Expression and refolding of recombinant human fibroblast procathepsin D.
Procathepsin D is a precursor of the human lysosomal protease cathepsin D. Due to its short half-life, procathepsin D is difficult to obtain in quantities sufficient to allow structural and enzymatic studies. To obtain large quantities of this precursor, procathepsin D was expressed using the T7 promoter vector pET3a in bacteria that carry a chromosomal copy of the T7 RNA polymerase gene under the control of the lac promoter. At high cell density in rich medium, basal levels of T7 RNA polymerase were sufficient to express recombinant procathepsin D without addition of an exogenous inducer of the lac promoter. The recombinant protein, constituting almost half of the total cell protein, accumulated in intracytoplasmic inclusion bodies and was isolated from the insoluble fraction of lysed cells. Antibodies prepared against the purified recombinant protein were shown to crossreact with native human placental and porcine spleen cathepsin D. Recombinant procathepsin D was solubilized in denaturants and was refolded. After extended preincubation of the denatured protein at acid pH to allow folding and activation of the zymogen, pepstatin inhibitable catalytic proteolysis was detected. These data demonstrated that the glycosylated aspartic protease, procathepsin D can be refolded and activated in an unglycosylated form and thus provides a system for the study of procathepsin D structure and function.
Conner
G E
GE
Department of Cell Biology and Anatomy, University of Miami School of Medicine, FL 33101.
Udey
J A
JA
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies; D001483:Base Sequence; D002402:Cathepsin D / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D003429:Cross Reactions; D004792:Enzyme Precursors / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D005260:Female; D005347:Fibroblasts / Q000201:enzymology*; D005822:Genetic Vectors; D006801:Humans; D008969:Molecular Sequence Data; D010920:Placenta / Q000201:enzymology; D011247:Pregnancy; D011401:Promoter Regions, Genetic; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000096:biosynthesis; D012995:Solubility; D013552:Swine
D000906:Antibodies; D004792:Enzyme Precursors; D011994:Recombinant Proteins; C058345:procathepsin D; D002402:Cathepsin D