Cloning and Aggregation Characterization of Rubber Elongation Factor and Small Rubber Particle Protein from Ficus carica
Cloning and Aggregation Characterization of Rubber Elongation Factor and Small Rubber Particle Protein from Ficus carica
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DOI：
10.1007/s12033-017-0051-6
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发表时间：
2018-02
影响因子：
2.6
通讯作者：
Saki Yokota;Yurina Suzuki;K. Saitoh;S. Kitajima;N. Ohya;T. Gotoh
中科院分区：
文献类型：
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作者：
Saki Yokota;Yurina Suzuki;K. Saitoh;S. Kitajima;N. Ohya;T. Gotoh
橡胶延伸因子（REF）和小橡胶颗粒蛋白（SRPP）是从巴西橡胶树（橡胶树;分别为HbREF和HbSRPP）中收获的主要乳胶蛋白。它们的氨基酸序列彼此具有高度同源性。本研究从无花果乳管中克隆了两个编码REF/SRPP家族蛋白的cDNA（FcREF/SRPP-1和-2）。这些蛋白质的氨基酸序列不仅彼此之间，而且与HbREF和HbSRPP具有高度的同源性。在大肠杆菌中表达重组FcREF/SRPP-1和-2，并使用刚果红结合试验、琼脂糖凝胶电泳和透射电子显微镜检查其聚集特性。FcREF/SRPP-1在PBS中孵育时形成原纤维，并生长为快速沉淀的微米级无定形聚集体。FcREF/SRPP-1的这些聚集特性与HbREF的那些聚集特性非常相似，尽管FcREF/SRPP-1聚集体的生长速率和尺寸劣于HbREF的那些。FcREF/SRPP-2在孵育期间也形成聚集体，但它们不沉淀，如对于HbSRPP所报道的。我们的结果表明，FcREF/SRPP-1和-2分别对应于HbREF和HbSRPP。这些聚集特性可以为将REF/SRPP家族蛋白分类为REF或SRPP提供有用的基准。
Rubber elongation factor (REF) and small rubber particle protein (SRPP) are major latex proteins harvested fromHevea brasiliensis(the rubber tree; HbREF and HbSRPP, respectively). Their amino acid sequences exhibit high homology with each other. In the present study, we cloned two cDNAs encoding REF/SRPP-family proteins (FcREF/SRPP-1 and -2) from the laticifers ofFicus carica(fig tree). The amino acid sequences of these proteins showed high homology not only with each other but also with HbREF and HbSRPP. Recombinant FcREF/SRPP-1 and -2 were expressed inE.coli,and their aggregation properties were examined using a Congo red binding assay, agarose gel electrophoresis, and transmission electron microscopy. FcREF/SRPP-1 formed fibrils when incubated in PBS, and grew to micrometer-sized amorphous aggregates that precipitated rapidly. These aggregation properties of FcREF/SRPP-1 are quite similar to those of HbREF, although the growth rate and size of FcREF/SRPP-1 aggregates were inferior to those of HbREF. FcREF/SRPP-2 also formed aggregates during the incubation, but they did not precipitate, as has been reported for HbSRPP. Our results suggest that FcREF/SRPP-1 and -2 correspond to HbREF and HbSRPP, respectively. These aggregation properties could provide useful benchmarks for classifying REF/SRPP-family proteins as REF or SRPP.