Advanced

Directed evolution of a type I antifreeze protein expressed in Escherichia coli with sodium chloride as selective pressure and its effect on antifreeze tolerance

Meijer, P J LU ; Holmberg, Niklas; Grundström, Gunilla and Bülow, L LU (1996) In Protein Engineering 9(11). p.4-1051
Abstract

Both freezing tolerance and NaCl tolerance are improved when antifreeze proteins are expressed as fusion proteins with two domains of staphylococcal protein A (SPA) in Escherichia coli. To characterize these properties further we created a randomly mutated expression library in E. coli, based on the winter flounder antifreeze protein HPLC-8 component gene. Low-fidelity PCR products of this gene were fused to the spa gene encoding two domains of the SPA. The library was screened for enhanced NaCl tolerance and four clones were selected. The freezing tolerance of each of the selected clones was enhanced to varying extents. DNA sequencing of the isolated mutants revealed that the amphiphilic properties of the native antifreeze protein were... (More)

Both freezing tolerance and NaCl tolerance are improved when antifreeze proteins are expressed as fusion proteins with two domains of staphylococcal protein A (SPA) in Escherichia coli. To characterize these properties further we created a randomly mutated expression library in E. coli, based on the winter flounder antifreeze protein HPLC-8 component gene. Low-fidelity PCR products of this gene were fused to the spa gene encoding two domains of the SPA. The library was screened for enhanced NaCl tolerance and four clones were selected. The freezing tolerance of each of the selected clones was enhanced to varying extents. DNA sequencing of the isolated mutants revealed that the amphiphilic properties of the native antifreeze protein were essentially conserved. Furthermore, by studying the primary sequence of the randomly mutated clones, in comparison with the degree of freezing tolerance, we have identified clues which help in understanding the relationship between salt and freezing tolerance.

(Less)
Please use this url to cite or link to this publication:
author
organization
publishing date
type
Contribution to journal
publication status
published
subject
keywords
Adaptation, Biological, Antifreeze Proteins, Escherichia coli, Evolution, Molecular, Freezing, Gene Expression, Gene Library, Glycoproteins, Mutagenesis, Recombinant Fusion Proteins, Selection, Genetic, Sequence Analysis, Sodium Chloride, Staphylococcal Protein A
in
Protein Engineering
volume
9
issue
11
pages
4 pages
publisher
Oxford University Press
external identifiers
  • scopus:0029851323
ISSN
0269-2139
DOI
10.1093/protein/9.11.1051
language
English
LU publication?
yes
id
64012519-7779-4c98-9304-f5c86c950b7f
date added to LUP
2016-04-18 16:01:41
date last changed
2017-05-02 19:05:19
@article{64012519-7779-4c98-9304-f5c86c950b7f,
  abstract     = {<p>Both freezing tolerance and NaCl tolerance are improved when antifreeze proteins are expressed as fusion proteins with two domains of staphylococcal protein A (SPA) in Escherichia coli. To characterize these properties further we created a randomly mutated expression library in E. coli, based on the winter flounder antifreeze protein HPLC-8 component gene. Low-fidelity PCR products of this gene were fused to the spa gene encoding two domains of the SPA. The library was screened for enhanced NaCl tolerance and four clones were selected. The freezing tolerance of each of the selected clones was enhanced to varying extents. DNA sequencing of the isolated mutants revealed that the amphiphilic properties of the native antifreeze protein were essentially conserved. Furthermore, by studying the primary sequence of the randomly mutated clones, in comparison with the degree of freezing tolerance, we have identified clues which help in understanding the relationship between salt and freezing tolerance.</p>},
  author       = {Meijer, P J and Holmberg, Niklas and Grundström, Gunilla and Bülow, L},
  issn         = {0269-2139},
  keyword      = {Adaptation, Biological,Antifreeze Proteins,Escherichia coli,Evolution, Molecular,Freezing,Gene Expression,Gene Library,Glycoproteins,Mutagenesis,Recombinant Fusion Proteins,Selection, Genetic,Sequence Analysis,Sodium Chloride,Staphylococcal Protein A},
  language     = {eng},
  number       = {11},
  pages        = {4--1051},
  publisher    = {Oxford University Press},
  series       = {Protein Engineering},
  title        = {Directed evolution of a type I antifreeze protein expressed in Escherichia coli with sodium chloride as selective pressure and its effect on antifreeze tolerance},
  url          = {http://dx.doi.org/10.1093/protein/9.11.1051},
  volume       = {9},
  year         = {1996},
}