A new method for the extracellular production of recombinant thermolysin by co-expressing the mature sequence and pro-sequence in Escherichia coli.

A new method for the extracellular production of recombinant thermolysin by co-expressing the mature sequence and pro-sequence in Escherichia coli. Research Abstract Details 

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A new method for the extracellular production of recombinant thermolysin by co-expressing the mature sequence and pro-sequence in Escherichia coli. Abstract Text:

Kiyoshi Yasukawa,Masayuki Kusano,Kuniyo Inouye,

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 NH(2)-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.

A new method for the extracellular production of recombinant thermolysin by co-expressing the mature sequence and pro-sequence in Escherichia coli. Publishing Authors By Initials

K Yasukawa,M Kusano,K Inouye,

For similar enzymes and coenzymes: enzymes: hydrolases: peptide hydrolases: endopeptidases: metalloendopeptidases: thermolysin research abstracts see: enzymes and coenzymes: enzymes: hydrolases: peptide hydrolases: endopeptidases: metalloendopeptidases: thermolysin research

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A new method for the extracellular production of recombinant thermolysin by co-expressing the mature sequence and pro-sequence in Escherichia coli. Journal Published:

PUBLICATION TYPE: Research Support, Non-U.S. Gov

Journal: Protein engineering, design & selection : PEDS

VOLUME: 20

Page Numbers: 375-83

Journal Abbreviation: Protein Eng. Des. Sel.

ISSN: 1741-0126

DAY: 6

MONTH: 07

YEAR: 2007

A new method for the extracellular production of recombinant thermolysin by co-expressing the mature sequence and pro-sequence in Escherichia coli. Information

Number of References:

LANGUAGE: eng

NlmUniqueID: 101186484

A new method for the extracellular production of recombinant thermolysin by co-expressing the mature sequence and pro-sequence in Escherichia coli. Keywords Mesh Terms:

KEYWORDS: Thermolysin

MESH TERMS: isolation & purification

Chemical & Substance for Abstract: A new method for the extracellular production of recombinant thermolysin by co-expressing the mature sequence and pro-sequence in Escherichia coli. Information

Substance Name: Thermolysin

Registry Number: EC 3.4.24.27

Grant and Affiliation Information for A new method for the extracellular production of recombinant thermolysin by co-expressing the mature sequence and pro-sequence in Escherichia coli.

AFFILIATION: Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan.

Country: England

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MEDLINETA: Protein Eng Des Sel

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