Pet28a tev vector

The gene encoding ChimT was commercially synthesized in the pET28a-TEV vector by GenScript^® (Piscataway, NJ, USA). The recombinant protein was expressed in E. coli Artic Express cells (DE3, Agilent Technologies, Santa Clara, CA, USA) adding 1 mM of isopropyl β-D-1-thiogalactopyranoside (IPTG; Sigma-Aldrich, St. Louis, MO, USA), with shaking at 100 rpm for 24 h at 12 °C. The bacterial cells were ruptured by five cycles of ultrasonication of 30 s. each (38 MHz) followed by six cycles of freezing and thawing. Debris was removed by centrifugation and ChimT was passed over a HisTrap HP affinity column connected to an AKTA system (GE Healthcare, Boston, MA, USA) and further purified on a Superdex™ 200 gel-filtration column (GE Healthcare Life Sciences, Boston, MA, USA). The purified protein was then passed over a polymyxin-agarose column (Sigma-Aldrich, St. Louis, MO, USA) to remove any residual endotoxin content: less than 10 ng of lipopolysaccharide per 1 mg of protein was detected with the Quantitative Chromogenic Limulus Amebocyte Kit (QCL-1000 model, BioWhittaker, Walkersville, MD, USA) following the manufacturer’s instructions.

Open reading frames encoding TgELC2 (TGME49_305050) and TgMLC1 (TGME49_257680) sub cloned via NdeI/XhoI restriction enzymes into pET28a(+)-TEV vector were purchased from GenScript. The TgELC1 gene was cloned, by extending the TGME49_269442 open reading frame (GenScript) into a pNIC28_Bsa4⁴⁸ vector via BsaI restriction sites. DNA sequences of PfELC (PF3D7_1017500), PfELC-N (residues 1–74), PfMTIP (PF3D7_1246400), PfMTIP-S (residues 60–204) and PfMTIP^77–204 were amplified from P. falciparum 3D7 cDNA and cloned into a pNIC28_Bsa4 vector via BsaI restriction sites. These constructs have an N-terminal TEV-cleavable His₆-tag. TgMLC1-S (residues 66–146) was sub cloned into a pNIC_CTHF⁴⁸ vector via the BfuI restriction site. The vector has a C-terminal TEV-cleavable His₆-tag and FLAG-tag. The sequence encoding TgMyoA-C was amplified by two complementary primers and cloned via NcoI/KpnI restriction enzymes into a pET_GB1 vector. This construct contains an N-terminal TEV-cleavable His-GB1 domain. Expression cassettes of His-TgELC1 and His-GB1-TgMyoA-C were then sub cloned via NdeI/XbaI restriction enzymes into a pPYC⁴⁹ vector. The His-GB1-TgMyoA-C gene was then cut by SpeI/XbaI restriction enzymes and inserted into SpeI-cut pPYC-His_TgELC1 to construct the co-expression vector pPYC with TgELC1 and TgMyoA-C.