High yield synthesis and characterization of phosphorylated recombinant human procathepsin D expressed in mammalian cells

• Published in: Protein expression and purification 2006, Vol.45 (1), p.157-167
• Main Authors: Démoz, Marina, Castino, Roberta, Follo, Carlo, Hasilik, Andrej, Sloane, Bonnie F., Isidoro, Ciro
• Format: Article
• Language: English
• Subjects: Animals; Cathepsin D - drug effects; Cathepsin D - isolation & purification; Cathepsin D - metabolism; Cell Line; Cell Line, Tumor; Cells, Cultured; Conformational epitopes; DNA, Complementary - genetics; Enzyme Precursors - drug effects; Enzyme Precursors - isolation & purification; Enzyme Precursors - metabolism; Haplorhini; HeLa Cells; Humans; Immune Sera - pharmacology; Lysosomes; Mice; Oligosaccharides - chemistry; Phosphorylation; Protein folding; Rats; Recombinant Proteins - drug effects; Recombinant Proteins - isolation & purification; Recombinant Proteins - metabolism; Sensitivity and Specificity; Structure-Activity Relationship; Vaccinia virus
• ISSN: 1046-5928; 1096-0279
• DOI: 10.1016/j.pep.2005.07.024

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 protein was successfully expressed in various human and non-human cells. It was correctly synthesized as a glycosylated 53 kDa precursor (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-affinity 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 specific for the N-terminus, the purified protein was judged to be in the inactive precursor form. During incubation at acid pH the purified 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 specifically 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 interesting protein.