Facilitation of Expression and Purification of an Antimicrobial Peptide by Fusion with Baculoviral Polyhedrin in Escherichia coli

Bacterial strains.

E. coli TOP10 [F⁻ mcrAΔ(mrr-hsdRMS-mcrBC)Φ80lacZΔM15 ΔlacX74 deoR recA1 araD139 Δ(ara-leu)7697 galU galK rpsL (Str^r) endA1 nupG] (Invitrogen) was used to construct the recombinant plasmid and to assay the antibacterial activity of purified recombinant Hal18 toward a representative gram-negative strain. E. coli BL21 [F⁻ ompT hsdS_B (r_B⁻ m_B⁻) gal dcm] (Novagen) was used as a host strain for expressing the recombinant Polh-Hal18 fusion protein. Staphylococcus aureus (ATCC 6538p) was used to assay the antibacterial activity of purified recombinant Hal18 toward a representative gram-positive strain.

Plasmid construction.

Plasmid pPAP was constructed to encode the Polh-Hal18 fusion protein (Fig. 1A). First, a superlinker sequence between the BamHI and PstI sites of pSE420 (Invitrogen) was inserted into the multicloning site of pTrcHisC (Invitrogen) to construct pMPL100 (4,570 bp). The polh gene (735 bp) was obtained from the A. californica nuclear polyhedrosis virus genome (Pharmingen) using PCR amplification (primers 5′-AAGCTAGCATGCCGGATTATTCATACCGTCC-3′ and 5′-GTAGATCTATACGCCGGACCAGTGAACAG-3′) with a DNA thermal cycler (Mastercycler Personal 5332; Eppendorf Scientific) and inserted into the NheI and BglII sites of pMPL100 to construct pPolh (5,220 bp). The Hal18-encoding sequence (68 bp) was constructed by the hybridization of two synthetic oligonucleotides containing optimized E. coli major codons, BglII- and PstI-cleaved asymmetric cohesive ends, a hydroxylamine cleavage site, and a stop codon (Fig. 1B). This assembled Hal18-encoding sequence was inserted into the BglII and PstI sites of pPolh to construct a Polh-Hal18-encoding vector we designated pPAP (5,140 bp). The proteins encoded by recombinant plasmid pPAP contained hexahistidine (His₆) tags at their N termini for protein purification, and their expression was under the control of isopropyl-β-d-thiogalactopyranoside (IPTG)-inducible trc promoters.

Media and culture conditions.

For strain construction and protein expression, E. coli cells were grown in Luria-Bertani (LB) medium. The constructed transformant harboring the plasmid was stored at −80°C. Culture experiments were performed with 2 liters of LB medium supplemented with 50 μg/ml ampicillin (Sigma) in a 7-liter bioreactor (BioTron) at 37°C, with shaking at 250 rpm. Cell growth was monitored by measuring the optical density at 600 nm (OD₆₀₀) using a UV-visible spectrophotometer (UV-1601PC; Shimadzu). When cultures reached an OD₆₀₀ of 0.6, 1 mM (final concentration) IPTG was added to the culture broth for induction of the recombinant Polh-Hal18 fusion protein. At 5 h postinduction, culture samples were harvested for subsequent purification.

Purification of Polh-Hal18 fusion protein.

Harvested cells were resuspended in 3 ml of lysis buffer (buffer A; 50 mM Tris-HCl, 1 mM EDTA, 50 mM NaCl, and 1 mM phenylmethylsulfonyl fluoride, pH 8.0) per gram of cells (wet weight) and then treated with 80 μl of 10-mg/ml lysozyme per gram of cells. Samples were then frozen overnight at −20°C, after which they were thawed in water. The suspensions were passed twice through a high-pressure disrupter (Microfluidizer M-110Y; Microfluidics) at a flow rate of 450 ml/min and a pressure of 10⁸ Pa.

The insoluble inclusion bodies were isolated by centrifugation at 14,000 × g for 30 min at 4°C and then resuspended in buffer B (100 mM NaH₂PO₄, 8 M urea, 10 mM Tris-HCl, pH 8.0). The resulting protein solution was clarified by centrifugation at 15,000 × g for 30 min at 4°C. The supernatant was affinity purified with Ni²⁺-nitrilotriacetate-agarose resin (QIAGEN) and eluted with an elution buffer (100 mM NaH₂PO₄, 8 M urea, 10 mM Tris-HCl, pH 5.9) containing 250 mM imidazole. The protein samples were dialyzed against 10 mM Tris-HCI (pH 12), lyophilized, and solubilized in distilled water to a final concentration of 15 mg/ml.

Cleavage and recovery of recombinant Hal18.

