Hernández-Rocamora Víctor M, Maestro Beatriz, Mollá-Morales Almudena, Sanz Jesús M
Instituto de Biología Molecular y Celular, Universidad Miguel Hernández, Avda Universidad s/n, Elche 03202, Spain.
Protein Eng Des Sel. 2008 Dec;21(12):709-20. doi: 10.1093/protein/gzn046. Epub 2008 Oct 7.
The C-LytA protein constitutes the choline-binding module of the LytA amidase from Streptococcus pneumoniae. Owing to its affinity for choline and analogs, it is regularly used as an affinity tag for the purification of proteins in a single chromatographic step. In an attempt to build a robust variant against thermal denaturation, we have engineered several salt bridges on the protein surface. All the stabilizing mutations were pooled in a single variant, C-LytAm7, which contained seven changes: Y25K, F27K, M33E, N51K, S52K, T85K and T108K. The mutant displays a 7 degrees C thermal stabilization compared with the wild-type form, together with a complete reversibility upon heating and a higher kinetic stability. Moreover, the accumulation of intermediates in the unfolding of C-LytA is virtually abolished for C-LytAm7. The differences in stability become more evident when the proteins are bound to a DEAE-cellulose affinity column, as most of wild-type C-LytA is denatured at approximately 65 degrees C, whereas C-LytAm7 may stand temperatures up to 90 degrees C. Finally, the change in the isoelectric point of C-LytAm7 enhances its solubility at acidic pHs. Therefore, C-LytAm7 behaves as an improved affinity tag and supports the engineering of surface salt bridges as an effective approach for protein stabilization.
C-LytA蛋白构成了肺炎链球菌LytA酰胺酶的胆碱结合模块。由于其对胆碱及其类似物具有亲和力，它经常被用作亲和标签，以便在单一色谱步骤中纯化蛋白质。为了构建一种抗热变性的强大变体，我们在蛋白质表面设计了几个盐桥。所有稳定突变都集中在一个单一变体C-LytAm7中，该变体包含七个变化：Y25K、F27K、M33E、N51K、S52K、T85K和T108K。与野生型相比，该突变体表现出7摄氏度的热稳定性，加热后具有完全可逆性和更高的动力学稳定性。此外，C-LytAm7几乎消除了C-LytA解折叠过程中中间体的积累。当蛋白质与DEAE-纤维素亲和柱结合时，稳定性差异变得更加明显，因为大多数野生型C-LytA在约65摄氏度时变性，而C-LytAm7可以承受高达90摄氏度的温度。最后，C-LytAm7等电点的变化提高了其在酸性pH值下的溶解度。因此，C-LytAm7表现为一种改进的亲和标签，并支持将表面盐桥工程作为蛋白质稳定化的有效方法。
