Xia Zhi-Jun, Liu Zhen, Kong Fan-Zhi, Fan Liu-Yin, Xiao Hua, Cao Cheng-Xi
Laboratory of Analytical Biochemistry and Bioseparation, State Key Laboratory of Microbial Metabolism, School of Life Science and Biotechnology, Shanghai Jiao Tong University, Shanghai, P. R. China.
Electrophoresis. 2017 Dec;38(24):3147-3154. doi: 10.1002/elps.201700187. Epub 2017 Sep 6.
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 pI 7.8. 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.
抗菌肽（AMPs）通常是具有针对病原体的广谱抗菌活性的小阳离子生物分子。从复杂样品中纯化它们对于研究其理化性质至关重要。在这项工作中，利用自由流区带电泳（FFZE）从酵母发酵液中纯化抗菌肽。同时，进行凝胶过滤色谱（GFC）作为比较。通过对两种方法得到的馏分进行SDS-PAGE分析来评估分离效率。我们的结果表明如下：（i）与GFC相比，FFZE的处理能力高出30倍以上；（ii）FFZE可实现87%的纯度和89%的回收率，而在GFC中，这些参数分别约为93%和82%；（iii）前者有2倍的稀释度，而后者有13倍的稀释度。此外，进行了Tricine-SDS-PAGE、非变性聚丙烯酰胺凝胶电泳（Native-PAGE）和凝胶等电聚焦（IEF）来表征纯化的抗菌肽。我们发现两种肽以一对的形式存在，分子量约为5.5和7.0 kDa，而等电点相同为7.8。通过标准琼脂扩散法证明这两种肽具有抗菌活性。因此，FFZE可用于连续纯化具有高生物活性的抗菌肽，这将导致其在临床和制药领域的广泛应用。
