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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

Construction of the recombinant expression vector pGAPMP1102. α-factor: native S. cerevisiae α-mating factor secretion signal, which can be self-cleaved by the endogenous Kex2 protease to leave a native sequence of target peptide; pAOX: methanol-inducible alcohol oxidase one promoter from P. pastoris; pGAP: glyceraldehyde-3-phosphate dehydrogenase promoter from P. pastoris; PTEF1: transcription elongation factor one gene promoter; PEM7: synthetic prokaryotic promoter; Zeocin: Zeocin resistance gene; pUC ori: replication and maintenance of the plasmid in E. coli; CYC1 TT: transcription termination region.
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Fig1: Construction of the recombinant expression vector pGAPMP1102. α-factor: native S. cerevisiae α-mating factor secretion signal, which can be self-cleaved by the endogenous Kex2 protease to leave a native sequence of target peptide; pAOX: methanol-inducible alcohol oxidase one promoter from P. pastoris; pGAP: glyceraldehyde-3-phosphate dehydrogenase promoter from P. pastoris; PTEF1: transcription elongation factor one gene promoter; PEM7: synthetic prokaryotic promoter; Zeocin: Zeocin resistance gene; pUC ori: replication and maintenance of the plasmid in E. coli; CYC1 TT: transcription termination region.

Mentions: As shown in Figure 1, the pGAPMP1102 plasmid contained an inserted target MP1102 fragment of 120 bp. All positive transformants were verified by sequencing. The correct pGAPMP1102 recombinant plasmid was linearized with AvrII and transformed into P. pastoris X-33 by electroporation. The positive transformants were screened by PCR using GAP gene-specific and MP1102 gene-specific primers, and an empty pPICZαA vector transformant was used as the negative control (data not shown).Figure 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)

Construction of the recombinant expression vector pGAPMP1102. α-factor: native S. cerevisiae α-mating factor secretion signal, which can be self-cleaved by the endogenous Kex2 protease to leave a native sequence of target peptide; pAOX: methanol-inducible alcohol oxidase one promoter from P. pastoris; pGAP: glyceraldehyde-3-phosphate dehydrogenase promoter from P. pastoris; PTEF1: transcription elongation factor one gene promoter; PEM7: synthetic prokaryotic promoter; Zeocin: Zeocin resistance gene; pUC ori: replication and maintenance of the plasmid in E. coli; CYC1 TT: transcription termination region.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig1: Construction of the recombinant expression vector pGAPMP1102. α-factor: native S. cerevisiae α-mating factor secretion signal, which can be self-cleaved by the endogenous Kex2 protease to leave a native sequence of target peptide; pAOX: methanol-inducible alcohol oxidase one promoter from P. pastoris; pGAP: glyceraldehyde-3-phosphate dehydrogenase promoter from P. pastoris; PTEF1: transcription elongation factor one gene promoter; PEM7: synthetic prokaryotic promoter; Zeocin: Zeocin resistance gene; pUC ori: replication and maintenance of the plasmid in E. coli; CYC1 TT: transcription termination region.
Mentions: As shown in Figure 1, the pGAPMP1102 plasmid contained an inserted target MP1102 fragment of 120 bp. All positive transformants were verified by sequencing. The correct pGAPMP1102 recombinant plasmid was linearized with AvrII and transformed into P. pastoris X-33 by electroporation. The positive transformants were screened by PCR using GAP gene-specific and MP1102 gene-specific primers, and an empty pPICZαA vector transformant was used as the negative control (data not shown).Figure 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