Serine peptidase, nucleic acid molecule and application thereof

Abstract

The invention belongs to the technical field of enzyme engineering and molecular biology, and particularly relates to serine peptidase, a nucleic acid molecule and application of the serine peptidase. According to the invention, three kinds of serine peptidase SpBcA, SpBcB and SpBcC are identified in B. cardium for the first time, and the structures and functions of the three kinds of serine peptidase SpBcA, SpBcB and SpBcC are analyzed. SpBcA and SpBcB have good serine peptidase activity, can tolerate high temperature (the optimum enzyme reaction temperature is 60 DEG C), and have good activity under an alkaline condition. It is further found that SpBcA is generated in the form of zymogen and is subjected to self-splitting. An inhibitory propeptide is arranged at the N end and can be used as a molecular chaperone to inhibit the activity of the molecular chaperone, and a novel activity mechanism is shown. In addition, the SpBcA serine peptidase can degrade various substrates such as casein, fibrinogen, antibacterial peptide LL-37, gelatin and the like. Therefore, as a novel serine peptidase, the serine peptidase has potential industrial and medical application values.

Description

Technical Field

[0001] The invention belongs to the technical field of enzyme engineering and molecular biology, and specifically relates to a serine peptidase, a nucleic acid molecule and applications thereof. Background Art

[0002] The information disclosed in the background of the invention is only intended to enhance the understanding of the overall background of the invention and should not be necessarily regarded as an acknowledgment or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art.

[0003] Serine peptidases, also known as serine proteases, are a class of proteolytic enzymes found in eukaryotes, prokaryotes and viruses, named after the serine in their active center. Serine peptidases play an important and extensive physiological role in biological organisms. They play an important role in embryonic development, tissue reconstruction, cell differentiation, angiogenesis and pathogen invasion. Serine peptidases play an important role in both medical and industrial fields due to their unique biological activity and broad application prospects. In the medical field, it has become an important drug for the treatment of many diseases; in the industrial field, it has become a key enzyme preparation for improving product quality and efficiency. With the continuous development of biotechnology, the application prospects of serine peptidases will be broader, bringing more benefits to human health and production and life.

[0004] The serine protease superfamily has many members, all of which contain Ser, His, and Asp in their active sites and have the same catalytic mechanism. At present, serine proteases are mainly classified by substrate specificity and the topological structure of the catalytic site. For example, based on substrate specificity, serine proteases can be divided into trypsin-like, chymosin-like, subtilisin-like, elastase-like, and kinase; based on the topological structure of the catalytic site, serine proteases can be divided into 13 families, including the serine peptidase S8 family. Among the serine peptidase S8 family, subtilisin has been the most widely studied and has become an important model system for protein engineering research. Subtilisin has a wide range of substrate specificity. Casein and Suc-Ala-Ala-Pro-Phe-NHPhNO 2 It is often used as a protein substrate and synthetic substrate for the analysis of subtilisin activity. Due to its wide substrate specificity, subtilisin has become an ideal candidate protein for substrate specificity engineering in proteolytic enzymes. In addition, by means of molecular biology and enzyme engineering technology, optimizing the substrate specificity and catalytic efficiency of S8 family serine peptidases or screening new S8 family serine peptidases still has broad development prospects in many fields. Summary of the invention

[0005] In view of the deficiencies of the prior art, the purpose of the present invention is to provide a novel S8 family serine peptidase, nucleic acid molecule and its application. Specifically, the inventors previously successfully isolated and identified a strain called Bergeyella.cardium from a positive blood culture of a patient with infective endocarditis, and named it B.cardium HPQL. Subsequently, the inventors also obtained the whole genome sequence of B.cardium HPQL. Based on whole genome screening, three possible S8 family serine peptidases were identified in B.cardium. They were named SpBcA ( S erine P eptidases from B .cardium), SpBcB and SpBcC. The N-terminus of SpBcA, SpBcB and SpBcC contains a subtilis-like serine protease domain of the KP-43 subfamily of the S8 serine peptidase family. Structural prediction and comparison show that the structures of these three proteins are different from each other and from other reported serine peptidases. The present invention expresses and purifies these proteins, and analyzes the activities and application potentials of these three serine proteases. Among them, SpBcA and SpBcB have good degradation ability for casein. The optimal reaction temperature of the enzyme is 60°C and it maintains good enzyme activity within the pH range of 7.0-9.5. At the same time, SpBcA can degrade fibrinogen. Further based on structural and biochemical analysis, the present invention studies and discovers a new self-cleavage regulation mechanism of the serine peptidase SpBcA. It can be seen that the serine peptidase from B.cardium is a new type of serine peptidase with potential industrial and medical application value. Based on the above research results, the present invention is completed.

[0006] In order to achieve the above technical objectives, the technical solution provided by the present invention is as follows:

[0007] The first aspect of the present invention provides a serine peptidase, wherein the serine peptidase has:

[0008] (a1) the amino acid sequence shown in any one of SEQ ID NOs. 1-3;

[0009] (a2) a protein derived from the amino acid sequence shown in any one of SEQ ID NOs. 1-3, wherein one or more amino acid residues are substituted and / or deleted and / or added and the protein has the same or similar activity as the amino acid sequence shown in any one of SEQ ID NOs. 1-3;

[0010] (a3) proteins encoded by other genes having an amino acid sequence composition similarity of more than 90% with that of SEQ ID NO. 1-3 and having the enzyme activity of any one of SEQ ID NO. 1-3.

[0011] In the present invention, the serine peptidase having the amino acid sequence shown in SEQ ID NO.1 is named SpBcA, the serine peptidase having the amino acid sequence shown in SEQ ID NO.2 is named SpBcB, and the serine peptidase having the amino acid sequence shown in SEQ ID NO.3 is named SpBcC. It should be noted that the SpBcC is inactive in vitro.

