Chaurasia Reetika, Liang Cathleen, How Kenneth, Vieira Dielson S, Vinetz Joseph M
Section of Infectious Diseases, Department of Internal Medicine, Yale School of Medicine, New Haven, CT, USA.
Protein J. 2023 Dec;42(6):792-801. doi: 10.1007/s10930-023-10152-2. Epub 2023 Sep 1.
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动蛋白质科学诸多方面发展的基础。这类蛋白通常用于在实验系统中实现功能蛋白的可视化，尤其是在细胞生物学中。生物技术中的一个重要问题是功能性可溶性蛋白的生产。在此，我们报告了在PF07598基因家族中使用可溶性、富含半胱氨酸的钩端螺旋体分泌外毒素（即所谓的毒力修饰（VM）蛋白）的mCherry融合蛋白。mCherry融合蛋白有助于对VM蛋白（LA3490和LA1402）的粉色菌落进行视觉检测，并在细胞裂解和后续色谱步骤中追踪它们。圆二色光谱分析证实了mCherry融合蛋白的稳定性和稳健性，其结构与AlphaFold结构预测结果相当。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，它在没有mCherry标签的情况下产生，这也加强了无融合蛋白的重组蛋白生产方案。当前的研究提供了合成50 - 125 kDa可溶性、富含半胱氨酸、经高效液相色谱（FPLC）纯化的高质量蛋白的方法，该蛋白带有或不带有mCherry标签。mCherry融合蛋白的使用实现了简化、高效的蛋白生产过程以及定性和定量的下游分析与功能研究。评估了故障排除和优化方法以克服重组蛋白表达和纯化中的困难，证明了生物技术在加速重组蛋白生产方面的实用性。
