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

Mao R, Teng D, Wang X, Zhang Y, Jiao J, Cao X, Wang J - BMC Microbiol. (2015)

Bottom Line: Six media were assayed for the improved yield of recombinant MP1102 (rMP1102).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.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory of Feed Biotechnology, Ministry of Agriculture, 12 Zhongguancun Nandajie St., Haidian District, Beijing, 100081, P. R. China. rain_mry@126.com.

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.

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Related in: MedlinePlus

Carbon sources and medium selection for the high-level production of rMP1102 at the shake flask level. A, B: Effects of different carbon sources on the total protein levels and antimicrobial activities of the fermentation supernatants of Pichia pastoris GAPMP1102; C: Total protein levels of the fermentation supernatants of Pichia pastoris GAPMP1102 in the different media; D Tricine-SDS-PAGE analysis of fermentation supernatant of Pichia pastoris GAPMP1102 in Med-1. Lane M: a total of 5 μl of protein molecular weight marker (from top to bottom: 40, 25, 15, 10, 4.6 and 1.7 kDa). Lanes 1–6: 10 μl of rMP1102 fermentation supernatants taken at 0, 24, 48, 72, 96, and 120 h of induction, respectively. The arrow indicates rMP1102. Each data point is the average of three replicates, and the error bars represent the standard deviation. Different lowercase letters (a, ab, b, c) above the bar indicate significant differences between groups (P < 0.05).
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Fig3: Carbon sources and medium selection for the high-level production of rMP1102 at the shake flask level. A, B: Effects of different carbon sources on the total protein levels and antimicrobial activities of the fermentation supernatants of Pichia pastoris GAPMP1102; C: Total protein levels of the fermentation supernatants of Pichia pastoris GAPMP1102 in the different media; D Tricine-SDS-PAGE analysis of fermentation supernatant of Pichia pastoris GAPMP1102 in Med-1. Lane M: a total of 5 μl of protein molecular weight marker (from top to bottom: 40, 25, 15, 10, 4.6 and 1.7 kDa). Lanes 1–6: 10 μl of rMP1102 fermentation supernatants taken at 0, 24, 48, 72, 96, and 120 h of induction, respectively. The arrow indicates rMP1102. Each data point is the average of three replicates, and the error bars represent the standard deviation. Different lowercase letters (a, ab, b, c) above the bar indicate significant differences between groups (P < 0.05).

Mentions: Unlike the AOX1 promoter, which is inactive on glucose and glycerol and requires methanol to initiate target gene expression, the GAP promoter constitutively expresses target proteins on different carbon sources [18]. The yield of rMP1102 increased with increasing carbon source concentrations until 40 g/l. The maximum total protein level, rMP1102 yield and antimicrobial activity were 67.8 mg/l, 27.8 mg/l and 6400 AU/ml, respectively, after 96 h of cultivation at an initial glucose concentration of 40 g/l (Figure 3A, B). Additionally, rMP1102 was not secreted into the medium at high levels when low concentrations (10 and 20 g/l) of any of the three carbon sources were used. However, the yields of rMP1102 decreased to 26.16 and 18.71 mg/l in glucose and maltose, respectively at concentrations of 50 g/l (P < 0.05), which suggested that excessive high carbon source concentrations did not benefit the production of rMP1102.Figure 3


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

Mao R, Teng D, Wang X, Zhang Y, Jiao J, Cao X, Wang J - BMC Microbiol. (2015)

Carbon sources and medium selection for the high-level production of rMP1102 at the shake flask level. A, B: Effects of different carbon sources on the total protein levels and antimicrobial activities of the fermentation supernatants of Pichia pastoris GAPMP1102; C: Total protein levels of the fermentation supernatants of Pichia pastoris GAPMP1102 in the different media; D Tricine-SDS-PAGE analysis of fermentation supernatant of Pichia pastoris GAPMP1102 in Med-1. Lane M: a total of 5 μl of protein molecular weight marker (from top to bottom: 40, 25, 15, 10, 4.6 and 1.7 kDa). Lanes 1–6: 10 μl of rMP1102 fermentation supernatants taken at 0, 24, 48, 72, 96, and 120 h of induction, respectively. The arrow indicates rMP1102. Each data point is the average of three replicates, and the error bars represent the standard deviation. Different lowercase letters (a, ab, b, c) above the bar indicate significant differences between groups (P < 0.05).
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4373065&req=5

Fig3: Carbon sources and medium selection for the high-level production of rMP1102 at the shake flask level. A, B: Effects of different carbon sources on the total protein levels and antimicrobial activities of the fermentation supernatants of Pichia pastoris GAPMP1102; C: Total protein levels of the fermentation supernatants of Pichia pastoris GAPMP1102 in the different media; D Tricine-SDS-PAGE analysis of fermentation supernatant of Pichia pastoris GAPMP1102 in Med-1. Lane M: a total of 5 μl of protein molecular weight marker (from top to bottom: 40, 25, 15, 10, 4.6 and 1.7 kDa). Lanes 1–6: 10 μl of rMP1102 fermentation supernatants taken at 0, 24, 48, 72, 96, and 120 h of induction, respectively. The arrow indicates rMP1102. Each data point is the average of three replicates, and the error bars represent the standard deviation. Different lowercase letters (a, ab, b, c) above the bar indicate significant differences between groups (P < 0.05).
Mentions: Unlike the AOX1 promoter, which is inactive on glucose and glycerol and requires methanol to initiate target gene expression, the GAP promoter constitutively expresses target proteins on different carbon sources [18]. The yield of rMP1102 increased with increasing carbon source concentrations until 40 g/l. The maximum total protein level, rMP1102 yield and antimicrobial activity were 67.8 mg/l, 27.8 mg/l and 6400 AU/ml, respectively, after 96 h of cultivation at an initial glucose concentration of 40 g/l (Figure 3A, B). Additionally, rMP1102 was not secreted into the medium at high levels when low concentrations (10 and 20 g/l) of any of the three carbon sources were used. However, the yields of rMP1102 decreased to 26.16 and 18.71 mg/l in glucose and maltose, respectively at concentrations of 50 g/l (P < 0.05), which suggested that excessive high carbon source concentrations did not benefit the production of rMP1102.Figure 3

Bottom Line: Six media were assayed for the improved yield of recombinant MP1102 (rMP1102).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.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory of Feed Biotechnology, Ministry of Agriculture, 12 Zhongguancun Nandajie St., Haidian District, Beijing, 100081, P. R. China. rain_mry@126.com.

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.

Show MeSH
Related in: MedlinePlus