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Production of a modified peptide clavanin in Pichia pastoris: cloning, expression, purification and in vitro activities.

Mulder KC, de Lima LA, Aguiar PS, Carneiro FC, Franco OL, Dias SC, Parachin NS - AMB Express (2015)

Bottom Line: Antimicrobial peptides are one of the most promising peptide-based drugs due to their enormous potential as novel biopharmaceuticals in both human and animal industries.In order to develop strategies to over produce such molecules, heterologous production of a modified version of clavanin A, here named clavanin MO (clavMO), was successfully achieved in the methylothopic yeast Pichia pastoris.ClavMO was fused to thioredoxin as a carrier protein and the construction was tested using two promoters, PAOX1 and PGAP, based on either induced or constitutive expression systems, respectively.

View Article: PubMed Central - PubMed

Affiliation: Centro de Análises Proteômicas e Bioquímicas, Pós-Graduação em Ciências Genômicas e Biotecnologia, Universidade Católica de Brasília, Brasília, DF, Brazil, kellylmulder@gmail.com.

ABSTRACT
Antimicrobial peptides are one of the most promising peptide-based drugs due to their enormous potential as novel biopharmaceuticals in both human and animal industries. In order to develop strategies to over produce such molecules, heterologous production of a modified version of clavanin A, here named clavanin MO (clavMO), was successfully achieved in the methylothopic yeast Pichia pastoris. ClavMO was fused to thioredoxin as a carrier protein and the construction was tested using two promoters, PAOX1 and PGAP, based on either induced or constitutive expression systems, respectively. After growth in 5 L Bioreactor, clavMO-thio was recovered and purified through size exclusion chromatography. Our findings show that both constitutive and inducible expression systems produce active clavMO fused to thioredoxin against both Gram-negative Klebsiella pneumoniae and Gram-positive Staphylococcus aureus microorganisms.

No MeSH data available.


Related in: MedlinePlus

Plasmid vector map. The constitutive system was developed using the plasmid pGAPZαB, which contains the PGAP promoter. The inducible system was constructed using the pPICZαA, which contains the PAOX promoter. HIS his-tag, Trx thioredoxin E. coli gene, Cla clavanin gene, AOX TT terminator.
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Fig1: Plasmid vector map. The constitutive system was developed using the plasmid pGAPZαB, which contains the PGAP promoter. The inducible system was constructed using the pPICZαA, which contains the PAOX promoter. HIS his-tag, Trx thioredoxin E. coli gene, Cla clavanin gene, AOX TT terminator.

Mentions: The thio-clavMO gene sequence was synthesized by Epoch Life Science (Additional file 1: Figure S1). To clone the gene into both plasmids pPICZαA and pGAPZαB, the restriction sites EcoRI and SacII were used, resulting in the expression vectors pPICZαA-clavMO and pGAPZαB-clavMO (Figure 1; Table 1). Both plasmids were firstly transformed into E. coli XL1-Blue strains and sequenced to confirm the insertion of the expression cassette.Figure 1


Production of a modified peptide clavanin in Pichia pastoris: cloning, expression, purification and in vitro activities.

Mulder KC, de Lima LA, Aguiar PS, Carneiro FC, Franco OL, Dias SC, Parachin NS - AMB Express (2015)

Plasmid vector map. The constitutive system was developed using the plasmid pGAPZαB, which contains the PGAP promoter. The inducible system was constructed using the pPICZαA, which contains the PAOX promoter. HIS his-tag, Trx thioredoxin E. coli gene, Cla clavanin gene, AOX TT terminator.
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4524883&req=5

Fig1: Plasmid vector map. The constitutive system was developed using the plasmid pGAPZαB, which contains the PGAP promoter. The inducible system was constructed using the pPICZαA, which contains the PAOX promoter. HIS his-tag, Trx thioredoxin E. coli gene, Cla clavanin gene, AOX TT terminator.
Mentions: The thio-clavMO gene sequence was synthesized by Epoch Life Science (Additional file 1: Figure S1). To clone the gene into both plasmids pPICZαA and pGAPZαB, the restriction sites EcoRI and SacII were used, resulting in the expression vectors pPICZαA-clavMO and pGAPZαB-clavMO (Figure 1; Table 1). Both plasmids were firstly transformed into E. coli XL1-Blue strains and sequenced to confirm the insertion of the expression cassette.Figure 1

Bottom Line: Antimicrobial peptides are one of the most promising peptide-based drugs due to their enormous potential as novel biopharmaceuticals in both human and animal industries.In order to develop strategies to over produce such molecules, heterologous production of a modified version of clavanin A, here named clavanin MO (clavMO), was successfully achieved in the methylothopic yeast Pichia pastoris.ClavMO was fused to thioredoxin as a carrier protein and the construction was tested using two promoters, PAOX1 and PGAP, based on either induced or constitutive expression systems, respectively.

View Article: PubMed Central - PubMed

Affiliation: Centro de Análises Proteômicas e Bioquímicas, Pós-Graduação em Ciências Genômicas e Biotecnologia, Universidade Católica de Brasília, Brasília, DF, Brazil, kellylmulder@gmail.com.

ABSTRACT
Antimicrobial peptides are one of the most promising peptide-based drugs due to their enormous potential as novel biopharmaceuticals in both human and animal industries. In order to develop strategies to over produce such molecules, heterologous production of a modified version of clavanin A, here named clavanin MO (clavMO), was successfully achieved in the methylothopic yeast Pichia pastoris. ClavMO was fused to thioredoxin as a carrier protein and the construction was tested using two promoters, PAOX1 and PGAP, based on either induced or constitutive expression systems, respectively. After growth in 5 L Bioreactor, clavMO-thio was recovered and purified through size exclusion chromatography. Our findings show that both constitutive and inducible expression systems produce active clavMO fused to thioredoxin against both Gram-negative Klebsiella pneumoniae and Gram-positive Staphylococcus aureus microorganisms.

No MeSH data available.


Related in: MedlinePlus