Expression and biochemical characterization of recombinant human epididymis protein 4
Expression and biochemical characterization of recombinant human epididymis protein 4
复制标题
DOI：
10.1016/j.pep.2014.08.004
复制
发表时间：
2014-10-01
影响因子：
1.6
通讯作者：
Gao, Xin
中科院分区：
文献类型：
--
作者：
Hua, Ling;Liu, Yunhui;Gao, Xin
乳清酸性蛋白(WAP)是一个重要的抗菌肽基因家族，具有重要的免疫功能。人附睾蛋白4(HE4)是一种含有两个乳清酸性蛋白四硫键核心(WFDC)结构域的124个氨基酸的多肽，其功能尚未得到很好的研究。在此，构建了编码HE4蛋白与IgG1Fc结构域融合的融合基因。重组HE4蛋白在巴斯德毕赤酵母和哺乳动物HEK293-F细胞中以分泌型蛋白形式表达，并进行了纯化。我们的数据表明，这两种表达系统产生的HE4蛋白既能与革兰氏阴性菌结合，也能与革兰氏阳性菌结合，但对金黄色葡萄球菌的生长有轻微的抑制作用。此外，HE4对胰酶、弹性蛋白酶、基质金属肽酶9和枯草芽孢杆菌的分泌性蛋白水解酶具有抑制活性。研究了糖基化对HE4生化特性的影响。LC-ESI-MS糖基化分析表明，巴斯德毕赤酵母表达的高甘露糖糖基化HE4的生物活性低于HEK293-F细胞表达的复杂糖基化HE4。并对其意义进行了讨论，为其在肿瘤发生发展和天然免疫系统中的重要作用提供了理论依据。(C)2014 Elsevier Inc.保留所有权利。
Whey acidic proteins (WAP) belong to a large gene family of antibacterial peptides that perform critical immune system functions. The function of human epididymis protein 4 (HE4), a 124-amino acid long polypeptide that has two whey acidic protein four-disulfide core (WFDC) domains, is not well studied. Here, a fusion gene encoding the HE4 protein fused to an IgG1 Fc domain was constructed. The recombinant HE4 protein was expressed as a secretory protein in Pichia pastoris and mammalian HEK293-F cells and was subsequently purified. Our data suggested that the HE4 protein produced by these two expression systems bound to both gram-negative and gram-positive bacteria, but demonstrated slightly inhibitory activity towards the growth of Staphylococcus aureus. Moreover, HE4 exhibited proteinase inhibitory activity towards trypsin, elastase, matrix metallopeptidase 9, and the secretory proteinases from Bacillus subtilis. The effects of glycosylation on the biochemical characterization of HE4 were also investigated. LC-ESI-MS glycosylation analysis showed that the high-mannose glycosylated form of HE4 expressed by P. pastoris has lower biological activity when compared to its complex-glycosylated form produced from HEK293-F cells. The implications of this are discussed, which may be provide theoretical basis for its important role in the development of cancer and innate immune system. (C) 2014 Elsevier Inc. All rights reserved.