title: 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.
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.
authors: 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.
authors: Zixin
authors: Z
authors: Peng
authors: Anru
authors: A
authors: Wang
authors: Qiuyue
authors: Q
authors: Feng
authors: Zhaoyue
authors: Z
authors: Wang
authors: Iskra Vitanova
authors: IV
authors: Ivanova
authors: Xiuping
authors: X
authors: He
authors: Borun
authors: B
authors: Zhang
authors: Weiping
authors: W
authors: Song
mesh_terms: D000821:Animal Feed / Q000032:analysis*; D000818:Animals; D001419:Bacteria / Q000187:drug effects; D001424:Bacterial Infections / Q000382:microbiology / Q000662:veterinary; D008826:Microbial Sensitivity Tests; D010843:Pichia / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D015202:Protein Engineering; D013552:Swine / Q000235:genetics / Q000254:growth & development* / Q000378:metabolism; D013553:Swine Diseases / Q000382:microbiology; D023083:beta-Defensins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism* / Q000494:pharmacology
chemical_list: D023083:beta-Defensins