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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

Effects of peptone and yeast extracts from Oxoid and HRBS (crude industry level) on the production of rMP1102. A, B: Effects of the peptone and yeast extracts on the total protein levels and antimicrobial activities of the fermentation supernatants of Pichia pastoris GAPMP1102. C: Tricine-SDS-PAGE analyses of the fermentation supernatants from Pichia pastoris GAPMP1102 in Med-1 supplemented with peptone and yeast extract from Oxoid. 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 the rMP1102 fermentation supernatants taken at 0, 24, 48, 72, 96, and 120 h of induction, respectively. D: Tricine-SDS-PAGE analysis of the fermentation supernatants from the Pichia pastoris GAPMP1102 in Med-1 supplemented with peptone and yeast extract from HRBS (crude industry level). 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). Lane 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, b, c) above the bar indicate significant differences between groups (P < 0.05).
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Fig4: Effects of peptone and yeast extracts from Oxoid and HRBS (crude industry level) on the production of rMP1102. A, B: Effects of the peptone and yeast extracts on the total protein levels and antimicrobial activities of the fermentation supernatants of Pichia pastoris GAPMP1102. C: Tricine-SDS-PAGE analyses of the fermentation supernatants from Pichia pastoris GAPMP1102 in Med-1 supplemented with peptone and yeast extract from Oxoid. 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 the rMP1102 fermentation supernatants taken at 0, 24, 48, 72, 96, and 120 h of induction, respectively. D: Tricine-SDS-PAGE analysis of the fermentation supernatants from the Pichia pastoris GAPMP1102 in Med-1 supplemented with peptone and yeast extract from HRBS (crude industry level). 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). Lane 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, b, c) above the bar indicate significant differences between groups (P < 0.05).

Mentions: To identify the best medium for the high-level production of rMP1102, six different media were examined. As shown in Figure 3C, the highest total protein and rMP1102 yield of 100.06 mg/l and 42.83 mg/l were observed with Med-1 following 120 h of cultivation. To further improve this yield and identify a suitable medium for rMP1102 production, yeast extracts and peptones from Oxoid and HRBS (crude industrial grades) were added. The higher total protein, rMP1102 yield and antimicrobial activity of 280.41 mg/l, 120.57 mg/l and 12800 AU/ml were observed following the addition of industrial yeast extract and peptone to Med-1 (Figure 4A, B); this yield was 2.80 times that observed with Med-1 alone. Additionally, the total protein, rMP1102 yield and antimicrobial activity were 190.26 mg/l, 78.01 mg/l and 9600 AU/ml following the use of Med-1 with added Oxoid yeast extract and peptone (Figure 4A, B). Tricine-SDS–PAGE revealed a target band of rMP1102 between 4.4 and 10.0 kDa that was more obvious following the use of Med-1 with HRBS yeast extract and peptone than the medium that contained Oxoid (Figure 4C, D), which suggesting that industrial yeast extract and peptone were more suitable for the production of rMP1102.Figure 4


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)

Effects of peptone and yeast extracts from Oxoid and HRBS (crude industry level) on the production of rMP1102. A, B: Effects of the peptone and yeast extracts on the total protein levels and antimicrobial activities of the fermentation supernatants of Pichia pastoris GAPMP1102. C: Tricine-SDS-PAGE analyses of the fermentation supernatants from Pichia pastoris GAPMP1102 in Med-1 supplemented with peptone and yeast extract from Oxoid. 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 the rMP1102 fermentation supernatants taken at 0, 24, 48, 72, 96, and 120 h of induction, respectively. D: Tricine-SDS-PAGE analysis of the fermentation supernatants from the Pichia pastoris GAPMP1102 in Med-1 supplemented with peptone and yeast extract from HRBS (crude industry level). 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). Lane 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, b, c) above the bar indicate significant differences between groups (P < 0.05).
© Copyright Policy - open-access
Related In: Results  -  Collection

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Fig4: Effects of peptone and yeast extracts from Oxoid and HRBS (crude industry level) on the production of rMP1102. A, B: Effects of the peptone and yeast extracts on the total protein levels and antimicrobial activities of the fermentation supernatants of Pichia pastoris GAPMP1102. C: Tricine-SDS-PAGE analyses of the fermentation supernatants from Pichia pastoris GAPMP1102 in Med-1 supplemented with peptone and yeast extract from Oxoid. 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 the rMP1102 fermentation supernatants taken at 0, 24, 48, 72, 96, and 120 h of induction, respectively. D: Tricine-SDS-PAGE analysis of the fermentation supernatants from the Pichia pastoris GAPMP1102 in Med-1 supplemented with peptone and yeast extract from HRBS (crude industry level). 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). Lane 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, b, c) above the bar indicate significant differences between groups (P < 0.05).
Mentions: To identify the best medium for the high-level production of rMP1102, six different media were examined. As shown in Figure 3C, the highest total protein and rMP1102 yield of 100.06 mg/l and 42.83 mg/l were observed with Med-1 following 120 h of cultivation. To further improve this yield and identify a suitable medium for rMP1102 production, yeast extracts and peptones from Oxoid and HRBS (crude industrial grades) were added. The higher total protein, rMP1102 yield and antimicrobial activity of 280.41 mg/l, 120.57 mg/l and 12800 AU/ml were observed following the addition of industrial yeast extract and peptone to Med-1 (Figure 4A, B); this yield was 2.80 times that observed with Med-1 alone. Additionally, the total protein, rMP1102 yield and antimicrobial activity were 190.26 mg/l, 78.01 mg/l and 9600 AU/ml following the use of Med-1 with added Oxoid yeast extract and peptone (Figure 4A, B). Tricine-SDS–PAGE revealed a target band of rMP1102 between 4.4 and 10.0 kDa that was more obvious following the use of Med-1 with HRBS yeast extract and peptone than the medium that contained Oxoid (Figure 4C, D), which suggesting that industrial yeast extract and peptone were more suitable for the production of rMP1102.Figure 4

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