Synthesis and expression of a gene encoding a 48-residue repeat in the Pseudomonas syringae ice nucleation protein.
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.
Hine
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Department of Biochemistry and Applied Molecular Biology, UMIST, Manchester, UK.
Brown
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D000595:Amino Acid Sequence; D001425:Bacterial Outer Membrane Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D001483:Base Sequence; D003001:Cloning, Molecular; D004269:DNA, Bacterial; D004926:Escherichia coli; D005813:Genes, Synthetic; D008969:Molecular Sequence Data; D011549:Pseudomonas / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism; D012091:Repetitive Sequences, Nucleic Acid
D001425:Bacterial Outer Membrane Proteins; D004269:DNA, Bacterial; D011993:Recombinant Fusion Proteins; C063113:ice nucleation protein