Home
Resources
Publications
Preparation of Active Recombinant Cathepsin K Expressed in Bacteria as Inclusion Body

Preparation of Active Recombinant Cathepsin K Expressed in Bacteria as Inclusion Body

Hyo-Sung Hwang, Hye-Shin Chung

Protein Expr Purif. 2002 Aug;25(3):541-6.

PMID: 12182837

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 38kDa which is in agreement with the predicted mass of the construct. Refolding was done by rapid dilution into 50mM Tris-HCl, pH 8.0 buffer containing 5mM EDTA, 10 mM GSH, 1mM 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 degrees 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.

Chemicals Related in the Paper:

Catalog Number	Product Name	Structure	CAS Number	Price
IAR42412307	Cathepsin S, Human, Recombinant, E. coli			Price
IAR42412319	Procathepsin K, Human, Recombinant, E. coli			Price

As a specialist in analytical chemical Products, Alfa Chemistry offers consumers a wealth of raw materials and a full range of technologies and services.

QUICK LINKS

HEADQUARTERS

Tel:

Fax:

Email: