One-step purification of cathepsin D by affinity chromatography using immobilized propeptide sequences.
In vivo, active cathepsin D proteinase is generated by removal of a 44-residue propeptide at its N-terminus. Here we report that mature cathepsin D and pseudocathepsin D (a partially activated form of cathepsin D with 25 amino acid residues removed from the propeptide) bind to the immobilized propeptide, while procathepsin D does not. The N-terminal 25 amino acid residues of the propeptide are sufficient for this binding. Based on this observation, a simple one-step procedure was developed to purify mature cathepsin D from whole cell extracts to near homogeneity. This method has the advantage over existing affinity-purification systems that active forms of the proteinase can be separated from inactive precursors and other aspartic proteinases. Furthermore, this technique was effective for pepsin as well, suggesting it may have general utility for all activated aspartic proteinases and perhaps other families of proteinases.
Wittlin
S
S
Novartis Pharma AG, Oncology, Basel, Switzerland. sergio.wittlin@pharma.novartis.com
Rösel
J
J
Stover
D R
DR
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D001943:Breast Neoplasms; D002402:Cathepsin D / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification*; D002417:Cattle; D002846:Chromatography, Affinity / Q000379:methods; D012085:Chymosin / Q000737:chemistry; D004792:Enzyme Precursors / Q000096:biosynthesis* / Q000302:isolation & purification; D005260:Female; D005982:Glutathione Transferase / Q000096:biosynthesis; D006801:Humans; D008969:Molecular Sequence Data; D010434:Pepsin A / Q000737:chemistry; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D012083:Renin / Q000737:chemistry; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013552:Swine; D014162:Transfection; D014407:Tumor Cells, Cultured
D004792:Enzyme Precursors; D011993:Recombinant Fusion Proteins; D005982:Glutathione Transferase; C058345:procathepsin D; D010434:Pepsin A; D012083:Renin; D012085:Chymosin; D002402:Cathepsin D