[0012] These three serine peptidases show certain similarities and have some common domains, including Peptidase_S8 domain, PKD domain and CTD domain. In addition to the shared domain, serine peptidases also contain specific additional domains: SpBcA contains a cleavage adhesin domain, which plays a role in cell adhesion, cell lysis and carbohydrate binding. SpBcB and SpBcC contain Choice_anch_J superfamily domain. In addition, SpBcA and SpBcB also have a sequence before the serine peptidase domain, while SpBcC lacks this sequence. Sequence alignment further shows that these three serine peptidases are similar to each other, especially on the serine peptidase domain, while the similarity decreases on the variable domain. Based on this information, the present invention believes that SpBcA, B and C are collateral relatives with potential different biological functions.

[0013] In (a2), the serine peptidase is a truncated variant of SpBcA, B and C, specifically SpBcA (30-601aa) (i.e., having the amino acid sequence from positions 30 to 601 as shown in SEQ ID NO.1), SpBcB (30-601aa) (i.e., having the amino acid sequence from positions 30 to 601 as shown in SEQ ID NO.2) and SpBc C (1-476aa) (i.e., having the amino acid sequence from positions 1 to 476 as shown in SEQID NO.3).

[0014] The study found that the SpBcA (30-601aa) has self-cleavage activity, and the study found that the serine peptidase can further enhance its activity after self-cleavage. At the same time, the protease activity of the serine peptidase SpBcA (30-601aa) depends on Ca 2+ The optimum temperature for the protease activity of SpBcA (30-601aa) is 60°C. SpBcA (30-601aa) has high activity in the pH range of 7 to 9.5.

[0015] The second aspect of the present invention provides a nucleic acid molecule, which can encode the above-mentioned serine peptidase.

[0016] The third aspect of the present invention provides a recombinant expression vector, which comprises the nucleic acid molecule described in the second aspect.

[0017] The fourth aspect of the present invention provides a host cell, which contains the recombinant expression vector described in the third aspect of the present invention or a chromosome in which the nucleic acid molecule described in the second aspect of the present invention is integrated, or is capable of expressing the serine peptidase described in the first aspect of the present invention.

[0018] The host cell may be a prokaryotic cell.

[0019] In another embodiment of the present invention, the host cell is a bacterial cell;

[0020] The bacterial cells are any one or more of Escherichia, Agrobacterium, Bacillus, Streptomyces, Pseudomonas or Staphylococcus;

[0021] In another specific embodiment of the present invention, the bacterial cell is Bergeyella, Escherichia coli (such as Escherichia coli BL21 (DE3)), Agrobacterium tumefaciens, Agrobacterium rhizogenes, Lactococcus lactis, Bacillus subtilis, Bacillus cereus or Pseudomonas fluorescens.

[0022] Therefore, the host cell may be a naturally occurring bacterium (B. cardium) or a genetically engineered bacterium obtained through artificial modification (such as genetically engineered Escherichia coli BL21 (DE3)).

[0023] Furthermore, the present invention also protects the fermentation product or metabolites of the host cell.

[0024] In the present invention, the term "fermented product" is used to refer to a fermentation product. The corresponding fermented product may be a liquid obtained from the process of fermenting and culturing host cells, and therefore, may also be referred to as a fermentation liquid; the liquid may contain host cells (cell bodies), but does not necessarily need to contain host cells (cell bodies). The liquid preferably contains metabolites produced by the host cells of the present invention, and the metabolites particularly include the above-mentioned serine peptidase.

[0025] The fifth aspect of the present invention provides a method for preparing the serine peptidase described in the first aspect of the present invention, comprising the steps of: culturing the host cell described in the fourth aspect of the present invention to express the serine peptidase; and isolating and purifying the serine peptidase.

[0026] The sixth aspect of the present invention provides the use of the above-mentioned serine peptidase, nucleic acid molecule, recombinant expression vector, host cell in any one or more of the following:

[0027] (a) Substrate degradation;

[0028] (b) Detection of pathogens or preparation of products for detection of pathogens;

[0029] (c) Research on the virulence mechanism of pathogens;

[0030] (d) Screening or preparing pathogen inhibitors.

[0031] Wherein, in application (a), the substrate includes but is not limited to casein, gelatin, fibrinogen and antimicrobial peptide LL-37.

[0032] In the applications (b) to (d), the pathogen may be Bergeyella sp, or further Bergeyella cardium.

[0033] In (b), the product may be a detection kit, a detection device or an equipment, which is not specifically limited herein.

[0034] In (d), the pathogen inhibitor may be in the form of any one or more of the following products, such as medicines, health products, foods, sanitary products or (environmental) disinfection products, without specific limitation.

[0035] Beneficial technical effects of one or more of the above technical solutions:

[0036] The above technical scheme identified three serine peptidases SpBcA, SpBcB and SpBcC for the first time and analyzed their protease activities, which belong to the S8 family of serine peptidases. SpBcA and SpBcB were successfully purified in vitro and were active. SpBcA contains an N-terminal domain located before the S8 serine peptidase domain. There is a peptide in the domain that passes through the active site of the serine peptidase domain. Self-cleavage to remove the N-terminal domain can activate SpBcA. Mutagenesis studies revealed the self-cleavage site of SpBcA (residues 116-120) and determined its self-cleavage regulatory mechanism. In addition, SpBcA can also degrade casein, fibrinogen, antimicrobial peptide LL-37 and gelatin. The above technical scheme lays a research foundation for its application in food, medicine and other fields. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0038] Figure 1 The domain organization and sequence alignment of SpBcA, SpBcB and SpBcC in the embodiments of the present invention

[0039] (a) Domains of SpBcA, SpBcB, and SpBcC. The color scheme is as follows: orange, serine peptidase signal peptide (SP); light orange, propeptide; light blue, serine peptidase S8 family domain in kp43 protease (peptidase_S8 domain); yellow, β-barrel domain; pink, repeat sequence in polycystic kidney disease 1 (PKD1) and other proteins (PKD); green, cleavage adhesin domain; magenta, secretion system C-terminal sorting domain (CTD); cyan, anchor selection J domain (choice-of-anchor J). (b) Multiple sequence alignment of SpBcA, SpBcB, and SpBcC by Jalview. The identity threshold for coloring is 60%.