The purified fusion proteins (15 mg/ml) were mixed with 3 volumes of hydroxylamine cleavage reaction buffer (0.22 M Tris base, 1.7 M hydroxylamine-HCl, 4.5 M guanidine-HCl, and 1% 1-propanol, pH 9.0, with the final solution adjusted to pH 8.8) (24) and incubated at 55°C for 24 h. The cleavage reaction samples were then dialyzed against 20 mM phosphate-buffered saline (PBS; pH 7.4) and centrifuged. The supernatant containing recombinant Hal18 was saved and lyophilized. Recombinant Hal18 was finally purified by reverse-phase high-pressure liquid chromatography (RP-HPLC) with the AKTAbasic system (Amersham Biosciences) using a 4.6- by 100-mm Chromolith performance RP18e column (Merck) with a linear gradient of 20 to 40% acetonitrile containing 0.1% (vol/vol) trifluoroacetic acid at a flow rate of 3 ml/min for 1 h. The absorbance at 215 nm was monitored. Each peak was collected, lyophilized, and tested for antimicrobial activity. Total protein amounts were quantified using a commercially available kit (Bio-Rad). The molecular weight was analyzed by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) on a Voyager system 4095 mass spectrometer (Applied Biosystems).

Chemical synthesis of control Hal18 peptide.

Standard Hal18 was chemically synthesized with an automated solid-phase peptide synthesizer (Pioneer; Applied Biosystems) (provided by the Korea Basic Science Institute) and purified to near homogeneity by RP-HPLC. Each peak was identified by MALDI-TOF mass spectrometry.

SDS-PAGE and Western blot analysis.

The cells were harvested by centrifugation at a maximal speed (13,000 rpm) for 10 min at 4°C, and the samples were mixed with protein sample buffer (0.5 M Tris-HCl [pH 6.8], 10% glycerol, 5% sodium dodecyl sulfate [SDS], 5% β-mercaptoethanol, 0.25% bromophenol blue) and heated to 100°C for 5 min. After centrifugation for 1 min, the samples were subjected to 12% (wt/vol) SDS-polyacrylamide gel electrophoresis (SDS-PAGE) or 16% (wt/vol) Tricine SDS-PAGE. The protein bands were detected by Coomassie blue staining (Bio-Rad), silver staining (Bio-Rad), or Western blotting. For Western blot analysis, the gel was transferred onto a nitrocellulose membrane (Amersham Pharmacia) using a Mini-Trans blot cell (Bio-Rad) and Bjerrum and Schafer-Nielsen transfer buffer (48 mM Tris, 39 mM glycine, 20% methanol) for 30 min at 15 V. Proteins of interest were detected using a polyclonal rabbit anti-His₆ antibody (1:2,500 [vol/vol]; Santa Cruz Biotechnology) and alkaline phosphatase-conjugated goat anti-rabbit IgG (1:50,000 [vol/vol]; Sigma). The membrane was then washed and developed colorimetrically with FAST Red TR/naphthol AS-MX (4-chloro-2-methylbenzene diazonium-3-hydroxy-2-naphthoic acid-2,4-dimethylanilide phosphate; Sigma). The membrane was scanned and its image was analyzed by Gel-Pro Analyzer software (Media Cybernetics).

Antimicrobial activity assays.

The antimicrobial activity of recombinant Hal18 was assayed using radial diffusion and MIC assays, with E. coli TOP10 serving as a representative gram-negative bacterium and S. aureus ATCC 6538p serving as a representative gram-positive bacterium. For the radial diffusion assay, the bacteria were cultured overnight in 3% tryptic soy broth (TSB; Difco), subcultured at 1:1,000 in prewarmed TSB, and grown for 2.5 h to mid-exponential growth phase at 37°C with shaking at 250 rpm. The bacterial concentration was adjusted to an OD₆₀₀ of 0.3, and 1% (vol/vol) culture broth was rapidly added to previously autoclaved, warm (42°C) PBS (pH 7.4) containing 3% powdered TSB and 1% (wt/vol) low-electroendosmosis type I agarose (Sigma). The bacteria were dispersed, and the agar was plated on 100-mm petri dishes. A 3-mm-diameter gel punch was used to make evenly spaced wells. For the assay, 10 μl of solution containing recombinant Hal18 (250 mM) in acidified distilled water (0.01% acetic acid) was added to each well. The plates were then incubated for 3 h at 37°C and overlaid with 10 ml of sterile agar (6% [wt/vol] TSB and 1% agarose). After the plates were incubated for 12 h at 37°C, the diameter of the clear zone surrounding each well was measured. For the MIC assay (14), the sample peptide was dissolved to various concentrations in deionized water and added to an equal volume of a test cell culture (10⁵ CFU/ml) with TSB medium in a 96-well tissue culture plate. The plate was incubated at 37°C overnight, and the lowest sample concentration yielding no bacterial growth was identified as the MIC.

Expression of Polh-Hal18 fusion protein.

