Comparison of inducible versus constitutive expression of plectasin on yields and antimicrobial activities in Pichia pastoris.

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 ＞ 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.

• Authors: Xin Chen, Yong-hao Hu, Wei-di Chen, Wen-di Li, Zi-chao Huang, Yun Li, Yu-wen Luo, Yu-xia Huang, Yi-tai Chen, Kai Wang, Ling Li
• Journal: Protein expression and purification (Protein Expr Purif) Vol. 118 Pg. 70-6 (Feb 2016) ISSN: 1096-0279 [Electronic] United States
• PMID: 26500192 (Publication Type: Comparative Study, Journal Article, Research Support, Non-U.S. Gov't)
• Copyright: Copyright © 2015 Elsevier Inc. All rights reserved.

Chemical References

• Anti-Bacterial Agents
• Peptides
• plectasin

Topics

• Anti-Bacterial Agents (biosynthesis, pharmacology)
• Bacteria (drug effects, growth & development)
• Gene Expression Regulation, Bacterial
• Humans
• Microbial Sensitivity Tests
• Peptides (genetics, metabolism, pharmacology)
• Pichia (classification, genetics, metabolism)