[0040] Figure 2 Expression and purification of serine peptidase guided by structure prediction in the present invention

[0041] (a) and (b) Predicted structures of SpBcA(30-601aa) and SpBcB(30-601aa), including the S8 serine peptidase domain and the auxiliary β-barrel domain. The structures are shown as cartoon representations. The serine peptidase domain, propeptide domain, and β-barrel domain are colored in light blue, light orange, and yellow, respectively. The residues in the active center are shown as sticks and colored in purple. (c) and (e) Domain organization of SpBcA(30-601aa) and SpBcB(30-601aa). (d) and (f) Purified serine peptidases of SpBcA(30-601aa) and SpBcB(30-601aa). CL: cell lysate, FT: flow-through, W1-W5: wash fractions 1-5, E1-E7: elution fractions 1-7.

[0042] Figure 3 The activity reaction conditions of the serine peptidase in the embodiment of the present invention are tested

[0043] (a) Analysis of the effect of ions on the activity of SpBcA (30-601aa) protease using azocasein as substrate. The vertical axis represents the UV absorbance of the protein hydrolysate at 450nm. The horizontal axis shows different ions: (-) means the reaction was carried out in the presence of the protease SpBcA (30-601aa) but without any ions in the reaction system; Ca 2 +, Mg2+, Mn2+, Zn2+ and EDTA represent the corresponding components added to the reaction system. Error bars represent standard deviations calculated from three experiments (n=3). Statistical significance is represented by p value, ns not significant, *(p<0.05), **(p<0.01).

[0044] (b) Analysis of Ca2+ activity of peptidase SpBcA (30-601aa) using azocasein as substrate 2 + optimal concentration. The vertical axis represents the UV absorbance of the protein hydrolysate at 450nm. The horizontal axis shows the Ca 2 + concentration). Error bars represent standard deviations calculated from three experiments (n=3). Statistical significance is indicated by p-values, *(p<0.05), **(p<0.01), ***(p<0.001).

[0045] (c) The optimal reaction temperature of SpBcA (30-601aa) was analyzed using azocasein as a substrate. The vertical axis represents the UV absorbance of the protein hydrolysate at 450nm. The horizontal axis shows the different temperatures used in the analysis. The error bars represent the standard deviation calculated from three experiments (n=3). Statistical significance is represented by p value, ns not significant, * (p<0.05), ** (p<0.01), *** (p<0.001).

[0046] (d) Analysis of the optimal reaction pH of peptidases using azocasein as a substrate. The vertical axis represents the UV absorbance of the protein hydrolysate at 450 nm. The horizontal axis shows the pH used in the analysis. The error bars represent the standard deviation calculated from three experiments (n=3).

[0047] Figure 4 The activity of the two serine peptidases activated by self-cleavage in the embodiment of the present invention

[0048] (a) and (b) Degradation of β-casein by SpBcA (30-601aa) (a) or SpBcB (30-601aa) (b). 100 μg of β-casein was mixed with different amounts of SpBcA and incubated for 30 minutes. The weight ratios of SpBcA (30-601aa) (enzyme, E) to β-casein (substrate, S) were 1:5, 1:10, 1:25, 1:50, 1:100 and 1:500 (E / S, enzyme / substrate), respectively. 10 μl of the sample was mixed with 5× SDS loading buffer and denatured at 100°C for 10 minutes to stop the reaction. The samples were then separated by SDS-PAGE.

[0049] (c) and (d) Analysis of the self-cleavage ability of SpBcA (30-601aa) and SpBcB (30-601aa). 10 μg of serine peptidases SpBcA (30-601aa) (c) and SpBcB (30-601aa) (d) were respectively dissolved in 110 μl of 100 mM Tris-HCl, 150 mM NaCl and 5 mM CaCl 2Pre-incubate at 37°C. Take 10 μl samples from the reaction mixture at 0, 5, 10, 15, 20, 30, and 60 minutes, and treat according to Figures (a), (b) and the Methods section.

[0050] (e) and (f) Protease assays of SpBcA(30-601aa)(e) or SpBcB(30-601aa)(f) without self-cleavage (time course experiments). 200 μg of β-casein was mixed with 20 μg of SpBcA(30-601aa)(e) and 20 μg of SpBcB(30-601aa)(f) in 220 μl of 100 mM Tris-HCl, 150 mM NaCl and 5 mM CaCl 2 20 μl samples were taken from the reaction mixture at 0, 5, 10, 15, 20, 30, and 60 minutes, and processed according to the Figures (a), (b) and the Methods section.

[0051] (g) and (h) SpBcA (30-601aa) (g) or SpBcB (30-601aa) (h) self-cleavage ability detection (time course experiment). 20 μg of SpBcA (30-601aa) (g) and SpBcB (30-601aa) (h) were preincubated at 37°C for 15 min, and then 200 μg of casein and 220 μl of 100 mM Tris-HCl, 150 mM NaCl and 5 mM CaCl were added. 2 20 μl samples were taken from the reaction mixture at 0, 5, 10, 15, 20, 30, and 60 minutes and processed as described in steps (a) and (b) and the methods section.

[0052] Figure 5 Purification and activity detection of SpBcA (30-601aa) mutants in the present invention

[0053] (a) The predicted structure of SpBcA (30-601aa), including the S8 serine peptidase domain, the auxiliary β-barrel domain, and the active site of the serine peptidase SpBcA (30-601aa): D133, H160, and S388. The coloring scheme of the SpBcA structure is similar to that of Figure 2 same.

[0054] (b) Purification of SpBcA variants and mutants, including SpBcA(104-601aa), SpBcA(30-601aa)-H160A, SpBcA(30-601aa)-mutation-A, SpBcA(30-601aa)-mutation-B and SpBcA(30-601aa)-mutation-AB.

[0055] (c) Degradation of β-casein by SpBcA(30-601aa), SpBcA(30-601aa)-H160A, SpBcA(30-601aa)-mut-A, SpBcA(30-601aa)-mut-B, and SpBcA(30-601aa)-mut-AB. 100 μg of β-casein was mixed with different amounts of serine peptidase and incubated for 30 min. The mass ratios of SpBcA(30-601aa), SpBcA(30-601aa)-H160A, SpBcA(30-601aa)-mut-A, SpBcA(30-601aa)-mut-B, and SpBcA(30-601aa)-mut-AB (enzyme, E) and β-casein (substrate, S) were 1:5 and 1:10 (E / S, enzyme / substrate), respectively.

