Study of a novel antiosteoporosis screening model targeted on cathepsin K.
OBJECTIVE
To establish an effective assay to access the effects of natural products on cathepsin K for screening antiosteoporosis drugs.


METHODS
To obtain the purified cathepsin K, we cloned the target fragment from the mRNA of human osteosacoma cell line MG63 and demonstrated its correctness through DNA sequencing. Cathepsin K was expressed in a high amount in E. coli after IPTG induction, and was purified to near homogenetity through resolution and column purification. The specificity of the protein was shown by Western blotting experiment. The biological activity of the components in the fermentation broth was assayed by their inhibitory effects on cathepsin K and its analog papain.


RESULTS
With the inhibition of papain activity as a screen index, the fermentation samples of one thousand strains of fungi were tested and 9 strains among them showed strong inhibitory effects. The crude products of the fermentation broth were tested for their specific inhibitory effects on the purified human cathepsin K, the product of fungi 2358 shows the highest specificity against cathepsin K.


CONCLUSIONS
The compounds isolated from fungi 2358 show the highest biological activity and are worth further structure elucidation and function characterization.
Yang
Jun
J
Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences, China. yangjunimb@hotmail.com
Shang
Guang-Dong
GD
Zhang
Yue-Qin
YQ
D001681:Biological Assay; D015153:Blotting, Western; D056657:Cathepsin K; D002403:Cathepsins / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000302:isolation & purification; D002460:Cell Line; D003001:Cloning, Molecular; D018076:DNA, Complementary; D004353:Drug Evaluation, Preclinical; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D015870:Gene Expression; D006801:Humans; D010024:Osteoporosis / Q000188:drug therapy*; D010206:Papain / Q000037:antagonists & inhibitors*; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid
D018076:DNA, Complementary; D002403:Cathepsins; D010206:Papain; C534130:CTSK protein, human; D056657:Cathepsin K