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

Research output: Contribution to journalArticle


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 J Meijer
  • Niklas Holmberg
  • Gunilla Grundström
  • L Bülow
External organisations
  • Lund University
Research areas and keywords

Subject classification (UKÄ) – MANDATORY

  • Medical Biotechnology
  • Biochemistry and Molecular Biology


  • 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
Original languageEnglish
Pages (from-to)1051-4
Number of pages4
JournalProtein Engineering
Issue number11
Publication statusPublished - 1996 Nov
Publication categoryResearch