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  • Title: Cynomolgus monkey (Macaca fascicularis) cathepsin K: cloning, expression, purification, and activation.
    Author: McQueney MS, Feild J, Hanning CR, Brun K, Ramachandran K, Connor J, Drake F, Jones CS, Amegadzie BY.
    Journal: Protein Expr Purif; 1998 Dec; 14(3):387-94. PubMed ID: 9882573.
    Abstract:
    Methodology for the production of recombinant active cynomolgus monkey (Macaca fascicularis) cathepsin K (EC 3.4.22.38) was elucidated. The cDNA encoding the cathepsin K was cloned from female M. cynomolgus monkey mRNA. The deduced amino acid sequence of M. cynomolgus preprocathepsin K from the cDNA sequence showed 94.2% identity to human preprocathepsin K. Sequence differences occurred only in the prepro- domains; the mature domains were identical. The recombinant M. cynomolgus cathepsin K was expressed as a secreted proenzyme using baculovirus-infected SF21 insect cells having the predicted N-terminus (LYPEEILDTH ellipsis ), indicating proper cleavage of the secretion sequence. Purified monkey procathepsin K was activated under autocatalytic conditions at pH 4.0. The mature enzyme was composed of mixture of enzymes having N-termini of Gly113 and Arg114. The molecular weight was determined to be 23,668.3 Da by MALDI-TOF-MS which is consistent with the absence of carbohydrate on the mature enzyme. These results indicate that monkey procathepsin K is able to autoactivate and produces a mature enzyme which is identical to that of human cathepsin K. Since the sequence of monkey and human mature cathepsin K are identical and the in vitro activation mechanisms appear to be indistinguishable, monkeys are predicted to be a good animal model for evaluating cathepsin K inhibitors in vivo as therapeutic agents for diseases characterized by excessive bone loss, such as osteoporosis.
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