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. 2025 Jan 18;17(6):5066–5074. doi: 10.1007/s12602-025-10456-y

Recombinant Expression of a New Antimicrobial Peptide Composed of hBD-3 and hBD-4 in Escherichia coli and Investigation of Its Activity Against Multidrug-Resistant Bacteria

Nianzhi Ning 1, Han Yan 1,2, Binwang Cao 1, Wenjing Yu 1, Liangyan Zhang 1, Deyu Li 1, Tao Li 1,✉, Xingxiao Zhang 2,✉, Hui Wang 1,✉
PMCID: PMC12634730  PMID: 39825027

Abstract

Human β-defensin (HBD) has been recognized as a promising antimicrobial agent due to its broad-spectrum antimicrobial activity against various pathogens. In our previous work, we engineered a chimeric human β-defensin, designated H4, by fusing human β-defensin 3 and human β-defensin 4, resulting in enhanced antimicrobial activity and salt stability. However, the high cost of chemical synthesis due to the relatively large number of amino acids in H4 has limited its applications. To reduce production costs, we aimed to develop an alternative method using a prokaryotic expression system. We first optimized the codon usage of the H4 gene for prokaryotic expression and then cloned it into the pET32a vector, incorporating thioredoxin and enterokinase cleavage sites to minimize toxicity in host cells. The resulting plasmid was transformed into E. coli BL21, yielding a fusion protein (TrxA-EK-H4). Correct cleavage of TrxA-EK-H4 required the addition of urea as a denaturant in the dialysis buffer. However, on-column enzymatic cleavage obviated the need for denaturants and yielded higher-purity rH4. The antibacterial activity of rH4 against multidrug-resistant Acinetobacter baumannii was comparable to that of chemically synthesized H4. This study demonstrates a valuable strategy for efficient purification of challenging proteins and has significant implications for future biotechnological applications.

Keywords: Human β-defensin, Prokaryotic expression system, Acinetobacter baumannii, Cell infection

Introduction

Infections caused by multidrug-resistant bacteria acquired during hospital stays can have devastating consequences for patients, including prolonged illness, increased risk of complications, and higher mortality rates due to delayed or ineffective treatment [1]. Acinetobacter baumannii is a significant pathogen that can cause a variety of serious infections, such as acquired pneumonia, sepsis, and wound infections [2]. Moreover, infections caused by A. baumannii are also associated with an alarming increase in antimicrobial resistance. Epidemiological studies have revealed that a substantial proportion of hospital-acquired A. baumannii infections exhibit multidrug resistance [3–5]. There is an urgent need for innovative treatment approaches for Acinetobacter baumannii.

Human β-defensins (HBDs) are cationic antimicrobial peptides primarily secreted by epithelial cells of the skin, gastrointestinal tract, and urogenital tract [6]. These peptides play a vital role in protecting the body against invasive microbial infections [7]. Previous studies have demonstrated that HBDs exhibit strong antimicrobial activity against multidrug-resistant Acinetobacter baumannii. By combining the sequences of human β-defensin-3 (hBD-3) and human β-defensin-4 (hBD-4), we designed a chimeric human β-defensin, designated H4. This hybrid peptide exhibited improved antimicrobial activity and salt tolerance compared to its natural counterparts, making it a promising candidate for antimicrobial applications [8]. However, the high cost of chemical synthesis, due to the 45 amino acids in H4, has limited its application, prompting the need for more cost-effective production methods.

Several methods can be employed to obtain antimicrobial peptides, including direct extraction from animals and plants. However, this approach has several drawbacks, such as high cost, low yields, labor-intensive procedures, and inadequate purity [9]. In contrast, chemical synthesis methods like solid-phase peptide synthesis and fragment condensation can also be used, although these approaches are often hindered by high synthesis costs and inefficient chemical reactions [10, 11]. More promisingly, genetic engineering technology offers a viable alternative for producing antimicrobial peptides, which may eventually become the preferred method for obtaining purified human β-defensins [12].

Genetic engineering technology employed in the production of antimicrobial peptides encompasses both eukaryotic and prokaryotic expression systems [13]. The most widely utilized host organisms for these expression systems are Escherichia coli and Pichia pastoris [14]. These hosts have enabled researchers to economically produce antimicrobial peptides. The prokaryotic expression system based on Escherichia coli has been the primary platform for the production of recombinant antimicrobial peptides, with most being produced using this system [15].

