Protein Expression and Purification 88 (2013) 47–53

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Protein Expression and Purification

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Cloning, expression and characterization of antimicrobial porcine b defensin1 in Escherichia coli

Chun-li Li ⇑, Ting-ting Xu, Rui-bo Chen, Xian-xian Huang, Yan-cong Zhao, Yuan-yuan Bao,Wei-dong Zhao, Zhen-yu ZhengDepartment of Animal and Veterinary Science, Henan Agricultural University, Zhengzhou 450002, Henan, The People’s Republic of China

a r t i c l e i n f o a b s t r a c t

Article history:Received 23 August 2012and in revised form 25 November 2012Available online 7 December 2012

Keywords:Antimicrobial activityPorcine b defensin 1Recombinant expressionE. coli

1046-5928/$ - see front matter � 2012 Elsevier Inc. Ahttp://dx.doi.org/10.1016/j.pep.2012.11.015

⇑ Corresponding author. Address: Department of AnHenan Agricultural University, No. 95, Wenhua RoadProvince, The People’s Republic of China. Fax: +86 37

E-mail address: hnclli@163.com (C.-l. Li).

Porcine b defensin 1 (pBD1) is a cationic antimicrobial peptide with three pairs of disulfide bonds.When expressed in insect cells, two polypeptides of different length (pBD138 and pBD142) accumu-lated, which differed by N-terminal truncation. However, only pBD142 was found in pigs. pBD142 hadstronger antimicrobial activity than pBD138, and thus could be a good candidate as a bactericidalagent for pigs. In this study, pBD142 gene, obtained by RT-PCR using the tongue total RNA as atemplate, was cloned into pET30a expression vector and transformed into Escherichia coli BL21(DE3) plysS. The recombinant pBD142 was expressed after induction by IPTG and purified by Histag affinity column with 90% purity. The recombinant pBD142 exhibited antimicrobial activity againstboth Gram-positive Staphylococcus aureus and Gram-negative E. coli including the multi-resistantE. coli. The minimum inhibitory concentrations (MICs) of recombinant pBD142 against tested bacteriawere 100 lg/mL for E. coli and 80 lg/mL for S. aureus. In addition, pBD142 showed low hemolyticactivity and high thermal stability. These properties are relevant for the biotechnological applicationsof the peptide.

� 2012 Elsevier Inc. All rights reserved.

Introduction

It is recognized that multi-resistant bacteria endangered thehealth of animal because of failure of the current antimicrobial re-agents [1–3]. It is a serious worldwide problem which is increasingand has implications for morbidity and mortality, including thepigs [3–5]. Therefore, it is very important to discover new effectiveantimicrobial reagents for pig diseases. Defensins are proposed assuch candidates for the treatments. Defensins are a family of lowmolecular weight peptides secreted by organism, and can protecthosts from a broad range of pathogens including bacteria, virusand fungus [6–9]. In addition, defensins have different antibacte-rial mechanisms against pathogens, which make them alternativesfor novel therapeutic drugs for infections [10–12].

Based on the spatial distribution of the six cysteine residues andtheir pairing of disulfide bonds, mammalian defensins are classifiedinto a, b and h defensins [7]. b defensins are the evolutionarily old-est one among them and represent the first-line defense to counter-act bacterial invasions [13]. For pigs, only b defensins have beenreported so far [13–14], and relatively little work has been done

ll rights reserved.

imal and Veterinary Science,, Zhengzhou 450002, Henan

1 63558180.

on the porcine b defensins [15]. Porcine b defensin 1 (pBD1)1 wasthe first discovered b defensin in pigs [16], and showed antimicrobialactivity against both gram-positive and gram-negative bacteria [17–18]. When expressed in insect cells, two polypeptides of differentlength accumulated. One was pBD138, the other was pBD142

