A method for conformational signal peptide-mediated secretion expression of heterologous proteins in escherichia coli

Abstract

The application discloses a method for conformational signal peptide-mediated secretion and expression of heterologous proteins in Escherichia coli, and specifically comprises the following steps: simultaneously fusing at least two heterologous proteins to a conformational signal peptide (such as sfGFP) and constructing a heterologous recombinant protein expression vector, wherein the conformational signal peptide has a beta-barrel structure, and the fusion sites are at least two of the amino terminal end, the carboxyl terminal end and a plurality of loop rings of the conformational signal peptide; and then transforming the heterologous recombinant protein expression vector into Escherichia coli and culturing the obtained genetic engineering strain for expression. According to the method, the extracellular secretion and expression of the heterologous proteins in the Escherichia coli can be realized without the mediation of a classical signal peptide, the sfGFP as a fusion tag has no influence on the function of the target protein, and the corresponding target protein can be quickly expressed and purified; and the method expands the secretion and expression path of the protein, can realize the simultaneous expression of a plurality of heterologous proteins, and can effectively improve the expression amount of the target protein.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of bioengineering technology, and particularly relates to a conformational signal peptide with a beta (β) barrel structure, and particularly to a method for mediating extracellular secretion expression of heterologous proteins in Escherichia coli by super-folded green fluorescent protein (sfGFP). BACKGROUND

[0002] Secretory expression of heterologous proteins in Escherichia coli can be divided into two ways: periplasmic secretion expression and extracellular secretion expression. Periplasmic secretion expression refers to the process of transporting recombinant proteins from cytoplasm to periplasmic space through a signal peptide. Periplasmic space is the oxidative gap between the inner and outer membranes of Escherichia coli, which is similar to the environment of endoplasmic reticulum in eukaryotic cells, which helps the correct folding and disulfide bond formation of nascent peptide chains, and thus obtains recombinant proteins with biological activity. Commonly used signal peptides include PhoA, OmpA, OmpT, LamB, 6-lactamase, enterotoxin ST-Ⅱ, LT-A, LT-B, Staphylococcus aureus protein A, and human growth hormone signal peptide, etc. These signal peptides can effectively transport the expressed proteins from cytoplasm to periplasm. Extracellular secretion expression refers to the process of secreting recombinant proteins to the outside of the cell through a specific secretion pathway. Gram-negative bacteria have seven major secretion systems, types I to VII, among which types II and V are most widely used. Extracellular secretion of recombinant proteins has significant advantages: since there are fewer proteins secreted by Escherichia coli itself, the purification process of recombinant proteins is more convenient. The common feature of these secretion systems is that the target protein is first transported from the cytoplasm to the periplasmic space through a signal peptide, and then folded and assembled into a functional form in the periplasmic space. However, due to the limited periplasmic space, disulfide bond mispairing may occur, resulting in a decrease in the content of correctly folded proteins. In addition, the blockage of the outer membrane also makes the yield of proteins secreted to the extracellular lower.

[0003] The beta-barrel structure is a common protein structure, usually formed by multiple anti-parallel beta-strands connected by hydrogen bonds, forming a hollow cylindrical or barrel-shaped structure. The beta-barrel structure has both structural stability and functional diversity. The beta-barrel structure can adapt to a variety of functions, from molecular channels, transport proteins to enzymes and fluorescent proteins, and has a wide range of application potential. The beta-barrel structure is particularly common in channel proteins in biological membranes and some fluorescent proteins. Among them, green fluorescent protein (GFP) is a protein that emits strong green fluorescence under ultraviolet light, which was first isolated and purified from Aequorea victoria by Shim et al. in 1962. Super-folded green fluorescent protein (sfGFP) is obtained by six rounds of mutations based on GFP. The mutation sites include S30R, Y39N, N105T, Y145F, I171V and A206V. The sfGFP significantly improves the folding rate and stability, and the folding rate is 3.5 times that of ordinary GFP. After the sfGFP is fused with many proteins, the fusion protein shows better fluorescence intensity, folding rate and solubility. Therefore, sfGFP has potential application value in constructing an efficient secretory expression system.

