毕赤酵母
抗菌肽
大肠杆菌
分子生物学
表达式向量
亲和层析
生物
蛋白酶
生物化学
色谱法
化学
肽
基因
重组DNA
酶
作者
Yingli Zhang,Zhongchen Li,Li Li,Ben Rao,Lixin Ma,Yaping Wang
出处
期刊:Microorganisms
[MDPI AG]
日期:2021-09-01
卷期号:9 (9): 1858-1858
被引量:6
标识
DOI:10.3390/microorganisms9091858
摘要
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.
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