Purification and Characterization of Aspartic Protease Derived from Sf9 Insect Cells

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.