Junqueira-de-Azevedo Inácio de L M, Pertinhez Thelma, Spisni Alberto, Carreño Flávia Regina, Farah Chuck S, Ho Paulo Lee
Centro de Biotecnologia, Instituto Butantan, São Paulo, Brazil.
Biochim Biophys Acta. 2003 May 30;1648(1-2):90-8. doi: 10.1016/s1570-9639(03)00111-0.
The EF-hand protein family is comprised of many proteins with conserved Ca(2+)-binding motifs with important biological roles in intracellular communication. During the generation of Expressed Sequence Tags (ESTs) from the venom glands of the Viperidae snake Bothrops insularis, we identified a cDNA coding for a putative Ca(2+) binding protein with four EF-hand motifs, named here calglandulin. The deduced amino acid sequence displayed the greatest sequence similarity with calmodulin (59%), followed by troponin-C (52%). The encoded polypeptide was first expressed in Escherichia coli as a 6XHis-tagged fusion protein. The expressed protein was purified by Ni(2+)-charged affinity chromatography and circular dichroism (CD) spectroscopy confirmed the prevalence of alpha-helix as observed in calmodulin/calmodulin-like proteins. A polyclonal antiserum was generated in mice using this recombinant calglandulin. To investigate the tissue-specific biological occurrence of this protein, this antiserum was used in Western blot experiments, which revealed an immunoreactive band in samples of venom gland extracts from different snakes, but not in the crude venom or in brain, heart and other tissues. This exclusive occurrence suggests a specialized function of calglandulin in snake venom glands.
EF 手型蛋白家族由许多具有保守钙结合基序的蛋白质组成，这些蛋白质在细胞内通讯中发挥着重要的生物学作用。在从岛蝰（Bothrops insularis）蛇毒腺中生成表达序列标签（EST）的过程中，我们鉴定出一个编码具有四个 EF 手型基序的假定钙结合蛋白的 cDNA，在此命名为钙腺蛋白（calglandulin）。推导的氨基酸序列与钙调蛋白的序列相似性最高（59%），其次是肌钙蛋白 C（52%）。编码的多肽首先在大肠杆菌中作为 6XHis 标签融合蛋白表达。表达的蛋白通过镍离子亲和色谱法纯化，圆二色性（CD）光谱证实了其具有与钙调蛋白/类钙调蛋白相似的α螺旋结构。使用这种重组钙腺蛋白在小鼠中制备了多克隆抗血清。为了研究该蛋白在组织中的特异性生物学存在情况，将这种抗血清用于蛋白质印迹实验，结果显示在不同蛇的毒腺提取物样本中出现了一条免疫反应带，但在粗毒、脑、心脏和其他组织中未出现。这种独特的存在表明钙腺蛋白在蛇毒腺中具有特殊功能。