(KNIGNSVSCLRNKGVCMPGKCAPKMKQIGTCGMPQVKCCKRK, under-lined was pBD138), which differed by N-terminal truncation.pBD142 had stronger antimicrobial activity than pBD138. However,only pBD142 was found in pigs [17], and mature pBD1 was 42 aminoacids according to the prediction of cleavage site between maturepeptide and signal peptide by software [19]. Although pBD1 genehad been expressed using a eukaryotic system and showed antimi-crobial activity, the yield of active product was lower and the growthof host cell was slower compared with that in a prokaryotic system(mainly in Escherichia coli) [17,20]. In addition, many kinds of defen-sins have been successfully expressed in E. coli [21–30]. In the presentstudy, pBD142 gene was cloned and expressed in E. coli BL21(DE3)plysS. The recombinant pBD142 was purified by affinity column,and then characterized by its antimicrobial activity against bacteria,hemolytic activity against erythrocytes and thermal stability. Our re-sults might give a deeper insight into the application of pBD142.

1 Abbreviations used: pBD1, porcine b defensin 1; LB, luria broth; MICs,minimalinhibitory concentrations; SD, standard deviation; ANOVA, analysis of variance; OD,optical density; sBD1, sheep beta defensin 1

48 C.-l. Li et al. / Protein Expression and Purification 88 (2013) 47–53

Materials and methods

Bacterial strain

E. coli DH5a (maintained in our laboratory) was used for clone.BL21 (DE3) plysS was used for expression (Invitrogen). The E. coliATCC 25922 and Staphylococcus aureus ATCC 25923 werepurchased from the Beijing Ordinary Microbiology Strain StoreCenter, Beijing, China, and E. coli isolate resistant to antibioticswhich was isolated and identified from the sick fowl was giftedby Dr. Gong-zheng Hu. The E. coli ATCC 25922, S. aureus ATCC25923 and E. coli isolate were used for testing antimicrobialactivity of pBD142.

Fig. 1. The construct of pET-p

Molecular cloning of pBD142 gene and construction of expressionplasmid

Total RNA samples were obtained from tongue tissue of 35-day-old healthy Yorkshire (Sus scrofa domistica) by Trizol methods[31]. The first strand cDNA was synthesized with RT-PCR Kitfollowing the manufacture’s instruction (Takara biotech Co. Ltd.Dalian, China). The double-strand cDNA was amplified usingforward primer 50-ATGAGACTCCACCGCCTCCT-30 and reverseprimer 50-CTGTGGGGTTGTTTCTTCACTTTC-30 for full length pBD1gene (designed according to Genbank accession No. AF031666.1).The product by PCR was cloned into pGEM-T easy vector, whichresulted in T-pBD1, then transformed into E. coli DH5a cells.

BD142 expression vector.

Fig. 2. SDS–PAGE analysis of expressed and purified recombinant pBD142. (A) SDS–PAGE analysis of expressed recombinant pBD142. Lane 1, 2 and 3 showed theexpressed protein after inducing BL-pET for 6 h, 4 h and 0 h as control, respectively.Lane 4 showed protein marker, Lane 5, 6 and 7 showed the expressed pBD142 afterinducing BL-pET-pBD1 for 0 h, 4 h and 6 h, respectively. The arrow indicted thetarget protein. (B) SDS–PAGE analysis of purified recombinant pBD142. Lane 1showed protein marker, Lane 2 and 3 showed purified pBD142. The arrow indictedthe target protein.

C.-l. Li et al. / Protein Expression and Purification 88 (2013) 47–53 49

The corresponding DNA sequence encoding pBD142 wasamplified by the same protocol using T-pBD1 as a template. For-ward primer was 50-GAATTCAAAAACATAGGAAATTCTGTTAGC-30

and reverse primer was 50-GCGGCCGCTTATTACTTCCTTTT-30. Twostop codons were added into the reverse primer (bold) and tworestriction sites EcoR I and Not I were in the forward and reverseprimers, respectively (underlined). The PCR product was clonedinto pGEM-T easy vector. Released by EcoR I/Not I digestion,pBD142 gene was inserted into EcoR I/Not I digested pET-30aexpression vector (Novagen), which resulted in plasmid pET-pBD1. The construct of expression vector was shown in Fig. 1.

