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Deletion of a cytotoxic, N-terminal putative signal peptide results in a significant increase in production yields in Escherichia coli and improved specific activity of Cel12A from Rhodothermus marinus

Wicher, K. B. ; Abou-Hachem, M. LU ; Halldórsdóttir, S. ; Thorbjarnadóttir, S. H. ; Eggertsson, G. ; Hreggvidsson, G. O. ; Nordberg Karlsson, E. LU and Holst, O. LU (2001) In Applied Microbiology and Biotechnology 55(5). p.578-584
Abstract

The thermostable cellulase Cel12A from Rhodothermus marinus was produced at extremely low levels when expressed in Escherichia coli and was cytotoxic to the cells. In addition, severe aggregation occurred when moderately high concentrations of the enzyme were heat-treated at 65°C, the growth optimum of R. marinus. Sequence analysis revealed that the catalytic module of this enzyme is preceded by a typical linker sequence and a highly hydrophobic putative signal peptide. Two deletion mutants lacking this hydrophobic region were cloned and successfully expressed in E. coli. These results indicated that the N-terminal putative signal peptide was responsible for the toxicity of the full-length enzyme in the host organism. This was further... (More)

The thermostable cellulase Cel12A from Rhodothermus marinus was produced at extremely low levels when expressed in Escherichia coli and was cytotoxic to the cells. In addition, severe aggregation occurred when moderately high concentrations of the enzyme were heat-treated at 65°C, the growth optimum of R. marinus. Sequence analysis revealed that the catalytic module of this enzyme is preceded by a typical linker sequence and a highly hydrophobic putative signal peptide. Two deletion mutants lacking this hydrophobic region were cloned and successfully expressed in E. coli. These results indicated that the N-terminal putative signal peptide was responsible for the toxicity of the full-length enzyme in the host organism. This was further corroborated by cloning and expressing the hydrophobic N-terminal domain in E. coli, which resulted in extensive cell lysis. The deletion mutants, made up of either the catalytic module of Cel12A or the catalytic module and the putative linker sequence, were characterised and their properties compared to those of the full-length enzyme. The specific activity of the mutants was approximately threefold higher than that of the full-length enzyme. Both mutant proteins were highly thermostable, with half-lives exceeding 2 h at 90°C and unfolding temperatures up to 103°C.

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organization
publishing date
type
Contribution to journal
publication status
published
subject
in
Applied Microbiology and Biotechnology
volume
55
issue
5
pages
7 pages
publisher
Springer
external identifiers
  • scopus:0035010839
  • pmid:11414324
ISSN
0175-7598
DOI
10.1007/s002530000559
language
English
LU publication?
yes
id
546d8c6f-1a9c-47b8-9b55-3605df3bb3e5
date added to LUP
2018-11-14 21:12:59
date last changed
2021-03-24 03:23:17
@article{546d8c6f-1a9c-47b8-9b55-3605df3bb3e5,
  abstract     = {<p>The thermostable cellulase Cel12A from Rhodothermus marinus was produced at extremely low levels when expressed in Escherichia coli and was cytotoxic to the cells. In addition, severe aggregation occurred when moderately high concentrations of the enzyme were heat-treated at 65°C, the growth optimum of R. marinus. Sequence analysis revealed that the catalytic module of this enzyme is preceded by a typical linker sequence and a highly hydrophobic putative signal peptide. Two deletion mutants lacking this hydrophobic region were cloned and successfully expressed in E. coli. These results indicated that the N-terminal putative signal peptide was responsible for the toxicity of the full-length enzyme in the host organism. This was further corroborated by cloning and expressing the hydrophobic N-terminal domain in E. coli, which resulted in extensive cell lysis. The deletion mutants, made up of either the catalytic module of Cel12A or the catalytic module and the putative linker sequence, were characterised and their properties compared to those of the full-length enzyme. The specific activity of the mutants was approximately threefold higher than that of the full-length enzyme. Both mutant proteins were highly thermostable, with half-lives exceeding 2 h at 90°C and unfolding temperatures up to 103°C.</p>},
  author       = {Wicher, K. B. and Abou-Hachem, M. and Halldórsdóttir, S. and Thorbjarnadóttir, S. H. and Eggertsson, G. and Hreggvidsson, G. O. and Nordberg Karlsson, E. and Holst, O.},
  issn         = {0175-7598},
  language     = {eng},
  month        = {06},
  number       = {5},
  pages        = {578--584},
  publisher    = {Springer},
  series       = {Applied Microbiology and Biotechnology},
  title        = {Deletion of a cytotoxic, N-terminal putative signal peptide results in a significant increase in production yields in Escherichia coli and improved specific activity of Cel12A from Rhodothermus marinus},
  url          = {http://dx.doi.org/10.1007/s002530000559},
  doi          = {10.1007/s002530000559},
  volume       = {55},
  year         = {2001},
}