title: Biochemical and preliminary X-ray characterization of the tumor-associated calcium signal transducer 2 (Trop2) ectodomain.
abstract: 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).
authors.affiliation: Department of Chemistry and Biochemistry, Faculty of Chemistry and Chemical Technology, University of Ljubljana, SI-1000 Ljubljana, Slovenia.
authors.forename: Tilen
authors.initials: T
authors.lastname: Vidmar
authors.forename: Miha
authors.initials: M
authors.lastname: Pavšič
authors.forename: Brigita
authors.initials: B
authors.lastname: Lenarčič
mesh_terms: D000951:Antigens, Neoplasm / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D015815:Cell Adhesion Molecules / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002850:Chromatography, Gel; D002942:Circular Dichroism; D003460:Crystallization; D004591:Electrophoresis, Polyacrylamide Gel; D006031:Glycosylation; D006801:Humans; D055503:Protein Multimerization; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D061987:Sf9 Cells
chemical_list: D000951:Antigens, Neoplasm; D015815:Cell Adhesion Molecules; D011994:Recombinant Proteins; C499380:TACSTD2 protein, human
keywords: Dimer; EpCAM; Extracellular domain; MultiBac; N-Glycosylation; Protein crystallization; Sf9 insect cells