%0 Journal Article
%T Expression, Purification, and Characterization of Ras Protein (BmRas1) from Bombyx mori
%A Yanping Quan
%A Guangqiang Liu
%A Wei Yu
%A Zuoming Nie
%A Jian Chen
%A Zhengbing Lv
%A Yaozhou Zhang
%J International Journal of Genomics
%D 2012
%I Hindawi Publishing Corporation
%R 10.1155/2012/747539
%X The Ras subfamily is the member of small G proteins superfamily involved in cellular signal transduction. Activation of Ras signaling causes cell growth, differentiation, and survival. Bombyx mori Ras-like protein (BmRas1) may belong to the Ras subfamily. It contained an H-N-K-Ras-like domain. The BmRas1 mRNA consisted of 1459£¿bp. The open reading frame contained 579£¿bp, encoding 192 amino acids. The protein had such secondary structures as ¦Á-helices, extended strand, and random coil. BmRas1 was expressed successfully in E. coli BL21. The recombinant protein was purified with metal-chelating affinity chromatography. The GTPase activity of purified protein was determined by FeSO4-(NH4)2MoO4 assay. The results showed that purified recombinant protein had intrinsic activity of GTPase. High titer polyclonal antibodies were generated by New Zealand rabbit immunized with purified protein. The gene expression features of BmRas1 at different stages and in different organs of the fifth instar larvae were analyzed by Western blot. The results showed that BmRas1 was expressed highly in three development stages including egg, pupae, and adult, but low expression in larva. BmRas1 was expressed in these tissues including head, malpighian tubule, genital gland, and silk gland. The purified recombinant protein would be utilized to further function studies of BmRas1. 1. Introduction Ras genes were first identified as homologues of rodent sarcoma virus genes. In 1982, human DNA sequences homologous to the transforming oncogenes of the v-Harvey (H-Ras) and Kirsten (K-Ras) rat sarcoma virus were identified in DNA sequences derived from a human bladder and a human lung cancer cell line, respectively. There are three mammalian Ras proteins: H-Ras, N-Ras, and K-Ras, which consisted of 188-189 amino acid (p21 proteins), encoded by three ras genes [1]. The Ras isoforms are highly homologous [2]. Ras proteins are positioned at the inner surface of the plasma membrane where they serve as binary molecular switches to transduce extracellular ligand-mediated stimuli into the cytoplasm to control signal transduction pathways that influence cell growth, differentiation, and apoptosis [3, 4]. The Ras protein is the prototype of the Ras superfamily of small GTPases, which share a high degree of sequence similarity and a common three-dimensional structure, called the GTP-binding domain. This domain enables them to act as molecular switches cycling between two defined conformational states: an inactive guanosine-diphosphate (GDP-) bound and an active guanosine-triphosphate-(GTP-) bound
%U http://www.hindawi.com/journals/ijg/2012/747539/