pGEM-T Easy Vector systems (Promega Corporation, Madison, WI, USA) were utilized to clone DEFB106. pTWIN1 (New England Biolabs, Inc., Beijing, China) was used as the expression vector. E. coli BL21 (DE3) [F-ompT hsdSB (rB-mB)-gal dcm (DE3); Stratagene, La Jolla, CA, USA] was used as a host for heterologous protein expression. E. coli K12D31, S. aureus CMCC26003 and C. albicans SC5314 were used for antimicrobial assays.

Human tissue array was purchased from Shanxi Chaoying Biotechnology Co., Ltd., (Xi’an, China). The epididymis slice was preserved by our laboratory, and was obtained from a healthy donor (age, 25 years) who died in a car accident. All the procedures were approved by the Ethics Committee of Shanghai Institutes for Biological Sciences Chinese Academy of Sciences (Shanghai, China) and informed written consent was obtained from the donor’s family.

The nucleotide sequence encoding the putative mature DEFB106 peptide containing 45 residues (FFDEKCNKLKGTCKNNCGKNE ELIALCQKSLKCCRTIQPCGSIID) was sub-cloned by PCR from the pGEM-DEFB106 plasmid and inserted into the pTWIN1 vector and digested with NcoI and XhoI (Fig. 1). The forward primer was 5′-GCGCCATGGACTTTTTTGATGAGAA-3′, (NcoI restriction site underlined) and the reverse primer was 5′-GGCCTCGAGTTAATCTATAATGCTC-3′ (XhoI restriction site underlined). Enzymes used in this study were purchased from Takara Co., Ltd. (Shiga, Japan).

All the experiments were conducted as in our previous study, but with minor modifications. (12). The vectors were confirmed with DNA sequencing and transformed into competent E. coli BL21 (DE3) cells. A single colony was incubated in 3 ml LB medium (Luria-Bertani broth) with 100 μg/ml ampicillin and cultured at 37°C and 83 × g in a shaking incubator overnight. The culture was transferred into 30 ml LB medium at a ratio of 2% (v/v). When the cell cultures (37°C, 83 × g) reached OD600 nm = 0.8, different concentrations of IPTG (0.3, 0.5 and 1.0 mM) were added to induce protein expression at different temperatures (37, 22 and 16°C). For a large scale culture, 250 ml LB medium was induced with 0.3 mM IPTG at OD600 = 0.8, 37°C. The cells were harvested by centrifugation (5,000 × g for 10 min at 4°C) and were re-suspended in 5 ml lysis buffer [20 mM sodium phosphate buffer, pH 8.5, 0.5 M NaCl, 0.1 mM EDTA and 0.1% Triton X-100 (v/v)] and lysed by sonication on ice. Following centrifugation at 13,000 × g for 30 min the supernatant was isolated and incubated with 1 ml chitin beads at 4°C for 30 min with gentle shaking. Subsequent to washes with 20 volumes of washing buffer (20 mM sodium phosphate buffer pH 8.0, 0.5 M NaCl, 0.1 mM EDTA and 0.1% Triton X-100), beads affiliated with protein samples were equally divided into 1.5 ml Eppendorf tubes, each containing 1.0 ml cleavage buffer (20 mM sodium phosphate buffer, 0.5 M NaCl and 0.1 mM EDTA) with varying pHs of 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0 and 7.5. Following incubation overnight (>18 h) at room temperature, beads were washed twice with cleavage buffer at the corresponding pHs, and the supernatant was collected after centrifugation at 500 × g for 5 min. A total quantity of 10 μl chitin beads were mixed with 10 μl 2X sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) loading buffer and bathed in boiling water for 10 min. Cleavage efficiency was estimated by SDS-PAGE and the Gel Image system version 4.00 (Tanon Science and Technology Co., Shanghai, China). For large-scale preparation of DEFB106, the culture volume was expanded to 1 liter and 5 ml beads were used. Following cleavage and elution, protein was concentrated by ultrafiltration with an Amicon Ultra 3K device (Millipore, Billerica, MA, USA) and applied to a Superdex-75 column by the Fast Protein Liquid Chromatography (FPLC) system (GE Healthcare, Wasukesha, WI, USA). Protein was eluted with 20 mM phosphate buffer, pH 7.2.

The molecular weight of the purified protein was determined by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectra recorded on a Bruker microFlex MALDI-TOF-MS spectrometer (ABI, Foster City, CA, USA). The peptide sample was mixed with an equal volume of matrix 2, 5-dihydroxybenzoic acid. The instrument was operated in the positive ion/linear mode with an accelerating voltage of 20 kV and scanning m/z range of 3,000–12,000.

