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Expression of mouse beta defensin 2 in escherichia coli and its broad-spectrum antimicrobial activity

Mature mouse beta defensin 2 (mBD2) is a small cationic peptide with antimicrobial activity. Here we established a prokaryotic expression vector containing the cDNA of mature mBD2 fused with thioredoxin (TrxA), pET32a-mBD2. The vector was transformed into Escherichia Coli (E. coli) Rosseta-gami (2) for expression fusion protein. Under the optimization of fermentation parameters: induce with 0.6 mM isopropylthiogalactoside (IPTG) at 34°C in 2×YT medium and harvest at 6 h postinduction, fusion protein TrxA-mBD2 was high expressed in the soluble fraction (>95%). After cleaved fusion protein by enterokinase, soluble mature mBD2 was achieved 6 mg/L with a volumetric productivity. Purified recombinant mBD2 demonstrated clear broad-spectrum antimicrobial activity for fungi, bacteria and virus. The MIC of antibacterial activity of against Staphylococcus aureus was 50 μg/ml. The MIC of against Candida albicans (C. albicans) and Cryptococcus neoformans (C. neoformans) was 12.5μg/ml and 25μg/ml, respectively. Also, the antimicrobial activity of mBD2 was effected by NaCl concentration. Additionally, mBD2 showed antiviral activity against influenza A virus (IAV), the protective rate for Madin-Darby canine kidney cells (MDCK) was 93.86% at the mBD2 concentration of 100 μg/ml. These works might provide a foundation for the following research on the mBD2 as therapeutic agent for medical microbes.


INTRODUCTION
Defensins are cationic antimicrobial peptides that exert a direct antimicrobial effect on invading microbes and are expressed by many different organisms (1). They have broadspectrum antimicrobial activity against a variety of organisms including bacteria, fungi, and enveloped viruses (2). Based on the spatial distribution of their six-cysteine residues and the connectivity of the disulfide bonds, defensins can be classified into three categories: -, -, and -defensins (3). -defensins are predominantly produced by barrier epithelial cells, they not only attack invading microbes, but may also modulate the 1181 Gong, T. et al. Expression of mouse beta defensin 2 in E. coli host's immune response and cell-mediated immunity via cytokine expression, providing an interface between innate and adaptive immune response (4). With the growing problem of pathogenic organisms which are resistant to conventional antibiotics, there is increased interest in the pharmacological application of antimicrobial peptides to treat infection.
Especially, with the increasing of occurrence of immunocompromised patients and increasing use of catheters and implants, the incidence of invasive fungal infections in humans has increased considerably. Candida bloodstream infections have steadily increased since the 1980s and account for 8-15% of all bloodstream infections (5). It had initiated a search for innate peptide antibiotics as alternative drug therapies because of the presence of fungal strains with multidrug resistance. Mouse -defensin 2 was firstly reported by Morrison et al. in 1999 (6), its gene sequence is similarly to another mouse and human airway beta defensins. In the past decade, the information on mBD2 function was very scarce, mBD2 was only reported to show activity against Staphylococcus aureus in vivo (7). The effects of mBD2 on pathogenic bacteria, fungi and virus had been poorly studied.
In the present paper, we established a prokaryotic expression system for producing recombinant mBD2 (rmBD2).
The conditions of cultivation and induction were optimized systematically for further improve mBD2 productivity. Purified rmBD2 showed not only antibacterial activity against Staphylococcus aureus and antifungal activity against C.
albicans and C. neoformans, but also antiviral activity against IAV. It might be as a therapeutic agent for the inhibition of microbe infection and avoiding the problems of acquired resistance.

Expression vector construction and protein expression
The cDNA for mature mBD2 was amplified using polymerase chain reaction (PCR) from the pcDNA3.

Product purification and analysis
Purification was performed on the ÄKTA Purifer system

Antibacterial assay
The antibacterial activity of rmBD2 was evaluated using a broth dilution assay (9).

Protein expression and purification
Because of its well-established expression systems, fast growth rate and low cost, the E. coli is used commonly as a host cell to express proteins. In our present study, the employed expression system is the E. coli pET/Origami, in which the E. coli Origami host strains have mutations in both the thioredoxin reductase and the glutathione reductase genes to enhance greatly cytoplasmic disulfide bond formation (13). Additionally, the Rosseta strains supply rare tRNAs to enhance the expression of eukaryotics proteins. In the expression vector pET32a-mBD2

Antifungal assays
Because of the toxicity of the currently used polyene antifungal drugs and the emergence of resistant candidal specie to azolebased agents, alternative treatment strategies are needed. Due to their unique mechanism of action, -defensins are expected to be ideal therapeutic agents for mitigating the problem of acquired resistance (19). Several plant defensins are inhibitory to C. albicans which can cause fatal infections in immunocompromised patients (20). In the present work, two assays were employed to examine the antifungal activity of In addition, the NaCl concentration also had an effect on the antifungal activity of mBD2. And the antifungal effect was suppressed by increasing NaCl concentration, when the concentration was 150 mM, rmBD2 was almost totally inactive ( Figure 4b). This salt-sensitive property was in accordance with another peptides isolated from mouse (9,16).