Hine A V, Brown T A
Department of Biochemistry and Applied Molecular Biology, UMIST, Manchester, UK.
Gene. 1994 May 3;142(1):73-8. doi: 10.1016/0378-1119(94)90357-3.
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.
这项工作的目的是构建丁香假单胞菌冰核蛋白InaZ的重复水结合结构域的短类似物。对这些类似物的结构分析可能会提供与冰核形成所依赖的蛋白质-水接触相关的数据。一个编码来自InaZ的48聚体重复序列的人工基因由四个寡脱氧核糖核苷酸合成，并连接到表达载体pGEX2T中。重组载体在大肠杆菌中克隆，并获得了谷胱甘肽S-转移酶融合蛋白。通过液滴冷冻试验检测时，这种融合蛋白显示出低水平的冰核活性。该融合蛋白可用凝血酶切割，这为将来以适合核磁共振光谱结构分析的量回收48聚体肽提供了一种方法。
