Preparation of active recombinant cathepsin K expressed in bacteria as inclusion body.
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.
Department of Microbiology, Hannam University, 133 Ojung-dong, Daeduk-ku, Daejon 306-791, Republic of Korea.
Hyo-Sung
HS
Hwang
Hye-Shin
HS
Chung
D002384:Catalysis; D056657:Cathepsin K; D002403:Cathepsins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D003094:Collagen / Q000378:metabolism; D004789:Enzyme Activation; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D006868:Hydrolysis; D002479:Inclusion Bodies / Q000201:enzymology*; D008970:Molecular Weight; D017510:Protein Folding; D011498:Protein Precursors / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D020673:Protein Renaturation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013997:Time Factors
D011498:Protein Precursors; D011994:Recombinant Proteins; D003094:Collagen; D002403:Cathepsins; C534130:CTSK protein, human; D056657:Cathepsin K