Comparison of inducible versus constitutive expression of plectasin on yields and antimicrobial activities in Pichia pastoris.
Protein Expr Purif
; 118: 70-6, 2016 Feb.
Article
in En
| MEDLINE
| ID: mdl-26500192
ABSTRACT
BACKGROUND:
Plectasin might serve as a substitute for traditional antibiotics, but its yields and antimicrobial activities warrant further investigation.OBJECTIVE:
To identify the influence of inducible versus constitutive expression of plectasin on yields and antimicrobial activities.METHODS:
Through SOE-PCR, a recombinant plectasin gene was generated and inserted into inducible (pPICZαA) and constitutive (pGAPZαA) vectors in order to create Pichia pastoris GS115 strains. After 120 h of fermentation, supernatants were purified by an AKTA purifier using nickel columns. Minimal inhibitory concentration (MIC) and inhibition zone assays were performed after Tricine-SDS-PAGE.RESULTS:
After 120 h of fermentation, the yield of constitutive plectasin (370 µg/ml) was much lower than that from inducible vector (880 µg/ml) (P < 0.05). However, constitutive strain reached its plateau phase faster and keep more consistent yield (P < 0.05). The MICs of inducible plectasin against Methicillin-resistant Staphylococcus aureus (MRSA) 15471118, vancomycin-resistant Enterococcus feces (VREF), and penicillin-resistant Streptococcus pneumonia (PRSP) 31355 were 64, 32, and 64 µg/ml, respectively, while those of constitutive plectasin were 4, 4, and 16 µg/ml. No significant differences were observed in antimicrobial activities between inducible and constitutive plectasin for MRSA 15471118, VREF and PRSP 31355 (all P ï¼ 0.05). However, constitutive plectasin had a larger inhibition zone than inducible plectasin with the same mass.CONCLUSIONS:
Although P. pastoris GS115 (pGAPZαA-Plectasin-GS115) had lower expression than P. pastoris GS115 (pPICZαA-plectasin-GS115), it reached the plateau phase faster, had steadier yields and showed superiority in antimicrobial activities. Therefore, pGAPZαA might be more suitable for expression of plectasin in GS115 compared with pPICZαA.Key words
Full text:
1
Collection:
01-internacional
Database:
MEDLINE
Main subject:
Peptides
/
Pichia
/
Anti-Bacterial Agents
Limits:
Humans
Language:
En
Journal:
Protein Expr Purif
Year:
2016
Document type:
Article