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.
- gene synthesis
- fusion protein
- protein purification
- synthetic oligodeoxyribonucleotides
- Western analysis
Hine, A. V., & Brown, T. A. (1994). Synthesis and expression of a gene encoding a 48-residue repeat in the Pseudomonas syringae ice nucleation protein. Gene, 142(1), 73-78. https://doi.org/10.1016/0378-1119(94)90357-3