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巴豆核糖体失活蛋白的纯化及其功能表征。

Purification and functional characterization of recombinant balsamin, a ribosome-inactivating protein from Momordica balsamina.

机构信息

Centre for Chemistry and Biotechnology, School of Life and Environment Sciences, Deakin University, Waurn Ponds, 75 Pigdons Road, Locked Bag 20000, Geelong, VIC 3220, Australia.

Centre for Molecular and Medical Research, School of Medicine, Deakin University, Waurn Ponds, 75 Pigdons Road, Locked Bag 20000, Geelong, VIC 3220, Australia.

出版信息

Int J Biol Macromol. 2018 Jul 15;114:226-234. doi: 10.1016/j.ijbiomac.2018.02.114. Epub 2018 Feb 19.

Abstract

Balsamin, a type I ribosome-inactivating protein (RIP), has been shown to inhibit HIV-1 replication at the translation step. Our recent studies have shown that balsamin also possess anti-tumor, antibacterial and DNase-like activity, however, the amount of natural balsamin in Momordica balsamina seeds is limited and preclinical studies require large quantities of pure, bioactive balsamin. Therefore, in this study, we cloned the balsamin gene, expressed it in E.coli BL21 (DE3) strain and purified it by nickel affinity chromatography. Functional analysis indicated that balsamin exhibits both RNA N-glycosidase activity, releasing the Endo-fragment from rabbit reticulocyte rRNA, and DNase-like activity, converting the supercoiled form of a plasmid into the linear form in a concentration-dependent manner. Analysis of secondary structure revealed that recombinant balsamin mainly consisted of α-helical and random coiled with minimal turns and β-sheets. Recombinant balsamin was found to be stable in the temperature range of 20-60 °C and pH range of 6-9. Antimicrobial assays showed that the minimum inhibitory concentrations of recombinant balsamin for various pathogens ranged between 1.56 and 12.5 μg/ml. Heterologous expression and purification of balsamin carries great importance as it provides an alternative approach for large-scale preparation of biologically active recombinant balsamin, which is difficult from its natural source.

摘要

巴拉斯明是一种 I 型核糖体失活蛋白 (RIP),已被证明可在翻译步骤中抑制 HIV-1 复制。我们最近的研究表明,巴拉斯明还具有抗肿瘤、抗菌和 DNA 酶样活性,然而,苦瓜种子中天然巴拉斯明的含量有限,临床前研究需要大量的纯、生物活性巴拉斯明。因此,在这项研究中,我们克隆了巴拉斯明基因,在大肠杆菌 BL21 (DE3) 菌株中表达,并通过镍亲和层析进行纯化。功能分析表明,巴拉斯明既具有 RNA N-糖苷酶活性,从兔网织红细胞 rRNA 中释放内酶片段,又具有 DNA 酶样活性,以浓度依赖的方式将质粒的超螺旋形式转化为线性形式。二级结构分析表明,重组巴拉斯明主要由α-螺旋和无规卷曲组成,很少有转角和β-折叠。重组巴拉斯明在 20-60°C 的温度范围和 6-9 的 pH 范围内稳定。抗菌试验表明,重组巴拉斯明对各种病原体的最小抑菌浓度在 1.56 至 12.5μg/ml 之间。巴拉斯明的异源表达和纯化具有重要意义,因为它为大规模制备生物活性重组巴拉斯明提供了一种替代方法,而从天然来源获得重组巴拉斯明则很困难。

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