title: Expression, purification and auto-activation of cathepsin E from insect cells.
abstract: Cathepsin E is an aspartic protease that belongs to the pepsin family. This protease is similar to cathepsin D but differs in its tissue distribution and cell localization. Elevated levels of this enzyme are linked to several tumors, including devastating pancreatic ductal adenocarcinoma. In this manuscript, we present a new protocol for the high-yield purification of recombinant human cathepsin E in the baculovirus expression system. The recombinant protein was produced by the Sf9 insect cell line and secreted into the medium in the form of an inactive zymogen. Procathepsin E was purified using ion-exchange and size exclusion chromatographies followed by pepstatin- and heparin-affinity chromatography steps. The zymogen was activated at an acidic pH, resulting in a high yield of the activated intermediate of cathepsin E. The enzymatic activity, stability, and molecular weight corresponded to those of cathepsin E. The new purification procedure will promote further studies of this enzyme to improve the understanding of its structure-function relationship and consequently enable the development of better therapeutic approaches.
authors.forename: Tajana Z
authors.initials: TZ
authors.lastname: Železnik
authors.forename: Vida
authors.initials: V
authors.lastname: Puizdar
authors.affiliation: Jozef Stefan Institute, Department of Biochemistry and Molecular and Structural Biology, Jamova 39, SI-1000 Ljubljana, Slovenia. iztok.dolenc@ijs.si.
authors.forename: Iztok
authors.initials: I
authors.lastname: Dolenc
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D020059:Cathepsin E / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D004792:Enzyme Precursors / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D004926:Escherichia coli; D057809:HEK293 Cells; D006801:Humans; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D061987:Sf9 Cells
chemical_list: D004792:Enzyme Precursors; D011994:Recombinant Proteins; C067581:procathepsin E; D020059:Cathepsin E