Gbp1-nt antibacterial protein and its efficient expression and purification method

Abstract

The application belongs to the technical field of protein purification, and provides GBP1-NT antibacterial protein and a high-efficiency expression and purification method thereof. Through accurate design and system optimization of a GBP1-N terminal 255-amino-acid fragment (GBP1-NT), the common industry problem of "poor stability and low yield" of natural antibacterial peptides is solved. The application not only realizes comprehensive quantitative breakthroughs in expression efficiency, antibacterial activity, stability and safety through accurate design of GBP1-NT, but also constructs an antibacterial peptide development strategy based on function analysis of natural immune effector molecular domains, provides a replicable and generalizable technical path for research and development of new antibacterial peptides, promotes the conversion process of natural immune molecules to clinical applications, and also provides a new path for sterilization by combining GBP1-NT protein and antibacterial peptides for sterilization.

Description

Technical Field

[0001] This invention belongs to the field of protein purification technology, and relates to GBP1-NT antimicrobial protein, specifically to GBP1-NT antimicrobial protein and its efficient expression and purification method. Background Technology

[0002] Globally, antibiotic resistance has become a major challenge threatening public health. Data from the World Health Organization shows that over one million people die annually from infections caused by drug-resistant bacteria. The development of traditional antibiotics lags far behind the rate of resistance development, thus necessitating the development of novel, highly effective, and low-resistance antimicrobial agents. Antimicrobial peptides, as a class of small molecule polypeptides widely present in organisms, exert their antimicrobial effects through multiple targets, such as disrupting bacterial cell membrane integrity, inhibiting the activity of key intracellular enzymes, or interfering with bacterial metabolism. They are less likely to induce resistance and have become an important research direction for replacing traditional antibiotics. Existing antimicrobial peptides mainly include naturally derived defensins, cathelicidins family peptides, and artificially modified peptides. Natural antimicrobial peptides are mostly isolated and purified from animals, plants, and microorganisms, exhibiting good biocompatibility, but generally suffer from poor stability, susceptibility to protease degradation, and short in vivo half-life. While artificially modified peptides have improved stability through amino acid substitution and cyclization modifications, they often suffer from increased cytotoxicity and high production costs, limiting their clinical translation and large-scale application.

[0003] Among innate immune effector molecules, guanylate-binding protein 1 (GBP1), a member of the interferon-induced GTPase family, has been found to possess potential antibacterial activity in recent years. Existing research indicates that GBP1 can inhibit intracellular bacteria by targeting the cell wall to lyse bacteria and recruiting non-classical inflammasomes. However, its application in the field of antimicrobial peptides is still in its early stages. Current research on GBP1 mainly focuses on its immunomodulatory functions, such as activating the NLRP3 inflammasome and mediating antimicrobial autophagy. The evaluation of its activity as an independent antimicrobial peptide, identification of its core functional domains, and structural optimization have not been thoroughly explored. Furthermore, the natural GBP1 protein suffers from problems such as large molecular weight, insufficient stability, and the tendency to form inclusion bodies during recombinant expression, making it difficult to directly apply as an antimicrobial peptide in clinical infection treatment and agricultural disease control.

[0004] Despite GBP1's potential as a novel antimicrobial peptide, existing technologies still face several key challenges: First, the mechanism of GBP1's antimicrobial activity is insufficiently understood. Its core functional domains and key amino acid sites for functioning as an antimicrobial peptide remain unclear, hindering targeted structural modification to enhance antimicrobial activity and reduce cytotoxicity. Second, efficient recombinant expression or chemical synthesis methods for GBP1 are lacking. Existing expression systems (such as the E. coli prokaryotic expression system) are prone to protein folding errors or loss of activity, while eukaryotic expression systems have high production costs, making large-scale production difficult. Third, research on GBP1's in vivo stability and bioavailability is inadequate. Natural GBP1 is easily degraded by proteases in vivo, has a short half-life, making it difficult to achieve effective antimicrobial concentrations, and its large molecular structure may result in poor tissue penetration, affecting in vivo antimicrobial efficacy.

