Purification and Characterization of Aspartic Protease Derived from Sf9 Insect Cells

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概要

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• An aspartic protease that is significantly produced by baculovirus-infected Spodoptera frugiperda Sf9 insect cells was purified to homogeneity from a growth medium. To monitor aspartic protease activity, an internally quenched fluoresce (IQF) substrate specific to cathepsin D was used. The purified aspartic protease showed a single protein band on SDS–PAGE with an apparent molecular mass of 40 kDa. The N-terminal amino acid sequence of the enzyme had a high homology to a Bombyx mori aspartic protease. The enzyme showed greatest affinity for the IQF substrate at pH 3.0 with a Km of 0.85 μM. The kcat and kcat⁄Km values were 13 s−1 and 15 s−1 μM−1 respectively. Pepstatin A proved to be a potent competitive inhibitor with inhibitor constant, Ki, of 25 pM.

• 2010-10-23

著者

• Takahashi S

  Akita Res. Inst. Food And Brewing Akita Jpn

• Gotoh Takeshi

  Department of Engineering in Applied Chemistry, Akita University

• Kikuchi Ken-Ichi

  Department of Engineering in Applied Chemistry, Akita University

• TAKAHASHI Saori

  Akita Research Institute of Food and Brewing

• Takahashi S

  Kyoto Univ. Kyoto Jpn

• Ono Hiroki

  Department of Materials-Process Engineering and Applied Chemistry for Environments, Akita University

• NIRASAWA Satoru

  Japan International Research Center for Agricultural Sciences

• Takahashi S

  Akita Res. Inst. Of Food And Brewing

• Takahashi Saori

  Akita Res. Inst. Of Food And Brewing

• Ono Hiroki

  Department Of Cardiovascular Medicine Kitakyushu Municipal Medical Center

• Kikuchi Ken-ichi

  Department Of Engineering In Applied Chemistry Akita University

• Gotoh Takeshi

  Department Of Engineering In Applied Chemistry Akita University

• Gotoh Takeshi

  Department Of Engineering In Applied Chemistry Graduate School Of Engineering And Resource Science A

• Kikuchi Ken-ichi

  Department Of Engineering In Applied Chemistry Graduate School Of Engineering And Resource Science A

• Nirasawa Satoru

  Post-harvest Science And Technology Division Japan International Research Center For Agricultural Sc

• Suzuki Takahito

  Biological Laboratory Faculty Of Science Nara Women's University

• Ono Hiroki

  Department Of Materials-process Engineering And Applied Chemistry For Environments Akita University

• Takahashi S

  Department Of Biology The University Of Tokyo

• Gotoh Takeshi

  Department Of Engineering And Applied Chemistry Graduate School Of Engineering And Resource Science

• Takahashi Satoru

  Institute Of Basic Medical Sceinces And Center For Tara

• KIKUCHI KEN-ICHI

  Department of Chemical Engineering for Resources, Akita University

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