Cynomolgus monkey (Macaca fascicularis) cathepsin K: Cloning, expression, purification, and activation

Methodology for the production of recombinant active cynomolgus monkey (Mac aca fascicularis) cathepsin It (EC 3.4.22.38) was elucidated, The cDNA enco ding the cathepsin K was cloned from female M. cynomolgus monkey mRNA The d educed amino acid sequence of M. cynomolgus preprocathepsin K from the cDNA sequence showed 94.2% identity to human preprocathepsin K. Sequence differ ences occurred only in the prepro-domains; the mature domains were identica l. The recombinant M. cynomolgus cathepsin K was expressed as a secreted pr oenzyme using baculovirus-infected SF21 insect cells having the predicted N -terminus (LYPEEILDTH...), indicating proper cleavage of the secretion sequ ence. Purified monkey procathepsin K was activated under autocatalytic cond itions at pH 4.0, The mature enzyme was composed of mixture of enzymes havi ng N-termini of Gly(113) and Arg(114). Th, molecular weight was determined to be 23,668.3 Da by MALDI-TOF-MS which is consistent with the absence of c arbohydrate on the mature enzyme, These results indicate that monkey procat hepsin K is able to autoactivate and produces a mature enzyme which is iden tical to that of human cathepsin K. Since the sequence of monkey and human mature cathepsin K are identical and the in vitro activation mechanisms app ear to be indistinguishable, monkeys are predicted to be a good animal mode l for evaluating cathepsin K inhibitors in vivo as therapeutic agents for d iseases characterized by excessive boneless, such as osteoporosis, (C) 1998 Academic Press.