Circular dichroism spectroscopy was performed on a Jasco J-810 spectropolarimeter (Tokyo, Japan) using a quartz cell of 1 mm path length at 25°C with a scanning range of 200–250 nm and scanning speed of 100 nm/min.

The antimicrobial activity of DEFB106 was determined using the colony forming unit (CFU) assay, as described previously (12,14). E. coli K12D31 grown in LB medium with 50 μg/ml streptomycin at 37°C, S. aureus CMCC26003 grown in Mueller-Hinton (MH) broth at 37°C and C. albicans SC5314 grown in YPD (2% tryptone, 1% yeast extract and 2% glucose, at 30°C) were used as the sensitive strains. When grown to the mid-phase, the strains were diluted to a concentration of 10⁶ CFU/ml in the cold sterile 10 mM sodium phosphate buffer (E. coli/S. aureus, pH 7.4; C. albican pH 6.8). When added to recombinant DEFB106 (10 mM PBS served as the negative control) of different concentrations, the strains were incubated for 3 h at 37°C (E. coli and S. aureus) and 30°C (C. albicans). The cultures were then serially diluted and cultured on LB (E. coli), MH (S. aureus) or YPD (C. albicans) agar plates in triplicate, respectively. Following incubation (E. coli and S. aureus at 37°C overnight, and C. albicans at 30°C for 24 h), the surviving colonies were manually counted, and the antimicrobial activity was calculated using the formula: % survival = (number of surviving colonies treatment with β-defensins/surviving colonies of control) × 100 (13).

Binding assays were performed in 96-well microplates at room temperature using the Octet Red 96 system (Fortebio, Menlo Park, CA, USA) (15,16). Prior to executing the binding assay, the recombinant protein was labeled with biotin at a molar ratio of 1:1 (biotin:protein) in 20 mM PBS at pH 7.2, and the unbound biotin was removed by Zeba™ spin desalting columns (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s instructions. In addition, Biosensor tips (Fortebio) were pre-wetted with PBS for 10 min. Subsequent to this, 50 μg/ml DEFB106-biotin, PBS and heparin (GE Healthcare Bio-Sciences AB, Uppsala, Sweden) or lipopolysaccharide [LPS, from E. coli O111:B4 (Sigma-Aldrich, St. Louis, MO, USA)] with serial dilutions were added to the 96-well microtiter plates at a volume of 200 μl/well. The subsequent measurement processes were all under computer control. Program procedures were established as follows: For the initial step, biosensors were washed in PBS buffer for 60 sec to form a baseline; DEFB106 labeled with biotin was loaded into biosensors for 450 sec; biosensors were moved into PBS buffer in another line of wells for 420 sec to remove any unbound DEFB106-biotin; the biosensors labeled with DEFB106-biotin were exposed to heparin or LPS of different concentrations for association, and were monitored for 300 sec; and then, the biosensors were moved back into PBS buffer to disassociate for another 300 sec. Data were fit globally and generated automatically by Octet User software (version 7.0; Fortebio).

To investigate whether the affinity of DEFB106 was based on its charges, 700 μg purified protein was incubated with 700 μl Heparin Sepharose CL-6B (GE Healthcare, Wasukesha, WI, USA) in 20 mM Tris-HCl, pH 7.4 at 4°C for 1 h. After washing three times with 0.5 ml buffer, heparin beads were divided into seven equal parts, and eluted by NaCl solution of different concentration ranging from 0 to 3.0 M. Then the beads were added to 100 μl SDS-PAGE loading buffer and analysed on 15% SDS-PAGE gels.

IHC was performed with human tissue arrays and human epididymis tissue sections to identify the distribution of DEFB106. Primary goat antibody against the C-terminus of DEFB106 was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA) and diluted with PBS containing 10% normal rabbit serum at a ratio of 1:50 and incubated with slices overnight at 4°C. The slices were washed five times every 5 min. The secondary antibody horseradish peroxidase-conjugated rabbit anti-goat (dilution 1:100; Boster Biological Technology, Ltd., Wuhan, China) was added and incubated with slices for 1 h at room temperature. The normal goat IgG served as a negative control. When stained with DAB (brown), the sections were counterstained with hematoxylin (blue).

