Purification and characterization of aspartic protease derived from Sf9 insect cells |
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Authors: | Gotoh Takeshi Ono Hiroki Kikuchi Ken-Ichi Nirasawa Satoru Takahashi Saori |
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Affiliation: | Department of Engineering in Applied Chemistry, Graduate School of Engineering and Resource Science, Akita University, Akita, Japan. tgotoh@ac5.as.akita-u.ac.jp |
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Abstract: | 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 K(m) of 0.85 μM. The k(cat) and k(cat)/K(m) values were 13 s(-1) and 15 s(-1) μM(-1) respectively. Pepstatin A proved to be a potent competitive inhibitor with inhibitor constant, K(i), of 25 pM. |
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