From Gibson Assembly to Cell-Free Expression: a guide to Recombinant Plasmid and Toscana Virus N Protein Production.

Toscana virus (TOSV) is a mosquito-borne phlebovirus endemic to the Mediterranean region and an important cause of arboviral infections in humans, particularly during the summer months. Although TOSV infections are often asymptomatic or mild, the virus can cause neuroinvasive disease, including meningitis and encephalitis, highlighting the need for rapid and reliable tools for its detection and characterization. N protein was identified as a promising diagnostic target for TOSV due to its accumulation in infected cells and its role as primary immune target. The aim of this study was to establish a rapid and easily adaptable workflow for the recombinant production of the nucleoprotein (N) of Toscana virus (TOSV). We introduced a Gibson® Assembly-based cloning strategy using the pT7CFE1-CHis plasmid, specifically selected for its compatibility with cell free protein expression systems. The Gibson® approach enabled fast and efficient construct generation, overcoming the constraints typically associated with classical restriction-based cloning. Subsequently, cell free in vitro synthesis produced the recombinant protein within 16hours, enabling an efficient production. The yield was sufficient to perform downstream assays, including Western blot, thereby demonstrating the suitability of this combined strategy for rapid antigen prototyping and preliminary characterization. Our results highlight the recombinant TOSV antigen to diagnostics to support timely and disease detection and control.