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 Km of 0.85μM. 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 25pM.