In this study, we developed and evaluated an Escherichia coli–based prokaryotic expression system for producing chimeric human β-defensin H4, and subsequently assessed its antimicrobial activity using both bactericidal assays and in vitro cell infection assay.

Materials and Methods

Bacterial Strains and Culture Condition

Acinetobacter baumannii strain MDR-ZJ06 was isolated from the intensive care unit of the first affiliated hospital at Zhejiang University in Hangzhou, China [16]. Acinetobacter baumannii ATCC19606 was obtained from the American Type Culture Collection (ATCC). For culturing, all E. coli and A. baumannii strains were grown in Luria–Bertani (LB) medium (comprising 5 g/L yeast extract, 10 g/L tryptone, 10 g/L NaCl at a pH 7.2–7.4) at 37 ℃.

Construction of the E. coli Expression Vector pET32a-H4

Based on our previous report on the H4 sequence, we performed codon optimization to enhance its expression in Escherichia coli. The optimized H4 gene was then synthesized and inserted downstream of the enterokinase cleavage site within the pET32a plasmid, allowing for fusion expression with the TrxA protein. The constructed plasmid was verified by sequencing and then transformed into E. coli BL21.

Optimization of Induction Conditions

To enhance the expression level of the recombinant protein TrxA-EK-H4, we investigated the effect of Isopropyl β-D-1-thiogalactopyranoside (IPTG) concentration on protein induction. Specifically, we evaluated the impact of 0, 0.1, 0.2, 0.3, and 0.4 mM IPTG on protein expression at a cultivation temperature of 15 °C and an agitation speed of 120 rpm. After a 16-h induction period, the bacteria cells were harvested by centrifugation and then lysed using a lysis buffer. The resulting supernatant and pellet fractions were collected by centrifugation at 8000 g for 5 min and then subjected to SDS-PAGE analysis.

Purification of TrxA-EK-H4 Protein

E. coli BL21 harboring the pET32a-H4 plasmid was cultured in Luria–Bertani (LB) medium supplemented with 50 µg/mL of ampicillin. IPTG induction occurred when the culture reached an optical density of 0.6 at 600 nm (OD600). The bacterial cells were harvested by centrifugation at 8000 g for 20 min and resuspended in a solution (20 mM NaH2PO4·H2O, 0.5 M NaCl, 5 mM imidazole, and 6 M urea, pH 7.4). Subsequently, the bacterial cells were lysed using a low-temperature, ultra-high-pressure cell disruption apparatus (JN-MiniPro), and the recombinant TrxA-EK-H4 protein was purified by affinity chromatography using HisTrap FF columns (Cytiva).

Purification and Identification of Recombinant H4 (rH4) protein

Enzyme Cleavage in Dialysis Buffer

The purified TrxA-EK-H4 fusion protein was re-natured through dialysis against 25 mM Tris–HCl (pH 8.0). The re-natured protein was then incubated with 100 units of enterokinase (EK, Solarbio, Beijing) in a buffer consisting of Tris–HCl and 4 M urea at 25 °C for 16 h or more. After cleavage, the resulting product was subjected to Tris-Tricine-SDS-PAGE analysis and N-terminal sequencing.

On-Column Enzymatic Cleavage

After purification, the TrxA-EK-H4 fusion protein was subjected to dialysis against binding buffer (20 mM NaH2PO4·H2O, 0.5 M NaCl, 0.5 mM imidazole, 6 M urea, pH 7.4) and re-bound to a HisTrap FF column. The column was then equilibrated with equilibrium buffer (25 mM Tris–HCl, pH 8.0). Next, 100 units of enterokinase (Solarbio, Beijing) were added to 3 mL of Tris–HCl (25 mM, pH 8.0) and injected into the column using a syringe. The mixture was incubated at 25 °C for 16 h to allow cleavage. The tag-free rH4 protein was then eluted with equilibrium buffer, while undigested proteins and TrxA tags were eluted with elution buffer (20 mM NaH2PO4·H2O, 0.5 M NaCl, 5 mM imidazole, 6 M urea, pH 7.4). The identity of the eluted protein was confirmed by Tris-Tricine-SDS-PAGE analysis and N-terminal sequencing. Finally, the purified recombinant H4 protein was subjected to dialysis renaturation against phosphate-buffered saline (PBS) to restore its native conformation. The purity of rH4 was analyzed by silver staining gel electrophoresis and evaluated using BandScan 5.0 software. To verify that rH4 was correctly cleaved, the N-terminal sequence of rH4 was analyzed using a Thermo Q-Exactive Orbitrap mass spectrometer coupled with an EASY-nLC 1200 system.

