Synthesis of biologically active Shiga toxins in cell-free systems

Shiga toxins (Stx) produced by pathogenic bacteria can cause mild to severe diseases in humans. Thus, the analysis of such toxins is of utmost importance. As an AB5 toxin, Stx consist of a catalytic A-subunit acting as a ribosome-inactivating protein (RIP) and a B-pentamer binding domain. In this study we synthesized the subunits and holotoxins from Stx and Stx2a using different cell-free systems, namely an E. coli- and CHO-based cell-free protein synthesis (CFPS) system. The functional activity of the protein toxins was analyzed in two ways. First, activity of the A-subunits was assessed using an in vitro protein inhibition assay. StxA produced in an E. coli cell-free system showed significant RIP activity at concentrations of 0.02 nM, whereas toxins synthesized in a CHO cell-free system revealed significant activity at concentrations of 0.2 nM. Cell-free synthesized StxA2a was compared to StxA2a expressed in E. coli cells. Cell-based StxA2a had to be added at concentrations of 20 to 200 nM to yield a significant RIP activity. Furthermore, holotoxin analysis on cultured HeLa cells using an O-propargyl-puromycin assay showed significant protein translation reduction at concentrations of 10 nM and 5 nM for cell-free synthesized toxins derived from E. coli and CHO systems, respectively. Overall, these results show that Stx can be synthesized using different cell-free systems while remaining functionally active. In addition, we were able to use CFPS to assess the activity of different Stx variants which can further be used for RIPs in general.