Departamento de Genética del Desarrollo y Fisiología Molecular, Instituto de Biotecnología. Universidad Nacional Autónoma de México, Cuernavaca, Morelos, 62210, Mexico.
Departamento de Fisiología, Facultad de Medicina, Universidad Nacional Autónoma de México, Ciudad de México, 04510, Mexico.
Protein Expr Purif. 2023 Jan;201:106172. doi: 10.1016/j.pep.2022.106172. Epub 2022 Sep 15.
Heterologous expression systems have been used as a powerful experimental strategy to study the function of many proteins, particularly ion transporters. For this experiment, it is fundamental to prepare an expression vector encoding a protein of interest. However, we encountered problems in vector preparation of the voltage sensor domain (VSD) of murine sperm-specific Na/H exchanger (sNHE) due to its severe toxicity to bacteria. We overcame the problems by insertion of an amber stop codon or a synthetic intron into the coding sequence of the VSD in the expression vectors. Both methods allowed us to express the protein of interest in HEK293 cells (combined with a stop codon suppression system for amber codon). The VSD of mouse sNHE generates voltage-dependent outward ionic currents, which is a probable cause of toxicity to bacteria. We propose these two strategies as practical solutions to study the function of any protein toxic to bacteria.
异源表达系统已被用作研究许多蛋白质（尤其是离子转运蛋白）功能的强大实验策略。对于这个实验，制备编码感兴趣蛋白质的表达载体是至关重要的。然而，由于鼠精子特异性 Na+/H+交换体（sNHE）的电压传感器域（VSD）对细菌具有严重毒性，我们在载体制备中遇到了问题。我们通过在表达载体的 VSD 编码序列中插入琥珀终止密码子或合成内含子来克服这些问题。这两种方法都允许我们在 HEK293 细胞中表达目的蛋白（与琥珀密码子的终止密码子抑制系统相结合）。鼠 sNHE 的 VSD 产生电压依赖性外向离子电流，这可能是细菌毒性的原因。我们提出这两种策略作为研究任何对细菌有毒性的蛋白质功能的实用解决方案。