Antibacterial Activity of rH4 Against A. baumannii MDR-ZJ06

The antibacterial activity of rH4 was evaluated using a modified microdilution assay [8]. Bacteria were cultured in an aerobic incubator at 37 ℃ until they reached an optical density of 0.6. The bacterial suspension was then diluted in PBS (pH 7.2). Each well received 50 µL of a 1000 CFU bacterial suspension and 50 µL of rH4 peptide at various concentrations. The plates were incubated at 37 °C for 2 h, after which 50 µL of the bacterial solution was plated on LB agar medium. Following incubation at 37 °C for 24 h, bactericidal activity was assessed by calculating the ratio of colony numbers in experimental groups to that of the control group (no peptide treatment). The 50% inhibitory concentration (IC50) was defined as the peptide concentration at which 50% of viable cells were killed. Each experiment was performed in triplicate.

Infection and Antibacterial Activity in A549

Human type II alveolar lung cells (A549) were cultured in a 75 cm2 flask (CAS, Shanghai, China) in DMEM medium without antibiotics, supplemented with 10% fetal bovine serum (Gibco). Prior to infection with Acinetobacter baumannii ATCC19606, the A549 cells were pre-incubated in the medium for 12 h. The cells (5 × 106 cells/mL) were harvested, diluted to a concentration of 2 × 105 cells/mL, and then 100 µL of the cell suspension was added to each well of a 96-well plate, resulting in approximately 2 × 104 cells per well. After a 12-h incubation period, the culture medium was removed, and the cells were washed twice with PBS. For bacterial infection, a serum-free DMEM solution was used at a multiplicity of infection (MOI) ratio of 1:100 (2 × 106 CFU/well). A control group without bacteria served as a positive control. Following a 2-h incubation period at 37 ℃, the cells were washed twice with PBS and then treated with different concentrations of defensins for 2 h at 37 ℃. A control group without defensins was used as a negative control, with three replicates in each group. After treatment, the cells were washed twice with PBS and lysed by adding 100 µL of a 0.5 × PBS solution containing 0.1% Triton X-100 for 10 min. The cells were then scraped with a pipette tip, and bacterial loads were enumerated on LB agar plate.

Statistical Methods

All data are presented as mean ± standard deviation (SD). Continuous variables were compared using Student’s t-test or Mann–Whitney U-test, as appropriate. All tests were two-tailed, with statistically significance defined as P < 0.05. Statistical analyses were performed using IBM SPSS Statistics version 19.0 and GraphPad Prism version 8.

Results

Plasmid Construction and Transformation

As illustrated in Fig. 1a, the fusion-expressed protein comprises the TrxA protein, a histidine tag, an enterokinase cleavage site, and the H4 peptide. The open reading frame of H4 is situated immediately downstream of the enterokinase cleavage site, ensuring the production of tag-free recombinant H4 protein following cleavage. Upstream of the enterokinase cleavage site lies the histidine tag, which facilitates affinity chromatography purification. The TrxA protein mitigates the toxicity of H4 to prokaryotic cells. After transformation, the pET32a-H4 plasmid was successfully introduced into Escherichia coli BL21 (Fig. 1b), and sequence analysis confirmed its accuracy (Fig. 1c).

Fig. 1.