[0056] (d) Degradation of β-casein by SpBcA (104-601aa). 100 μg of β-casein was mixed with different amounts of SpBcA (104-601) and incubated for 30 min. The mass ratios of SpBcA (enzyme, E) to β-casein (substrate, S) were 1:5, 1:10, 1:25, 1:50, 1:100 and 1:500 (enzyme / substrate), respectively.

[0057] (e) The cleavage site of the propeptide (30-601aa) of SpBcA. Figure 2 Colored in the same way. The cleavage site is indicated in cyan.

[0058] Figure 6 Comparison of the SpBcAN propeptide and the subtilisin I9 domain in the embodiments of the present invention

[0059] (a) Superposition of the structures of SpBcA in complex with (Ser221Cys)-ubtilisin E-peptide (PDB code: 1SCJ). Superposition of the structures of SpBcAS8-serine peptidase domain in complex with (Ser221Cys)-ubtilisin E-propeptide (PDB code: 1SCJ). Figure 2 The I9 and S8 domains of 1SCJ are shown in pink and grey, respectively. The two structures are shown side by side in the overlay of panels (b) and (c).

[0060] (b) Structure of the SpBcAN terminal propeptide and S8 serine peptidase domain.

[0061] (c) Structure of the (Ser221Cys)-Subtilisin E-propeptide complex.

[0062] Figure 7 In the present invention, fibrinogen, antimicrobial peptide LL-37, gelatin and hemoglobin are substrates of serine peptidase SpBcA (30-601aa).

[0063] (a) SpBcA (30-601aa) degrades fibrinogen. 220 μg of fibrinogen and 22 μg of serine peptidase SpBcA (30-601aa) were mixed in 110 μl of 100 mM Tris-HCl, 150 mM NaCl and 5 mM CaCl 2 At the indicated time points, aliquots (10 μl) were taken from the reaction mixture and processed as described in the Methods section.

[0064] (b) SpBcA (30-601aa) degrades the antimicrobial peptide LL-37. In 110 μl of 100 mM Tris-HCl, 150 mM NaCl and 5 mM CaCl 2 In solution, 20 μg of antimicrobial peptide LL-37 was pre-incubated with 0.2 μg of serine peptidase SpBcA (30-601aa). At the indicated time points, aliquots (10 μl) were taken from the reaction mixture and processed as described in the Methods section.

[0065] (c) SpBcA (30-601aa) degrades gelatin. Gelatin was pre-incubated with serine peptidase SpBcA (30-601aa). (+) is the positive control provided by the kit.

[0066] (d) Degradation of hemoglobin by SpBcA (30-601aa). 110 μg of hemoglobin was mixed with serine peptidase 11 μg of SpBcA (30-601aa) in 110 μl of 100 mM Tris-HCl, 150 mM NaCl and 5 mM CaCl 2 At the indicated time points, aliquots (10 μl) were taken from the reaction mixture and processed according to the methods section. DETAILED DESCRIPTION

[0067] It should be noted that the following detailed descriptions are all illustrative and intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs.

[0068] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit the exemplary embodiments according to the present invention. As used herein, unless the context clearly indicates otherwise, the singular form is also intended to include the plural form. In addition, it should be understood that when used in this specification,

[0069] When the terms “include” and / or “comprises” are used, it indicates that features, steps, operations, devices, components and / or their combinations are present.

[0070] The present invention is further described in conjunction with specific examples. The following examples are only for explaining the present invention and are not intended to limit the content thereof. If the specific experimental conditions are not specified in the examples, they are usually carried out according to conventional conditions or the conditions recommended by the reagent company; the reagents and consumables used in the following examples, unless otherwise specified, can all be obtained from commercial sources.

[0071] The present invention is further explained by the following examples, but they are not intended to limit the present invention. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The test methods for the specific conditions in the following examples are usually carried out under normal conditions.

[0072] Example

[0073] method

[0074] Bacterial strains, plasmids and growth conditions

[0075] Table 1 lists the bacterial strains and plasmids used in this application. B. cardium HPQL was grown in Columbia blood agar (Thermo Fisher Scientific) and cultured at 35°C for 72 hours. E. coli DH5α and BL21 (DE3) were used as hosts for cloning and protein expression, respectively. E. coli strains were grown in Luria-Bertani (LB) medium at 37°C with or without the addition of ampicillin (benzylpenicillin) (100 mg / mL) as needed.

[0076] Table 1: Strains and plasmids used in the study

[0077]

[0078]

[0079] Gene cloning and protein expression

[0080] Bergeyella cardium and C-terminal domains were input, and the genes encoding SpBcA, B, and C were identified by searching the National Center for Biotechnology Information Conserved Domain Database (https: / / www.ncbi.nlm.nih.gov / Structure / cdd / wrpsb.cgi). The full-length target gene was amplified using genomic DNA from B. cardium HPQL strain as a PCR template and cloned into the BamHI (Takara, #1605) and XhoI (Takara, #1605) restriction enzyme sites of the pGEX-6p-2 vector to obtain a protein with a GST tag and a protease cleavage site at the N-terminus of the protein. The cloning method for truncated variants was the same, except that the plasmid containing the full-length gene was used as a PCR template. The catalytic mutant and inhibitory mutant of SpBcA were generated by site-directed mutagenesis PCR (Quick Change). The primers used are listed in Table 2.

[0081] The protein was expressed in E. coli BL21 (DE3) in LB medium (Hopebio, #HB0128) supplemented with 100 mg / ml ampicillin (Solarbio, #A8180) overnight. 0.6 L of E. coli BL21 (DE3) transformed with the corresponding plasmid was cultured at 37°C until OD600 reached 0.6-0.8. IPTG (Sigma, #I6758) was added at a final concentration of 0.3 mM at 20°C to induce protein expression. After 14 hours of induction, the cells were harvested by centrifugation at 4000 rpm for 15 minutes. The bacterial pellet was used for protein purification.

