Expression and refolding of recombinant human fibroblast procathepsin D

Gregory E Conner, J. A. Udey

Research output: Contribution to journalArticle

21 Citations (Scopus)

Abstract

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 was 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.

Original languageEnglish
Pages (from-to)1-9
Number of pages9
JournalDNA and Cell Biology
Volume9
Issue number1
StatePublished - May 2 1990

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Fibroblasts
Cathepsin D
Recombinant Proteins
Peptide Hydrolases
Enzyme Precursors
Inclusion Bodies
procathepsin D
Population Groups
Proteolysis
Half-Life
Proteins
Swine
Spleen
Cell Count
Bacteria
Acids
Antibodies
Genes
bacteriophage T7 RNA polymerase

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics
  • Cell Biology

Cite this

Expression and refolding of recombinant human fibroblast procathepsin D. / Conner, Gregory E; Udey, J. A.

In: DNA and Cell Biology, Vol. 9, No. 1, 02.05.1990, p. 1-9.

Research output: Contribution to journalArticle

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