Extracellular production of recombinant thermolysin expressed in Escherichia coli, and its purification and enzymatic characterization.
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.
Inouye
Kuniyo
K
Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan. inouye@kais.kyoto-u.ac.jp
Minoda
Masashi
M
Takita
Teisuke
T
Sakurama
Haruko
H
Hashida
Yasuhiko
Y
Kusano
Masayuki
M
Yasukawa
Kiyoshi
K
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
D001426:Bacterial Proteins; D011994:Recombinant Proteins; D013820:Thermolysin