LeSBT1, a subtilase from tomato plants - Overexpression in insect cells, purification, and characterization

The cDNA of a tomato subtilase designated LeSBT1 was cloned from a tomato f lower cDNA library. The deduced amino acid sequence indicated for LeSBT1 th e structure of a prepro-protein targeted to the secretory pathway by virtue of an amino-terminal signal peptide. LeSBT1 was expressed in the baculovir us/insect cell system and a processed 73-kDa form of LeSBT1, lacking both s ignal peptide and prodomain, was purified to homogeneity from culture super natants, This 73-kDa LeSBT1, however, lacked proteolytic activity. Zymogen activation to yield 68-kDa LeSBT1 required the additional processing of an amino-terminal autoinhibitory peptide in a strictly pH-dependent manner. Ma ture 68-kDa LeSBT1 showed highest activity at acidic pH consistent with its presumed localization in the apoplast of the plant cell. In comparison to other plant subtilases, LeSBT1 exhibited a narrower substrate specificity i n that it cleaves only polypeptide substrates preferentially but not exclus ively carboxyl-terminal of glutamine residues. The possible involvement of LeSBT1 in selective proprotein processing is discussed with reference to th e related mammalian proprotein convertases.