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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

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Abstract

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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Acknowledgments

We thank Dong Wu, Fei Xie, Yakun Wu and Huixin Chen for technical assistance. Grants from the Beijing Nova program of the Beijing Municipal Science & Technology Commission (no. Z111105054511018), the Major State Basic Research Development Program of China (973 Program) (no. 2012CB723702) and the Postdoctoral Science Foundation of Beijing Haidian supported this work.

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Authors and Affiliations

  1. State Key Laboratory of Direct-Fed Microbial Engineering, No. B-3 Northern Territory of Zhongguancun Dongsheng Science and Technology Park, Haidian District, Beijing, 100192, People’s Republic of China

    Zixin Peng, Anru Wang, Qiuyue Feng & Weiping Song

  2. CAS Key Laboratory of Microbial Physiological and Metabolic Engineering, Institute of Microbiology, Chinese Academy of Sciences, No. 1 Beichen West Road, Chaoyang District, Beijing, 100101, People’s Republic of China

    Zixin Peng, Zhaoyue Wang, Xiuping He & Borun Zhang

  3. Biological Faculty of Sofia University, Sofia, Bulgaria, 1000

    Iskra Vitanova Ivanova

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  1. Zixin Peng
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Correspondence to Anru Wang or Zhaoyue Wang.

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Anru Wang and Zhaoyue Wang contributed equally to this work.

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Peng, Z., Wang, A., Feng, Q. et al. 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. Appl Microbiol Biotechnol 98, 5487–5497 (2014). https://doi.org/10.1007/s00253-014-5560-7

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