title: Extracellular production of recombinant thermolysin expressed in Escherichia coli, and its purification and enzymatic characterization.
abstract: Thermolysin is a representative zinc metalloproteinase derived from Bacillus thermoproteolyticus and a target in protein engineering to understand the catalytic mechanism and thermostability. Extracellular production of thermolysin has been achieved in Bacillus, but not in Escherichia coli, although it is the most widely used as a host for the production of recombinant proteins. In this study, we expressed thermolysin as a single polypeptide pre-proenzyme in E. coli under the original promoter sequences in the npr gene, the gene from B. thermoproteolyticus, which encodes thermolysin. Active mature thermolysin (34.6 kDa) was secreted into the culture medium. The recombinant thermolysin was purified to homogeneity by sequential column chromatography procedures of the supernatant with hydrophobic-interaction chromatography followed by affinity chromatography. The purified recombinant product is indistinguishable from natural thermolysin from B. thermoproteolyticus as assessed by hydrolysis of N-[3-(2-furyl)acryloyl]-glycyl-L-leucine amide and N-carbobenzoxy-L-asparatyl-L-phenylalanine methyl ester. The results demonstrate that our expression system should be useful for structural and functional analysis of thermolysin.
authors.affiliation: Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan. inouye@kais.kyoto-u.ac.jp
authors.forename: Kuniyo
authors.initials: K
authors.lastname: Inouye
authors.forename: Masashi
authors.initials: M
authors.lastname: Minoda
authors.forename: Teisuke
authors.initials: T
authors.lastname: Takita
authors.forename: Haruko
authors.initials: H
authors.lastname: Sakurama
authors.forename: Yasuhiko
authors.initials: Y
authors.lastname: Hashida
authors.forename: Masayuki
authors.initials: M
authors.lastname: Kusano
authors.forename: Kiyoshi
authors.initials: K
authors.lastname: Yasukawa
mesh_terms: D001407:Bacillus / Q000201:enzymology / Q000235:genetics* / Q000302:isolation & purification; D001426:Bacterial Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D002384:Catalysis; D002853:Chromatography, Liquid / Q000379:methods; D004926:Escherichia coli; D015870:Gene Expression; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D013820:Thermolysin / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification
chemical_list: D001426:Bacterial Proteins; D011994:Recombinant Proteins; D013820:Thermolysin