title: A 38 kDa precursor protein of aqualysin I (a thermophilic subtilisin-type protease) with a C-terminal extended sequence: its purification and in vitro processing.
abstract: The precursor of aqualysin I, an extracellular subtilisin-type protease produced by Thermus aquaticus, consists of four domains: an N-terminal signal peptide, an N-terminal pro-sequence, a protease domain, and a C-terminal extended sequence. In an Escherichia coli expression system for the aqualysin I gene, a 38 kDa precursor protein consisting of the protease domain and the C-terminal extended sequence is accumulated in the membrane fraction and processed to a 28 kDa mature enzyme upon heat treatment at 65 degrees C. The 38 kDa precursor protein is separated as a soluble form from denatured E. coli proteins after heat treatment. Accordingly, purification of the 38 kDa proaqualysin I was performed using chromatography. The purified precursor protein gave a single band on SDS-polyacrylamide gels. The precursor protein exhibited proteolytic activity comparable to that of the mature enzyme. The purified precursor protein was processed to the mature enzyme upon heat treatment. The processing was inhibited by diisopropyl fluorophosphate. The processing rate increased upon either the addition of mature aqualysin I or upon an increase in the concentration of the precursor, suggesting that the cleavage of the C-terminal extended sequence occurs through an intermolecular self-processing mechanism.
authors: Department of Biotechnology, University of Tokyo, Japan.
authors: K
authors: K
authors: Kurosaka
authors: T
authors: T
authors: Ohta
authors: H
authors: H
authors: Matsuzawa
mesh_terms: D000595:Amino Acid Sequence; D008969:Molecular Sequence Data; D011498:Protein Precursors / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D012697:Serine Endopeptidases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D013381:Subtilisins / Q000737:chemistry
chemical_list: D011498:Protein Precursors; D012697:Serine Endopeptidases; D013381:Subtilisins; C055102:aqualysin I