Expression and purification of the recombinant pBD142

pET-pBD1 was transformed into E. coli BL21(DE3) pLysS cells toproduce the expression strain BL-pET-pBD1. A single colony ofE. coli BL-pET-pBD1 cells was inoculated into 2 mL Luria broth(LB) medium containing 100 lg/mL kanamycin at 37 �C overnight.Then the cells were inoculated into 200 mL fresh LB medium with100 lg/mL kanamycin until mid-log phase (absorbance at 600 nmabout 0.6–1.0) by addition of 1 mM final concentration of IPTG forinduction. The cells were cultivated for additional 4–6 h and har-vested by centrifugation at 8000g for 5 min.

The E. coli cells were re-suspended in 1/10 cold buffer (20 mMTris–HCl, 0.5 mM NaCl, 10 mM b mercaptoethanol, pH 8.0). Thenbacteria were lysed by sonication for 30 min and centrifuged at12,000g for 20 min at 4 �C to remove cell debris. The supernatantwas collected and was applied on the Ni–NTA agarose column(His tag affinity column) to purify recombinant protein. The targetprotein was eluted from the resin with Tris gradient buffer (20 mMTris–HCl, 0.5 mM NaCl, 10 mM b mercaptoethanol, pH 8.0) con-taining 0 mM, 20 mM, 60 mM, 120 mM imidazole, respectively.The purified protein was dialyzed against 10 mM phosphate buffer(2.7717 g Na2HPO4�12H2O and 0.35256 g NaH2PO4�2H2O dissolvedin 1000 mL distilled water, pH 7.4) at 4 �C overnight and concen-trated with polyethylene glycol 8000. The concentration of proteinwas determined by UV absorption at 280 nm and 260 nm (refer-ence 320 nm) by spectrophotometer (2800 UV/VIS, ShangHai).

Antimicrobial activity assay

The antimicrobial activity of pBD142 was evaluated by 96-wellturbidimetric method as described previously with some modifica-tions [27,32]. The bacterial cells were cultured in LB at 37 �C over-night, then diluted to 105–106 CFU/mL. 100 lL diluted bacteriawere mixed with 100 lL pBD142 solution (final concentrationswere 0–100 lg/mL) in polypropylene 96-well microtiter plate.The growth of bacteria was measured as increase of the opticaldensity at 630 nm (reference 405 nm) by a microplate reader (StatFax 2100, Awarenss Technology Inc. USA) after incubation at 37 �Cfor different time. Minimal inhibitory concentrations (MICs) weredefined as the lowest concentration of pBD142 at which no micro-bial growth by visual analysis at 37 �C for 16 h. All assays were car-ried out in triplicates. All values were expressed asmean ± standard deviation (SD).

Hemolytic activity of pBD142

The hemolytic activity of pBD142 was tested as described previ-ously with some modifications [33]. In brief, six milliliter porcinefresh blood was centrifuged at 1800g for 10 min at room tempera-ture. The pellet was washed three times with saline solution andre-suspended in same solution (in a 100-fold diluted concentra-tion). Aliquots (100 lL suspension) were added to 100 lL differentconcentrations of pBD142 (final concentrations were 0–100 lg/mL)in 96-well microtiter plate. After incubated at 37 �C for 1 h, the

plate was centrifuged at 1800g for 5 min, and 100 lL supernatantsof each well were transferred to a new 96-well microtiter plateseparately. Extractions were measured at 450 nm (reference630 nm) using a microplate reader. The percentage hemolysiswas calculated by comparison to the negative and positive controlcontaining no peptide or 1% Triton 100 [34–35].

Thermal stability of recombinant pBD142

The recombinant protein was expressed as described above, thecells were suspended in 1/10 cooled buffer (20 mM Tris–HCl,0.5 mM NaCl, pH 8.0) and lysed by sonication. After centrifugation,the supernatant was collected as cell crude extract and separatedinto different eppendorf tubes. The supernatant was treated at dif-ferent temperatures in water bath such as at 60 �C for 10 min and30 min, 80 �C for 10 min and 30 min, 100 �C for 10 min and 30 min,respectively. The purified pBD142 by Ni–NTA agarose column wasalso treated at different temperatures in water bath as describedabove.

