The initial toxic effects of the Ctx multimer could already be detected at 4 nM. The LT complex induced cell death at higher concentrations, starting at an initial concentration of 5 nM. A functional analysis was performed using cell-based assays, thereby demonstrating that the LT complex induced the characteristic cell elongation of target cells after 24 h. Through an iterative optimization procedure, the synthesis of the individual AB5 toxins was established, and the formation of multimeric structures could be shown by autoradiography. Here, we present the cell-free synthesis and functional characterization of two AB5 toxins, namely the cholera toxin (Ctx) and the heat-labile enterotoxin (LT), using two eukaryotic cell-free systems based on Chinese hamster ovary (CHO) and Spodoptera frugiperda (Sf21) cells. The open nature of cell-free systems allows for the addition of supplements affecting protein concentration and folding. As a cell lysate, rather than viable cells, is used, the toxic effects on the host organism can be circumvented. Cell-free protein synthesis (CFPS) represents a versatile key technology for the production of toxic proteins.
0 Comments
Leave a Reply. |