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PEDS Advance Access originally published online on July 6, 2007
Protein Engineering Design and Selection 2007 20(8):375-383; doi:10.1093/protein/gzm031
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© The Author 2007. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org

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

Kiyoshi Yasukawa, Masayuki Kusano and Kuniyo Inouye1

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

1 To whom correspondence should be addressed. E-mail: inouye{at}kais.kyoto-u.ac.jp

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.

Keywords: autocatalytic digestion activity/Escherichia coli/metalloproteinase/pro-sequence/thermolysin

Received March 12, 2007; revised May 1, 2007; accepted May 25, 2007.


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M. Kusano, K. Yasukawa, and K. Inouye
Insights into the Catalytic Roles of the Polypeptide Regions in the Active Site of Thermolysin and Generation of the Thermolysin Variants with High Activity and Stability
J. Biochem., January 1, 2009; 145(1): 103 - 113.
[Abstract] [Full Text] [PDF]


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