After heat treatment, the sample was centrifuged again at12,000g for 10 min to remove the precipitates, respectively. Thesupernatant was collected and analyzed by SDS–PAGE. The activityof purified pBD142 after heating at 80 �C for 30 min was measuredby 96-well turbidimetric method described above.

Statistical analysis

Analysis of variance (ANOVA) was performed by software SPSS17.0. LSD or Dunnett method was used to compare treatmentmeans. Significance was defined at P < 0.05.

Results

Cloning of pBD142 gene and construction of expression vector

Full length of pBD1 cDNA was obtained by RT-PCR using tonguetotal RNA as a template. Positive recombinant plasmid T-pBD1was confirmed by PCR and sequencing. The result showed thecDNA sequence was same as expected (Genbank accession No.AF031666.1).

Mature pBD1 (pBD142) was amplified by PCR using T-pBD1 vec-tor as a template, and cloned into expression vector pET30a be-tween EcoR I and Not I restriction sites. The recombinant plasmidpET-pBD1 was confirmed by PCR and EcoR I/Not I digestion. The

Fig. 3. Antimicrobial activity of pBD142 against E. coli, S. aureus and multi-resistant E. coli, respectively. Different bacteria (E. coli, S. aureus and multi-resistant E. coli) werediluted to 105–106, then treated with different concentrations of pBD142 (0–100 lg/mL) in 96-well plate, respectively. The growth of bacteria was measured by optical densityat 630 nm (reference 405 nm) by a microplate reader after incubation for different time. All assays were carried out in triplicates. Data represent mean ± standard deviation.

Fig. 4. Hemolytic activity of pBD142. Fresh porcine blood was washed three timesand re-suspended in a 100-fold diluted concentration. Then the sample wasincubated with different pBD142 concentrations (0–100 lg/mL) in 96-well micro-titer plate (total volume was 200 lL). After incubated at 37 �C for 1 h, the plate wascentrifuged and 100 lL supernatants of each well were transferred to a new 96-wellmicrotiter plate separately. The release of hemoglobin from porcine erythrocyteswas measured at 450 nm (reference 630 nm) by a microplate reader. The percent-age hemolysis was calculated by the equation: hemolysis (%) = (OD of peptide-treated sample � OD of buffer-treated sample)/(OD of 1% Triton-100-treatedsample � OD of buffer-treated sample). All assays were carried out in triplicates.Data represent mean ± standard deviation.

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sequencing result showed that mature pBD1 DNA was 146 bp inlength including the enzyme site (data not shown) and recombi-nant plasmid pET-pBD1 was successfully constructed.

Expression and purification of the recombinant pBD142

The BL-pET-pBD1 was induced with 1 mM IPTG for 4–6 h andpBD142 was successfully expressed as a fusion protein with Histag in N-terminal, as showed in Fig. 2A. The molecular weight ofexpressed pBD142 was about 11.7 kDa according to analysis byGel-ProAnalyzer (4.0), a value close to the theoretical mass of10.235 kDa. The amino acid sequence of the recombinant pBD1was predicted as following:MHHHHHHSSGLVPRGSGMKETAAAKFERQHMDSPDLGTDDDDKAMAKNIGNSVSCLRNKGVCMPGKCAPKMKQIGTCGMPQVKCCKRK (underlined was the sequence of maturepBD142).

The fusion protein was easily purified using affinity column.Most of the protein was eluted from the column when the imidaz-ole concentration was 120 mM and showed high purity of greaterthan 90% (Fig. 2B).