[0005] Furthermore, the balancing issue between cytotoxicity and antimicrobial activity, a common problem in the development of existing antimicrobial peptides, has not yet been effectively resolved in GBP1 research. How to retain its antimicrobial activity while reducing damage to mammalian cells through reasonable structural modification remains a critical technical bottleneck that urgently needs to be overcome. Therefore, addressing the shortcomings of existing antimicrobial peptide and GBP1 research, this invention provides a novel antimicrobial peptide based on GBP1 protein and its efficient expression and purification method. Through structural modification, activity optimization, and efficient preparation techniques, it solves problems such as poor stability, high cytotoxicity, and high production costs, possessing significant theoretical and practical value. Summary of the Invention

[0006] The purpose of this invention is to provide GBP1-NT antimicrobial protein and its efficient expression and purification method, which combines GBP1-NT antimicrobial protein with bacitracin for sterilization, and provides a new efficient purification method and sterilization means for GBP1-NT antimicrobial protein.

[0007] To achieve the above objectives, the technical solution of the present invention is as follows:

[0008] This invention provides a method for expressing and purifying GBP1-NT antimicrobial protein. The method specifically includes: cloning the base sequence corresponding to amino acids 1-255 of human GBP1 protein into the BamHI and EcoRI restriction sites of the pGEX-6P-1 vector, transforming it into competent cells, picking single clones and transferring them to 10 mL LB medium containing ampicillin, culturing overnight at 37°C and 200 rpm, transferring them 1:100 to 1 L LB medium, and continuing to culture at 37°C and 200 rpm for 3-4 h until OD600 = 0.8, adding 0.1 mM IPTG, inducing at 16°C and 130 rpm for 14-16 h, harvesting the cells, resuspending the cells in Lysis buffer, sonicating and centrifuging, collecting the supernatant and passing it through a pre-packed GST column, eluting with an AKTA protein purification system, then adding PreScission protease at a mass ratio of 30:1 and digesting overnight at 4°C, followed by purification again on a GST column, and then concentrating the obtained protein by ultrafiltration.

[0009] The present invention also provides a method for obtaining GBP1-NT antimicrobial protein by the purification method described above.

[0010] The present invention also provides the application of the GBP1-NT antimicrobial protein as described above in sterilization.

[0011] Preferably, the effective bactericidal concentration of the GBP1-NT antimicrobial protein is not less than 50 μg / mL.

[0012] The present invention also provides the application of GBP1-NT antimicrobial protein, with or without bacitracin, in sterilization.

[0013] Preferably, the concentration of the bacitracin is not less than 100 μg / mL.

[0014] The beneficial effects of this invention are:

[0015] This invention solves the common industry problem of "poor stability and low yield" of natural antimicrobial peptides by precisely designing and systematically optimizing the first 255 amino acid fragments of the N-terminus of GBP1-NT. Its production efficiency and stability have been tested and found to be at a high level. Compared to natural antimicrobial peptides, GBP1-NT achieves a significant increase in soluble expression levels through a prokaryotic expression system incorporating a GST soluble tag, and high-purity protein can be obtained solely through affinity chromatography, greatly shortening the purification cycle. Compared to artificially modified peptides, GBP1-NT does not require complex chemical synthesis and modification; it can be directly expressed through genetic engineering, reducing production costs and exhibiting virtually no cytotoxicity, significantly improving biosafety and achieving the dual goals of low toxicity and high efficiency. Compared to full-length GBP1, GBP1-NT does not rely on cumbersome and costly mammalian cell eukaryotic expression systems; highly active protein can be obtained solely through an E. coli prokaryotic expression system. Furthermore, this innovative approach combines GBP1-NT with bacitracin, achieving synergistic bactericidal effects through a dual mechanism of "outer membrane disruption + intracellular inhibition." This successfully overcomes the technical limitation of bacitracin's inability to act on Gram-negative bacteria, providing a novel combination therapy for the clinical treatment of drug-resistant bacteria. In summary, this invention not only achieves a comprehensive quantitative breakthrough in expression efficiency, antibacterial activity, stability, and safety through the precise design of GBP1-NT, but also constructs a set of antimicrobial peptide development strategies based on the functional analysis of the structural domains of innate immune effector molecules. This provides a replicable and scalable technical path for the research and development of novel antimicrobial peptides, promoting the translation of innate immune molecules into clinical applications. Attached Figure Description

[0016] Figure 1 This is the antibacterial effect of hGBP1-NT in this invention;

[0017] Figure 2 This relates to the antibacterial effects of hGBP2 and mGBP2-NT proteins in this invention;

[0018] Figure 3 This invention demonstrates the antibacterial effects of GBP1-NT proteins truncated to different lengths.

