Optimization of expression conditions for a novel NZ2114-derived antimicrobial peptide-MP1102 under the control of the GAP promoter in Pichia pastoris X-33

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• 作者：Ruoyu Mao ; Da Teng ; Xiumin Wang ; Yong Zhang ; Jian Jiao ; Xintao Cao…
• 关键词：MP1102 ; GAP promoter ; Pichia pastoris ; Fermentation
• 刊名：BMC Microbiology
• 出版年：2015
• 出版时间：December 2015
• 年：2015
• 卷：15
• 期：1
• 全文大小：2,250 KB
• 参考文献：1. Zasloff, M (2002) Antimicrobial peptides of multicellular organisms. Nature 415: pp. 389-95 CrossRef
  2. Mygind, PH, Fischer, RL, Schnorr, KM, Hansen, MT, S?nksen, CP, Ludvigsen, S (2005) Plectasin is a peptide antibiotic with therapeutic potential from a saprophytic fungus. Nature 437: pp. 975-80 CrossRef
  3. Zhang, J, Yang, Y, Teng, D, Tian, Z, Wang, S, Wang, J (2011) Expression of plectasin in Pichia pastoris and its characterization as a new antimicrobial peptide against Staphyloccocus and Streptococcus. Protein Exprs Purif 78: pp. 189-96 CrossRef
  4. Schneider, T, Kruse, T, Wimmer, R, Wiedemann, I, Sass, V, Pag, U (2010) Plectasin, a fungal defensin, targets the bacterial cell wall precursor Lipid II. Science 328: pp. 1168-72 CrossRef
  5. Andes, D, Craig, W, Nielsen, LA, Kristensen, HH (2009) In vivo pharmacodynamic characterization of a novel plectasin antibiotic, NZ2114, in a murine infection model. Antimicrob Agents Chemother 53: pp. 3003-9 CrossRef
  6. Ostergaard, C, Sandvang, D, Frimodt-Moller, N, Kristensen, HH (2009) High cerebrospinal fluid (CSF) penetration and potent bactericidal activity in CSF of NZ2114, a novel plectasin variant, during experimental pneumococcal meningitis. Antimicrob Agents Chemother 53: pp. 1581-5 CrossRef
  7. Brinch, KS, Tulkens, PM, Bambeke, F, Frimodt-Moller, N, Hoiby, N, Kristensen, HH (2010) Intracellular activity of the peptide antibiotic NZ2114: studies with Staphylococcus aureus and human THP-1 monocytes, and comparison with daptomycin and vancomycin. J Antimicrob Chemother 65: pp. 1720-4 CrossRef
  8. Xiong, YQ, Hady, WA, Deslandes, A, Rey, A, Fraisse, L, Kristensen, H-H (2011) Efficacy of NZ2114, a novel plectasin-derived cationic antimicrobial peptide antibiotic, in experimental endocarditis due to methicillin-resistant Staphylococcus aureus. Antimicrob Agents Chemother 55: pp. 5325-30 CrossRef
  9. Waterham, HR, Digan, ME, Koutz, PJ, Lair, SV, Cregg, JM (1997) Isolation of the Pichia pastoris glyceraldehyde-3-phosphate dehydrogenase gene and regulation and use of its promoter. Gene 186: pp. 37-44 CrossRef
  10. Wang, Q, Zhao, LL, Sun, JY, Liu, JX, Weng, XY (2012) Enhancing catalytic activity of a hybrid xylanase through single substitution of Leu to Pro near the active site. World J Microbiol Biotechnol 28: pp. 929-35 CrossRef
  11. Khasa, Y, Khushoo, A, Srivastava, L, Mukherjee, K (2007) Kinetic studies of constitutive human granulocyte-macrophage colony stimulating factor (hGM-CSF) expression in continuous culture of Pichia pastoris. Biotechnol Lett 29: pp. 1903-8 CrossRef
  12. Wu, JM, Lin, JC, Chieng, LL, Lee, CK, Hsu, TA (2003) Combined use of GAP and AOX1 promoter to enhance the expression of human granulocyte-macrophage colony-stimulating factor in Pichia pastoris. Enzyme Microb Technol 33: pp. 453-9 CrossRef
  13. Hong, I-P, Lee, S-J, Kim, Y-S, Choi, S-G (2006) Recombinant expression of human cathelicidin (hCAP18/LL-37) in Pichia pastoris. Biotechnol Lett 29: pp. 73-8 CrossRef
  14. Hsu, K-H, Pei, C, Yeh, J-Y, Shih, C-H, Chung, Y-C, Hung, L-T (2009) Production of bioactive human α-defensin 5 in Pichia pastoris. J Gen Appl Microbiol 55: pp. 395-401 CrossRef
  15. Huang, Y (2012) Secretion and activity of antimicrobial peptide cecropin D expressed in Pichia pastoris
• 刊物主题：Microbiology; Biological Microscopy; Fungus Genetics; Parasitology; Virology; Life Sciences, general;
• 出版者：BioMed Central
• ISSN：1471-2180

文摘

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.