Preparation of active recombinant cathepsin K expressed in bacteria as inclusion body

https://doi.org/10.1016/S1046-5928(02)00033-5Get rights and content

Abstract

Human cathepsin K (EC 3.4.22.38) is a member of the cysteine protease family with high primary sequence homology to cathepsins S, L, and B. It has been shown that cathepsin K plays a major role in the resorption of the bone matrix by osteoclasts. Cathepsin K has a potential as a drug target for the diseases related to bone matrix metabolism such as osteoporosis. We have expressed recombinant human procathepsin K in Escherichia coli as inclusion bodies. Purified procathepsin K had size of 38 kDa which is in agreement with the predicted mass of the construct. Refolding was done by rapid dilution into 50 mM Tris–HCl, pH 8.0 buffer containing 5 mM EDTA, 10 mM GSH, 1 mM GSSG, 0.7 M l-arginine, 0.5 M NaCl, and 1% CHAPS and further dialysis against 25 mM Tris–HCl, pH 8.0 containing 0.5 M NaCl. Mature active cathepsin K was prepared from refolded procathepsin K by incubating at 40 °C in pH 4.0 buffers with or without pepsin or cysteine. The presence of pepsin or cysteine in autocatalysis buffer did not have effect on the degree of conversion of nascent to mature cathepsin K, but reduced the autocatalysis time slightly. Proteolytic activity was confirmed using synthetic substrate, and Western blotting identified mature cathepsin K. Active cathepsin K had type I and II collagenolytic activities which could be inhibited by E-64.

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Materials

The E. coli strain, JM109[recA1 endA1 gyrA96 thi-1 hsdR17 supE44 relA1 Δ(lac proAB)/F(traD36 proAB lacIq lacZΔM15)], was used as a expression host. Expression vector with procathepsin K gene inserted into pQE-30 (Qiagen, USA) was a kind gift from Dr. Stephen Weiss in University of Michigan. E. coli culture medium was Luria–Bertani (LB; 1% Bacto peptone, 0.5% NaCl, and 0.5% yeast extract) containing ampicillin (50μg/ml). Prestained molecular weight markers were purchased from Gibco BRL

Expression, purification, and refolding of recombinant protein procathepsin K

Recombinant human procathepsin K was abundantly expressed in E. coli with N-terminal His-tag (Fig. 1). Recombinant protein expressed with IPTG induction showed approximate molecular weight of 38 kDa in SDS–PAGE gel, which corresponds to the size of procathepsin K. Recombinant procathepsin K was expressed as insoluble inclusion body and could be dissolved in denaturing buffer. This is advantageous in isolating recombinant insoluble procathepsin K from soluble proteins, since separation of

Acknowledgements

This work has been supported by Hannam University.

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