[0004] The beta-barrel structure in sfGFP is composed of 11 anti-parallel beta-strands, forming a solid barrel-shaped framework; the center of the barrel contains an alpha-helix, which wraps around the chromophore responsible for absorbing and emitting fluorescence. The inventors' previous studies have shown that by fusing certain heterologous protein genes to the carboxy terminus of super-folded green fluorescent protein and constructing a heterologous protein recombinant expression vector, the heterologous protein can be expressed extracellularly in Escherichia coli after the recombinant expression vector is transformed into Escherichia coli, without the need for signal peptide mediation. However, with the development of technology, higher requirements are put forward for the secretion expression of heterologous proteins, such as higher expression amount, fusion expression of multiple heterologous proteins, etc. SUMMARY

[0005] Therefore, the present application utilizes the beta-barrel structure of sfGFP to improve the strategy of sfGFP-mediated secretion expression of heterologous proteins in Escherichia coli, to effectively solve the deficiencies in current technology, and to further promote the development of large-scale production. The technical solutions of the present application are as follows:

[0006] The first aspect of the present application provides a method for conformational signal peptide-mediated secretion expression of heterologous proteins in Escherichia coli, comprising the following steps:

[0007] S1, simultaneously fusing at least two heterologous proteins to a conformational signal peptide having a beta-barrel structure, the fusion sites being at least two of the amino terminus, the carboxyl terminus, and the plurality of Loop loops of the conformational signal peptide, and constructing a heterologous recombinant protein expression vector;

[0008] S2, transforming the heterologous recombinant protein expression vector into E. coli, culturing the obtained genetically engineered strain to express, and obtaining the fusion protein.

[0009] In the method of the present application, the heterologous proteins in step S1 can be the same heterologous protein or different heterologous proteins. When they are the same heterologous protein, by integrating them into different positions of the conformational signal peptide, the expression amount of the protein can be effectively improved. When they are different heterologous proteins, by the method of the present application, a plurality of proteases in one reaction can be integrated into the same sfGFP scaffold to form a complex with multiple functions.

[0010] In the method of the present application, the conformational signal peptide is super-folded green fluorescent protein. Through analysis and verification by the inventors, the following three Loop loops can be used as sites for integrating heterologous proteins: Loop 1 with the sequence TTGKL (SEQ ID NO. 1), Loop 2 with the sequence VEDGS (SEQ ID NO. 2), and Loop 3 with the sequence GPVLL (SEQ ID NO. 3).

[0011] In some embodiments of the present application, the heterologous proteins are nanobody Fu2 and nanobody ANTE, both of which can neutralize SARS-CoV-2. After fusing nanobody Fu2 and nanobody ANTE to sfGFP (at the amino terminus and the carboxyl terminus of sfGFP, respectively), the obtained fusion protein can not only be expressed and secreted extracellularly in E. coli, but also has a better affinity with SARS-CoV-2 than nanobody Fu2 or nanobody ANTE alone.

[0012] In other embodiments of the present application, the heterologous protein is sweet protein monellin. The data of the examples show that by fusing monellin to any of Loop 1, Loop 2, and Loop 3 of sfGFP, extracellular secretion expression can be achieved, and sfGFP as a fusion tag does not affect its activity. In addition, by integrating monellin into different positions of sfGFP, such as simultaneously integrating it into Loop 2 and Loop 3, the expression amount of the protein can be effectively improved.

[0013] In the method of the present application, the construction method of the heterologous recombinant protein expression vector can be constructed by using conventional techniques in the art, such as in some embodiments of the present application, the expression vector is constructed by homologous recombination based on the pET23a vector.

[0014] The second aspect of the present application provides a fusion protein prepared according to the method of secreting and expressing the heterologous protein in E. coli mediated by the conformational signal peptide, which at least includes the following:

[0015] The fusion protein Fu2-sfGFP-ANTE has an amino acid sequence as shown in SEQ ID NO. 7;

[0016] The fusion protein sfGFP2-monellin-sfGFP2-3-monellin-sfGFP3 has an amino acid sequence as shown in SEQ ID NO. 12.

[0017] Compared with the prior art, the present application has the following advantages:

[0018] (1) Self-secretion does not require signal peptide mediation. Compared with the conventional E. coli extracellular secretion expression which requires the guidance of a classic signal peptide, the present application uses a conformational signal peptide as a fusion tag, which does not affect its β-barrel structure, and realizes the extracellular secretion expression of heterologous proteins in E. coli by self-secretion.