[0082] Table 2: PCR primers used in the study

[0083]

[0084]

[0085] Protein purification

[0086] The bacterial pellet obtained from 0.6 liters of liquid culture was resuspended in 40 mL of GST lysis buffer (50 mM Tris-HCl, 500 mM NaCl, 10% glycerol and 1 mM DTT, pH 7.4). After mixing the bacterial suspension, the cells were lysed by ultrasound at a power of 300 W for 30 minutes, with a working time of 3 seconds and a pause of 7 seconds. The cell lysate was clarified by centrifugation (11000 rpm, 1 hour, 4°C) and filtered with a 0.22-μm syringe filter (Millipore, #SLGPR33RB). The filtered supernatant was incubated with GST-Tag purification resin (BeyoGold, #P2251) at 4°C for 1 hour with rotation. Then, the supernatant was discarded and the GST-Tag purification resin was washed at least 3 times with GST wash buffer (50 mM Tris-HCl, 500 mM NaCl, 10 mM glycerol and 1 mM DTT, pH 7.4). Finally, the target protein bound to the GST-Tag purification resin was eluted with GST elution buffer (50mM Tris-HCl, 500mM NaCl and 10mM GSH, pH 8.0). The eluate was transferred to a 1.5mL centrifuge tube, snap-frozen in liquid nitrogen, and stored at -80°C. Before biochemical detection, the protein was thawed on ice and concentrated to an appropriate concentration using a 15ml 10KD cut-off ultrafiltration centrifuge tube (Millipore, #UFC901008). The purity of the obtained protein was verified by SDS-PAGE electrophoresis.

[0087] Predictions from structural models

[0088] The structural models of SpBcA, B and C were predicted using the trRosseta online server (https: / / yanglab.qd.sdu.edu.cn / trRosetta / ). In brief, the protein sequences of SpBcA, B and C were used as input sequences for structure prediction and predictions were performed under default parameter settings. The highest-ranked model in each prediction result, model 1, was used for further analysis. The TM score reflects the confidence of the overall structure prediction. A TM score (0-1) above 0.5 generally indicates that the model topology is correct. The prediction confidence of each residue is represented by the LDDT score (0-100 points) of each residue in the B-factor column in the structure PDB file. The structure figure was made using PyMOL (Schroedinger).

[0089] Protease activity assay

[0090] Serine peptidase activity was detected using bovine β-casein (Millipore Sigma, #C6905), bovine plasma fibrinogen (MeilunBio, #MB5809), human antimicrobial peptide LL-37 (AnaSpec, #As-61302) and hemoglobin (Sigma, #H7379). Bovine β-casein was used as substrate, and in a reaction system containing 110 μl reaction buffer (100 mM Tris-HCl (pH 7.4), 150 mM NaCl and 5 mM CaCl2), 20, 10, 4, 2, 1 and 0.2 μg were incubated with 100 μg β-casein at a mass ratio of serine peptidase to substrate of 1:5, 1:10, 1:25, 1:50, 1:100 and 1:50, respectively, and incubated at 37°C for 30 min, and 10 μl of sample was mixed with 5×SDS loading buffer. The samples were denatured by incubation at 100°C for 10 minutes, electrophoresed on 15% SDS polyacrylamide gel (Epizyme Biotech, #PG114), and stained with Coomassie Brilliant Blue staining reagent (Solarbio, #P1305).

[0091] For time course experiments, proteolysis is stopped at specific time points. Figure 4 For the self-cleavage experiment in c, 10 μg of SpBcA (30-601aa) or SpBcB (30-601aa) was added to 110 μl of reaction buffer and incubated at 37°C. 10 μl samples were collected at 0, 5, 10, 15, 20, 25, 30, 45, 60, and 90 min. Figure 4 For the time course experiments in e and f, 200 μg of casein was mixed with 220 μl of reaction buffer, and the mixture was added to 20 μg of SpBcA (30-601aa) or SpBcB (30-601aa) and incubated at 37°C. 20 μl samples were collected at 0, 5, 10, 15, 20, 30 and 60 min. Figure 4 For the time course experiment in g, 20 μg of SpBcA (30-601aa) or SpBcB (30-601aa) were pre-incubated at 37°C for 15 min before self-cleavage. Then, 200 μg of casein was mixed with 220 μl of reaction buffer, and the mixture was added to the self-cleaved SpBcA (30-601aa) ( Figure 4 g) or SpBcB(30-601aa)( Figure 4h) and incubated at 37°C for different times. 20 μl samples were taken at 0, 5, 10, 15, 20, 30 and 60 min time points. In all time course experiments, protein samples were mixed with 5× SDS loading buffer and further denatured by incubation at 100°C for 10 min to completely stop the proteolytic process.

[0092] When the antimicrobial peptide LL-37, hemoglobin, and fibrinogen were used as substrates for the assay, the substrate was incubated with SpBcA (30-601aa) at a serine peptidase:substrate weight ratio of 1:100 (LL-37) or 1:10 (fibrinogen and hemoglobin) at 37°C in 110 μl of buffer (100 mM Tris, 150 mM NaCl, 5 mM CaCl) at the indicated time points. 2 , pH 7.4). In the time course experiment, proteolysis was stopped at specific time points (LL-37: 0, 5, 10, 20, 30, 60, 120, 240 and 480 minutes; hemoglobin: 0, 5, 15, 30, 60, 120, 240 and 480 minutes; bovine plasma fibrinogen: 0, 5, 15, 30, 60, 120, 240, 480, 720, 1440, 2160 and 3600 minutes). 20 μl of hemoglobin and fibrinogen samples were analyzed by 15% SDS-PAGE, while LL-37 was analyzed by 4-20% Precast Protein Plus Gel (YEASEN, #36270ES10) and then Coomassie staining.

