Hirakane Makoto, Taniguchi Masahiro, Yoshinaga Sosuke, Misumi Shogo, Terasawa Hiroaki
Department of Structural BioImaging, Faculty of Life Sciences, Kumamoto University, Chuo-ku, Kumamoto 862-0973, Japan.
Department of Environmental and Molecular Health Sciences, Faculty of Life Sciences, Kumamoto University, Chuo-ku, Kumamoto 862-0973, Japan.
Protein Expr Purif. 2014 Apr;96:20-5. doi: 10.1016/j.pep.2014.01.010. Epub 2014 Jan 31.
Pheromones are species-specific chemical signals that regulate a wide range of social and sexual behaviors in many animals. In mice, the male-specific peptide ESP1 (exocrine gland-secreting peptide 1) is secreted into tear fluids and enhances female sexual receptive behavior. ESP1 belongs to the ESP family, a multigene family with 38 genes in mice. ESP1 shares the highest homology with ESP4. ESP1 is expressed in the extraorbital lacrimal gland, whereas ESP4 is expressed in some exocrine glands. Thus, ESP4 is expected to have a function that has not been elucidated yet. Large amounts of the purified ESP4 protein are required for structural and biochemical studies. Here we present an expression and purification scheme for the recombinant ESP4 protein. The N-terminally histidine-tagged ESP4 fusion protein was expressed in Escherichia coli as inclusion bodies, which were solubilized and purified by nickel affinity chromatography. The histidine tag was cleaved with thrombin and removed by a second nickel affinity chromatography step. The ESP4 protein was isolated with high purity by reversed-phase chromatography. For NMR analyses, we prepared a stable isotope-labeled ESP4 protein. Three repeated freeze-drying steps after the reversed-phase chromatography were required, to remove a volatile contaminating compound and to obtain an NMR spectrum with a homogeneous line shape. AMS-modification and far-UV CD spectroscopic analyses suggested that ESP4 has an intramolecular disulfide bridge and a helical structure, respectively. The present study provides a powerful tool for structural and biochemical studies of ESP4, leading toward the elucidation of the roles of the ESP family members.
信息素是物种特异性的化学信号,可调节许多动物的广泛社会行为和性行为。在小鼠中,雄性特异性肽ESP1(外分泌腺分泌肽1)分泌到泪液中,增强雌性的性接受行为。ESP1属于ESP家族,这是一个在小鼠中有38个基因的多基因家族。ESP1与ESP4的同源性最高。ESP1在外眶泪腺中表达,而ESP4在一些外分泌腺中表达。因此,预计ESP4具有尚未阐明的功能。结构和生化研究需要大量纯化的ESP4蛋白。在此,我们提出了一种重组ESP4蛋白的表达和纯化方案。N端带有组氨酸标签的ESP4融合蛋白在大肠杆菌中作为包涵体表达,通过镍亲和层析进行溶解和纯化。组氨酸标签用凝血酶切割,并通过第二步镍亲和层析去除。通过反相层析以高纯度分离出ESP4蛋白。为了进行核磁共振分析,我们制备了稳定同位素标记的ESP4蛋白。反相层析后需要进行三个重复的冻干步骤,以去除挥发性污染化合物并获得具有均匀线形的核磁共振谱。AMS修饰和远紫外圆二色光谱分析分别表明ESP4具有分子内二硫键和螺旋结构。本研究为ESP4的结构和生化研究提供了有力工具,有助于阐明ESP家族成员的作用。