[0019] (2) Strong self-secretion ability. Compared with the conventional E. coli extracellular secretion method which is difficult to realize the secretion of exogenous proteins rich in cysteine, the present application can realize the secretion expression of nanobodies rich in cysteine, and the fusion protein is secreted into the culture medium and the outer membrane.

[0020] (3) Little effect on the function of part of the target protein. The fusion tag protein often affects the function of the target protein connected thereto, and the target protein with function can be obtained only after subsequent enzymatic cleavage and purification steps, which has the problems of complex operation process and low yield of target protein. In the present application, sfGFP as a fusion tag does not affect the function of the target protein.

[0021] (4) Rapid optimization of expression conditions. Since the fusion of heterologous proteins with sfGFP does not affect the luminescence of sfGFP, by virtue of this property of sfGFP, the fluorescence intensity of the extracellular secretion fusion protein is measured to quickly determine whether the fusion protein is expressed and find the optimal expression conditions.

[0022] (5) Rich secretory expression pathway. By fusing heterologous proteins to the N- or C-terminus of sfGFP and several different Loop loops, the secretory expression of fusion proteins is achieved. Unlike the classic signal peptide, by fusing this specific beta-barrel structure of super-fold green fluorescent protein as a new type of signal peptide, the secretory expression of heterologous proteins can be achieved by fusing heterologous proteins to different positions of sfGFP, realizing the multi-use of a conformational signal peptide, increasing the means of heterologous protein secretory expression, and increasing the candidate for secretory expression of certain heterologous proteins.

[0023] (6) Integrated expression of multiple heterologous proteins. Simultaneous fusion of heterologous proteins to the N- and C-terminus of sfGFP or Loop loop to achieve secretory expression of fusion proteins, which facilitates the integration of multiple proteases in one reaction into the same sfGFP scaffold to form a complex with multiple functions.

[0024] (7) Improve the expression amount of heterologous proteins. By integrating the same heterologous protein into different positions of sfGFP, the expression amount of the protein can be effectively improved, and the economic benefit of protein expression can be improved. BRIEF DESCRIPTION OF DRAWINGS

[0025] Figure 1 The design schematic diagram of the heterologous recombinant protein expression vector used in the embodiments of the application.

[0026] Figure 2 The SDS-PAGE detection result diagram of the fusion proteins sfGFP-Fu2 and Fu2-sfGFP and sfGFP-ANTE in Example 1.

[0027] Figure 3 The SDS-PAGE detection result diagram of the fusion proteins sfGFP-Fu2 and Fu2-sfGFP and sfGFP-ANTE after enzyme digestion in Example 1.

[0028] Figure 4 The comparison diagram of the affinity of fusion proteins sfGFP-Fu2 and Fu2-sfGFP and sfGFP-ANTE with SARS-CoV-2 before and after enzyme digestion by Elisa in Example 1.

[0029] Figure 5 The comparison diagram of the affinity of nanobody Fu2, fusion protein Fu2-sfGFP and SARS-CoV-2 in Example 1.

[0030] Figure 6 The comparison diagram of the affinity of nanobody ANTE, fusion protein sfGFP-ANTE and SARS-CoV-2 in Example 1.

[0031] Figure 7Figure of SDS-PAGE detection result of fusion protein Fu2-sfGFP-ANTE in Example 2.

[0032] Figure 8 Figure of comparison of affinity of nanobody Fu2, ANTE and fusion protein Fu2-sfGFP- ANTE with SARS-CoV-2 by Elisa in Example 2.

[0033] Figure 9 Figure of expression of fusion protein sfGFP-monellin, sfGFP1-monellin-sfGFP1, sfGFP2-monellin-sfGFP2 and sfGFP3-monellin-sfGFP3 and purification of monellin after enzyme digestion by SDS-PAGE in Example 3.

[0034] Figure 10 Figure of SDS-PAGE detection result of fusion protein sfGFP2-monellin-sfGFP2-3-monellin-sfGFP3 in Example 4.

