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Production, purification, and crystallization of human interleukin‐1β converting enzyme derived from an Escherichia coli expression system
Author(s) -
Malinowski John J.,
Grasberger Bruce L.,
Trakshel Gary,
Huston Edward E.,
Banks Tracey M.,
Brake Patricia G.,
Ciccarelli Richard B.,
Jones Barry N.,
Koehn James A.,
Kratz Diane,
Lundberg Nicole,
Stevis Panayiotis E.,
Helaszek Carla T.,
Ator Mark A.,
Small Wood Angela M.,
Stams Travis,
Rubin Byron,
Alexander Richard S.
Publication year - 1995
Publication title -
protein science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.353
H-Index - 175
eISSN - 1469-896X
pISSN - 0961-8368
DOI - 10.1002/pro.5560041021
Subject(s) - escherichia coli , thioredoxin , biochemistry , affinity chromatography , recombinant dna , fusion protein , enzyme , protease , biology , chemistry , microbiology and biotechnology , gene
Interleukin‐1β converting enzyme (ICE) is a cysteine protease that catalyzes the conversion of the inactive precursor form of IL‐1β to an active mature form. The mature form of IL‐1β is involved in mediating inflammatory responses and in the progression of autoimmune diseases. We recently reported on the production of active human ICE in insect cells using the baculovirus expression system (Wang XM et al., 1994, Gene 145 :273–277). Because the levels of expression achieved with this system were limiting for the purpose of performing detailed biochemical and biophysical studies, we examined the production of ICE in Escherichia coli . By using a tac promoter‐based expression system and fusion to thioredoxin we were able to recover high levels of active ICE protein. The expressed protein, which was distributed between the soluble and insoluble fractions, was purified to homogeneity from both fractions using a combination of classical and affinity chromatography. Comparisons of ICE derived from both fractions indicated that they were comparable in their specific activities, subunit composition, and sensitivities to specific ICE inhibitors. The combined yields of ICE obtained from the soluble and insoluble fractions was close to 1 mg/L of induced culture. Recombinant human ICE was crystallized in the presence of a specific ICE inhibitor in a form suitable for X‐ray crystallographic analysis. This readily available source of ICE will facilitate the further characterization of this novel and important protease.