A method for secreting expression of a mussel adhesion protein in bacillus subtilis

Abstract

This invention discloses a method for secreting mussel adhesion proteins in Bacillus subtilis. Mussel adhesion proteins have significant application value, but the yield of naturally derived mussel adhesion proteins is extremely low, and they are difficult to secrete extracellularly during heterologous expression. Therefore, this invention uses the chaperone protein Spy from Escherichia coli to mediate the secretory expression of mussel adhesion proteins, achieving highly efficient secretory expression of mussel adhesion proteins Mfp3 and Mfp5 in the food-grade host Bacillus subtilis.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and discloses a method for secreting and expressing mussel adhesion proteins in Bacillus subtilis. Background Technology

[0002] Mussel foot protein (MIP) can adhere to the surfaces of various materials in humid environments, exhibiting excellent adhesion, water resistance, and biocompatibility, making it a potential adhesive material. However, the yield of MIP extracted directly from natural mussels is extremely low; approximately 1 g of MIP can be extracted from 10,000 purple mussels (Cristian et al. 1991. Comp. Comp. Biochem. Physiol. B. 98:569-572. Doi: 10.1016 / 0305-0491(91)90255-C.), which is insufficient to meet actual production needs. Heterologous protein expression is the only way to achieve large-scale production of MIP.

[0003] Currently, some prokaryotic and eukaryotic hosts have been used to attempt to express mussel adhesion proteins. Researchers have only achieved intracellular expression of mussel adhesion proteins in *E. coli* (Yang et al. 2013. *Biofouling.* 29:483-90. Doi: 10.1080 / 08927014.2013.782541.) and *Pichia pastoris* (Filpula et al. 1990 *Biotechnol Prog.* 6:171-7. Doi: 10.1021 / bp00003a001.). Intracellular expression of mussel adhesion proteins requires complex purification processes such as cell disruption, increasing production costs. In contrast, secretory expression (expressing the target protein extracellularly) has advantages in terms of purification efficiency and production cost. However, the sequence and structural characteristics of mussel adhesion proteins pose significant challenges to their secretory expression. For example, the mussel adhesion protein Mfp1 contains multiple repeating decapeptides; and the mussel adhesion proteins Mfp3 and Mfp5 have high levels of specific amino acids (60 mol% of Mfp3 is composed of glycine, tryptophan, arginine, and asparagine) (Silverman et al. 2007. Mar. Biotechnol. 9:661-81. Doi:10.1007 / s10126-007-9053-x.). This leads to the easy formation of inclusion bodies during the recombinant expression of mussel adhesion proteins. Bacillus subtilis, as a recognized food-grade strain, has advantages such as good safety and strong protein secretion capacity in the production of heterologous proteins (Su et al. 2020. Microb Cell Fact. 19:173. Doi: 10.1186 / s12934-020-01436-8.). To date, there are no reports on the expression of mussel adhesion proteins in Bacillus subtilis. To improve the yield of recombinant mussel adhesion proteins and obtain a safe expression host, it is necessary to achieve secretory expression of mussel adhesion proteins in Bacillus subtilis. Spy protein is an ATP-independent molecular chaperone in Escherichia coli that can prevent protein aggregation, aid protein folding, and enhance protein stability (Quan et al.. 2011. Nat Struct Mol Biol.18: 262–269. Doi: org / 10.1038 / nsmb.2016). However, whether Spy can promote protein secretion has not been reported. This invention fuses Spy with mussel adhesion proteins to achieve secretory expression of mussel adhesion proteins in Bacillus subtilis. Summary of the Invention

[0004] Although mussel adhesive protein has significant application value, the yield of naturally derived mussel adhesive protein is extremely low. During heterologous expression, the mussel adhesive protein is difficult to secrete extracellularly into the host cell. Therefore, this invention fuses the chaperone protein Spy from *Escherichia coli* with the mussel adhesive protein for expression, enabling Spy to assist in the secretion of the mussel adhesive protein, thus achieving secretory expression of the mussel adhesive protein in the food-grade host *Bacillus subtilis*.