[0035] Figure 11 Results of determination of sweetness value of sweet protein by electronic tongue in the present application. DETAILED DESCRIPTION

[0036] The technical solutions of the present application will be described clearly and completely in combination with examples and drawings. It should be understood that the specific embodiments described herein are only used to illustrate and explain the present application, and are not used to limit the present application.

[0037] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present application belongs. The term "comprising" and any variation thereof in the specification and claims of the present application is intended to cover not exclusively including.

[0038] The following examples relate to the expression vectors of heterologous recombinant proteins pET23a-sfGFP-Fu2, pET23a-Fu2-sfGFP, pET23a-sfGFP-ANTE, pET23a-Fu2-sfGFP-ANTE, pET23a-sfGFP-monellin, pET23a-sfGFP1-monellin-sfGFP1, pET23a-sfGFP2-monellin-sfGFP2, pET23a-sfGFP3-monellin-sfGFP3 and pET23a-sfGFP2-monellin-sfGFP23-monellin-sfGFP3, which are prepared by homologous recombination, and their structures are shown in Figure 1 SEQ ID NO. 13, the nucleotide sequence of Fu2 is shown in SEQ ID NO. 14, the nucleotide sequence of ANTE is shown in SEQ ID NO. 15, and the nucleotide sequence of monellin is shown in SEQ ID NO. 16; in the recombinant plasmid pET23a-sfGFP-Fu2, sfGFP and Fu2 are connected by a sequence shown in SEQ ID NO. 17, and the target gene Fu2 has an HA tag at the tail end; in the recombinant plasmid pET23a-Fu2-sfGFP, Fu2 and sfGFP are connected by a sequence shown in SEQ ID NO. 18; in the recombinant plasmid pET23a-sfGFP-ANTE, sfGFP and ANTE are connected by a sequence shown in SEQ ID NO. 17, and the target gene Fu2 has an HA tag at the tail end; in the recombinant plasmid pET23a-Fu2-sfGFP-ANTE, Fu2 and sfGFP are connected by a sequence shown in SEQ ID NO. 19, and sfGFP and ANTE are connected by a sequence shown in SEQ ID NO. 20, and ANTE has an HA tag at the tail end; in the recombinant plasmid pET23a-sfGFP-monellin, sfGFP and monellin are connected by a sequence shown in SEQ ID NO. 20; in the recombinant plasmids pET23a-sfGFP1-monellin-sfGFP1, pET23a-sfGFP2-monellin-sfGFP2, pET23a-sfGFP3-monellin-sfGFP3 and pET23a-sfGFP2-monellin-sfGFP23-monellin-sfGFP3, monellin is connected to different Loop loops of sfGFP at both ends by sequences shown in SEQ ID NO. 21-22.

[0039] Example 1

[0040] In this example, sfGFP is used as a conformational signal peptide to mediate the extracellular secretion expression of nanobody Fu2 or ANTE in E. coli BL21 (DE3), as follows:

[0041] (1) Nanobody Fu2 is fused to the N- or C-terminus of sfGFP.

[0042] The constructed recombinant plasmid pET23a-sfGFP-Fu2 and pET23a-Fu2-sfGFP are transformed into E. coli competent cells BL21 (DE3) strain, which is incubated at 37°C overnight to obtain the recombinant strain. A single colony is picked and inoculated into 100 mL of LB liquid medium (the concentration of ampicillin is 50 μg / mL), which is incubated at 37°C with shaking. When the OD 600 is about 0.6, 0.5 mM IPTG is added, and the culture is induced at 18°C or 28°C for 18-42 hours with shaking. After the culture is completed, the bacterial cells and the supernatant of the culture medium are collected by centrifugation at 12000 rpm and 4°C. The bacterial cells are washed with TEN buffer (50 mM Tris-HCl, 5 mM EDTA, 50 mM NaCl, pH 8.0) at 4°C for more than 4 hours, and the proteins secreted by the strain are directly obtained from the culture medium or TEN buffer. SDS-PAGE is used to analyze the secretion expression of the fusion protein.

[0043] (2) Nanobody ANTE is fused to the C-terminus of sfGFP.

