Grant-in-Aid for Scientific Research (B)
|Allocation Type||Single-year Grants|
|Research Institution||Osaka University of Pharmaceutical Sciences|
ISHIDA Toshimasa Osaka University of Pharmaceutical Sciences, Professor, 薬学部, 教授 (00111021)
TOMOO Koji Osaka University of Pharmaceutical Sciences, research associate, 薬学部, 助手 (70257898)
IN Yasuko Osaka University of Pharmaceutical Sciences, research associate, 薬学部, 助手 (50257896)
|Project Period (FY)
1994 – 1996
Completed(Fiscal Year 1996)
|Budget Amount *help
¥6,400,000 (Direct Cost : ¥6,400,000)
Fiscal Year 1996 : ¥1,400,000 (Direct Cost : ¥1,400,000)
Fiscal Year 1995 : ¥1,600,000 (Direct Cost : ¥1,600,000)
Fiscal Year 1994 : ¥3,400,000 (Direct Cost : ¥3,400,000)
|Keywords||cathepsin L / procathepsin L / recombinant / expression / X-ray analysis / NMR / inhibitor / molecular design|
In the term of 1994-1996, we went ahead with the research project according to the following research plans :
(1) Large scale expression of rat cathepsin L in Escherichia coli.
(2) Isolation and purification of recombinant rat cathepsin L from Escherichia coli.
(3) Identification of active conformation of recombinant rat cathepsin L by the comparison of native one.
(4) X-Ray crystal structure determination of recombinant rat cathepsin L.
(5) Preparation and physicochemical characterization of recombinant rat cathepsin L-inhibitor complex.
(6) X-Ray crystal structure determination of recombinant rat cathepsin L and its complex with inhibitor.
(7) NMR solution conformation of recombinant rat cathepsin L and its complex with inhibitor.
(8) Model building and characterization of cathepsin L active site and elucidation of the catalytic mechanism at atomic level.
(9) Molecular design and inhibitory measurement of cathepsin L-specific inhibitor based on the results of (4) - (8).
We succeeded already in
the research plans of (1) - (3), and are now vigorously studying the projects of (4) - (7) at the same time, with hoping these accomplishments in 1997. The main reason why we could not complete the research project within 1994-1996 is due to the instability of recombinant cathepsin L.Although structure determinations by X-ray diffraction and NMR methods require the stability of sample for a long time, rat cathepsin L itself decomposes to peptide fragments within few days due to the high autocatalytic activity. In order to overcome such trouble, we prepared (i) two kinds of recombinant procathepsin L from E.coli., which are resistant against the autocatalysis, and (ii) cathepsin L-inhibitor complex, where the inhibitor was complexed with recombinant cathepsin L at its expression state. Although the tertiary structure of mature cathepsin L itself could not be analyzed, we believe that the structure analyzes of the procathepsin Ls and cathepsin L-inhibor complex would not affect the accomplishment of this research project, seriously, i.e., the molecular design of cathepsin L-specific inhibitor. At present, the tertiary structures of a procathepsin L and cathepsin L-inhibitor complex are being analyzed by X-ray diffraction and various 2D NMR spectroscopy methods, and we expect the completion in 1997. Less