The full length kpsS1 gene was cloned initially into a pET-15b vector to encode a recombinant protein with an N-terminal hexahistidine tag, however expression trials performed in E. coli under a variety of conditions yielded only insoluble protein. We therefore opted to construct a fusion protein (Supplementary Table -3), a strategy which has previously been used successfully in the production of challenging GTs. We assembled a plasmid encoding the 86-amino acid colicin E9 immunity protein Im9, bearing an N-terminal His tag, fused to KpsS1, and this Im9-KpsS1 fusion was then taken forward for expression trials in E. coli BL21 (DE3) cells. Again, under many conditions the fusion was insoluble, however low levels of soluble Im9-KpsS1 (Mw: 68.9 kDa) was detected in cell lysate at 4 h post induction (Supplementary Figure ).