[0044] The constructed recombinant plasmid pET23a-sfGFP-ANTE is transformed into E. coli competent cells BL21 (DE3) strain, which is incubated at 37°C overnight to obtain the recombinant strain. A single colony is picked and inoculated into 100 mL of LB liquid medium (the concentration of ampicillin is 50 μg / mL), which is incubated at 37°C with shaking. When the OD 600 is about 0.6, 0.5 mM IPTG is added, and the culture is induced at 18°C or 28°C for 18-42 hours with shaking. After the culture is completed, the bacterial cells and the supernatant of the culture medium are collected by centrifugation at 12000 rpm and 4°C. The bacterial cells are washed with TEN buffer (50 mM Tris-HCl, 5 mM EDTA, 50 mM NaCl, pH 8.0) at 4°C for more than 4 hours, and the proteins secreted by the strain are directly obtained from the culture medium or TEN buffer. SDS-PAGE is used to analyze the secretion expression of the fusion protein.

[0045] The expression of fusion protein sfGFP-Fu2 (amino acid sequence as shown in SEQ ID NO. 4) and Fu2-sfGFP (amino acid sequence as shown in SEQ ID NO. 5) is as shown in Figure 2 a, 2b and 2d, the fusion protein sfGFP-Fu2 and Fu2-sfGFP were detected in the culture supernatant and outer membrane, and the protein band (44 kDa in molecular weight) was consistent with the actual situation. With the extension of the induction time, the protein content of the fusion protein secreted into the culture medium gradually increased. Among them, Figure 2 a is the expression of sfGFP-Fu2 in the culture medium, Figure 2 b is the expression of Fu2-sfGFP in the culture medium, Figure 2 d1 is the expression of Fu2-sfGFP outer membrane protein under 18°C and 28°C conditions, Figure 2 d2 is the expression of sfGFP-Fu2 outer membrane protein under 18°C and 28°C conditions.

[0046] The expression of fusion protein sfGFP-ANTE (amino acid sequence as shown in SEQ ID NO. 6) is as shown in Figure 2 c and 2d, the fusion protein sfGFP-ANTE was detected in the culture supernatant and outer membrane, and the protein band (70.9 kDa in molecular weight) was consistent with the actual situation. With the extension of the induction time at 28°C, the protein content of the fusion protein secreted into the culture medium gradually increased. Among them, Figure 2 c is the expression of sfGFP-ANTE in the culture medium, Figure 3 d3 is the expression of sfGFP-ANTE outer membrane protein under 18°C and 28°C conditions.

[0047] For fusion proteins sfGFP-Fu2, Fu2-sfGFP and sfGFP-ANTE, HRV 3C protease was used to remove sfGFP at 4°C overnight, and the target protein was obtained by Ni-NTA purification method, as shown in Figure 3 . Figure 3 1 is the antibody part Fu2 of Fu2-sfGFP after HRV 3C protease digestion, Figure 3 2 is the antibody part Fu2 of sfGFP-Fu2 after HRV 3C protease digestion, Figure 4-6 3 is the antibody part ANTE of sfGFP-ANTE after HRV 3C protease digestion.

[0048] The affinity of the nanobody before and after enzyme digestion was compared by Elisa method. Specifically, SARS-CoV-2 coronavirus spike glycoprotein S1 was diluted to a final concentration of 1 μg / ml or 10 -7 mol / L, coated on a 96-well plate, and incubated overnight at 4°C, with maltose-binding protein (MBP) as a negative control; the sample was washed with PBST, and blocked with 1% BSA in PBST buffer at room temperature for 1 h; the nanobody was serially diluted to different concentration gradients, and incubated at room temperature for 1 h. The sample was washed, incubated with HRP-conjugated Mouse anti HA-Tag mAb and TMB substrate, and the absorbance value was measured at 450 nm using a microplate reader. The results are shown in Figure 1 It was found that the presence or absence of sfGFP had no significant effect on the affinity of the antibody.