The recombinant Polh-Hal18 fusion protein was successfully expressed and showed an apparent molecular mass of about 33.6 kDa (Fig. 2), which was in good agreement with the predicted size (∼28.6 kDa for Polh, ∼2 kDa for Hal18, and ∼3 kDa for the hexahistidine affinity ligand). The expression level of the Polh-Hal18 fusion protein reached about 40% of the total cellular proteins at 5 h postinduction (Fig. 2A, lane W). Previous results from our lab (34) and others (15) have indicated that Polh forms inclusion bodies in E. coli. To directly confirm that this was the case for the recombinant Polh-Hal18 fusion protein, cells were disrupted by sonication, and SDS-PAGE and Western blot analyses were performed on whole-cell lysates, insoluble cell debris, and soluble supernatant samples (Fig. 2). The Polh-Hal18 fusion band was clearly detected with a high density in the insoluble cell debris fraction (Fig. 2A and B, lanes IS) but was not detected in the soluble supernatant fraction (Fig. 2A and B, lanes S), demonstrating that the Polh-Hal18 fusion protein was contained in the insoluble inclusion body fraction.

Purification of recombinant Hal18.

Because the Polh-Hal18 fusion protein was completely insoluble in E. coli, the isolation of inclusion bodies allowed us to recover the protein with an initial purity of about 65% (data not shown). The Polh-Hal18 fusion protein included a His₆ affinity tag, and thus affinity purification under denaturing conditions was used to obtain samples with a higher purity. We found it difficult to elute the Polh-Hal18 fusion protein under low-pH conditions (pH 4.5), and thus 250 mM imidazole was added to the elution buffer (Fig. 3A, lane A). This allowed us to purify the recombinant Polh-Hal18 fusion protein with an ∼86% purity (Table 1).

The purified Polh-Hal18 fusion protein was cleaved by hydroxylamine in freshly prepared cleavage buffer (pH 9.0) containing 4.5 M guanidine-HCl. Optimization experiments showed that <50% of the fusion protein was cleaved in older buffer, guanidine-HCl-free buffer, or buffer that contained 6 M urea instead of guanidine-HCl (data not shown). Under the optimized conditions, most of the Polh-Hal18 fusion protein was successfully cleaved (Fig. 3B, lane 2). After dialysis of the protein against 20 mM PBS (pH 7.4), we removed the guanidine-HCl and decreased the pH to 7.4, causing the larger Polh-containing fragments to become insoluble while the smaller Hal18 peptides remained soluble. This special property of Polh allowed us to separate cleaved recombinant Hal18 peptides from precipitated Polh proteins by simple centrifugation (Fig. 3B, lanes 3 and 4). From approximately 30 mg of input purified fusion protein, we recovered about 0.82 mg of cleaved recombinant Hal18 showing a broad band upon gel analysis and having an ∼47% yield and ∼25% purity (Fig. 3B, lane 3, and Table 1). We then performed RP-HPLC to further purify the recombinant Hal18 to a higher degree than that obtained by simple dialysis and centrifugation (Fig. 3B, lane 4). The resulting peptide had a molecular mass of ∼2 kDa, which was almost identical to that of the synthetic version (1.93 kDa) (Fig. 3B, lane 5). The full isolation process generated 0.52 mg of recombinant Hal18, with a 30% recovery yield and 91% purity, from approximately 30 mg of input purified fusion protein, including 1.76 mg (theoretical value) of the recombinant Hal18 peptide (Table 1).

Antimicrobial activity of recombinant Hal18.

The bactericidal activity of recombinant Hal18 cleaved from the fusion protein and recovered by dialysis and centrifugation was qualitatively determined with a radial diffusion assay using E. coli as a representative gram-negative bacterium. The soluble supernatant from the dialysis and centrifugation step (soluble Hal18 peptide) showed a bactericidal activity (Fig. 4, spot 1) comparable to that of standard synthetic Hal18 (spot 2). In contrast, no bactericidal activity was seen for the insoluble pellet from the dialysis and centrifugation step (spot 3), the affinity-purified recombinant Polh-Hal18 fusion protein (spot 6), or the other negative controls (spots 4, 5, and 7). These results confirm that biologically active cleaved recombinant Hal18 peptides were present in the soluble fraction after dialysis and centrifugation. Similar results were obtained for radial diffusion assays using the gram-positive bacterium S. aureus (data not shown).

We then performed a MIC assay to quantitatively investigate the antimicrobial activity of recombinant Hal18. Recombinant Hal18 recovered by simple dialysis and centrifugation yielded MICs of 20 mM and 10 mM for E. coli and S. aureus, respectively (Table 2). The MICs of recombinant Hal18 further purified by RP-HPLC were 5 mM for E. coli and 1.25 mM for S. aureus. When we compared the MICs of RP-HPLC-purified recombinant Hal18 to those of peptides recovered by simple dialysis and centrifugation, we found that the MIC of the RP-HPLC-purified Hal18 was fourfold higher for E. coli and eightfold higher for S. aureus. Importantly, the biological activity of recombinant Hal18 was almost identical to that of the chemically synthesized Hal18 peptide used as a positive control (Table 2).