Synthesis and expression of a gene encoding a 48-residue repeat in the Pseudomonas syringae ice nucleation protein

Anna V. Hine, Terence A. Brown

Research output: Contribution to journalArticle

Abstract

The aim of this work was to construct short analogues of the repetitive water-binding domain of the Pseudomonas syringae ice nucleation protein, InaZ. Structural analysis of these analogues might provide data pertaining to the protein-water contacts that underlie ice nucleation. An artificial gene coding for a 48-mer repeat sequence from InaZ was synthesized from four oligodeoxyribonucleotides and ligated into the expression vector, pGEX2T. The recombinant vector was cloned in Escherichia coli and a glutathione S-transferase fusion protein obtained. This fusion protein displayed a low level of ice-nucleating activity when tested by a droplet freezing assay. The fusion protein could be cleaved with thrombin, providing a means for future recovery of the 48-mer peptide in amounts suitable for structural analysis by nuclear magnetic resonance spectroscopy.
Original languageEnglish
Pages (from-to)73-78
Number of pages6
JournalGene
Volume142
Issue number1
DOIs
Publication statusPublished - 13 May 1994

Keywords

  • gene synthesis
  • fusion protein
  • protein purification
  • synthetic oligodeoxyribonucleotides
  • Western analysis

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