[0019] Figure 4 This describes the bactericidal effect of hGBP1-NT combined with bacitracin in this invention. Detailed Implementation

[0020] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0021] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0022] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0023] Example

[0024] 1 Experimental Methods

[0025] 1.1 Protein purification

[0026] The base sequence corresponding to amino acids 1-255 of the human GBP1 protein (hereinafter referred to as hGBP1-NT) was cloned into the BamHI and EcoRI restriction sites of the pGEX-6P-1 vector. Amino acids 1-253 of the human or mouse GBP2 proteins (hGBP2-NT and mGBP2) were cloned into the expression vector using the same method. The plasmid was transformed into BL21(DE3) competent cells, and single colonies were picked and cultured overnight at 37°C and 200 rpm in 10 mL LB medium containing ampicillin. The plasmid was then transferred 1:100 to 1 L LB medium and cultured at 37°C and 200 rpm for 3-4 h until OD600 = 0.8. IPTG was added to a final concentration of 0.1 mM, and the cells were induced at 16°C and 130 rpm for 14-16 h before harvesting. The bacterial cells were resuspended in Lysis buffer (50 mM Tris-HCl, 150 mM NaCl, pH=8.5, 1% Triton X-100), sonicated, centrifuged, and the supernatant was passed through a pre-packed GST column. The cells were eluted using an AKTA protein purification system, and then PreScission protease was added at a mass ratio of 30:1 and digested overnight at 4°C. The cells were then purified again on a GST column, and the obtained protein was concentrated by ultrafiltration.

[0027] 1.2 In vitro sterilization test

[0028] Escherichia coli (ATCC11775) was cultured overnight in LB medium. After 1:60 transfer, the culture was continued until the OD600 reached approximately 0.6. 100 μL of the bacterial culture was serially diluted 10-fold, plated overnight, and colony forming units (CFU) were counted. After determining the CFU, the bacterial culture was diluted to 1 x 10⁻⁶ with PBS (pH 7.4). 8 CFU / mL. Figure 1The bacterial culture was incubated in vitro with 50 μg / mL bovine serum albumin (BSA), hGBP1-FL, hGBP1-NT, and hGBP1-CT in the presence or absence of 2 mM GTP. The reaction conditions were 37°C in a shaker at 150 rpm for 2 h. Subsequently, the six groups of samples were serially diluted 10-fold with PBS (pH 7.4). 10 μL of each sample was taken from each gradient and dropped onto LB or LB0N50 solid medium plates and incubated overnight at 37°C. Figure 2 The bacterial culture was incubated in vitro with 50 μg / mL BSA, hGBP1-NT, hGBP2-NT, and mGBP2-CT in the presence or absence of 2 mM GTP. Subsequent experimental procedures were the same as those described above. Figure 1 Same. LB solid medium formula: tryptone 10 g / L, yeast extract 5 g / L, sodium chloride 10 g / L, agar 20 g / L; LB0N50 solid medium formula: tryptone 5 g / L, yeast extract 2.5 g / L, agar 20 g / L.

[0029] 1.3 Amino acid length optimization

[0030] hGBP1 was truncated at the N-terminus every 5 amino acids (1-235, 1-240, 1-245, 1-250, 1-255, 1-260, 1-265, 1-270 amino-terminus), and purified using the same method as in 1.1. The purified protein was subjected to a sterilization test using the same method as in 1.2, and the experimental results were presented as percentages.

