Recombinant fluorescent fusion proteins are fundamental to advancing many aspects of protein science. Such proteins are typically used to enable the visualization of functional proteins in experimental systems, particularly cell biology. An important problem in biotechnology is the production of functional, soluble proteins. Here we report the use of mCherry-fusions of soluble, cysteine-rich, Leptospira-secreted exotoxins in the PF07598 gene family, the so-called virulence modifying (VM) proteins. The mCherry fusion proteins facilitated the visual detection of pink colonies of the VM proteins (LA3490 and LA1402) and following them through lysis and sequential chromatography steps. CD-spectroscopy analysis confirmed the stability and robustness of the mCherry-fusion protein, with a structure comparable to AlphaFold structural predictions. LA0591, a unique member of the PF07598 gene family that lacks N-terminal ricin B-like domains, was produced without mCherry tag that strengthens the recombinant protein production protocol without fusion protein as well. The current study provides the approaches for the synthesis of 50–125 kDa soluble, cysteine-rich, high-quality fast protein liquid chromatography (FPLC)-purified protein, with and without a mCherry tag. The use of mCherry-fusion proteins enables a streamlined, efficient process of protein production and qualitative and quantitative downstream analytical and functional studies. Approaches for troubleshooting and optimization were evaluated to overcome difficulties in recombinant protein expression and purification, demonstrating biotechnology utility in accelerating recombinant protein production.

重组荧光融合蛋白是推进蛋白质科学许多方面的基础。此类蛋白质通常用于实现实验系统中功能蛋白质的可视化，特别是细胞生物学。生物技术的一个重要问题是功能性可溶性蛋白质的生产。在这里，我们报告了 PF07598 基因家族中可溶性、富含半胱氨酸、钩端螺旋体分泌的外毒素的 mCherry 融合体的使用，即所谓的毒力修饰 (VM) 蛋白。mCherry 融合蛋白有助于视觉检测 VM 蛋白（LA3490 和 LA1402）的粉红色菌落，并通过裂解和连续层析步骤跟踪它们。CD 光谱分析证实了 mCherry 融合蛋白的稳定性和稳健性，其结构与 AlphaFold 结构预测相当。LA0591 是 PF07598 基因家族的一个独特成员，缺乏 N 端蓖麻毒素 B 样结构域，在生产时没有 mCherry 标签，该标签也增强了无需融合蛋白的重组蛋白生产方案。目前的研究提供了合成 50-125 kDa 可溶性、富含半胱氨酸、高质量快速蛋白质液相色谱 (FPLC) 纯化蛋白质（带或不带 mCherry 标签）的方法。mCherry 融合蛋白的使用可以简化、高效的蛋白质生产过程以及定性和定量下游分析和功能研究。对故障排除和优化方法进行了评估，以克服重组蛋白表达和纯化中的困难，证明生物技术在加速重组蛋白生产方面的实用性。