Expression of a soluble and active <i>Pf</i>-apiTyrRS in <i>E</i>. <i>coli</i>.

(A) The overall organization of Pf-apiTyrRS, compared to TyrRSs from humans (mitochondria and cytosol), bacteria, and archaea. Each structural domain is given in a specific color: blue for mitochondrial and apicoplast targeting signals, black for catalytic domains, green for the canonical α-helical anticodon-binding domains, purple for anticodon-binding domains homologous to TrpRS [31], and grey for additional C-terminal domains (S4-like in bacteria and organelles; EMAPII-like in vertebrates [32]). Plasmodium-specific idiosyncratic insertions are shown in yellow. Red dots indicate the position of the signature sequences present in the catalytic domains of class I aaRSs. (B) Coomassie-stained gel (loading control) and the corresponding Western blot show Pf-apiTyrRS expressed from the Plasmodium wild-type nucleotide sequence (Endo), the optimized gene sequence for E. coli expression (Opt), and the harmonized gene sequence (Harm). T stands for Total extract, and S stands for Soluble extract (supernatant of the centrifuged total extract). On the coomassie-stained gel, overexpression of the Harm Pf-TyrRS is indicated with a red asterisk. Additional bands potentially correspond to degradation products. On the Western Blot, 6-His tagged proteins were specifically detected with a mouse anti-penta-his tag antibody (Qiagen). (C) Aminoacylations of E. coli tRNATyr (0.8 μM) performed with Pf-apiTyrRSs (100 nM) produced from the three gene variants were measured after 5, 10 and 15 min incubation. Controls (without tRNA) were substracted. Results are an average of three independent experiments. *significant at pS1 Table).