Expression of human cathepsin K in Pichia pastoris and preliminary crystallographic studies of an inhibitor complex

Abstract

Cathepsin K is a cysteine protease of the papain family, which is predominantly expressed in osteoclasts, and is regarded as a key protease in bone remodeling. To facilitate structural studies of the protein, the wild-type sequence of the protease has been mutated so as to replace a potential N-glycosylation site. We have expressed the mutant human cathepsin K to 190 mg/5 L using the Pichia pastoris expression system. Cathepsin K was inactivated with the mechanism-based inhibitor, APC3328, and crystallized from magnesium formate. A 2.2 8, X-ray data set has been collected on crystals belonging to space group P 2 1 2~2~. with a = 41.66 A, b = 5 1.41 A, and c = 107.72 A. There is most likely one molecule per asymmetric unit.

Effects of Cysteine Proteases on the Structural and Mechanical Properties of Collagen Fibers

“…Proteases-Human cathepsins K, V, S, and L were expressed in Pichia pastoris using the pPIC9K vector (32,33). Cathepsin proteins were purified by chromatography using N-butyl-Sepharose and SP-Sepharose (Amersham Biosciences) (34), and their active site concentrations were determined by E-64 titration as described previously (35).…”

Dipeptide-derived nitriles containing additional electrophilic sites: Potentially irreversible inhibitors of cysteine proteases

“…Cathepsin L from bovine liver and recombinant human cathepsin S were purchased from Calbiochem, Darmstadt, Germany. Recombinant human cathepsin K (expressed in Pichia pastoris) was a gift from D. Brömme 15 . The substrates, Z-Phe-Arg-NHNp, Z-Phe-Arg-NHMec, Z-Val-Val-Arg-NHMec, and Z-LeuArg-NHMec, were from Bachem, Bubendorf, Switzerland.…”

Glycosaminoglycan-Mediated Loss of Cathepsin K Collagenolytic Activity in MPS I Contributes to Osteoclast and Growth Plate Abnormalities

“…Recombinant human cathepsin K was expressed using the Pichia pastoris expression system as previously described. 18 Total volume of each reaction was 100 l, digestions were performed at 28°C for 4 hours. Cathepsin K collagen digestions were performed in the presence of 800 nmol/L chondroitin 4-sulfate (Sigma-Aldrich, St-Louis, MO) (chondroitin 4-sulfate concentration is based on an average molecular weight of 30 kDa).…”