[0005] The objective of this invention can be achieved through the following technical solutions:

[0006] A mussel adhesion protein expression cassette includes a Spy encoding gene (spy) and a mussel adhesion protein gene. The Spy encoding gene sequence is shown in SEQ ID NO.5, and the mussel adhesion protein gene is selected from any one of mfp3, mfp5, mfp6, vfp5, or vfp3; preferably mfp3 or mfp5. The mfp3 gene sequence is shown in SEQ ID NO.8, and the mfp5 gene sequence is shown in SEQ ID NO.10.

[0007] As a preferred embodiment of the present invention, the mussel adhesion protein expression cassette is composed of P 43 Promoter (SEQ ID NO.2), P cryIIIa Promoter (SEQ ID NO.3), signal peptide SP AprE The coding region of Spy (SEQ ID NO.4), the coding gene of Spy (SEQ ID NO.5), the coding region of the linker peptide (SEQ ID NO.6), and the TEV protease cleavage site CS TEV It consists of a coding region (SEQ ID NO.7), a mussel adhesion protein gene (SEQ ID NO.8 or 10), and a T0 transcription terminator (SEQ ID NO.9). The P used in this invention... 43 The promoter is derived from Bacillus subtilis 168, P cryIIIa The promoter is derived from Bacillus thuringiensis, and the two together form P 4C The promoter, together, initiates the expression of the fusion protein; via the signal peptide SP AprE Secretory expression of the fusion protein was achieved, and a linker peptide was added between the chaperone protein and the target protein to improve the secretion efficiency of the fusion protein. Finally, a TEV protease cleavage site was added between the chaperone protein Spy and the target protein Mfp to remove the chaperone protein Spy.

[0008] As a preferred embodiment of the present invention, the mussel protein expression cassette sequence is shown in SEQ ID NO.1 or SEQ ID NO.11. The mussel protein expression cassette of the present invention can be synthesized by a gene company.

[0009] A recombinant fragment of mussel adhesion protein, characterized by comprising an upstream homologous arm LF-bleomycin resistance gene Zeo-the mussel adhesion protein expression cassette-a downstream homologous arm RF.

[0010] A genetically engineered Bacillus subtilis bacterium containing the mussel adhesion protein expression cassette or the recombinant fragment of the mussel adhesion protein.

[0011] As a preferred embodiment of the present invention, the Bacillus subtilis genetically engineered strain is constructed by the following method: the recombinant fragment of the mussel adhesion protein is transformed into Bacillus subtilis PD8 competent cells, and the Bacillus subtilis genetically engineered strain is obtained after screening with bleomycin.

[0012] As a further preferred embodiment of the present invention, the total genome of Bacillus subtilis PD8 (Zhao et al.. 2019. Biotechnol Bioeng. 116:2052-2060. Doi: 10.1002 / bit.26992.) was used as a template (GenBank accession number: NC_000964.3). Primers P3 / P4 and P7 / P8 amplified the upstream homologous arm LF and the downstream homologous arm RF, respectively. The pUB110 plasmid (Gryczan et al.. 1980. J Bacteriol. 141:246-253. Doi: Using 10.1128 / jb.141.1.246-253.1980 as a template, primers P5 / P6 amplified the bleomycin (Zeocin) resistance gene. An overlap extension PCR fusion fragment LF, the bleomycin resistance gene Zeo, and the Spy-Mfp expression cassette SM and RF were then used to construct the fragment LF-Zeo-SM-RF. This fragment was transformed into competent cells of Bacillus subtilis PD8 (Anagnostopoulos et al. 1961. JBacteriol. 81:741-746. Doi: 10.1016 / B978‐012373944‐5.00036‐5). After selection with 20 ppm bleomycin, the target protein expression strains PD8SM3 and PD8SM5 were obtained. In both strains, the Spy-mMfp expression cassette was integrated into the host's amyE site. The primers used in this invention are shown in Table 1.

[0013] Table 1 Primers used in this invention

[0014]

[0015] The application of the mussel adhesion protein expression cassette and the recombinant fragment of the mussel adhesion protein described in this invention in the secretion and expression of mussel adhesion protein using Bacillus subtilis.

[0016] A method for expressing mussel adhesion proteins using Bacillus subtilis secretion includes the following steps:

[0017] (1) Construct the genetically engineered Bacillus subtilis strain described above;

[0018] (2) After fermenting the Bacillus subtilis genetically engineered bacteria in TB medium for 32-40 h, the supernatant of the medium is collected by centrifugation, and the mussel adhesion protein is present in the supernatant of the medium.

[0019] As a preferred embodiment of the present invention, the method further includes step (3) purification of the target protein Spy-Mfp: the supernatant of the culture medium is precipitated with saturated ammonium sulfate, incubated at 4°C for 12 h, the precipitate is collected by centrifugation, and the target protein Spy-Mfp is purified by nickel column purification.

[0020] As a preferred embodiment of the present invention, the method further includes step (4) shearing of Mfp: the purified target protein Spy-Mfp is refolded in TEV buffer, the supernatant is collected by centrifugation, and TEV protease is added. After incubation at 4°C for 12 h or at 30°C for 1-2 h, the supernatant is collected by centrifugation to obtain the individual mussel adhesion protein Mfp.

[0021] Beneficial effects:

[0022] This invention utilizes the chaperone protein Spy from *Escherichia coli* to mediate the secretory expression of mussel adhesion proteins, providing a method for the efficient secretory expression of mussel adhesion proteins Mfp3 and Mfp5 in the food-grade host *Bacillus subtilis*. SDS-PAGE analysis showed that the recombinant mussel adhesion proteins were electrophoretically pure (no obvious contaminating protein bands on the SDS-PAGE gel), with a yield of approximately 120 mg / L. Attached Figure Description

[0023] Figure 1 Construction process of engineered bacteria in this invention

[0024] A: Construction of the Spy-Mfp expression cassette; B: Construction of the expression strain

[0025] Figure 2 Spy-mediated Mfp secretion expression

[0026] The five-pointed star in the image represents the Spy-Mfp fusion protein.

[0027] Figure 3 Mfp cleavage and purification

[0028] M: Maker; 1: PD8SM3 supernatant; 2: PD8SM3 purification; 3: PD8SM3 digestion; 4: Mfp3; 5: PD8SM5 supernatant; 6: PD8SM5 purification; 7: PD8SM5 digestion; 8: Mfp5; 9: TEV protease; The pentagram indicates the digested Mfp3.

[0029] Specific Implementation Cases

[0030] Example 1: Secretory Expression of Spy-Mfp3

[0031] Using genomic DNA of strain PD8 as a template, the upstream homologous arm LF and the downstream homologous arm RF were amplified using primers P3 / P4 and P7 / P8, respectively. Using pUB110 plasmid (Gryczan et al. 1980. J Bacteriol. 141:246-253. Doi:10.1128 / jb.141.1.246-253.1980) as a template, the bleomycin (Zeo) resistance gene was amplified using primers P5 / P6. The Spy-Mfp3 expression cassette (synthesized by a gene company and with the sequence shown in SEQ ID) was amplified using primers P1 / P2. As shown in NO.1); then, these four fragments (LF, bleomycin resistance gene, spy-mfp3 expression cassette, and RF) were fused by overlap extension PCR to form the fragment LF-Zeo-SM3-RF; the LF-Zeo-SM3-RF fragment was transformed into Bacillus subtilis PD8 competent cells, and strain PD8SM3 was obtained after selection with 20 ppm bleomycin. After fermentation of strain PD8SM3 in TB medium for 36 h, the supernatant was collected by centrifugation and analyzed by 15% SDS-PAGE. The results showed that Spy can mediate the secretion of Mfp3 into the supernatant of the culture medium ( Figure 2 ).

[0032] Example 2: Secretory Expression of Spy-Mfp5

[0033] The construction method of the expression strain was the same as in Example 1. The upstream homologous arm LF, the downstream homologous arm RF, the bleomycin resistance gene, and the Spy-Mfp5 expression cassette (synthesized by a gene company, sequence shown in SEQ ID NO. 11) were amplified separately. These four fragments were then fused by overlap extension PCR to form the fragment LF-Zeo-SM5-RF. This fragment was chemically transformed into Bacillus subtilis PD8 competent cells, and strain PD8SM5 was obtained after selection with 20 ppm bleomycin. After fermentation in TB medium for 36 h, the supernatant was collected by centrifugation and analyzed by 15% SDS-PAGE. The results showed that Spy could mediate the secretion of Mfp5 into the culture supernatant. Figure 2 ).