Fig. 1

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Construction and verification of the prokaryotic expression vector for recombinant H4 (rH4). a A fusion expression scheme was designed to express rH4 protein in the pET32a plasmid. b Successful transformation of the pET32a-H4 plasmid into E. coli BL21(DE3) was confirmed by PCR, with Lane 1 showing the PCR product and Lane M serving as a marker. c Sequencing identification of the rH4 PCR product revealed the correct DNA sequence (highlighted in red), which includes the enterokinase site (marked in yellow)

Expression and Purification of rH4

The SDS-PAGE analysis showed that the expression level of TrxA-EK-H4 was highest when it was cultured at 15 °C, 120 rpm and induced by 0.4 mM IPTG in E. coli BL21 (Fig. 2a). After on-column enzymatic cleavage in 25 mM Tris–HCl buffer, a highly purified monomer of rH4 with a purity of 97.8% was successfully obtained (Fig. 2b). However, after purification of the TrxA-EK-H4 protein in Tris–HCl, enterokinase cleavage failed to yield the correct rH4 band (around 6 kDa). This was possibly due to the enterokinase being unable to access the target cleavage site. The addition of 4 M urea to the buffer resulted in the appearance of the rH4 band (Fig. 2c). These results indicate that a certain concentration of urea increases the accessibility of potentially hindered enterokinase cleavage site, thereby facilitating the cleavage of target protein. The purity of rH4 obtained by on-column cleavage was significantly higher than that obtained by in-dialysate buffer (Fig. 2d). Silver-stained gel showed that the purity of rH4 obtained by on-column cleavage reached up to 99.2%, which was similar to the purity (99.0%) of synthesized H4 (Fig. 2e). To verify that rH4 was correctly cleaved, N-terminal sequencing of rH4 was performed. The result showed that the N-terminal sequence of rH4 was GIINTLQK, which is consistent with the theoretical sequence, indicating that the cleavage was accurate (Fig. 3). Through on-column cleavage, approximately 14.6 mg of rH4 could be obtained from a 1-L fermentation culture with an overall recovery ratio of 57%.

Fig. 2.

Fig. 2

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Expression and identification of rH4 protein. a Induction of fusion proteins expression with varying IPTG concentrations. Lane M denotes the protein marker. Lanes 1–3 display the TrxA-EK-H4 expression under an IPTG concentration of 0 mM, including the total, supernatant, and sediment fractions. Lanes 4–6 show TrxA-EK-H4 expression at an IPTG concentration of 0.1 mM, including the total, supernatant, and sediment fractions. Lanes 7–9 show the expression of TrxA-EK-H4 at an IPTG concentration of 0.2 mM, including the total, supernatant, and sediment fractions. Lanes 10–12 show the expression of TrxA-EK-H4 at an IPTG concentration of 0.3 mM, including the total, supernatant, and sediment fractions. Lanes 13–15 show the TrxA-EK-H4 expression at an IPTG concentration of 0.4 mM, including the total, supernatant, and sediment fractions. b Tris-Tricine-SDS-PAGE analysis of the Trx-EK-H4 after on-column enterokinase cleavage. Lane M represents the protein marker, while Lane 1 shows the synthetic H4. Lanes 2 and 3 display the Tris–HCl equilibrium buffer after cleavage, and Lane 4 shows the impure TrxA-EK-H4 protein. Lanes 5–7 show undigested Trx-EK-H4 and Trx protein after cleavage, while Lane 8 represents the on-column cleavage production of rH4. c Effect of urea concentration on enterokinase cleavage. Lane 1 displays the TrxA-EK-H4. Lanes 2–8 show the digested protein at various urea concentrations (1, 0.01, 0.1, 2, 4 and 6 M, respectively). Lanes 9 represents the synthetic H4. d Tris-Tricine-SDS-PAGE analysis of TrxA-EK-H4 and its cleavage production. Lane 1 displays the TrxA-EK-H4. Lane 2 shows TrxA-EK-H4 digested in Tris–HCl equilibration buffer containing 4 M urea. Lane 3 shows the protein after cleavage and re-loading with the nickel column. Lane 4 shows the on-column enzymatic cleavage rH4. Lane 5 represents the synthetic H4. e Silver staining gel for purity analysis of rH4. Lane M represents the protein marker, while lane 1 shows the synthetic H4. Lane 2 shows the on-column enterokinase cleavage rH4, and lane 3 displays the Trx-EK-H4 protein

Fig. 3.

