Protein Expression and PuriWcation 45 (2006) 157–167 www.elsevier.com/locate/yprep 1046-5928/$ - see front matter  2005 Elsevier Inc. All rights reserved. doi:10.1016/j.pep.2005.07.024 High yield synthesis and characterization of phosphorylated recombinant human procathepsin D expressed in mammalian cells Marina Démoz a , Roberta Castino a , Carlo Follo a , Andrej Hasilik b , Bonnie F. Sloane c,d , Ciro Isidoro a,¤ a Dipartimento di Scienze Mediche, Università “A. Avogadro,” via Solaroli 17, 28100 Novara, Italy b Institut für Physiologische Chemie, Philipps-Universität, Marburg D-35033, Germany c Department of Pharmacology, Wayne State University, Detroit, MI 48201, USA d Barbara Ann Karmanos Cancer Institute, Wayne State University, Detroit, MI 48201, USA Received 18 April 2005, and in revised form 6 July 2005 Available online 19 August 2005 Abstract We used a vaccinia virus expression system for the production of recombinant human cathepsin D (CD), a lysosomal protease implicated in various patho-physiological processes including cancer, neurodegeneration, and development. The recombinant pro- tein was successfully expressed in various human and non-human cells. It was correctly synthesized as a glycosylated 53 kDa precur- sor (proCD rec ) that reacted with a polyclonal antibody against residues 7–21 of the propeptide sequence. In contrast to the control, in cells infected with the recombinant virus proCD rec was largely secreted into the culture medium, although it contained high-mannose oligosaccharides with uncovered mannose-6-phosphate residues. Intracellular proCD rec was processed into the 48 kDa intermediate single-chain and the 31 plus 13 kDa double-chain forms, however, the processing was slower than in normal cells. A method based on Pepstatin A-aYnity chromatography allowed to isolate the recombinant protein from the medium of infected cells. Based on its latency in activity assay at acid pH and on its reactivity with antibodies speciWc for the N-terminus, the puriWed protein was judged to be in the inactive precursor form. During incubation at acid pH the puriWed proCD rec underwent autocatalytic processing and acquired pepstatin A-sensitive enzyme activity, as expected for correctly folded proCD. Antiserum raised in rabbits against proCD rec speciWcally reacted with human, but not with mouse proCD under non-denaturing conditions. We conclude that our vaccinia virus- directed proCD rec displays structural and functional features resembling those of native human proCD. This system can therefore be exploited for the synthesis of large quantities of human proCD, allowing further studies on the structure and function of this interest- ing protein.  2005 Elsevier Inc. All rights reserved. Keywords: Lysosomes; Vaccinia virus; Protein folding; Conformational epitopes Cathepsin D (CD) 1 is an aspartic endopeptidase nor- mally located in endosomal and lysosomal organelles and ubiquitously expressed in mammalian cells [1]. While in lysosomes CD is mainly involved in the degra- dation of (auto) phagocytosed proteins, within endo- somes this protease aVects the limited proteolysis of various substrates (such as pro-hormones and pro- enzymes), which results in their biological activation [2]. CD-mediated proteolysis has been shown to be impor- tant in various patho-physiological conditions including tissue homeostasis and organ development [3], neurode- generative diseases [4–7], ageing [8,9], and cancer [10–12]. In certain conditions, CD is translocated into the cytosol where it exerts a pro-apoptotic activity [13,14]. This activ- ity has been demonstrated directly by microinjecting CD * Corresponding author. Fax: +39 0321 620421. E-mail address: isidoro@med.unipmn.it (C. Isidoro). 1 Abbreviations used: CD, cathepsin; M6P, mannose-6-phosphate; Pst, pepstatin A; BrdU, 5-bromodeoxyuridine; TK, thymidine kinase.