Biochemical and preliminary X-ray characterization of the tumor-associated calcium signal transducer 2 (Trop2) ectodomain

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• 作者：Tilen Vidmar ; Miha Pav?i? ; Brigita Lenar?i?
• 关键词：Extracellular domain ; Sf9 insect cells ; N-Glycosylation ; Dimer ; Protein crystallization ; EpCAM ; MultiBac
• 刊名：Protein Expression and Purification
• 出版年：2013
• 出版时间：September, 2013
• 年：2013
• 卷：91
• 期：1
• 页码：69-76
• 全文大小：1041 K

文摘

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^¦¤/N). Recombinant protein was purified from cell culture supernatants using two subsequent nickel ion-affinity chromatographies with a final yield of 15-17 mg 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^¦¤/N exhibit similar biochemical properties, however the solubility of Trop2EC^¦¤/N is much lower (less than 1 mg/ml). For the purpose of structural studies, we crystallized the glycosylated form gTrop2EC. The native dataset was collected with a resolution of 2.94 ? 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).