2006年4月
Extracellular production of recombinant thermolysin expressed in Escherichia coli, and its purification and enzymatic characterization
PROTEIN EXPRESSION AND PURIFICATION
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- 巻
- 46
- 号
- 2
- 開始ページ
- 248
- 終了ページ
- 255
- 記述言語
- 英語
- 掲載種別
- 研究論文(学術雑誌)
- DOI
- 10.1016/j.pep.2005.07.023
- 出版者・発行元
- ACADEMIC PRESS INC ELSEVIER SCIENCE
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. (c) 2005 Elsevier Inc. All rights reserved.
- リンク情報
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- DOI
- https://doi.org/10.1016/j.pep.2005.07.023
- J-GLOBAL
- https://jglobal.jst.go.jp/detail?JGLOBAL_ID=200902260990859756
- PubMed
- https://www.ncbi.nlm.nih.gov/pubmed/16169746
- Web of Science
- https://gateway.webofknowledge.com/gateway/Gateway.cgi?GWVersion=2&SrcAuth=JSTA_CEL&SrcApp=J_Gate_JST&DestLinkType=FullRecord&KeyUT=WOS:000236828100010&DestApp=WOS_CPL
- ID情報
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- DOI : 10.1016/j.pep.2005.07.023
- ISSN : 1046-5928
- J-Global ID : 200902260990859756
- PubMed ID : 16169746
- Web of Science ID : WOS:000236828100010