Xin Chen1, Yong-hao Hu2, Wei-di Chen2, Wen-di Li2, Zi-chao Huang2, Yun Li1, Yu-wen Luo1, Yu-xia Huang1, Yi-tai Chen1, Kai Wang1, Ling Li3. 1. Department of Respiratory Medicine, Zhujiang Hospital, Southern Medical University, Guangzhou 510280, China. 2. Biosafety Level-3 Laboratory, Guangdong Provincial Key Laboratory of Tropical Disease Research, School of Public Health and Tropical Medicine, Southern Medical University, Guangzhou 510515, China. 3. Biosafety Level-3 Laboratory, Guangdong Provincial Key Laboratory of Tropical Disease Research, School of Public Health and Tropical Medicine, Southern Medical University, Guangzhou 510515, China. Electronic address: liling@smu.edu.cn.
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.
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.