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Acknowledgments
We thank Dr. Hitoshi Shimano and Dr. Hiroaki Suzuki for their helpful discussions. We also thank Mrs. Emiko Noguchi for her excellent assistance. This work was supported by NLG919 Genome Network Project and the Grants-in-Aid for Scientific Research (C) (19590933) from the Ministry of Education, Science, Sports and Culture, Japan. This work was also supported by the Naito foundation and Astellas foundation for Research on Metabolic Disorders.
Keywords
EstE5; Esterase/lipase; EstE5–PMSF; Charge-relay system; Active site inhibition
Introduction
Fig. 1.
The suggested molecular mechanism of covalent bonding between EstE5 and PMSF. This follows the typical mechanism of serine protease and PMSF.
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Materials and methods
Protein production and purification. EstE5 was cloned, purified and crystallized from the metagenome library (Accession No. DQ842023) as described previously [10]. Briefly, hexahistidine-tagged EstE5 was cloned into the pET21a vector and expressed in Escherichia coli BL21(DE3). The protein was then purified using a two-step purification protocol primary meristems consisted of affinity and size-exclusion chromatography. After the Ni-affinity chromatography step, the protein was purified on a HiLoad 26/60 Superdex-200 prep-grade column (GE Healthcare) using an elution buffer composed of 10 mM Tris–HCl (pH 8.0), 50 mM NaCl, and 2 mM DTT. The purified protein was then concentrated to 5 mg/ml for inhibition experiments and to 30 mg/ml for crystallization.