Protease II from Moraxella lacunata: cloning, sequencing, and expression of the enzyme gene, and crystallization of the expressed enzyme.
A gene coding for protease II (basic amino acid specific oligoendopeptidase) from Moraxella lacunata was cloned and expressed in Escherichia coli DH1. The transformant harboring a hybrid plasmid, pMPROII-12, with a 3.0-kbp insert at the PvuII-SacI site in pUC19, showed 23-fold higher enzyme activity than M. lacunata. The expressed enzyme from E. coli DH1/pMPROII-12 was purified by 40-80% ammonium sulfate fractionation, chromatography on DEAE-Toyopearl, and Sephadex G-150 gel filtration. The enzyme was most active at pH 6.5 and stable at pH 6.5-9.5. It had an optimum temperature of 35 degrees C for 5 min of reaction and was stable to up to 35 degrees C for 30 min at pH 7.0. Its molecular weight was estimated to be 80,000 by SDS-PAGE and gel-filtration analyses. It enzyme was inhibited by diisopropyl fluorophosphate (DFP) and classified as a serine endoprotease. Its amino acid sequence was 38% homologous to that of the E. coli protease II. By alignment with other members of the prolyl endopeptidase family, the amino acid residues involved in the catalytic triad were deduced to be Ser-534, Asp-619, and His-654. The enzyme was crystallized by the hanging drop vapor diffusion method using PEG 4000 as precipitant.
Yoshimoto
T
T
School of Pharmaceutical Sciences, Nagasaki University.
Tabira
J
J
Kabashima
T
T
Inoue
S
S
Ito
K
K
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D003460:Crystallization; D004926:Escherichia coli; D015964:Gene Expression Regulation, Bacterial / Q000502:physiology*; D015971:Gene Expression Regulation, Enzymologic / Q000502:physiology*; D005798:Genes, Bacterial; D008666:Metalloendopeptidases / Q000096:biosynthesis / Q000235:genetics*; D008969:Molecular Sequence Data; D009016:Moraxella; D011994:Recombinant Proteins / Q000096:biosynthesis; D013379:Substrate Specificity
D011994:Recombinant Proteins; D008666:Metalloendopeptidases; C022653:auR protein, Staphylococcus aureus