Comparison of antimicrobial peptide purification via free-flow electrophoresis and gel filtration chromatography
Comparison of antimicrobial peptide purification via free-flow electrophoresis and gel filtration chromatography
复制标题
自由流动电泳和凝胶过滤层析纯化抗菌肽的比较
DOI：
10.1002/elps.201700187
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发表时间：
2017
期刊：
影响因子：
2.9
通讯作者：
Cao Cheng-Xi
中科院分区：
文献类型：
--
作者：
Xia Zhi-Jun;Liu Zhen;Kong Fan-Zhi;Fan Liu-Yin;Xiao Hua;Cao Cheng-Xi
抗菌肽通常是一类具有广谱抗菌活性的阳离子生物小分子。从复杂的样品中提纯它们对于研究它们的物理化学性质是必不可少的。采用自由流区带电泳法(FFZE)从酵母菌发酵液中分离纯化AMPs。同时，采用凝胶过滤层析(GFC)进行比较。通过对两种方法得到的组分进行SDS-PAGE分析，评价了分离效率。结果表明：(I)FFZE的处理能力是GFC的30多倍；(Ii)FFZE的纯度和回收率分别为87%和89%，而GFC的这两个参数分别约为93%和82%；(Iii)前者的∼稀释倍数为2倍，后者的∼稀释倍数为13倍。进一步用Tricine-SDS-PAGE、Native-PAGE和凝胶IEF对纯化的AMPs进行了表征。我们发现两个多肽以成对形式存在，分子质量分别为5.5kDa和7.0kDa，而相同的∼为7.8.通过标准化琼脂扩散法证明这两个多肽具有抑菌活性。因此，FFZE可用于连续纯化具有较高生物活性的AMPs，在临床和药学领域具有广泛的应用前景。
Antimicrobial peptides (AMPs) are usually small and cationic biomolecules with broad‐spectrum antimicrobial activities against pathogens. Purifying them from complex samples is essential to study their physiochemical properties. In this work, free‐flow zone electrophoresis (FFZE) was utilized to purify AMPs from yeast fermentation broth. Meanwhile, gel filtration chromatography (GFC) was conducted for comparison. The separation efficiency was evaluated by SDS‐PAGE analysis of the fractions from both methods. Our results demonstrated as follows: (i) FFZE had more than 30‐fold higher processing capacity as compared with GFC; (ii) FFZE could achieve 87% purity and 89% recovery rate while in GFC these parameters were about 93 and 82%, respectively; (iii) the former had ∼2‐fold dilution but the latter had ∼13‐fold dilution. Furthermore, Tricine‐SDS‐PAGE, Native‐PAGE, and gel IEF were carried out to characterize the purified AMPs. We found that two peptides existed as a pair with the molecular mass of ∼5.5 and 7.0 kDa, while the same pI7.8. These two peptides were proved to have the antimicrobial activity through the standardized agar diffusion method. Therefore, FFZE could be used to continuously purify AMPs with high bioactivity, which will lead to its wide application in the clinical and pharmaceutical fields.