2007年8月
A new method for the extracellular production of recombinant thermolysin by co-expressing the mature sequence and pro-sequence in Escherichia coli
PROTEIN ENGINEERING DESIGN & SELECTION
- ,
- ,
- 巻
- 20
- 号
- 8
- 開始ページ
- 375
- 終了ページ
- 383
- 記述言語
- 英語
- 掲載種別
- 研究論文(学術雑誌)
- DOI
- 10.1093/protein/gzm031
- 出版者・発行元
- OXFORD UNIV PRESS
Thermolysin, a representative zinc metalloproteinase from Bacillus thermoproteolyticus, is synthesized as inactive pre-proenzyme and receives autocatalytic cleavage of the peptide bond linking the pro- and mature sequences. The conventional expression method for recombinant thermolysin requires the autocatalytic cleavage, so that production of a mutant thermolysin is affected by its autocatalytic digestion activity. In this study, we have established a new expression method that does not require the autocatalytic cleavage. The mature sequence of thermolysin containing an NH2-terminal pelB leader sequence and the pre-prosequence of thermolysin were co-expressed constitutively in Escherichia coli as independent polypeptides under the original promoter sequences in the npr gene which encodes thermolysin. Unlike the conventional expression method, not only the wild-type thermolysin but also mutant thermolysins [E143A (Glu143 is replaced with Ala), N112A, N112D, N112E, N112H, N112K and N112R] were produced into the culture medium. The wild-type enzyme expressed in the present method was indistinguishable from that expressed in the conventional method based on autocatalytic cleavage, as assessed by hydrolysis of N-[3-(2-furyl)acryloyl]-glycyl-L-leucine amide and N-carbobenzoxy-L-aspartyl-L-phenylalanine methyl ester. The present method should be useful especially for preparation of active-site mutants of thermolysin, which might have suppressed autocatalytic digestion activity. The results also demonstrate clearly that the covalent linking between the pro- and mature sequences is not necessary for the proper folding of the mature sequence by the propeptide in thermolysin.
- リンク情報
-
- DOI
- https://doi.org/10.1093/protein/gzm031
- J-GLOBAL
- https://jglobal.jst.go.jp/detail?JGLOBAL_ID=200902282374118223
- PubMed
- https://www.ncbi.nlm.nih.gov/pubmed/17616558
- Web of Science
- https://gateway.webofknowledge.com/gateway/Gateway.cgi?GWVersion=2&SrcAuth=JSTA_CEL&SrcApp=J_Gate_JST&DestLinkType=FullRecord&KeyUT=WOS:000250197900002&DestApp=WOS_CPL
- ID情報
-
- DOI : 10.1093/protein/gzm031
- ISSN : 1741-0126
- eISSN : 1741-0134
- J-Global ID : 200902282374118223
- PubMed ID : 17616558
- Web of Science ID : WOS:000250197900002