Antimicrobial activity of pBD142

Antibacterial activity of recombinant pBD142 was tested by tur-bidimetric method. The growth of bacteria was measured by opti-cal density (OD) at 630 nm (reference 405 nm) at different timepoints (Fig. 3). The value of OD gradually increased with time ex-cept for at high pBD142 concentrations (at or above 80 lg/mL),and reduced with increasing pBD142 concentrations at the sametime points (P < 0.05). Significant difference was not only observedbetween the control and other different pBD142 concentrations byDunnett method for all the tested bacteria (P < 0.05), but also ob-served between high concentrations (at or above 80 lg/mL) andother different concentrations (P < 0.05). Furthermore, pBD142

showed similar activity against E. coli and multi-resistant E. coli(P > 0.05), and showed stronger antimicrobial activity against S.aureus than E. coli (P < 0.05). The results showed that MICs of re-combinant pBD142 were 100 lg/mL (about 10 lM) for E. coli and80 lg/mL (about 8 lM) for S. aureus.

Hemolytic activity of pBD142

Erythrocytes were collected from fresh porcine blood and incu-bated with different pBD142 concentrations in saline solution at37 �C for 1 h. The recombinant pBD142 had slight hemolytic activity

in vitro (Fig. 4). Less than 11% hemolysis was found at all differentconcentrations (0–100 lg/mL). The result indicated the fusion re-combinant pBD142 had low hemolytic activity.

Thermal stability of recombinant pBD142

In order to investigate thermal stability of recombinant pBD142,both cell crude extract and purified pBD142 were treated with dif-ferent temperatures for different duration, respectively. Heatingthe cell free extract significantly reduced protein concentrationto 47–52% (Fig. 5). There was no significant difference among the

Fig. 5. The protein concentrations of cell crude extract and purified pBD142 afterdifferent heat treatments. The black columns represented protein concentrations ofcell crude extract, while the blank ones represented the concentrations of purifiedpBD142. 1 showed the samples without heat treatment as control. 2–7 showed thesamples with different heat treatments, which were 60 �C for 10 min, 60 �C for30 min, 80 �C for 10 min, 80 �C for 30 min, 100 �C for 10 min, and 100 �C for 30 min,respectively. Different letters from the control at the top of column indictedsignificant differences between the heat treatment and the control for cell crudeextract (P < 0.05). Same letter indicted no differences between the heat treatmentand control for purified pBD142 (P > 0.05).

Fig. 7. Antibacterial activity of the purified pBD142 against S. aureus after heattreatment. The purified recombinant pBD142 (100 lg/mL) was heated at 80 �C for30 min, then cultivated with S. aureus in 96-well plate. The purified recombinantpBD142 (100 lg/mL) without heat treatment or no peptide was used as control,respectively. The growth of bacteria was measured by optical density at 630 nm(reference 405 nm) by a microplate reader after incubation for different time,respectively. All assays were carried out in triplicates. Data represent mean ± stan-dard deviation.

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samples after different heat treatments, but significant differencewas found between those with and without heat treatment(P < 0.05). SDS–PAGE analysis showed that most of the denaturedprotein in cell crude extract was not pBD142 but other protein, asshown in Fig. 6A.

The concentrations of purified pBD142 after different heat treat-ments were shown in Fig. 5. No significant difference was foundamong all the samples no matter with or without heat treatment(P > 0.05). SDS–PAGE analysis also proved it. Therefore the residualprotein did not change much (Fig. 6B).

The purified recombinant pBD142 (100 lg/mL) were heated at80 �C for 30 min and cultivated with S. aureus cells for 24 h. The re-sults indicated that the growth of bacteria was inhibited and thevalue of OD did not change much after 24 h (Fig. 7). There was

Fig. 6. SDS–PAGE analysis of cell crude extracts and purified pBD142 after different htreatments. Lane 1 showed the protein in cell crude exact without heat treatment. Lane 2treatments, which were 60 �C for 10 min, 60 �C for 30 min, 80 �C for 10 min, 80 �C for 30marker. The arrow indicted the target protein. (B) SDS–PAGE analysis of purified pBDtreatment. Lane 2–3 and lane 5–8 showed the residual protein after purified pBD142 w100 �C for 10 min, and 100 �C for 30 min, respectively. Lane 4 showed protein marker. T

no significant difference between the samples with and withoutheat treatment (P > 0.05). These data showed that the recombinantpBD142 protein expressed in E. coli cells was thermo-stable.

