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Recombinant human progranzyme 3 expressed in Escherichia coli for analysis of its activation mechanism
Author(s) -
Hirata Yukiyo,
Inagaki Hirofumi,
Kawada Tomoyuki
Publication year - 2010
Publication title -
microbiology and immunology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.664
H-Index - 70
eISSN - 1348-0421
pISSN - 0385-5600
DOI - 10.1111/j.1348-0421.2009.00187.x
Subject(s) - recombinant dna , periplasmic space , biology , ctl* , cathepsin b , cathepsin , cathepsin l , biochemistry , microbiology and biotechnology , escherichia coli , cathepsin d , enzyme , cytotoxic t cell , gene , in vitro
Gr3 is reported to play an important role in defense against viral infection. Although it is known that Gr3 is synthesized as a proenzyme and activated in the cytotoxic granules of NK cells and CTL, the activation mechanism is not clearly understood. In an attempt to analyze the activation mechanism of human Gr3, a recombinant pro‐Gr3 was expressed in the periplasm of E. coli and purified to homogeneity. On SDS‐PAGE the recombinant pro‐Gr3 showed a slightly higher molecular weight than the enzymatically active Gr3, because the former possesses a small propeptide at its N‐terminal. The recombinant pro‐Gr3 was enzymatically inactive. It could be activated by treatment with cathepsin C, which concomitantly decreased the molecular weight to that of active Gr3. The proteolytic reaction of cathepsin C did not continue after one dipeptide had been removed, indicating that the recombinant pro‐Gr3 had the native conformation without any refolding process. The recombinant pro‐Gr3 would be a valuable tool for analyzing the activation mechanism and exploring other activating enzymes besides cathepsin C.