The structure of the expression plasmid, pTWIN1 is shown in Fig. 1. The nucleotide sequence encoding mature DEFB106 was sub-cloned and inserted into the C-terminus of Ssp DnaB intein. The amino acid sequence of the final eluted polypeptide is shown beneath the plasmid map in Fig. 1. The first four amino acids, GRAM (glycine, arginine, alanine, methionine), were derived from pTWIN1 and the first glycine among them, adjacent to the intein, increased the cleavage efficiency of the fusion protein. In addition, one aspartate was added at the N-terminum of the mature DEFB106 to avoid frame shift.

The concentration of IPTG and the temperature were optimized to maximize the expression of the DEFB106 fusion protein. SDS-PAGE analysis showed that the DEFB106 fusion protein was highly soluble independent of the IPTG concentration and culture temperature (16, 22 and 37°C). The maximum expression yield was induced with 0.3 mM IPTG at 37°C (Fig. 2).

As the auto-cleavage mediated by intein is pH sensitive (17), it was determined that the optimal pH for auto-cleavage of intein-DEFB106 ranged from 6.0 to 6.5 (Fig. 3A). This finding was consistent with findings of previous studies, but different from that of other β-defensins, which range from pH 5.0 to 6.0 (12,13). The PAGE gel image analysis showed that ~80% of DEFB106 fusion protein was cleaved (Fig. 3B). The purity of the released DEFB106 protein was determined by Tricine-SDS-PAGE (Fig. 3B), a useful tool for the analysis of small molecular proteins with different charges (18). Following one step affinity purification, the purity of DEFB106 was up to 80%, and reached 95% with a yield of 3–5 mg/l after FPLC, analyzed by computer gray scan (Fig. 3B).

To analyze the quality of the recombinant protein, the molecular mass of the purified DEFB106 was identified by the MALDI-TOF mass spectrometry. As shown in Fig. 3C, the measured molecular mass was 5561.9, which was in accordance with the predicted theoretical molecular mass of 5562.6 Da. The predominant structure of DEFB106 in 20 mM phosphate buffer at pH 6.5 was estimated by Circular dichroism spectra (Fig. 3D) to be β-sheet (approximately accounted for 66.5%), which conformed to the typical β-sheet structure of β-defensins.

A broad spectrum of antimicrobial activity is a common feature of active β-defensins. The antimicrobial activity of the purified DEFB106 against E. coli K12D31, S. aureus and C. albicans was determined. The growth of all three bacterial strains was suppressed with increasing concentrations of DEFB106 protein (Fig. 4). E. coli K12D31 (LD₅₀ = 30 μg/ml) exhibited increased sensitivity to DEFB106 compared with C. albicans (LD₅₀ = 50 μg/ml) and S. aureus (LD₅₀ = 60 μg/ml).

Certain antimicrobial peptides have been shown to bind heparin and LPS (19). Moreover, it has been hypothesized that the affinity of several β-defensins and related peptides for heparin-derived disaccharide may be correlated with their antimicrobial activity (20). As defensins are cationic peptides, whereas heparin and LPS have negative charges, there may be high affinity between peptides and polysaccharides. The affinity of DEFB106 for heparin and LPS was measured based on the Bio-layer interferomertry (BLI) instrument (Octet; ForteBio). To deduce the binding affinity via kinetic rate constants (K_D = k_off/k_on, where K_D is the equilibrium dissociation constant, k_on the association rate constant, and k_off the dissociation rate constant), biosensor arrays were loaded with 50 μg/ml DEFB106-biotin and moved to heparin (concentrations ranged from 100 to 3.125 nM) or LPS (concentrations ranged from 1,000 to 62.5 nM), respectively. As shown in Fig. 5A and B, the affinity of DEFB106 binding with heparin and LPS was determined to be K_D = 5.08E-11 M and K_D = 1.90E-08 M. The affinity of DEFB106 for heparin was higher than that for LPS. The affinity of DEFB106 protein for heparin beads was also approved by its binding and elution from heparin beads with NaCl solution as shown in Fig. 5C, which demonstrated that the negative charges may be predominantly responsible for the affinity.

The localization of native DEFB106 protein was important in the investigation of the physiological and pathological functions. By using an antibody that recognized the C-terminus of DEFB106, IHC was used to identify the distribution of DEFB106 protein on human tissue arrays. Notably, native DEFB106 signals were observed in the bone marrow and skin, and particularly in the nuclei of epididymal cells (Fig. 6). In addition, DEFB106 signals in the skin appeared to be in the nuclei of hair follicle peripheral cells. As the high intensity of DEFB106 signals in the epididymis were consistent with the high mRNA level, IHC of human epididymis tissue slides were further stained to analyze the distribution of DEFB106. The positive signals were detected through the whole tissue from caput to cauda regions. The signals in the cauda region were more intensive than in the caput and corpus.