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 cytotoxic T-lymphocytes, was produced in its catalytically active form by recombinant technology using Bacillus subtilis as host. The enzyme displays 40-45% identity to other members of the human granzyme group, and 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 chromatography. Initial characterization reveals a protein of approximately 28 kDa that is specifically labelled by [3H]di-isopropyl fluorophosphate. Measurements of Kcat/K(m) for single-residue thioester substrates show approximately a two-fold preference for a Lys versus Arg residue at Pl. No activity was observed on ester substrates with various other residues at the Pl position. Using oligopeptide substrates, the enzyme displays peptidolytic activity C-terminal to both Lys and Arg residues with comparable rates of hydrolysis. Likewise, substrate hydrolysis is blocked most efficiently by inhibitors that contain Lys or Arg at position Pl. The availability of the cloned enzyme will facilitate the analysis of biological roles for this novel granzyme, and differentiate its activity from that of other granzymes.
Babé
L M
LM
AxyS Pharmaceuticals, South San Francisco, CA 94080, USA.
Yoast
S
S
Dreyer
M
M
Schmidt
B F
BF
D000595:Amino Acid Sequence; D001120:Arginine; D001412:Bacillus subtilis / Q000235:genetics*; D001483:Base Sequence; D053818:Chymases; D004795:Enzyme Stability; D006801:Humans; D006868:Hydrolysis; D008239:Lysine; D008969:Molecular Sequence Data; D010455:Peptides / Q000378:metabolism; D011480:Protease Inhibitors / Q000494:pharmacology; D011994:Recombinant Proteins / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism; D012697:Serine Endopeptidases / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism*; D013379:Substrate Specificity; D053802:Tryptases
D010455:Peptides; D011480:Protease Inhibitors; D011994:Recombinant Proteins; D001120:Arginine; D012697:Serine Endopeptidases; C083294:chymase 2; D053818:Chymases; C509223:Tpsab1 protein, rat; C509221:Tpsb2 protein, rat; D053802:Tryptases; D008239:Lysine