[0049] Example 2

[0050] In this example, sfGFP was used as a conformational signal peptide to mediate the extracellular secretion expression of nanobody Fu2 and ANTE in E. coli BL21(DE3). The results of the recombinant plasmid used are shown in Figure 7 , i.e., the nanobody Fu2 and ANTE were fused to the N-terminus and C-terminus of sfGFP, respectively. In this example, the fusion protein Fu2-sfGFP-ANTE (amino acid sequence shown as SEQ ID NO. 7) was prepared by the following steps:

[0051] First, the constructed recombinant plasmid pET23a-Fu2-sfGFP-ANTE was transformed into E. coli competent cells BL21(DE3) strain, and incubated at 37°C overnight to obtain the recombinant strain. Second, a single colony was picked and inoculated into 100 mL of LB liquid medium (the concentration of ampicillin was 50 μg / mL), and incubated at 37°C with shaking. When the OD 600 was about 0.6, 0.5 mM IPTG was added, and the culture was induced at 18°C or 28°C with shaking for 18 to 42 hours. After the culture was completed, the bacterial cells and the culture medium supernatant were collected by centrifugation at 12000 rpm at 4°C. The bacterial cells were washed with TEN buffer (50 mM Tris-HCl, 5 mM EDTA, 50 mM NaCl, pH 8.0) at 4°C for more than 4 hours, and the protein secreted by the strain was directly obtained from the culture medium or TEN buffer. SDS-PAGE was used to detect and analyze the secretion expression of the fusion protein.

[0052] The secretion expression of the fusion protein Fu2-sfGFP-ANTE is shown in Figure 8As shown, the fusion protein Fu2-sfGFP-ANTE was detected in the outer membrane with a protein band consistent with the actual (molecular weight size of 97.9 kDa), and the protein content of the fusion protein secreted into the outer membrane was more when the bacteria were induced at 28°C than when induced at 18°C.

[0053] Using nanobody Fu2 and ANTE as controls, the size of the affinity of the fusion protein Fu2-sfGFP-ANTE for SARS-CoV-2 was detected by Elisa method (see Example 1). The results are shown in Figure 1 As shown, the resulting Fu2-sfGFP-ANTE antibody has better affinity for SARS-CoV2 S1 protein by connecting two different nanobodies to the two ends of sfGFP.

[0054] Example 3

[0055] In this example, sfGFP was used as a conformational signal peptide to mediate the extracellular secretion expression of sweet protein monellin in E. coli BL21 (DE3), and one sfGFP was integrated with heterologous protein monellin at one position. Referring to Figure 9 , this example lists the following four cases: ① heterologous protein monellin fused to the C-terminus of sfGFP, corresponding to the construction of recombinant plasmid pET23a-sfGFP-monellin; ② heterologous protein monellin fused to Loop1 of sfGFP, corresponding to the construction of recombinant plasmid pET23a-sfGFP1-monellin-sfGFP1; ③ heterologous protein monellin fused to Loop2 of sfGFP, corresponding to the construction of recombinant plasmid pET23a-sfGFP2-monellin-sfGFP2; ④ heterologous protein monellin fused to Loop3 of sfGFP, corresponding to the construction of recombinant plasmid pET23a-sfGFP3-monellin-sfGFP3.

[0056] (1) Preparation and detection of fusion protein sfGFP-monellin.

[0057] First, the constructed recombinant plasmid pET23a-sfGFP-monellin was transformed into E. coli competent cells BL21 (DE3) strain, incubated at 37°C overnight, and the recombinant strain was obtained. Second, a single colony was picked and inoculated into 100 mL of LB liquid medium (the concentration of ampicillin was 50 μg / mL), and incubated at 37°C with shaking. When the OD 600When the pH reached approximately 0.6, 0.5 mM IPTG was added to a final concentration, and the mixture was incubated at 18°C ​​with shaking for 18 hours to induce fusion. After incubation, the bacterial cells and culture supernatant were collected separately by centrifugation at 12000 rpm and 4°C. The bacterial cells were washed with TEN buffer (50 mM Tris-HCl, 5 mM EDTA, 50 mM NaCl, pH 8.0) at 4°C for more than 4 hours, and the secreted protein was directly obtained from the TEN buffer. SDS-PAGE was used to analyze the secretion and expression of the fusion protein.

[0058] like Figure 9 As shown in Figure 1, the fusion protein sfGFP-monellin (amino acid sequence as shown in SEQ ID NO.8) was detected in the outer membrane as a protein band (molecular weight of 50 kDa) that matches the actual protein. Figure 9 In a, 2 represents the removal of monellin from sfGFP by SUMO protease overnight at 4°C.

[0059] (2) Preparation and detection of fusion proteins sfGFP-monellin-sfGFP (Loop1, 2, 3).