[0031] 1.4 hGBP1-NT combined with bacitracin

[0032] Escherichia coli (ATCC11775) was cultured overnight in LB medium. After 1:60 transfer, the culture was continued until the OD600 reached approximately 0.6. 100 μL of the bacterial culture was then serially diluted 10-fold, plated overnight, and colony-forming units (CFU) were counted. After determining the CFU, the bacterial culture was diluted to 1×10⁻⁶ with PBS (pH 7.4). 8 CFU / mL. Figure 4The bacterial culture was incubated in vitro with 50 μg / mL bovine serum albumin (BSA), hGBP1-FL, and hGBP1-NT in the presence or absence of 2 mM GTP at 37 °C and 150 rpm for 2 h. Each group was then incubated with 100 μg / mL bacitracin (purchased from HARVEYBIO, catalog number AT0245, CAS number: 1405-87-4) for another 2 h. Subsequently, the five samples were serially diluted 10-fold with PBS (pH 7.4). 10 μL of each dilution was dropped onto LB agar plates and incubated overnight at 37 °C to verify the bactericidal effect of hGBP1-NT combined with bacitracin.

[0033] 2. Experimental Results

[0034] 2.1 GBP1-NT protein has a good effect against Gram-negative bacteria.

[0035] Compared to the full-length and CT proteins, the hGBP1-NT protein has a cytotoxic effect on Escherichia coli, impairing its growth under low-salt conditions. Figure 1 The addition of GTP reverses this effect, indicating that the binding sites of hGBP1-NT protein, GTP, and bacteria overlap, and there is competition between them. GTP binding occurs before bacterial binding. Figure 1 Furthermore, similar truncation and antibacterial experiments were performed on hGBP2 and mGBP2, which are homologous to hGBP1, and similar results were obtained. Figure 2 This indicates that the antibacterial activity of GBP family NT proteins is widespread.

[0036] 2.2 Shortened GBP1-NT proteins of different lengths all exhibited antibacterial effects.

[0037] The above results show that the NT truncation is 1-255 amino acids. To determine whether this length is the optimal antibacterial length, NT proteins of different lengths were truncated. It was found that shorter proteins had weaker antibacterial effects, while longer proteins, due to having 1-255 amino acids, showed similar antibacterial effects. Figure 3 Therefore, GBP1-NT has antibacterial activity within a certain range, with the first 255 amino acids exhibiting the strongest effect.

[0038] 2.3 The bactericidal effect of hGBP1-NT combined with bacitracin

[0039] hGBP1-NT alone only showed impaired E. coli growth on LB0N50 medium, while when used in combination with bacitracin, a reduction in the number of E. coli was observed on normal LB medium, indicating that the combination of the two can directly kill E. coli. Figure 4 This enhances the body's resistance to Gram-negative bacteria.

[0040] In summary, through precise design and systematic optimization of the first 255 amino acid fragments of GBP1-N (GBP1-NT), the GBP1-NT antimicrobial protein was obtained, providing a replicable and scalable technical path for the development of novel antimicrobial peptides. Furthermore, the combined use of GBP1-NT protein and antimicrobial peptides for sterilization provides a new approach to sterilization.

[0041] The above-described embodiments are merely preferred embodiments of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the appended claims.

Claims

1. A method for expressing and purifying GBP1-NT antimicrobial protein, characterized in that, The method specifically includes: cloning the base sequence corresponding to amino acids 1-255 of human GBP1 protein into the BamHI and EcoRI restriction sites of the pGEX-6P-1 vector, transforming it into competent cells, picking single clones and transferring them to 10 mL LB medium containing ampicillin, culturing overnight at 37°C and 200 rpm, transferring them 1:100 to 1 L LB medium, and continuing to culture at 37°C and 200 rpm for 3-4 h until OD600=0.8, adding 0.1 mM IPTG, inducing at 16°C and 130 rpm for 14-16 h, harvesting the cells, resuspending the cells in Lysis buffer, sonicating and centrifuging, collecting the supernatant and passing it through a GST pre-packed column, eluting with an AKTA protein purification system, then adding PreScission protease at a mass ratio of 30:1 and digesting overnight at 4°C, followed by purification again on a GST column, and then ultrafiltration concentration of the obtained protein.

2. Obtain GBP1-NT antimicrobial protein by the purification method according to claim 1.

3. The application of the GBP1-NT antimicrobial protein as described in claim 2 in sterilization.

4. The application according to claim 3, characterized in that, The effective bactericidal concentration of the GBP1-NT antimicrobial protein is not less than 50 μg / mL.

5. The application of GBP1-NT antimicrobial protein combined with bacitracin as described in claim 2 in sterilization.

6. The application according to claim 5, characterized in that, The concentration of bacitracin is not less than 100 μg / mL.

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