[0093] Gelatin zymography assay

[0094] Gelatin zymogram analysis was performed according to the kit (Real-Times (Beijing) Biotechnology Co. Ltd., #710278). In short, an SDS-PAGE gel containing collagenase substrate gelatin was prepared, and a sample containing a protease was electrophoresed in the gel. During the electrophoresis, SDS reversibly binds to the protease in the sample, causing the hydrogen bonds and hydrophobic interactions in the protease to be destroyed, thereby preventing them from degrading gelatin. After the electrophoresis is completed, the gel is incubated with a reactivation solution to restore the activity of the protease so that the gelatin in the gel is hydrolyzed at its migration position. After Coomassie brilliant blue staining, the gel is deeply stained due to the presence of substrate protein, forming a dark background. However, at the position of the protease band, the substrate is degraded by the protease and cannot be stained with Coomassie brilliant blue, thereby forming a clear white area. In this way, the size and activity (zymogram) of the protease can be displayed simultaneously, and the intensity is proportional to the enzyme activity.

[0095] Optimize reaction pH and enzyme activity temperature

[0096] 100 μg of casein was used as substrate to optimize the reaction pH and enzyme activity temperature. The optimal pH was determined by measuring the activity of the recombinant protein in 100 mM sodium acetate buffer at pH 5.0, 5.5, 6.0 and 6.5 and in 50 mM Tris-HCl buffer at pH 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 10.5 and 11.0. The reaction temperature was 37 ° C and the reaction time was 1 hour. The effect of temperature on protease activity was evaluated at 30, 37, 40, 50, 55, 60, 65 and 70 ° C. After incubation, the samples were then denatured at 100 ° C for 10 minutes, electrophoresed on 15% SDS polyacrylamide gel (EpizymeBiotech, #PG114), and stained with Coomassie Brilliant Blue (Solarbio, #P1305). All experiments were repeated 3 times.

[0097] Effects of metal cations on the activity of recombinant proteases

[0098] Check metal cation Ca 2+ Mg 2+ , Mn 2+ 、Zn 2+ Effect of EDTA on the protease activity of recombinant proteins. 100 μg of casein was used as a substrate. Reactions containing 10 μg of purified recombinant protein and casein were incubated at 37°C for 1 h with different metal ions or EDTA in 100 mM Tris-HCl and 150 mM NaCl buffer (pH 7.4). After incubation, the samples were then denatured at 100°C for 10 minutes, electrophoresed on a 15% SDS polyacrylamide gel (Epizyme Biotech, #PG114), and stained with Coomassie Brilliant Blue (Solarbio, #P1305). All experiments were repeated 3 times.

[0099] Recombinant protein Ca 2+ Optimal concentration for enzyme activity

[0100] Reactions containing 10 μg of purified recombinant protein and casein (100 μg) were incubated at 37°C in the presence of different concentrations of Ca 2+ The samples were incubated in 100 mM Tris-HCl and 150 mM NaCl buffer (pH 7.4) for 1 hour (0 mM, 1 mM, 5 mM, 10 mM, 20 mM and 40 mM, respectively). After incubation, the samples were denatured at 100°C for 10 minutes, electrophoresed on 15% SDS polyacrylamide gel, and stained with Coomassie Brilliant Blue reagent (Solarbio, #P1305). All experiments were repeated 3 times.

[0101] result

[0102] Identification of three serine peptidases as potential carriers of T9SS

[0103] Bioinformatics analysis was performed based on the Biotechnology Information Conserved Domain Database (https: / / www.ncbi.nlm.nih.gov / Structure / cdd / wrpsb.cgi), and three genes encoding CTD structural proteins (QHN65200.1, QHN65156.1, and QHN66091.1) were identified in the whole genome sequence of B.cardidium. The S8 family serine peptidase encoding domain was identified in the three identified proteins. They were named SpBcA (Serine Peptidases from B.cardidium) (QHN65200.1), SpBcB (QHN65156.1), and SpBcC (QHN66091.1), respectively.

[0104] These three serine peptidases show certain similarities and have some common domains, including the Peptidase_S8 domain, the PKD domain, and the CTD domain ( Figure 1 a). Peptidases_S8_KP43 proteases are members of the serine peptidase S8 or protease family with an Asp / His / Ser catalytic triad. The CTD domain targets the protein to the T9SS secretion system and is cleaved by a C-terminal signal serine peptidase after secretion. The PKD domain is associated with polycystic kidney disease 1 and plays a role in cell-cell / matrix interactions.

[0105] In addition to the shared domains, serine peptidases contain specific additional domains: SpBcA contains a lytic adhesin domain that plays a role in cell adhesion, cell lysis, and carbohydrate binding. SpBcB and SpBcC contain the Choice_anch_J superfamily domain ( Figure 1 a). In addition, SpBcA and SpBcB have a sequence before the serine peptidase domain, while SpBcC lacks this sequence. Sequence alignment further showed that the three serine peptidases are similar to each other, especially in the serine peptidase domain, while the similarity decreases in the variable domain ( Figure 1 b) Based on this information, the present invention believes that SpBcA, B and C are secretory proteins of T9SS and are paralogous relatives with potentially different biological functions.

[0106] Expression and purification of serine peptidase guided by structure prediction

[0107] To further characterize these serine peptidases, this study cloned these three serine peptidases and expressed them heterologously in Escherichia coli. This study first attempted to express and purify the full-length proteins, but only the serine peptidase SpBcC could be expressed and purified in very low amounts, and SpBcC was inactive in vitro. SpBcA and SpBcB were not dissolved. In order to obtain soluble proteins for biochemical studies, various protein truncation methods were tried. Based on the structural prediction of trrossetta, a series of truncation variants of the three serine peptidases were designed, including the predicted S8 serine peptidase domain and its auxiliary domain, namely SpBcA (30-601aa), SpBcB (30-601aa) ( Figure 2 ac,e) and SpBcC (1-476aa). The present invention attempts to express and purify these truncated variants. The truncated variants of SpBcA and SpBcB were successfully expressed and purified ( Figure 2 d,f); SpBcC(1-476aa) is not expressed. During the purification process, non-specific protein bands were noted in the elution fraction of the GST batch purification of SpBcA(30-601aa). To verify the identity of these bands, mass spectrometry experiments were performed. Mass spectrometry analysis showed that these bands were degradation products of SpBcA(30-601aa). This ruled out the possibility of E. coli contamination and suggested that SpBcA(30-601aa) might be active.

[0108] Analysis of the optimal reaction conditions of SpBcA in vitro

[0109] The present invention further studies the activity characteristics of serine peptidase SpBcA (30-601aa), including the effects of ions, temperature and pH on its activity. Experiments show that the protease activity of serine peptidase SpBcA (30-601aa) depends on Ca 2+ , independent of other divalent cations such as Mg 2+ , Mn 2+ and Zn 2+ , ( Figure 3 a, b). The optimal temperature for the activity of SpBcA (30-601aa) protease is 60℃ ( Figure 3 c). The optimal pH for SpBcA activity is between 7.5 and 9 ( Figure 3 d).

[0110] SpBcA and SpBcB serine peptidases are active

[0111] Next, the present invention tested whether the serine peptidase was active using casein as a substrate. Different concentrations of SpBcA (30-601aa) and SpBcB (30-601aa) were added to the reaction system, and the degradation of the substrate casein was proportional to the concentration of the serine peptidase ( Figure 4 a, b). This indicates that both serine peptidases are active. However, SpBcB (30-601aa) has lower protease activity in degrading casein than SpBcA (30-601aa) ( Figure 4 a, b).

[0112] Self-cleavage activates SpBcA serine peptidase activity

[0113] In the proteolytic studies using casein as substrate, it was observed that the molecular weight of serine peptidase SpBcA (30-601aa) decreased, while some nonspecific bands appeared for SpBcB (30-601aa), as shown by the bands on SDS-PAGE ( Figure 4 a, b). Since it has been demonstrated that subtilisin is a pro-protease whose N-terminal propeptide sequence inhibits its protease activity and is activated upon self-cleavage, it was suspected that the serine peptidases SpBcA (30-601aa) and SpBcB (30-601aa) might also undergo self-cleavage. To test this hypothesis, self-cleavage assays were performed using GST-tagged serine peptidases SpBcA (30-601aa) and SpBcB (30-601aa) as respective substrates and the protease itself instead of casein. After incubation, samples were taken at different time points. Indeed, a clear shift of the protein band after incubation of SpBcA (30-601aa) was observed. Mass spectrometry analysis of the downshifted band confirmed that this band was indeed SpBcA. After approximately 15 minutes, the self-cleavage of SpBcA (30-601aa) was complete ( Figure 4 c). On the other hand, SpBcB (30-601aa) also underwent self-degradation, but there was no typical single band ( Figure 4 d).

[0114] We further tested whether autolysis activated SpBcA(30-601aa) and SpBcB(30-601aa) by comparing casein degradation before and after preincubation with serine peptidases SpBcA(30-601aa) and SpBcB(30-601aa) ( Figure 4 eh). They were divided into two groups: In reaction 1, serine peptidases SpBcA (30-601aa) and SpBcB (30-601aa) reacted directly with casein ( Figure 4 e,f); In reaction 2, serine peptidases SpBcA (30-601aa) and SpBcB (30-601aa) were pre-incubated in the reaction system for 15 min for self-cleavage, and then casein was added to start casein digestion ( Figure 4 g,h). The study found that compared with the control without self-cleavage ( Figure 4e) Compared with the casein degradation bands of SpBcA (30-601aa) after cleavage, the degradation bands of casein were more obvious ( Figure 4 g), indicating that autocleavage indeed activated the serine peptidase SpBcA (30-601aa). However, for SpBcB (30-601aa), pre-incubation with autocleavage did not enhance its protease activity, as the control reaction ( Figure 4 f) and self-cleavage reaction ( Figure 4 h). Due to the lower activity of serine peptidase SpBcB (30-601aa), the serine peptidase SpBcA (30-601aa) was focused on in subsequent studies. Since the activity of serine peptidase SpBcA (30-601aa) is enhanced by autocleavage, serine peptidase SpBcA (30-601aa) was pre-incubated in the absence of substrate in subsequent experiments to fully activate the protease before the protease assay.

[0115] Regulatory mechanisms of serine peptidase activity

[0116] The serine peptidase SpBcA (30-601aa) contains a conserved catalytic triad (Asp133, His160, and Ser388) similar to subtilisin and other known members of the S8 protease family ( Figure 5 a). To verify the hypothesis, the active site of serine peptidase SpBcA (30-601aa) was first verified. A serine peptidase mutant with a predicted catalytic center mutation (H160A, Figure 5 b) and use casein ( Figure 5 c) As substrates, protease assays were performed using native serine peptidase SpBcA (30-601aa) and its mutant (H160A). The results showed that the catalytic mutant was unable to degrade casein and fibrinogen, indicating a loss of serine peptidase activity. These results confirm the identity of the active center of serine peptidase SpBcA.

[0117] Interestingly, SpBcA contains an N-terminal sequence that processes a serine peptidase domain, as shown in the N-terminal propeptide of subtilisin (I9 domain). However, the SpBcA propeptide has no sequence or structural similarity to the I9 domain, as can be seen from the structural superposition of SpBcA in complex with (Ser221Cys)-subtilisin E-propeptide ( Figure 6 ac). According to structural prediction, the N-terminal domain of serine peptidase SpBcA (30-601aa) contains a peptide segment that passes through the active site of serine peptidase. Therefore, it is assumed that the N-terminal sequence may inhibit the activity of serine peptidase SpBcA, and cutting off this domain may eliminate this inhibition.

[0118] To investigate the effect of the N-terminal domain on serine peptidase activity, truncated variants lacking the N-terminal domain were made and their serine peptidase activity was tested. Surprisingly, when the N-terminal domain of serine peptidase SpBcA (104-601aa) was deleted ( Figure 5 b), the serine peptidase still showed no activity towards casein ( Figure 5 d). It is suspected that the N-terminal domain may be necessary for the correct folding of the protein. To further verify the inhibitory effect of the N-terminal domain, it was attempted to detect whether inhibition of self-cleavage would inhibit the protease activity of SpBcA (30-601aa). In order to block self-cleavage, possible self-cleavage sites were searched on SpBcA (30-601aa). Based on structural prediction, it was speculated that the polypeptide TSNA (100-103) ( Figure 5 e) may be a potential self-cleavage site. Therefore, TSNA was replaced with AAAA (SpBcA(30-601aa)-mut-A) by site-directed mutagenesis. Figure 5 b) However, when its activity was tested using casein as a substrate, the mutant was still able to degrade casein ( Figure 5 c). Since a previous paper reported that there are two self-cleavage sites on the serine peptidase NbSLP1, and mutations in both sites are necessary to inhibit self-cleavage, other possible cleavage sites were also sought on the serine peptidase of the present invention. Residues 116-120 (TSPGL) ( Figure 5 e) may be an additional cleavage site since it also contains a "TS" sequence.