[0034] Example 3: Shearing and purification of Mfp

[0035] To purify the fusion proteins Spy-Mfp3 or Spy-Mfp5, the expression strains were fermented in TB medium for 36 h, and the supernatant was collected by centrifugation. The supernatant was precipitated with saturated ammonium sulfate, incubated at 4 °C for 12 h, and then centrifuged at 12,000 rpm for 20 min to collect the precipitate. The precipitate was resuspended in 5 mL of buffer A. The steps for purifying the target protein using a nickel column are as follows: First, equilibrate the column with five times the volume of the resin in buffer A (10 mM imidazole, 8 M urea, 100 mM NaH2PO4, 10 mM Tris-HCl, pH=7.9). Then, load 5 mL of protein sample. Next, add buffer B (30 mM imidazole, 8 M urea, 100 mM NaH2PO4, 10 mM Tris-HCl, pH=6.3) to elute impurities. Finally, elute with buffer C (300 mM imidazole, 8 M urea, 100 mM NaH2PO4, 10 mM Tris-HCl, pH=4.5) to obtain the target protein. The purified protein was renatured in TEV buffer (purchased from Shanghai Yuanye Biotechnology Co., Ltd.) for 12 h, then centrifuged at 12,000 rpm for 20 min to collect the supernatant. TEV protease (purchased from Shanghai Yuanye Biotechnology Co., Ltd.) was added according to the instructions, and the mixture was incubated at 4℃ for 12 h or 30℃ for 1-2 h. The supernatant was then collected by centrifugation and analyzed by 15% SDS-PAGE. TEV protease can be used to cleave the chaperone protein Spy (…). Figure 3 The Mfp cells were then purified using a nickel column to obtain individual Mfp cells. The purification rate of Mfp3 was 93%, with a yield of 120 mg / L fermentation broth supernatant; the purification rate of Mfp5 was 90%, with a yield of 113 mg / L fermentation broth supernatant.

Claims

1. A mussel adhesive protein expression cassette, characterized in that, The mussel adhesion protein expression cassette sequence is shown in SEQ ID NO.1 or SEQ ID NO.

11.

2. A recombinant fragment of mussel adhesion protein, characterized in that... It consists of an upstream homologous arm LF - the bleomycin resistance gene Zeo - the mussel adhesion protein expression cassette as described in claim 1 - and a downstream homologous arm RF.

3. A genetically engineered Bacillus subtilis bacterium, characterized in that... Contains the mussel adhesion protein expression cassette of claim 1 or the mussel adhesion protein recombinant fragment of claim 2.

4. The Bacillus subtilis genetically engineered bacterium according to claim 3, characterized in that... The recombinant fragment of mussel adhesion protein described in claim 2 was transformed into Bacillus subtilis PD8 competent cells, and the Bacillus subtilis genetically engineered bacteria were obtained after screening with bleomycin.

5. The application of the mussel adhesion protein expression cassette of claim 1 and the recombinant fragment of mussel adhesion protein of claim 2 in the secretion and expression of mussel adhesion protein using Bacillus subtilis.

6. A method for expressing mussel adhesion protein using Bacillus subtilis secretion, characterized in that... Includes the following steps: (1) Construct the Bacillus subtilis genetically engineered strain as described in claim 3 or 4; (2) After fermenting the Bacillus subtilis genetically engineered bacteria according to claim 3 or 4 in TB medium for 32-40 h, the supernatant of the medium is collected by centrifugation, and the mussel adhesion protein is present in the supernatant of the medium.

7. The method according to claim 6, characterized in that... It also includes step (3) purification of the target protein Spy-Mfp: the supernatant of the culture medium is precipitated with saturated ammonium sulfate, incubated at 4°C for 12 h, the precipitate is collected by centrifugation, and the target protein Spy-Mfp is purified by nickel column.

8. The method according to claim 7, characterized in that It also includes step (4) Mfp shearing: the purified target protein Spy-Mfp is refolded in TEV buffer, then the supernatant is collected by centrifugation, and TEV protease is added. After incubation at 4℃ for 12 h or at 30℃ for 1-2 h, the supernatant is collected by centrifugation to obtain the individual mussel adhesion protein Mfp.

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