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一种快速筛选、表达和纯化抗菌肽的方法。

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

作者信息

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

机构信息

State 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.

National Biopesticide Engineering Technology Research Center, Hubei Biopesticide Engineering Research Center, Hubei Academy of Agricultural Sciences, Biopesticide Branch of Hubei Innovation Centre of Agricultural Science and Technology, Wuhan 430064, China.

出版信息

Microorganisms. 2021 Sep 1;9(9):1858. doi: 10.3390/microorganisms9091858.

DOI:10.3390/microorganisms9091858
PMID:34576753
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC8469748/
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 and expression vectors. Twenty and ten 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 expression system, and one AMP (13) was purified using the expression system. Inhibition zone and minimum inhibitory concentration (MIC) tests confirmed that one ⌐-derived and two -derived AMPs have the inhibition activity. The MIC of AMP 13 and 16 from was 24.2 μM, and the MIC of AMP 13 from 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.

摘要

在本研究中，开发了一种用于抗菌肽（AMPs）快速筛选、表达和纯化的方法。使用SLOPE方法将AMPs基因与耐热CL7标签融合，并克隆到 和 表达载体中。构建了20个 和10个 表达载体。对表达上清液进行加热，去除杂蛋白，通过镍亲和（Ni-NTA）色谱法纯化融合蛋白。使用人鼻病毒（HRV）3C蛋白酶在柱上消化融合蛋白，释放并进一步纯化AMPs。使用 表达系统纯化了5种AMPs（1、2、6、13、16），使用 表达系统纯化了1种AMPs（13）。抑菌圈和最低抑菌浓度（MIC）测试证实，一种源自 的和两种源自 的AMPs具有抑菌活性。源自 的AMPs 13和16的MIC为24.2 μM，源自 的AMPs 13的MIC为8.1 μM。原核和真核表达系统的结合扩大了所开发方法的通用性，便于筛选大量具有生物活性的AMPs，建立AMPs文库，并通过工业化生物学方法生产AMPs。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/6581/8469748/fa40b7f85a1b/microorganisms-09-01858-g013.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/6581/8469748/3328b2306001/microorganisms-09-01858-g001.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/6581/8469748/3b902f74077b/microorganisms-09-01858-g002.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/6581/8469748/7330f9a21763/microorganisms-09-01858-g003.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/6581/8469748/589881e6bc9a/microorganisms-09-01858-g004.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/6581/8469748/81bd672937e1/microorganisms-09-01858-g005.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/6581/8469748/80e6d172660d/microorganisms-09-01858-g006.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/6581/8469748/c66b4d4dc72c/microorganisms-09-01858-g007.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/6581/8469748/831f5f13feff/microorganisms-09-01858-g008.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/6581/8469748/aef94d688997/microorganisms-09-01858-g009.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/6581/8469748/2ff705dca13d/microorganisms-09-01858-g010.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/6581/8469748/1d8989baa793/microorganisms-09-01858-g011.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/6581/8469748/4f8f06ddf754/microorganisms-09-01858-g012.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/6581/8469748/fa40b7f85a1b/microorganisms-09-01858-g013.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/6581/8469748/3328b2306001/microorganisms-09-01858-g001.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/6581/8469748/3b902f74077b/microorganisms-09-01858-g002.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/6581/8469748/7330f9a21763/microorganisms-09-01858-g003.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/6581/8469748/589881e6bc9a/microorganisms-09-01858-g004.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/6581/8469748/81bd672937e1/microorganisms-09-01858-g005.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/6581/8469748/80e6d172660d/microorganisms-09-01858-g006.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/6581/8469748/c66b4d4dc72c/microorganisms-09-01858-g007.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/6581/8469748/831f5f13feff/microorganisms-09-01858-g008.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/6581/8469748/aef94d688997/microorganisms-09-01858-g009.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/6581/8469748/2ff705dca13d/microorganisms-09-01858-g010.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/6581/8469748/1d8989baa793/microorganisms-09-01858-g011.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/6581/8469748/4f8f06ddf754/microorganisms-09-01858-g012.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/6581/8469748/fa40b7f85a1b/microorganisms-09-01858-g013.jpg

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