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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.

Show MeSH

Related in: MedlinePlus

Effects of different pHs on the yield of rMP1102 via fed-batch fermentation. A, C, E, G, I: Tricine-SDS-PAGE analyses of the fermentation supernatants of P. pastoris GAPMP1102 in pHs of 5.0, 5.5, 6.0, 6.5 and 7.0, respectively. 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 1–7: Supernatants (10 μl each) harvested at 0, 24, 48, 72, 96, 108 and 120 h of cultivation, respectively. The arrow indicates rMP1102. B, D, F, H, J: Cell wet weights and total protein levels of the fermentation supernatants of P. pastoris GAPMP1102 in pHs of 5.0, 5.5, 6.0, 6.5 and 7.0, respectively. K: Antimicrobial activity of the fermentation supernatants of P. pastoris GAPMP1102 in pHs of 5.0, 5.5, 6.0, 6.5 and 7.0, respectively. L: Tricine-SDS-PAGE analysis of the fermentation supernatant of P. pastoris AOXMP1102 in pH 5.0. 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: supernatants (10 μl each) harvested at 0, 24, 48, 72, 96, and 120 h of cultivation, 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, d) above the bar indicate significant differences between groups (P < 0.05).
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Fig5: Effects of different pHs on the yield of rMP1102 via fed-batch fermentation. A, C, E, G, I: Tricine-SDS-PAGE analyses of the fermentation supernatants of P. pastoris GAPMP1102 in pHs of 5.0, 5.5, 6.0, 6.5 and 7.0, respectively. 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 1–7: Supernatants (10 μl each) harvested at 0, 24, 48, 72, 96, 108 and 120 h of cultivation, respectively. The arrow indicates rMP1102. B, D, F, H, J: Cell wet weights and total protein levels of the fermentation supernatants of P. pastoris GAPMP1102 in pHs of 5.0, 5.5, 6.0, 6.5 and 7.0, respectively. K: Antimicrobial activity of the fermentation supernatants of P. pastoris GAPMP1102 in pHs of 5.0, 5.5, 6.0, 6.5 and 7.0, respectively. L: Tricine-SDS-PAGE analysis of the fermentation supernatant of P. pastoris AOXMP1102 in pH 5.0. 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: supernatants (10 μl each) harvested at 0, 24, 48, 72, 96, and 120 h of cultivation, 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, d) above the bar indicate significant differences between groups (P < 0.05).

Mentions: The experiments involving the fermentation of rMP1102 in 5-l fermenters focused on pH because pH is the key factor that influences the yields of target proteins [19]. The total protein level, rMP1102 yield and antimicrobial activity of the fermentation supernatant increased from 807.42 mg/l, 367.59 mg/l and 38,400 AU/ml, respectively, at pH 5.0 to the maximum values of 1213.64 mg/l, 538.17 mg/l and 153,600 AU/ml, respectively, at pH6.5 (Table 1). However, the corresponding values decreased to 1127.38 mg/l, 503.92 mg/l and 102,400 AU/ml at pH 7.0, which was not the perfect condition for the growth of the host and target peptide production in P. pastoris. This pattern of productivity paralleled the absolute antimicrobial activity values. The lowest productivity was 104464 AU/mg rMP1102, which was observed at the pH of 5.0, and the maximum max value occurred in pH 6.5 and was 285412 AU/mg rMP1102. However, productivity did not increase when the pH was raised to 7.0 (Table 1). Moreover, a wider target band resulted from pH 6.5 compared to the other pH values (Figure 5A, C, E, G, I). Additionally, the cell wet weights and total protein levels increased with the process of cultivation in all pH values (Figure 5B, D, F, H, J). The total protein level increased to the maximums at 84 and 96 h in the pHs of 5.0, 5.5, and 6.0. However, the highest total protein level and antimicrobial activity occurred at 120 h at the pHs of 6.5 and 7.0, which suggested that these conditions were beneficial for the continuous accumulation of rMP1102, whereas the target peptides accumulated faster in the low 5.0, 5.5, and 6.0 pH conditions (Figure 5B, D, F, H, J). rMP1102 was also expressed in P. pastoris via the inducible method at the 5-l fermenter level. The maximum total protein level was 695 mg/l at 120 h (accepted by Appl Microbiol Biotechnol, DOI 10.1007/s00253-015-6394-7), which was more than 70% reduced compared to the constitutive expression at pH 6.5. Furthermore, another non-target band appeared above rMP1102 between 3.3 and 5.8 kDa. This band might have represented incomplete cleavage by Kex2 due to the large amount of target peptides that were induced by methanol in a short time [20]. In contrast, there only the target rMP1102 was expressed in each of the assays that used the GAP promoter (Figure 5A, C, E, G, I). Very few or no other bands appeared around rMP1102, which was convenient for the process of purification.Table 1


