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

Anna V. Hine; Terence A. Brown

doi:10.1016/0378-1119(94)90357-3

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 journal › Article › peer-review

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 language	English
Pages (from-to)	73-78
Number of pages	6
Journal	Gene
Volume	142
Issue number	1
DOIs	https://doi.org/10.1016/0378-1119(94)90357-3
Publication status	Published - 13 May 1994

Keywords

gene synthesis
fusion protein
protein purification
synthetic oligodeoxyribonucleotides
Western analysis

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10.1016/0378-1119(94)90357-3

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

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.",

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year = "1994",

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T1 - Synthesis and expression of a gene encoding a 48-residue repeat in the Pseudomonas syringae ice nucleation protein

AU - Hine, Anna V.

AU - Brown, Terence A.

PY - 1994/5/13

Y1 - 1994/5/13

N2 - 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.

AB - 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.

KW - gene synthesis

KW - fusion protein

KW - protein purification

KW - synthetic oligodeoxyribonucleotides

KW - Western analysis

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U2 - 10.1016/0378-1119(94)90357-3

DO - 10.1016/0378-1119(94)90357-3

M3 - Article

SN - 0378-1119

VL - 142

SP - 73

EP - 78

JO - Gene

JF - Gene

IS - 1

ER -

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

Abstract

Keywords

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