A Method for Rapid Screening, Expression, and Purification of Antimicrobial Peptides.

Yingli Zhang,Zhongchen Li,Yaping Wang,Ben Rao,Lixin Ma,Li Li

doi:10.3390/microorganisms9091858

Yingli Zhang, Zhongchen Li + Show 4 more

Open Access

https://doi.org/10.3390/microorganisms9091858

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Abstract

In this study, a method for the rapid screening, expression and purification of antimicrobial peptides (AMPs) was developed. AMP genes were fused to a heat-resistant CL7 tag using the SLOPE method, and cloned into Escherichia coli and Pichia pastoris expression vectors. Twenty E. coli and ten P. pastoris expression vectors were constructed. Expression supernatants were heated, heteroproteins were removed, and fusion proteins were purified by nickel affinity (Ni-NTA) chromatography. Fusion proteins were digested on the column using human rhinovirus (HRV) 3C protease, and AMPs were released and further purified. Five AMPs (1, 2, 6, 13, 16) were purified using the E. coli expression system, and one AMP (13) was purified using the P. pastoris expression system. Inhibition zone and minimum inhibitory concentration (MIC) tests confirmed that one P. pastoris¬-derived and two E. coli-derived AMPs have the inhibition activity. The MIC of AMP 13 and 16 from E. coli was 24.2 μM, and the MIC of AMP 13 from P. pastoris was 8.1 μM. The combination of prokaryotic and eukaryotic expression systems expands the universality of the developed method, facilitating screening of a large number of biologically active AMPs, establishing an AMP library, and producing AMPs by industrialised biological methods.

Highlights

Accepted: 22 August 2021Antimicrobial peptides (AMPs) are small, naturally occurring peptides 5–100 amino acids in length, and possess high structural diversity [1,2,3]
We developed a method for rapid screening, expression and purification of antimicrobial peptides (AMPs)
The AMP sequences chosen in this experiment were selected from an AMP library, The

Summary

Introduction

Antimicrobial peptides (AMPs) are small, naturally occurring peptides 5–100 amino acids in length, and possess high structural diversity [1,2,3]. AMPs are produced in various eukaryotic and prokaryotic organisms, and some affect the innate immune systems of bacteria, plants, insects and mammals. AMPs have the advantages of low molecular weight, high heat resistance, broad-spectrum antimicrobial activity and, importantly, they show minimal antimicrobial resistance, making them a promising treatment for antimicrobial therapy [4]. AMPs protect against a wide variety of pathogens, including prokaryotic microorganisms, fungi, viruses, protozoa, insect cells and cancer cells. Advantages include antibacterial activity, degradability in vivo and, because they are from natural sources, they are generally safe and have non-toxic effects and, are applicable in foods and feed additives

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