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 different pHs on the yield of rMP1102 via fed-batch fermentation. A, C, E, G, I: Tricine-SDS-PAGE analyses of the fermentation supernatants of P. pastoris GAPMP1102 in pHs of 5.0, 5.5, 6.0, 6.5 and 7.0, respectively. 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 1–7: Supernatants (10 μl each) harvested at 0, 24, 48, 72, 96, 108 and 120 h of cultivation, respectively. The arrow indicates rMP1102. B, D, F, H, J: Cell wet weights and total protein levels of the fermentation supernatants of P. pastoris GAPMP1102 in pHs of 5.0, 5.5, 6.0, 6.5 and 7.0, respectively. K: Antimicrobial activity of the fermentation supernatants of P. pastoris GAPMP1102 in pHs of 5.0, 5.5, 6.0, 6.5 and 7.0, respectively. L: Tricine-SDS-PAGE analysis of the fermentation supernatant of P. pastoris AOXMP1102 in pH 5.0. 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: supernatants (10 μl each) harvested at 0, 24, 48, 72, 96, and 120 h of cultivation, 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, d) 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

Fig5: Effects of different pHs on the yield of rMP1102 via fed-batch fermentation. A, C, E, G, I: Tricine-SDS-PAGE analyses of the fermentation supernatants of P. pastoris GAPMP1102 in pHs of 5.0, 5.5, 6.0, 6.5 and 7.0, respectively. 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 1–7: Supernatants (10 μl each) harvested at 0, 24, 48, 72, 96, 108 and 120 h of cultivation, respectively. The arrow indicates rMP1102. B, D, F, H, J: Cell wet weights and total protein levels of the fermentation supernatants of P. pastoris GAPMP1102 in pHs of 5.0, 5.5, 6.0, 6.5 and 7.0, respectively. K: Antimicrobial activity of the fermentation supernatants of P. pastoris GAPMP1102 in pHs of 5.0, 5.5, 6.0, 6.5 and 7.0, respectively. L: Tricine-SDS-PAGE analysis of the fermentation supernatant of P. pastoris AOXMP1102 in pH 5.0. 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: supernatants (10 μl each) harvested at 0, 24, 48, 72, 96, and 120 h of cultivation, 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, d) above the bar indicate significant differences between groups (P < 0.05).
Mentions: The experiments involving the fermentation of rMP1102 in 5-l fermenters focused on pH because pH is the key factor that influences the yields of target proteins [19]. The total protein level, rMP1102 yield and antimicrobial activity of the fermentation supernatant increased from 807.42 mg/l, 367.59 mg/l and 38,400 AU/ml, respectively, at pH 5.0 to the maximum values of 1213.64 mg/l, 538.17 mg/l and 153,600 AU/ml, respectively, at pH6.5 (Table 1). However, the corresponding values decreased to 1127.38 mg/l, 503.92 mg/l and 102,400 AU/ml at pH 7.0, which was not the perfect condition for the growth of the host and target peptide production in P. pastoris. This pattern of productivity paralleled the absolute antimicrobial activity values. The lowest productivity was 104464 AU/mg rMP1102, which was observed at the pH of 5.0, and the maximum max value occurred in pH 6.5 and was 285412 AU/mg rMP1102. However, productivity did not increase when the pH was raised to 7.0 (Table 1). Moreover, a wider target band resulted from pH 6.5 compared to the other pH values (Figure 5A, C, E, G, I). Additionally, the cell wet weights and total protein levels increased with the process of cultivation in all pH values (Figure 5B, D, F, H, J). The total protein level increased to the maximums at 84 and 96 h in the pHs of 5.0, 5.5, and 6.0. However, the highest total protein level and antimicrobial activity occurred at 120 h at the pHs of 6.5 and 7.0, which suggested that these conditions were beneficial for the continuous accumulation of rMP1102, whereas the target peptides accumulated faster in the low 5.0, 5.5, and 6.0 pH conditions (Figure 5B, D, F, H, J). rMP1102 was also expressed in P. pastoris via the inducible method at the 5-l fermenter level. The maximum total protein level was 695 mg/l at 120 h (accepted by Appl Microbiol Biotechnol, DOI 10.1007/s00253-015-6394-7), which was more than 70% reduced compared to the constitutive expression at pH 6.5. Furthermore, another non-target band appeared above rMP1102 between 3.3 and 5.8 kDa. This band might have represented incomplete cleavage by Kex2 due to the large amount of target peptides that were induced by methanol in a short time [20]. In contrast, there only the target rMP1102 was expressed in each of the assays that used the GAP promoter (Figure 5A, C, E, G, I). Very few or no other bands appeared around rMP1102, which was convenient for the process of purification.Table 1

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