Background: 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.
Results: The mCherry fusion proteins facilitated the production of the VM proteins (LA3490 and LA1402) by enabling the visual detection of pink colonies 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 as a tagless protein that strengthens the recombinant protein production protocol. The current study provides the approaches for the synthesis of 50-125 kDa soluble, cysteine-rich, high-quality mCherry tagged or tagless fast protein liquid chromatography (FPLC)-purified protein.
Conclusions: 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 systemically evaluated to overcome difficulties in recombinant protein expression and purification, demonstrating biotechnology utility in accelerating recombinant protein production.
Keywords: PF07598 gene family; Soluble protein; VM proteins; fluorescence intensity; mCherry.