Molecular Cloning, Expression and Characterization of the Porcine &amp;#946; Defensin 2 in E. coli

Li, Chunli; Zhao, Yan-cong; Song, Xin; Huang, Xian-xian; Zhao, Weidong

doi:10.2174/0929866511320060010

Cited by 17 publications

(35 citation statements)

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“…pBD2 is highly expressed in the kidney, liver, and tongue (Chen et al, 2010). Moreover, pBD2 shows high antimicrobial activity against bacteria, including multi-resistant bacteria, which makes it a good candidate for antibiotics (Zhang et al, 2000;Veldhuizen et al, 2008;Li et al, 2013).…”

Section: Introductionmentioning

confidence: 99%

Codon optimization enhances the expression of porcine β-defensin-2 in Escherichia coli

Gao

Zhao

et al. 2015

Genet. Mol. Res.

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ABSTRACT. Porcine β-defensin-2 (pBD2) is a cationic antimicrobial peptide that has therapeutic potential. The amount of pBD2 in nature is limited, and the expression of pBD2 in Escherichia coli is low, probably because a different gene codon is used by prokaryotic organisms to that used by eukaryotes. Codon preference optimization is one of the ways to increase heterologous expression of pBD2. To achieve high expression of pBD2, the pBD2 gene was redesigned according to the preferred codon in E. coli without altering the amino acid sequence. The optimized gene was inserted into expression vector pET-30a and transformed into E. coli BL21 (DE3) plysS. Our results showed that pBD2 was expressed as His-Tag fusion protein at a level that was approximately 4-6 times greater than from the native gene, based on total protein expression. Expressed fusion pBD2 showed antimicrobial activity against both E. coli and Staphylococcus aureus. Moreover, pBD2 showed weak hemolytic activity and strong heat resistance. These results indicate that fusion pBD2 is functional and has similar properties to those of pBD2 from the native gene. Our current study demonstrated that codon optimization could enhance pBD2 expression in E. coli without altering its function. Therefore, the expression of 4979 Codon optimization enhances pBD2 expression in E. coli ©FUNPEC-RP www.funpecrp.com.br Genetics and Molecular Research 14 (2): 4978-4988 (2015) pBD2 after codon optimization in heterologous host cells might be useful and is worthy of further research.

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Section: Introductionmentioning

confidence: 99%

Codon optimization enhances the expression of porcine β-defensin-2 in Escherichia coli

Gao

Zhao

et al. 2015

Genet. Mol. Res.

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“…Studies showed pBD2 to have high antimicrobial activity against bacteria, including multidrug-resistant bacteria isolated from sick fowl (Veldhuizen et al, 2008;Li et al, 2013a), which makes it good candidate for antibiotic development. Furthermore, pBD2 showed low hemolytic activity, high salt-tolerance, and strong thermal stability, which are important properties for its practical application (Li et al, 2013a). However, the anti-bacterial mechanism of pBD2 and its role in the immune system are unclear.…”

Section: Discussionmentioning

confidence: 99%

“…His-pBD2 was expressed by inducing the Escherichia coli strain BL21(DE3)-pET-pBD2, which was constructed in our laboratory, with isopropyl β-D-1-thiogalactopyranoside (IPTG), and the protein was purified as previously described (Li et al, 2013a;Gao et al, 2015). The purified his-pBD2 was analyzed by SDS-PAGE, and used for the preparation of antibodies.…”

Section: Expression and Purification Of His-pbd2mentioning

confidence: 99%

“…Based on BL21(DE3)-pET-pBD2, which was constructed in our lab, his-pBD2 was expressed and purified as previously described (Li et al, 2013a;Gao et al, 2015). The molecular weight of purified his-pBD2 was about 12 kDa, and the purity was greater than 90% according to Gel-ProAnalyzer (4.0) analysis (Figure 1).…”

Section: Purification Of His-pbd2mentioning

confidence: 99%

“…Few studies have been conducted on porcine defensins (Shi et al, 1999;Zhang et al, 2000;Sang et al, 2009). Previous research showed that porcine β-defensin 2 (pBD2) has high antimicrobial activity against Gram-negative and Gram-positive bacteria, including multidrug-resistant bacteria isolated from sick fowl, which makes it a good candidate for antibiotic development (Zhang et al, 2000;Veldhuizen et al, 2008;Li et al, 2013a). However, the antibacterial mechanism of pBD2 and its role in the immune system, remain unclear.…”

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Preparation of polyclonal antibody against porcine beta defensin 2 and identification of its distribution in tissues of pig

Bao¹,

Li²,

Zhang³

et al. 2015

Genet. Mol. Res.

