Article ID | Journal | Published Year | Pages | File Type |
---|---|---|---|---|
1927626 | Archives of Biochemistry and Biophysics | 2006 | 9 Pages |
Abstract
Human granzyme 3 (Gr3) is a serine protease contained in the granules of natural killer cells and cytotoxic T lymphocytes. To elucidate the biochemical and physiological characteristics of Gr3, we attempted to prepare an enzymatically active recombinant human Gr3 without refolding and proteolytic activation. An expression vector was constructed, in which the pre-/pro-peptide coding sequence of Gr3 was replaced with the bacterial pelB leader sequence. The resultant expression product was a fully active protease in the periplasmic fraction of Escherichia coli and was purified to homogeneity. The purified enzyme effectively hydrolyzed Z-Lys-SBzl, a conventionally used substrate of Gr3. In addition, it also hydrolyzed the peptide substrate library FRETS-25Xaa series, required basic amino acid residues, Arg or Lys, at the P1 position, and most efficiently hydrolyzed the carboxylic side of Phe-Tyr-Argâ (P3-P2-P1) sequence of the 475 tripeptide combinations.
Keywords
Related Topics
Life Sciences
Biochemistry, Genetics and Molecular Biology
Biochemistry
Authors
Yukiyo Hirata, Hirofumi Inagaki, Takako Shimizu, Qing Li, Noriyuki Nagahara, Masayasu Minami, Tomoyuki Kawada,