Fig. 3

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MS/MS spectrum of N-terminal peptide of rH4. The purified rH4 was digested by Trypsin, and the resulting N-terminal peptides were identified by mass spectrometry. The MS/MS spectrum confirmed that TrxA-EK-rH4 had been correctly cleaved by enterokinase

Bactericidal Activity of rH4 Against A. baumannii

The bactericidal activity of recombinant H4 (rH4) and synthetic H4 was evaluated against multidrug-resistant A. baumannii MDR-ZJ06. The results show that both rH4 and synthetic H4 exhibit similar potency, with IC50 values of 2.85 ± 1.05 µg/mL and 3.67 ± 0.62 µg/mL, respectively (P = 0.655). This suggests that the bactericidal activity of rH4 is comparable to that of synthetic H4 against this clinically relevant strain of A. baumannii (Fig. 4).

Fig. 4.

Fig. 4

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Antibacterial activity of rH4 and synthetic H4 against multidrug-resistant A. baumannii. Approximately 1 × 103 CFU bacterial cells were used in each experiment, and the experiments were repeated three times. The results are presented as the mean ± standard error of the mean (SEM). There was no significant difference in antibacterial activity between rH4 and synthetic H4 against strain MDR.ZJ-06 (P = 0.655). ns, no significant difference

Antibacterial Activity on Infected Cells

We further evaluated the bactericidal activity of rH4 using an Acinetobacter baumannii infection model in A549 cells [8]. In this model, approximately 2500 ± 320 ATCC19606 bacteria adhered to the surface of A549 cells after a 2-h infection. Treatment with 10 µg/mL rH4 resulted in nearly complete eradication of the infecting bacteria. As the concentration of rH4 and synthetic H4 decreased, the number of surviving bacteria on the cell surface increased. Notably, there was no significant difference in the IC50 values for cell infection between rH4 and synthetic H4 (3.17 ± 0.45 µg/mL vs. 2.77 ± 0.63 µg/mL, P = 0.861) (Fig. 5). These results indicate that rH4 and synthetic H4 exhibit similar bactericidal activity against A. baumannii adhering to the cell surface.

Fig. 5.

Fig. 5

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Bactericidal activity of rH4 against A. baumannii in A549 infection model. All experiments were conducted three times, and the results were reported as the mean ± SEM. There was no significant difference in antibacterial activity between rH4 and synthetic H4 against A. baumannii in A549 infection model (P = 0.861). In the PBS control group, the mean bacterial count per well of infected cells was 2500 ± 320 CFU. ns, no significant difference

Discussion

Human β-defensins play a crucial role in eliminating pathogenic microorganisms within the body by exerting lethal effects through their biological activity [17]. Our previous research demonstrated that fusing human β-defensin 3 and human β-defensin 4 resulted in the creation of a chimeric human β-defensin, designated as H4. Notably, this chimeric defensin exhibited enhanced antimicrobial activity and improved salt stability, making it a promising candidate for antimicrobial therapy [8]. However, the high cost of chemical synthesis has limited its application. To overcome this limitation, we explored the use of prokaryotic expression systems for peptide production, which offer a cost-effective alternative. The primary objective of this study was to utilize this system for the production of recombinant human β-defensin H4.

Escherichia coli is the most widely used prokaryotic host organism for peptide expression systems [18–20]. The pET32a contains a thioredoxin protein in its multicloning site region, which can help mitigate the toxic effects of expressed protein on the host cell. By fusing this thioredoxin protein with the target peptide, it is possible to reduce the toxicity associated with the expression of certain proteins. This approach has been successfully employed for expressing bacterial toxins, such as antimicrobial peptide CM4 [21] and mHD5 [22]. Therefore, we selected the pET32a plasmid as a suitable vector for expressing the H4 in this study.

For human β-defensins, researchers found that the number of amino acids has a significant impact on its activity. Specifically, Tao Li and his colleagues observed that truncating varying numbers of amino acids at the N-terminus of human β-defensin 3 resulted in substantial changes to its antimicrobial activity and salt tolerance [23]. Given this finding, it is crucial to remove the His tag from the expressed rH4 protein. To achieve this, we utilized enterokinase cleavage site, a commonly employed method for purifying fusion-expressed proteins that enables the production of completely pure target proteins [24]. In this study, an enterokinase cleavage site was inserted between H4 and TrxA, allowing us to obtain tag-free H4 through enzymatic cleavage.

