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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

Chromatogram profile of supernatant of strain X-33/pPic-clavMO. 5 mg/mL of protein extract using a Hiload™ 16/60 Superdex™ 75 prep grade column (GE Healthcare) originating 24 fractions eluted in approximately 5 mL and monitored at 280 nm. The silver-stained gel shows the protein recovered from fractions 21 and 22 after purification process. MW molecular weight (kD).
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Fig4: Chromatogram profile of supernatant of strain X-33/pPic-clavMO. 5 mg/mL of protein extract using a Hiload™ 16/60 Superdex™ 75 prep grade column (GE Healthcare) originating 24 fractions eluted in approximately 5 mL and monitored at 280 nm. The silver-stained gel shows the protein recovered from fractions 21 and 22 after purification process. MW molecular weight (kD).

Mentions: Figure 4 shows the profile of the P. pastoris supernatant containing X33/pPICZαA-clavMO after 70 h of cultivation. From purification of 5 mg/mL of protein extract into a size exclusion chromatography, seven fractions were collected, numbered 16–22, and were selected for further analysis. After lyophilization, these samples were used to perform antibacterial assays, and two fractions, 21 and 22, were shown to have antibacterial activity. SDS-PAGE analyses of theses samples had shown the protein band corresponding to the molecular mass of clavMO but with different purity degrees (Figure 4).Figure 4


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)

Chromatogram profile of supernatant of strain X-33/pPic-clavMO. 5 mg/mL of protein extract using a Hiload™ 16/60 Superdex™ 75 prep grade column (GE Healthcare) originating 24 fractions eluted in approximately 5 mL and monitored at 280 nm. The silver-stained gel shows the protein recovered from fractions 21 and 22 after purification process. MW molecular weight (kD).
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig4: Chromatogram profile of supernatant of strain X-33/pPic-clavMO. 5 mg/mL of protein extract using a Hiload™ 16/60 Superdex™ 75 prep grade column (GE Healthcare) originating 24 fractions eluted in approximately 5 mL and monitored at 280 nm. The silver-stained gel shows the protein recovered from fractions 21 and 22 after purification process. MW molecular weight (kD).
Mentions: Figure 4 shows the profile of the P. pastoris supernatant containing X33/pPICZαA-clavMO after 70 h of cultivation. From purification of 5 mg/mL of protein extract into a size exclusion chromatography, seven fractions were collected, numbered 16–22, and were selected for further analysis. After lyophilization, these samples were used to perform antibacterial assays, and two fractions, 21 and 22, were shown to have antibacterial activity. SDS-PAGE analyses of theses samples had shown the protein band corresponding to the molecular mass of clavMO but with different purity degrees (Figure 4).Figure 4

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