title: Optimization of expression conditions for a novel NZ2114-derived antimicrobial peptide-MP1102 under the control of the GAP promoter in Pichia pastoris X-33.
abstract: BACKGROUND
  The infections caused by antibiotic multidrug-resistant bacteria seriously threaten human health. To prevent and cure the infections caused by multidrug-resistant bacteria, new antimicrobial agents are required. Antimicrobial peptides are ideal therapy candidates for antibiotic-resistant pathogens. However, due to high production costs, novel methods of large-scale production are urgently needed.
  
  
  RESULTS
  The novel plectasin-derived antimicrobial peptide-MP1102 gene was constitutively expressed under the control of the GAP promoter. The optimum carbon source and concentration were determined, and 4% glucose (w/v) was initially selected as the best carbon source. Six media were assayed for the improved yield of recombinant MP1102 (rMP1102). The total protein and rMP1102 yield was 100.06 mg/l and 42.83 mg/l, which was accomplished via the use of medium number 1. The peptone and yeast extract from Hongrun Baoshun (HRBS, crude industrial grade, Beijing, China) more effectively improved the total protein and the yield of rMP1102 to 280.41 mg/l and 120.57 mg/l compared to 190.26 mg/l and 78.01 mg/l that resulted from Oxoid (used in the research). Furthermore, we observed that the total protein, antimicrobial activity and rMP1102 yield from the fermentation supernatant increased from 807.42 mg/l, 384,000 AU/ml, and 367.59 mg/l, respectively, in pH5.0 to 1213.64 mg/l, 153,600 AU/ml and 538.17 mg/ml, respectively in pH 6.5 in a 5-l fermenter. Accordingly, the productivity increased from 104464 AU/mg rMP1102 in pH 5.0 to a maximum of 285412 AU/mg rMP1102 in pH 6.5. Finally, the recombinant MP1102 was purified with a cation-exchange column with a yield of 376.89 mg/l, 96.8% purity, and a molecular weight of 4382.9 Da, which was consistent with its theoretical value of 4383 Da.
  
  
  CONCLUSIONS
  It's the highest level of antimicrobial peptides expressed in Pichia pastoris using GAP promoter so far. These results provide an economical method for the high-level production of rMP1102 under the control of the GAP promoter.
authors.affiliation: Key Laboratory of Feed Biotechnology, Ministry of Agriculture, 12 Zhongguancun Nandajie St., Haidian District, Beijing, 100081, P. R. China. rain_mry@126.com.|Gene Engineering Laboratory, Feed Research Institute, Chinese Academy of Agricultural Sciences, 12 Zhongguancun Nandajie St., Haidian District, Beijing, 100081, P. R. China. rain_mry@126.com.
authors.forename: Ruoyu
authors.initials: R
authors.lastname: Mao
authors.affiliation: Key Laboratory of Feed Biotechnology, Ministry of Agriculture, 12 Zhongguancun Nandajie St., Haidian District, Beijing, 100081, P. R. China. tengda@caas.cn.|Gene Engineering Laboratory, Feed Research Institute, Chinese Academy of Agricultural Sciences, 12 Zhongguancun Nandajie St., Haidian District, Beijing, 100081, P. R. China. tengda@caas.cn.
authors.forename: Da
authors.initials: D
authors.lastname: Teng
authors.affiliation: Key Laboratory of Feed Biotechnology, Ministry of Agriculture, 12 Zhongguancun Nandajie St., Haidian District, Beijing, 100081, P. R. China. wangxiumin@caas.cn.|Gene Engineering Laboratory, Feed Research Institute, Chinese Academy of Agricultural Sciences, 12 Zhongguancun Nandajie St., Haidian District, Beijing, 100081, P. R. China. wangxiumin@caas.cn.
authors.forename: Xiumin
authors.initials: X
authors.lastname: Wang
authors.affiliation: Key Laboratory of Feed Biotechnology, Ministry of Agriculture, 12 Zhongguancun Nandajie St., Haidian District, Beijing, 100081, P. R. China. zhangyong198729@163.com.|Gene Engineering Laboratory, Feed Research Institute, Chinese Academy of Agricultural Sciences, 12 Zhongguancun Nandajie St., Haidian District, Beijing, 100081, P. R. China. zhangyong198729@163.com.
authors.forename: Yong
authors.initials: Y
authors.lastname: Zhang
authors.affiliation: Key Laboratory of Feed Biotechnology, Ministry of Agriculture, 12 Zhongguancun Nandajie St., Haidian District, Beijing, 100081, P. R. China. jiaojian0101@163.com.|Gene Engineering Laboratory, Feed Research Institute, Chinese Academy of Agricultural Sciences, 12 Zhongguancun Nandajie St., Haidian District, Beijing, 100081, P. R. China. jiaojian0101@163.com.
authors.forename: Jian
authors.initials: J
authors.lastname: Jiao
authors.affiliation: Key Laboratory of Feed Biotechnology, Ministry of Agriculture, 12 Zhongguancun Nandajie St., Haidian District, Beijing, 100081, P. R. China. xintao496@sina.com.|Gene Engineering Laboratory, Feed Research Institute, Chinese Academy of Agricultural Sciences, 12 Zhongguancun Nandajie St., Haidian District, Beijing, 100081, P. R. China. xintao496@sina.com.
authors.forename: Xintao
authors.initials: X
authors.lastname: Cao
authors.affiliation: Key Laboratory of Feed Biotechnology, Ministry of Agriculture, 12 Zhongguancun Nandajie St., Haidian District, Beijing, 100081, P. R. China. 2681298635@qq.com.|Gene Engineering Laboratory, Feed Research Institute, Chinese Academy of Agricultural Sciences, 12 Zhongguancun Nandajie St., Haidian District, Beijing, 100081, P. R. China. 2681298635@qq.com.
authors.forename: Jianhua
authors.initials: J
authors.lastname: Wang
mesh_terms: D023181:Antimicrobial Cationic Peptides / Q000096:biosynthesis* / Q000235:genetics; D002244:Carbon / Q000378:metabolism; D002681:China; D003470:Culture Media / Q000737:chemistry; D015870:Gene Expression; D008969:Molecular Sequence Data; D010455:Peptides / Q000235:genetics; D010843:Pichia / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics; D017422:Sequence Analysis, DNA
chemical_list: D023181:Antimicrobial Cationic Peptides; D003470:Culture Media; D010455:Peptides; D011994:Recombinant Proteins; C504857:plectasin; D002244:Carbon