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A Method for Rapid Screening, Expression, and Purification of Antimicrobial Peptides
Yingli Zhang1, Zhongchen Li1, Li Li1
1State Key Laboratory of Biocatalysis and Enzyme, Engineering Hubei Collaborative Innovation Center for Green Transformation of Bio-Resources, Hubei Key Laboratory of Industrial Biotechnology, Biology Faculty of Hubei University, Hubei University, Wuhan 430062, China.
A new method rapidly screens and purifies antimicrobial peptides (AMPs) using both bacterial (Escherichia coli) and yeast (Pichia pastoris) expression systems. This approach identifies potent AMPs for potential therapeutic applications.
Area of Science:
- Biotechnology and Molecular Biology
- Antimicrobial Peptide Discovery
- Protein Expression and Purification
Background:
- Antimicrobial peptides (AMPs) are crucial components of innate immunity with broad-spectrum activity.
- Efficient methods for screening, expressing, and purifying AMPs are essential for drug development.
- Current methods can be time-consuming and may not cover the full range of potential AMP candidates.
Purpose of the Study:
- To develop a rapid and versatile method for screening, expression, and purification of AMPs.
- To utilize both prokaryotic (Escherichia coli) and eukaryotic (Pichia pastoris) expression systems for AMP production.
- To identify and characterize AMPs with significant antimicrobial activity.
Main Methods:
- Genes encoding AMPs were fused to a heat-resistant CL7 tag using the SLOPE method.
- Cloning into Escherichia coli and Pichia pastoris expression vectors, followed by expression and heat treatment.
- Purification of fusion proteins via nickel affinity chromatography, on-column digestion with HRV 3C protease, and subsequent AMP purification.
Main Results:
- Successfully purified five AMPs using the E. coli system and one AMP using the P. pastoris system.
- Antimicrobial activity confirmed for two E. coli-derived AMPs and one P. pastoris-derived AMP via inhibition zone and MIC tests.
- Minimum inhibitory concentrations (MICs) determined, with P. pastoris-derived AMP 13 showing potent activity (8.1 μM).
Conclusions:
- The developed method enables rapid screening and purification of AMPs using a dual prokaryotic-eukaryotic expression system approach.
- This strategy facilitates the establishment of AMP libraries and the industrial-scale production of bioactive AMPs.
- The combined expression systems enhance the universality and efficiency of AMP discovery and development.
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