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
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 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 path ogenic bacteria (minimal inhibitory concentration ”MIC” 32128 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 ℃ 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.
Porcine β-defensin 2 Pichiapastoris Expression Purification Antimicrobial activity Stability
Zixin Peng Anru Wang Qiuyue Feng Zhaoyue Wang Iskra Vitanova Ivanova Xiuping He Borun Zhang Weiping Song
State Key Laboratory of Direct-Fed Microbial Engineering,No. B-3 Northern Territory of Zhongguancun State Key Laboratory of Direct-Fed Microbial Engineering,No. B-3 Northern Territory of Zhongguancun CAS Key Laboratory of Microbial Physiological and Metabolic Engineering. Institute of Microbiology, Biological Faculty of Sofia University. Sofia Bulgaria 1000
国内会议
2015第二届北京保护健康协会微生态学学术交流会暨微生态产业研讨会(北京)
北京
英文
222-232
2015-12-01（万方平台首次上网日期，不代表论文的发表时间）