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Heterologous expression of human granzyme K in Bacillus subtilis and characterization of its hydrolytic activity in vitro
Author(s) -
Bab Lilia M.,
Yoast Sienna,
Dreyer Mark,
Schmidt Brian F.
Publication year - 1998
Publication title -
biotechnology and applied biochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.468
H-Index - 70
eISSN - 1470-8744
pISSN - 0885-4513
DOI - 10.1111/j.1470-8744.1998.tb01383.x
Subject(s) - bacillus subtilis , biochemistry , granzyme , recombinant dna , enzyme , heterologous expression , enzyme kinetics , protease , proteases , chemistry , serine protease , biology , serine , microbiology and biotechnology , in vitro , cytotoxic t cell , active site , bacteria , perforin , genetics , gene
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 [ 3 H]di‐isopropyl fluorophosphate. Measurements of k cat / K m for single‐residue thioester substrates show approximately a two‐fold preference for a Lys versus Arg residue at P1. No activity was observed on ester substrates with various other residues at the P1 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 P1. 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.