes an Hbp signal peptide, the Hbp signal peptide has an amino acid sequence as shown in SEQ ID NO.

13.

7. A method for preparing the autotransporter secretion system according to any one of claims 1 to 6, characterized in that: The method comprises: The signal peptide region of the original expression vector is replaced with the functional unit to obtain a recombinant expression vector.

8. An exogenous protein, characterized in that The exogenous protein comprises the autotransporter secretion system according to any one of claims 1 to 5 and a target gene carried on the autotransporter secretion system; wherein the target gene comprises at least one of the following: Mouse Cd3e gene, mouse Prl gene, human WNT3A gene, human WNT5A gene, and rat Zg16 gene; When the target gene is a mouse Cd3e gene, the mouse Cd3e gene has a nucleotide sequence shown in SEQ ID NO.14; When the target gene is a mouse Prl gene, the mouse Prl gene has a nucleotide sequence shown in SEQ ID NO.15; In the case where the target gene is a human WNT3A gene, the human WNT3A gene has a nucleotide sequence shown in SEQ ID NO.16; In the case where the target gene is a human WNT5A gene, the human WNT5A gene has a nucleotide sequence shown in SEQ ID NO.17; When the target gene is rat Zg16 gene, the rat Zg16 gene has a nucleotide sequence shown in SEQ ID NO.

18.

9. A method for preparing the exogenous protein according to claim 8, characterized in that: The method comprises: The autotransporter secretion system is double-digested to obtain a digestion product; The target gene is subjected to the double enzyme digestion to obtain a gene fragment; Using a preset amplification primer set to perform PCR amplification on the gene fragment to obtain an amplified product; Performing DNA ligation on the enzyme-cut product and the amplified product to obtain a recombinant expression vector; Using host bacteria to transfect the recombinant expression vector to obtain recombinant cells; The recombinant cells are cultured, and then a large number of recombinant cells obtained by culture are subjected to enzyme digestion and purification to obtain foreign proteins.

10. The method according to claim 9, characterized in that The preset amplification primer set includes a first primer set, a second primer set, a third primer set, a fourth primer set and a fifth primer set, the upstream primer of the first primer set has a nucleotide sequence as shown in SEQ ID NO.19, and the downstream primer of the first primer set has a nucleotide sequence as shown in SEQ ID NO.20; The upstream primer of the second primer set has a nucleotide sequence as shown in SEQ ID NO.21, and the downstream primer of the second primer set has a nucleotide sequence as shown in SEQ ID NO.22; The upstream primer of the third primer set has a nucleotide sequence as shown in SEQ ID NO.23, and the downstream primer of the third primer set has a nucleotide sequence as shown in SEQ ID NO.24; The upstream primer of the fourth primer set has a nucleotide sequence as shown in SEQ ID NO.25, and the downstream primer of the fourth primer set has a nucleotide sequence as shown in SEQ ID NO.26; The upstream primer of the fifth primer set has a nucleotide sequence as shown in SEQ ID NO.27, and the downstream primer of the fifth primer set has a nucleotide sequence as shown in SEQ ID NO.28.

Images