During the enzyme cleavage step, we used two methods to cleave the fusion protein: column-based enzyme cleavage and dialysis-based enzyme cleavage in a container. The latter method involved dialyzing the fusion protein into the enzyme cleavage system, followed by cleavage in a container. In contrast to previous studies [22, 25], we found that cleavage in dialysis buffer lacking denaturants, such as urea, failed to yield the target protein band. This may be due to the DDDDK site being sterically hindered. In contrast, on-column cleavage in the absence of denaturants not only yielded the correct rH4 protein but also required fewer steps and resulted in higher protein purity.

MDR ZJ-06, a multidrug-resistant Acinetobacter baumannii clinical isolate recovered from blood, has been sequenced and exhibits resistance to carbapenems, cephalosporins, penicillins, β-lactamase inhibitor combinations, aminoglycosides, quinolones, chloramphenicol, trimethoprim/sulfamethoxazole, and minocycline, while remaining susceptible only to colistin [26]. Bactericidal assays showed that rH4 exhibited antibacterial activity against MDR ZJ-06 strain similar to that of synthetic H4. Using a cell infection model, we found that rH4 also exhibited similar bactericidal activity against adherent MDR ZJ-06 cells as synthetic H4, with no significant difference. These experimental results indicate that rH4 retained its fundamental biological activity. Previous study has demonstrated that linear variant of HBD-3 without disulfide bonds exhibit higher antimicrobial activity against certain bacteria, such as Proteus mirabilis, Escherichia coli, and Pseudomonas aeruginosa, compared to native HBD-3 [27, 28]. Proper folding is a common issue for proteins with multiple cysteine bridges when expressed in bacteria. Our engineered H4 based on HBD-3 was also difficult to form disulfide bonds when expressed in Escherichia coli. However, the antimicrobial activity of recombinant H4 was similar to that of synthesized H4, indicating that disulfide bond formation did not have a significant impact on the antimicrobial activity of H4 against multidrug-resistant Acinetobacter baumannii.

The development of peptide-based drugs faces major challenges, including formulation and delivery issues, as well as high production costs. In designing and developing peptide-based drugs, biological factors that affect peptide stability and bioavailability must be taken into consideration; for example, mucosal pH values and the presence of host or microbial proteases that can degrade candidate peptides [29]. The H4, a type of host-derived cationic antimicrobial peptide, also encounters similar challenges in antibacterial applications. Optimization will be explored in future studies, and we envision that this process might include (I) utilizing nano-carriers for delivery and conducting therapeutic efficacy studies in mouse infection models [30]; (II) developing compounds using H4 combined with non-biological materials to create antibacterial coatings [31]; (III) exploring methods for scale recombinant production[32]; and (IV) evaluating long-term stability and safety profiles.

In summary, our study demonstrated that recombinant human β-defensin (rH4) could be efficiently expressed using a prokaryotic expression system and purified to high purity through column-based enzymatic cleavage. The resulting rH4 exhibited antibacterial activity against multidrug-resistant similar Acinetobacter baumannii to that of synthetic H4. Our study provides a valuable strategy for the efficient purification of similar proteins that are challenging to cleave.

Author Contribution

HW, XXZ and TL designed and supervised the experiments. NZN, HY, BWC, WJY, LYZ, and DYL performed the experiment and data analysis. NZN and HY wrote the manuscript. All authors contributed to the article and approved the submitted version.

Funding

Funded by the State Key Laboratory of Pathogen and Biosecurity (SKLPBS2228).

Data Availability

No datasets were generated or analysed during the current study.

Declarations

Ethical Approval

Not required.

Conflict of Interest

The authors declare no competing interests.

Footnotes

Authorship notes: Nianzhi Ning, Han Yan, and Binwang Cao contributed equally to this work. Hui Wang, Xingxiao Zhang, and Tao Li contributed equally to this work. The order of authorship is based on contribution.

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Contributor Information

Tao Li, Email: litaobmi@126.com.

Xingxiao Zhang, Email: zhangxingxiao2017@163.com.

Hui Wang, Email: wanghui_dyx@hotmail.com.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Data Availability Statement

No datasets were generated or analysed during the current study.

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