Mouse and rat cystatin C: Escherichia coli production, characterization and tissue distribution

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Abstract

Recombinant mouse (Mus musculus) and rat (Rattus norvegicus) cystatin C were produced by expression in Escherichia coli, isolated and functionally characterized. The mouse and rat inhibitors were both fully active in titrations of papain. Determination of equilibrium constants for dissociation (Ki) for their complexes with the target proteinase, cathepsin B, produced values not largely different from that for human cystatin C (Ki 0.07-0.13 nM). Rabbit antisera against mouse and rat cystatin C were produced and used for improved affinity purification of the recombinant inhibitors. Affinity purified immunoglobulins isolated from the antiserum against mouse cystatin C were used for construction of a sensitive enzyme-linked immunosorbent assay. The assay was used to demonstrate a high degree of immunological cross-reactivity between mouse and rat cystatin C and could be used for cystatin C quantification in mouse and rat tissue homogenates. All tissues analyzed contained cystatin C, with a relative content very similar to that of human tissues. For all species, brain tissue contained the highest cystatin C amounts and liver the lowest, whereas kidney, spleen and muscle tissues were intermediate in content. In the mouse, a notable high cystatin C content in parotid gland tissue was observed. The high degree of similarity in distribution pattern and functional properties for mouse, rat and human cystatin C indicates that a murine model should be relevant for studies of the human disease, hereditary cystatin C amyloid angiopathy.

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Research areas and keywords

Subject classification (UKÄ) – MANDATORY

  • Hematology
  • Medicinal Chemistry
  • Pharmacology and Toxicology

Keywords

  • Cathepsin B, cysteine proteinase inhibitor, cysteine proteinases, E. coli expression, enzymelinkedimmunosorbent assay, hereditary cystatin C amyloid angiopathy, amyloidosis
Original languageEnglish
Pages (from-to)303-311
JournalComparative Biochemistry and Physiology - Part B: Biochemistry & Molecular Biology
Volume114
Issue number3
Publication statusPublished - 1996
Publication categoryResearch
Peer-reviewedYes