High-level expression, purification and characterisation of porcine β-defensin 2 in Pichia pastoris

来源 :2015中国畜牧生物科技大会 | 被引量 : 0次 | 上传用户:muyue3122
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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 codonoptimised protein corresponding to mature pBD2cDNA 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 ofprobiotics 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 ℃ 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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