Institute of Biochemistry, Food Science and Nutrition, The Robert H. Smith Faculty of Agriculture, Food and Environment, The Hebrew University of Jerusalem, P.O. Box 12, Rehovot 76100, Israel.
Appl Biochem Biotechnol. 2012 Apr;166(7):1703-10. doi: 10.1007/s12010-012-9571-5. Epub 2012 Feb 22.
The innate immunity utilizes a battery of broad-spectrum antibacterial cationic polypeptides (3-5 kDa), among them defensins. In humans, defensins are the first line of defense against pathogens and their expression has been implicated in several diseases. The antibacterial activity of defensins is generally ascribed to their overall positive charge, which enables them to disrupt bacterial membrane integrity and function, but their active surface has not been fully elucidated. To perform structural and functional assays, an efficient, high-yield, easy-to-use expression and purification system must be established. Up to now, most efforts to obtain larger quantities of active recombinant defensins have been only moderately successful. Herein, we report the establishment of an efficient, high-yield expression and purification system for human defensin 5 (HD-5). Using site-directed mutagenesis, we pinpoint several arginine residues and Y27 as important for HD-5 antibacterial activity. Our expression and purification system can be harnessed for structure/activity relationship studies of defensins in particular and small polypeptides in general.
先天免疫系统利用了一系列广谱的抗细菌阳离子多肽（3-5 kDa），其中包括防御素。在人类中，防御素是抵御病原体的第一道防线，其表达与多种疾病有关。防御素的抗菌活性通常归因于其整体正电荷，这使它们能够破坏细菌膜的完整性和功能，但它们的活性表面尚未完全阐明。为了进行结构和功能分析，必须建立一个高效、高产、易于使用的表达和纯化系统。到目前为止，获得大量活性重组防御素的大部分努力只是取得了中等程度的成功。本文报告了一种高效、高产的人防御素 5（HD-5）表达和纯化系统的建立。通过定点突变，我们确定了几个精氨酸残基和 Y27 对 HD-5 抗菌活性很重要。我们的表达和纯化系统可用于防御素特别是小肽的结构/活性关系研究。