[0060] First, the constructed recombinant plasmids pET23a-sfGFP1-monellin-sfGFP1, pET23a-sfGFP2-monellin-sfGFP2, and pET23a-sfGFP3-monellin-sfGFP3 were transformed into *E. coli* competent cells BL21(DE3) and cultured overnight at 37°C to obtain three recombinant strains. Next, single colonies were picked from each strain and inoculated into 100 mL of LB liquid medium (ampicillin concentration 50 μg / mL), and cultured with shaking at 37°C. OD 600 When the pH reached approximately 0.6, 0.5 mM IPTG was added to a final concentration, and the mixture was incubated at 18°C ​​with shaking for 18 hours to induce fusion. After incubation, the bacterial cells and culture supernatant were collected separately by centrifugation at 12000 rpm and 4°C. The bacterial cells were washed with TEN buffer (50 mM Tris-HCl, 5 mM EDTA, 50 mM NaCl, pH 8.0) at 4°C for more than 4 hours, and the secreted protein was directly obtained from the TEN buffer. SDS-PAGE was used to analyze the secretion and expression of the fusion protein.

[0061] like Figure 9b, 9c, 9d, fusion protein sfGFP1-monellin-sfGFP1 (amino acid sequence as shown in SEQ ID NO. 9), sfGFP2-monellin-sfGFP2 (amino acid sequence as shown in SEQ ID NO. 10), sfGFP3-monellin-sfGFP3 (amino acid sequence as shown in SEQ ID NO. 11) are all detected in the outer membrane protein band (41 kDa in size) consistent with the actual. Among them, Figure 9 b is sfGFP1-monellin-sfGFP1, i.e. monellin on Loop1 ring, Figure 9 c is sfGFP2-monellin-sfGFP2, i.e. monellin on Loop2 ring, Figure 9 d is sfGFP3-monellin-sfGFP3, i.e. monellin on Loop3 ring.

[0062] For fusion proteins sfGFP-monellin, sfGFP1-monellin-sfGFP1, sfGFP2-monellin-sfGFP2, SUMO protease or HRV 3C protease is used to remove sfGFP at 4°C overnight, and the target protein is obtained by Ni-NTA purification method, such as Figure 1 a, 9b, 9c, lane 2 (i.e. lane 1 is the outer membrane protein before enzyme digestion, and lane 2 is the monellin part after enzyme digestion).

[0063] Example 4

[0064] In this example, sfGFP is used as a conformational signal peptide to mediate the extracellular secretion of sweet protein monellin in E. coli BL21 (DE3). However, unlike Example 3, the same sfGFP is used as a scaffold to integrate monellin into multiple positions of sfGFP. Specifically, the heterologous protein monellin is fused to Loop2 and Loop3 of sfGFP, and the recombinant plasmid pET23a-sfGFP2-monellin-sfGFP23-monellin-sfGFP3 (see Figure 10 ) is constructed. The fusion protein sfGFP2-monellin-sfGFP2-3-monellin-sfGFP3 is prepared by the following steps:

[0065] First, the constructed recombinant plasmid pET23a-sfGFP2-monellin-sfGFP23-monellin-sfGFP3 (amino acid sequence as shown in SEQ ID NO. 12) was transformed into E. coli competent cells BL21 (DE3) strain, and incubated at 37°C overnight to obtain the recombinant strain. Second, a single colony was picked and inoculated into 100 mL of LB liquid medium (the concentration of ampicillin was 50 μg / mL), and incubated at 37°C with shaking. When the OD 600 was about 0.6, 0.5 mM IPTG was added, and the culture was induced at 18°C for 18 hours with shaking. After the culture was completed, the bacterial cells and the supernatant were collected by centrifugation at 12000 rpm at 4°C. The bacterial cells were washed with TEN buffer (50 mM Tris-HCl, 5 mM EDTA, 50 mM NaCl, pH 8.0) at 4°C for more than 4 hours, and the protein secreted by the strain was directly obtained from the TEN buffer. SDS-PAGE was used to detect and analyze the secreted expression of the fusion protein.

[0066] As shown in Figure 11 , the fusion protein sfGFP2-monellin-sfGFP2-3-monellin-sfGFP3 was detected in the outer membrane, and the actual protein band (molecular weight size of 54.7 kDa) was consistent. Compared with Example 3, the integration of monellin into different positions of sfGFP can effectively improve the expression amount of the protein.

