HETEROLOGOUS EXPRESSION OF HUMAN GRANZYME-K IN BACILLUS-SUBTILIS AND CHARACTERIZATION OF ITS HYDROLYTIC ACTIVITY IN-VITRO

Human granzyme K, a serine protease found in secretory granules of cyt otoxic T-lymphocytes, was produced in its catalytically active form by recombinant technology using Bacillus subtilis as host. The enzyme di splays 40-45% identity to other members of the human granzyme group, a nd its closest homologue (75% identity) is the rat tryptase RNK-tryp2, The recombinant protein can be recovered in its mature form from the bacterial culture supernatant and purified by cation exchange chromato graphy, Initial characterization reveals a protein of approximately 28 kDa that is specifically labelled by [H-3]di-isopropyl fluorophosphat e. Measurements of k(cat)/K-m for single-residue thioester substrates show approximately a two-fold preference for a Lys versus Arg residue at PI. No activity was observed on ester substrates with various other residues at the PI position. Using oligopeptide substrates, the enzym e displays peptidolytic activity C-terminal to both Lys and Arg residu es with comparable rates of hydrolysis. Likewise, substrate hydrolysis is blocked most efficiently by inhibitors that contain Lys or Arg at position PI. The availability of the cloned enzyme will facilitate the analysis of biological roles for this novel granzyme, and differentia te its activity from that of other granzymes.