Vidmar Tilen, Pavšič Miha, Lenarčič Brigita
Department of Chemistry and Biochemistry, Faculty of Chemistry and Chemical Technology, University of Ljubljana, SI-1000 Ljubljana, Slovenia.
Protein Expr Purif. 2013 Sep;91(1):69-76. doi: 10.1016/j.pep.2013.07.006. Epub 2013 Jul 17.
Trop2 is a stem/progenitor cell marker, which is also upregulated in several human carcinomas. The largest part of the molecule, recognized by several monoclonal antibodies, is represented by the extracellular part (ectodomain) and is composed of three modules. The aim of our work was to prepare the ectodomain of Trop2 in quantities sufficient for structural and functional studies. We used the Spodoptera frugiperda (Sf9) insect cell expression system to prepare the Trop2 ectodomain (Trop2EC) in two forms - wt glycosylated (gTrop2EC) and mutant non-glycosylated form (Trop2EC(Δ/N)). Recombinant protein was purified from cell culture supernatants using two subsequent nickel ion-affinity chromatographies with a final yield of 15-17mg of purified recombinant protein per liter of culture. Size-exclusion chromatography together with MALS and chemical crosslinking were used to demonstrate for the first time that the Trop2 ectodomain forms a dimer. Both gTrop2EC and Trop2EC(Δ/N) exhibit similar biochemical properties, however the solubility of Trop2EC(Δ/N) is much lower (less than 1mg/ml). For the purpose of structural studies, we crystallized the glycosylated form gTrop2EC. The native dataset was collected with a resolution of 2.94Å and will be used in ongoing work for phasing and structure solution to further understand the role of Trop2 and the structure-function relation between Trop2 and the epithelial cell adhesion molecule (EpCAM).
Trop2是一种干细胞/祖细胞标志物，在几种人类癌症中也有上调。该分子的最大部分可被几种单克隆抗体识别，由细胞外部分（胞外域）构成，由三个模块组成。我们工作的目的是制备足够数量的Trop2胞外域，用于结构和功能研究。我们使用草地贪夜蛾（Sf9）昆虫细胞表达系统制备两种形式的Trop2胞外域（Trop2EC）——野生型糖基化形式（gTrop2EC）和突变型非糖基化形式（Trop2EC(Δ/N)）。通过两次连续的镍离子亲和色谱从细胞培养上清液中纯化重组蛋白，每升培养物最终纯化重组蛋白的产量为15 - 17毫克。尺寸排阻色谱结合多角度激光光散射和化学交联首次证明Trop2胞外域形成二聚体。gTrop2EC和Trop2EC(Δ/N)都表现出相似的生化特性，然而Trop2EC(Δ/N)的溶解度要低得多（小于1毫克/毫升）。为了进行结构研究，我们使糖基化形式的gTrop2EC结晶。收集到分辨率为2.94Å的天然数据集，将用于正在进行的相位分析和结构解析工作，以进一步了解Trop2的作用以及Trop2与上皮细胞粘附分子（EpCAM）之间的结构 - 功能关系。