[0119] To verify the hypothesis, the present invention constructed two other mutants: SpBcA (30-601aa)-mut-B, in which residues 116-120 (TSPGL) were mutated to AAAAA, and the double-site mutant SpBcA (30-601aa)-mut-AB, in which peptides 100-103aa and 116-120aa were mutated to alanine ( Figure 5 b). The protease activity using casein as substrate showed that the activity of SpBcA(30-601aa)-mut-B was significantly reduced, while the activity of SpBcA(30-601aa)-mut-AB was between SpBcA(30-601aa)-mut-A and SpBcA(30-601aa)-mut-B ( Figure 5 c). This indicates that SpBcA residues 116-120aa (TSPGL) may be the self-cleavage site. In summary, the present invention has discovered an inhibitory domain that is different from the inhibitory domains of other proteases, revealing the autoinhibitory mechanism of serine peptidase SpBcA.

[0120] Analysis of the ability of SpBcA to degrade different substrates in vitro

[0121] Since the serine peptidase SpBcA (30-601aa) has the highest activity among these three proteins, the degradation ability of SpBcA on different substrates was tested. Its ability to degrade human hemoglobin, fibrinogen, gelatin (a key component of host cells) and the antimicrobial peptide LL-37 in the antimicrobial peptide family was evaluated. The results showed that the serine peptidase SpBcA (30-601aa) could significantly degrade fibrinogen ( Figure 7 a) LL-37( Figure 7 b) and gelatin ( Figure 7 c). In addition, fibrinogen began to be significantly degraded within 5 minutes. In contrast, hemoglobin was cleaved much less efficiently ( Figure 7 d).

[0122] In summary, the present invention identified three subtilisin-like proteases SpBcA, SpBcB and SpBcC in B.cardium, and analyzed their substrates, protein structures and functions. SpBcA and SpBcB have good serine peptidase activity, can tolerate high temperatures (the optimal enzyme reaction temperature is 60°C) and have good activity under alkaline conditions. Further, the present invention found that SpBcA is produced in the form of a zymogen and undergoes self-cleavage. There is an inhibitory propeptide at the N-terminus, which, as a molecular chaperone, can inhibit its activity and show a new activity mechanism. In addition, SpBcA serine peptidase can degrade a variety of substrates such as casein, fibrinogen, antimicrobial peptide LL-37, gelatin, etc. It can be seen that the serine peptidase from B.cardium is a new type of serine peptidase with potential industrial and medical application value.

[0123] Amino acid sequence information used in the present invention:

[0124] SbC

[0125]

[0126] SpBcB

[0127]

[0128] SpBcC

[0129]

[0130] Matters not covered by the present invention are known technologies.

[0131] The above embodiments are only for illustrating the technical concept and features of the present invention, and their purpose is to enable people familiar with the technology to understand the content of the present invention and implement it accordingly, and they cannot be used to limit the protection scope of the present invention. Any equivalent changes or modifications made according to the spirit of the present invention should be included in the protection scope of the present invention.

Claims

1. A serine peptidase, characterized in that The serine peptidase has: (a1) the amino acid sequence shown in any one of SEQ ID NOs. 1-3; (a2) a protein derived from the amino acid sequence shown in any one of SEQ ID NOs. 1-3, wherein one or more amino acid residues are substituted and / or deleted and / or added and the protein has the same or similar activity as the amino acid sequence shown in any one of SEQ ID NOs. 1-3; (a3) proteins encoded by other genes having an amino acid sequence composition similarity of more than 90% with that of SEQ ID NO. 1-3 and having the enzyme activity of any one of SEQ ID NO. 1-3.

2. The serine peptidase according to claim 1, characterized in that In (a2), the serine peptidase is a truncated variant of serine peptidase, specifically SpBcA (30-601aa) (i.e., having the amino acid sequence from positions 30 to 601 as shown in SEQ ID NO.1), SpBcB (30-601aa) (i.e., having the amino acid sequence from positions 30 to 601 as shown in SEQ ID NO.2) and SpBcC (1-476aa) (i.e., having the amino acid sequence from positions 1 to 476 as shown in SEQ ID NO.3).

3. A nucleic acid molecule, characterized in that The nucleic acid molecule encodes the serine peptidase according to claim 1 or 2.

4. A recombinant expression vector, characterized in that: The recombinant expression vector comprises the nucleic acid molecule of claim 3.

5. A host cell, characterized in that The host cell contains the recombinant expression vector of claim 4 or has the nucleic acid molecule of claim 3 integrated into its chromosome, or expresses the serine peptidase of any one of claims 1-2.

6. A method for preparing the serine peptidase according to claim 1, characterized in that: include: Cultivating the host cell according to claim 5 to express the serine peptidase; and isolating and purifying the serine peptidase.

7. Use of the serine peptidase according to claim 1 or 2, the nucleic acid molecule according to claim 3, the recombinant expression vector according to claim 4, or the host cell according to claim 5 in any one or more of the following: (a) Substrate degradation; (b) Detection of pathogens or preparation of products for detection of pathogens; (c) Research on the virulence mechanism of pathogens; (d) Screening or preparing pathogen inhibitors.

8. The use according to claim 7, characterized in that In application (a), the substrate includes but is not limited to casein, gelatin, fibrinogen and LL-37.

9. The use according to claim 7, characterized in that: In the applications (b) to (d), the pathogen is Bergeyella sp, further Bergeyella cardium.

10. The use according to claim 7, characterized in that: In (b), the product is a detection kit, detection device or equipment; In the above (d), the pathogen inhibitor is a medicine, a health product, a food, a sanitary product or an (environmental) disinfection product.

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