Discussion

In the last few years, some antimicrobial peptides were success-fully expressed in heterologous host cells, including E. coli, themost commonly used host cell because of its fast growth rate, eas-ily purified procedure, high yield of target protein and low cost.Porcine b defensin 1 is one of 30 porcine antimicrobial peptideswith a wide range of antimicrobial activity. In this study, pBD142

was successfully expressed in E. coli and had antimicrobial activityagainst the Gram-positive S. aureus and Gram-negative E. coli

eat treatments. (A) SDS–PAGE analysis of cell crude extracts after different heat–4 and lane 6–8 showed the residual protein in cell crude exact after different heatmin, 100 �C for 10 min, and 100 �C for 30 min, respectively. Lane 5 showed protein142 after different heat treatments. Lane 1 showed purified pBD142 without heatas treated at 60 �C for 10 min, 60 �C for 30 min, 80 �C for 10 min, 80 �C for 30 min,he arrow indicted the target protein.

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including the multi-resistant E. coli. The multi-resistant E. coli wasagainst cephalosporins (cefotaxime, cefoperazone), aminoglyco-sides (gentamycin, amikacin), and fluoroquinolones (ciprofloxacin,enrofloxacin) [36], which made pBD142 a promising candidate toprevent and cure porcine diseases caused by bacteria infection,especially for diseases caused by multi-resistant bacteria.

The minimum concentrations that required to inhibit bacteriawere 100 lg/mL for E. coli and 80 lg/mL for S. aureus, at whichthe recombinant pBD142 could inhibit the growth of bacteria for16 h or even longer. Although it was difficult to compare our re-sults with other pBD1 activity due to different experiment meth-ods and different tested strains [17,20], all the data showed thatpBD142 had antimicrobial activity against both Gram-negative bac-teria and Gram-positive one.

In the present study, the recombinant pBD142 was not cleavedby enterokinase to release the mature pBD142. It was reported thatmature hBD3 (human b defensin 3) and fusion hBD3 (with His tag)had similar antimicrobial activity against E. coli and S. aureus in ourlab [37]. The mature sheep beta defensin 1 (sBD1) and the sBD1with His tag on its C-terminus had similar antimicrobial activityagainst E. coli, S. aureus, Proteus vulgaris, Pseudomonas aeruginosa,and Shigella flexneri [38]. Others also reported that fusion defensinshad antimicrobial activities [39–42]. In addition, if the step ofcleavage and purification was deleted, the yield of pBD142 wouldincrease, which would reduce the cost and easily scale up. Somereported that N-terminal of defensins were important for theiractivities against microbes. If His tag or other tags were in theN-terminal, the peptides would lose their activities [21,24,43].Other reports suggested that N-terminal was not crucial for theactivities of defensins [37–42,44]. For pBD142, whether the fusionpart of pBD142 could potentially have an effect on activity neededfurther study.

Compared with the antibacterial activity of pBD142, the hemo-lytic activity of pBD142 was low, which was important for its usefor antimicrobial reagents. The recombinant pBD142 showed highthermal stability, which made easier for the fusion protein to bepurified when the cell free extract was heated first. Furthermore,it was a great advantage for the protein to be applied because ofits thermal stability.

Conclusion

In this study, pBD142 was successful cloned and transformedinto E. coli. The expressed target protein was purified by affinitycolumn with 90% purity and showed antimicrobial activity againstGram-positive S. aureus and Gram-negative E. coli including themulti-resistant E. coli. In addition, pBD142 showed low hemolyticactivity and high thermal stability. These results suggested thatpBD142 could be used as a potential antibacterial reagent in pigs.

Acknowledgments

The authors deeply appreciate Professor. Gong-zheng Hu forproviding the multi-resistant E. coli. The study is supported by Na-tional Natural Science Foundation of China (No. 31072015).

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