Department of Plant Breeding and Biotechnology, the University of Tabriz, Tabriz, Iran.
Genetics and Agricultural Biotechnology Institute of Tabarestan, Sari Agricultural Sciences and Natural Resources University, Sari, Iran.
Int J Biol Macromol. 2021 Mar 31;174:352-361. doi: 10.1016/j.ijbiomac.2021.01.129. Epub 2021 Jan 23.
In this study, encoding sequence of a new type 2 RIP (pebulin) was isolated and cloned from dwarf elder (Sambucus ebulus L.) native to the northern regions of Iran. The nucleotide sequence of pebulin was ligated to the pET-28a(+) expression plasmid and cloned into the E. coli strain BL21 (DE3) in order to express heterologously of recombinant protein. The recombinant pebulin protein was mainly produced in the form of insoluble inclusion bodies probably because to absence of N-glycosylation process in E. coli. Therefore, in order to increase the expression of recombinant protein in soluble form, co-expression of the target protein with the pG-Tf2 chaperone plasmid and incubation of bacterial culture under low temperature were used to enhance solubility and accumulation of recombinant protein. After purification of the recombinant protein using affinity chromatography method, the bioactivity of pebulin was analyzed by hemagglutination, anticancer, and antifungal assays. The results of the hemagglutination assay showed that purified pebulin agglutinated erythrocytes in all human blood groups. In addition, pebulin considerably inhibited the proliferation of cancer cell lines MCF-7 and HT-29 in a time- and dose-dependent manner and indicated remarkably growth-inhibiting effect against the plant pathogenic fungi such as Alternaria solani and Fusarium oxysporum.
在这项研究中，从原产于伊朗北部地区的矮小接骨木（Sambucus ebulus L.）中分离并克隆出一种新型 2 型 RIP（pebulin）的编码序列。 pebulin 的核苷酸序列被连接到 pET-28a(+)表达质粒上，并克隆到 E. coli 菌株 BL21 (DE3)中，以异源表达重组蛋白。重组 pebulin 蛋白主要以不溶性包涵体的形式产生，可能是因为 E. coli 中缺乏 N-糖基化过程。因此，为了增加重组蛋白以可溶性形式的表达，将目标蛋白与 pG-Tf2 伴侣质粒共表达，并在低温下培养细菌，以提高重组蛋白的可溶性和积累。使用亲和层析法纯化重组蛋白后，通过血凝、抗癌和抗真菌测定分析 pebulin 的生物活性。血凝测定的结果表明，纯化的 pebulin 能够凝集所有人类血型的红细胞。此外，pebulin 以时间和剂量依赖的方式显著抑制 MCF-7 和 HT-29 癌细胞系的增殖，并对植物病原真菌如茄病镰刀菌和尖孢镰刀菌表现出显著的生长抑制作用。
