Varejão Nathalia, Almeida Marcius da Silva, De Cicco Nuccia N T, Atella Geórgia C, Coelho Luana C B B, Correia M A Tereza S, Foguel Debora
Instituto de Bioquímica Médica, Programa de Biologia Estrutural, Centro Nacional de Ressonância Magnética Nuclear de Macromoléculas, Universidade Federal do Rio de Janeiro, Av. Bauhínia, 400, 21941-590, Rio de Janeiro, RJ, Brazil.
Biochim Biophys Acta. 2010 Sep;1804(9):1917-24. doi: 10.1016/j.bbapap.2010.06.004. Epub 2010 Jun 9.
CRAMOLL 1 is a mannose/glucose isolectin isolated from Cratylia mollis seeds. This lectin has 82% sequence identity with Con A and essentially the same quaternary structure. As with Con A, CRAMOLL 1 seems to undergo complex post-translational processing which makes it difficult to the use of traditional molecular cloning for heterologous expression. Here we report the expression and purification of functional recombinant CRAMOLL 1 (rCRAMOLL 1) in Escherichia coli. This was accomplished by introducing a chemically synthesized DNA encoding the mature CRAMOLL 1 amino acid sequence into a bacterial expression vector under T7 promoter control. Most of the recombinant lectin was found in insoluble aggregates (inclusion bodies), but we were able to recover reasonable amounts of soluble lectin in the active form by decreasing the protein induction temperature. The recombinant lectin was purified to homogeneity with one-step affinity chromatography. The plant CRAMOLL 1 (pCRAMOLL 1) and rCRAMOLL 1 share several physicochemical properties such as molecular mass, charge density and secondary and tertiary structures. However, pCRAMOLL 1 has a lower thermodynamic stability than rCRAMOLL 1 when probed by acidification, high temperature or high hydrostatic pressure, and this is probably caused by the presence of tetramers composed of fragmented monomers, which are formed in the plant cotyledon but absent from the recombinant protein. rCRAMOLL 1 behaves identically to its plant counterpart with respect to its specificity for monosaccharides, and to its agglutinating activities against rabbit erythrocytes and Trypanosoma cruzi epimastigote cells.
CRAMOLL 1是一种从毛蟹爪兰种子中分离得到的甘露糖/葡萄糖异凝集素。这种凝集素与伴刀豆球蛋白A有82%的序列同一性，并且具有基本相同的四级结构。与伴刀豆球蛋白A一样，CRAMOLL 1似乎经历了复杂的翻译后加工过程，这使得利用传统分子克隆进行异源表达变得困难。在此，我们报道了功能性重组CRAMOLL 1（rCRAMOLL 1）在大肠杆菌中的表达和纯化。这是通过将编码成熟CRAMOLL 1氨基酸序列的化学合成DNA引入受T7启动子控制的细菌表达载体中来实现的。大部分重组凝集素存在于不溶性聚集体（包涵体）中，但通过降低蛋白质诱导温度，我们能够回收相当数量的活性形式的可溶性凝集素。重组凝集素通过一步亲和层析纯化至均一。植物CRAMOLL 1（pCRAMOLL 1）和rCRAMOLL 1具有若干物理化学性质，如分子量、电荷密度以及二级和三级结构。然而，当通过酸化、高温或高静水压进行检测时，pCRAMOLL 1的热力学稳定性低于rCRAMOLL 1，这可能是由于存在由片段化单体组成的四聚体，这些四聚体在植物子叶中形成，但在重组蛋白中不存在。rCRAMOLL 1在对单糖的特异性以及对兔红细胞和克氏锥虫前鞭毛体细胞的凝集活性方面与其植物对应物表现相同。