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ABSTRACT. Porcine β-defensin 2 (pBD2) is an antimicrobial peptide in pigs that plays an important role in the immune system by preventing bacterial invasion. To produce an anti-pBD2 antibody, which is not commercially available, we expressed and purified a soluble, his-tagged version of pBD2 (his-pBD2). Purified pBD2 was injected into New Zealand white rabbits to generate polyclonal antiserum. Anti-pBD2 antibodies were purified by ammonium sulfate precipitation, followed by diethylaminoethyl cellulose ionexchange chromatography. The purified polyclonal antibody showed high sensitivity, with a titer as high as 204,800 by enzyme-linked immunosorbent assay, and it also showed high specificity for both his-pBD2 and native pBD2, as assessed by western blotting. Furthermore, immunohistochemistry analysis using the purified antibody revealed that pBD2 protein is distributed in the tongue, liver, kidney, small intestine, and large intestine of pigs. These results indicate that the prepared polyclonal antibody will be a useful tool for further studies of the function and mechanism of pBD2.

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High-level expression, purification and characterisation of porcine β-defensin 2 in Pichia pastoris and its potential as a cost-efficient growth promoter in porcine feed

Peng

An-ru²,

Feng³

et al. 2014

Appl Microbiol Biotechnol

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Porcine β-defensin 2 (pBD2), a recently discovered porcine defensin that is produced by the intestine, exerts antimicrobial activities and innate immune effects that are linked to intestinal diseases in pigs. Here, we report a codon-optimised protein corresponding to mature pBD2 cDNA that was expressed and purified in Pichia pastoris yeast. The highest amount of secreted protein (3,694.0 mg/L) was reached 144 h into a 150-h induction during high-density cultivation. Precipitation followed by gel exclusion chromatography yielded 383.7 mg/L purified recombinant pBD2 (rpBD2) with a purity of ~93.7 %. Two recombinant proteins of 5,458.5 and 5,258.4 Da were detected in the mass spectrum due to variation in the amino-terminus. The rpBD2 exhibited high antimicrobial activity against a broad range of pig pathogenic bacteria (minimal inhibitory concentration [MIC] 32-128 μg/mL); the highest activity was observed against Salmonella choleraesuis, Staphylococcus aureus and Streptococcus suis (MIC 32-64 μg/mL). However, rpBD2 also inhibited the growth of probiotics such as Lactobacillus plantarum, Bacillus subtilis and Saccharomyces cerevisiae, but at lower efficacies than the pathogens. Purified or unpurified rpBD2 also maintained high activity over a wide range of pH values (2.0-10.0), a high thermal stability at 100 °C for 40 min and significant resistance to papain, pepsin and trypsin. In addition, the activity of rpBD2 towards S. aureus was unaffected by 10 mM dithiothreitol (DTT) and 20 % dimethyl sulphoxide (DMSO). Our results suggest that pBD2 could be produced efficiently in large quantities in P. pastoris and be a substitute for traditional antibiotics for growth promotion in the porcine industry.

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Molecular Cloning, Expression and Characterization of the Porcine β Defensin 2 in E. coli

Cited by 17 publications

References 0 publications

Codon optimization enhances the expression of porcine β-defensin-2 in Escherichia coli

Codon optimization enhances the expression of porcine β-defensin-2 in Escherichia coli

Preparation of polyclonal antibody against porcine beta defensin 2 and identification of its distribution in tissues of pig

High-level expression, purification and characterisation of porcine β-defensin 2 in Pichia pastoris and its potential as a cost-efficient growth promoter in porcine feed

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