[0067] The activity of monellin in Examples 3-4 was determined by human tongue tasting and electronic tongue detection. Specifically, the sweet protein was gradiently diluted and dispensed into disposable paper cups. The panel members slowly put the sample into their mouths, and after 10 s or so in the mouth, they spit it out. The concentration was repeatedly adjusted until the lowest concentration at which the sweetness could be tasted was found. The experiment was repeated for three days, and the average value was taken as the final result. Sucrose gradient solutions were measured by electronic tongue, and the concentrations were 0, 0.6%, 2.4%, 5%, and 10%, respectively. According to the experimental results, a sucrose concentration standard curve was drawn. The sweetness value of the protein sample was calculated by the sucrose concentration standard curve (see ​ , the correlation coefficient was 0.9856). The detection results showed that the sweetness threshold of the secreted monellin protein reached 5 mg / L, which was 5000 times sweeter than sucrose of the same mass.

[0068] In summary, sfGFP as a conformational signal peptide mediates the extracellular secretion of heterologous proteins in E. coli BL21(DE3) in three steps: first, the fusion protein is synthesized in the cytoplasm and translocates to the periplasm; second, the fusion protein in the periplasm is localized to the outer membrane; and finally, the fusion protein is released from the outer membrane into the culture medium. Moreover, the present application shows that sfGFP as a conformational signal peptide with a β-barrel structure can successfully secrete heterologous proteins into the outer membrane and the culture medium after fusing one or more foreign proteins, without changing its conformation, without any signal peptide, and without the help of any secretion pathway; this provides a new idea for using a conformational signal peptide with a β-barrel structure for efficient expression, or for establishing a complex of integrated various proteases for biological catalytic synthesis, etc.

[0069] It should be noted that the above examples are only some of the embodiments of the present application but not all the embodiments, and are only used to illustrate the technical solutions of the present application but not to limit; based on the examples in the present application, all other examples obtained by those skilled in the art without creative labor belong to the scope of protection of the present application.

Claims

1. A method for conformational signal peptide mediated secretion expression of heterologous proteins in E. coli, characterized in that, The conformational signal peptide is a super-folded green fluorescent protein, and the method comprises the following steps: S1, simultaneously fusing at least two same or different heterologous proteins to a conformational signal peptide and constructing a heterologous recombinant protein expression vector, the conformational signal peptide has a beta-barrel structure, and the fusion sites are at least two of the amino terminal, the carboxyl terminal and the plurality of Loop loops of the conformational signal peptide; wherein the Loop loop of the super-folded green fluorescent protein is selected from Loop1 with the sequence shown in SEQ ID NO. 1, Loop2 with the sequence shown in SEQ ID NO. 2 and Loop3 with the sequence shown in SEQ ID NO. 3; S2, transforming the heterologous recombinant protein expression vector into E. coli, culturing the obtained genetically engineered strain for expression, and obtaining a fusion protein; The heterologous proteins are nanobody Fu2 and nanobody ANTE, the nanobody Fu2 and ANTE are respectively fused to the amino terminal and the carboxyl terminal of the super-folded green fluorescent protein, and the amino acid sequence of the obtained fusion protein is shown in SEQ ID NO. 7; or, The heterologous protein is monellin, which is fused to the Loop loop of the super-folded green fluorescent protein, and the amino acid sequence of the obtained fusion protein is shown in SEQ ID NO.

12.

2. The method of claim 1, wherein, In step S1, the pET23a is used as a basic vector, and the heterologous recombinant protein expression vector is constructed by homologous recombination.

3. A fusion protein Fu2-sfGFP-ANTE, characterized in that, Prepared by the method of claim 1, and the amino acid sequence is shown in SEQ ID NO.

7.

4. A fusion protein sfGFP2-monellin-sfGFP2-3-monellin-sfGFP3, characterized in that, Prepared by the method of claim 1, and the amino acid sequence is shown in SEQ ID NO.

12. Prepared by the method of claim 1, and the amino acid sequence is shown in SEQ ID NO.

7. Prepared by the method of claim 1, and the amino acid sequence is shown in SEQ ID NO. 12.

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