Aktaprime chromatography system

Antigen production and purification was carried out at the Instituto Carlos Chagas (ICC-Paraná), Universidade Federal de Minas Gerais (UFMG) and Instituto Oswaldo Cruz (IOC-Rio de Janeiro). Escherichia coli BL21 (DE3) pLysS (Invitrogen) was transformed with pRSET plasmids (Invitrogen) containing the Lci1, Lci2, Lci4, Lci5, Lci8 or Lci12 L. infantum gene insert [11 (link)]. Transformed bacteria were grown in Lysogeny broth medium and induced using 0.1 mM of isopropyl β- d -thiogalactoside overnight at 15°C to express Lci1, Lci4, Lci5, or at 37°C for 3h for Lci2 and Lci12 expression. Affinity chromatography was used to purify the proteins rLci1, rLci2, rLci5 and rLci8 from the soluble extract of proteins, while the crude extract was used to purify the rLci4 and rLci12 proteins, both using a HisTrap HP column (GE Healthcare, Piscataway, NJ) connected to an AKTAprime chromatography system (GE Healthcare, USA). rLci2 and rLci8 were submitted to a second purification step by ion-exchange chromatography using a Hitrap Q HP column (GE Healthcare, Piscataway, NJ) connected to an AKTAprime chromatography system.

The lysate was injected into a HiTrap^TM Chelating HP column (5 ml; GE Healthcare) connected to an AKTA prime chromatography system (GE Healthcare). The system was equilibrated with lysis buffer and the column washed until a baseline level was reached. The column was then washed with wash buffer (20 mM Tris–HCl pH 7.6, 10 mM MgCl₂, 150 mM KCl, 30 mM NH₄Cl and 5 mM imidazole) and the 70S ribosome was eluted using an elution buffer containing a high concentration of imidazole (20 mM Tris–HCl pH 7.6, 10 mM MgCl₂, 150 mM KCl, 30 mM NH₄Cl and 150 mM imidazole).
To separate the ribosomal subunits, the 70S ribosome eluent was dialyzed 4 times for 10 min with a ‘SUB’ buffer that comprised less Mg²⁺ (20 mM Tris–HCl pH 7.6, 1 mM MgCl₂, 150 mM KCl, 30 mM NH₄Cl). The HisTrap^TM HP column was then equilibrated with SUB buffer, the sample injected, and the flow-through collected. As the tetra (His)₆ tag is present only on the 50S subunit, it was trapped in the column whereas the free 30S ribosomal subunit was eluted in the flow-through. The eluted 30S ribosomal subunit was then concentrated using sucrose-cushion centrifugation or 30-kDa cut-off Amicon concentrators (Millipore) to a final concentration of 70 μM, flash frozen and stored in aliquots at −80 °C.

L. acidophilus cells were collected and sonicated by the method described in “Cell disruption” section. The supernatant solution was salted out with ammonium sulfate (10–60%). Precipitates were collected by centrifugation, resuspended in 5 mL of TD buffer (10 mM Tris-HCl (pH 7.8) and 1 mM dithiothreitol (DTT)), and filtered through a 0.45-µm filter cassette. Solutions were purified by ultrafiltration (Amicon Ultra 100-kDa filters; Merck) and collected upper phase of the filtration cassette. Chromatography was performed on an AKTAprime chromatography system (GE Healthcare, Little Chalfont, UK) (Fig. 1c). The crude purified solution was run through a HiPrep Q FF 16/10 strong anion-exchange chromatography column (GE Healthcare) with TD buffer, and the NaCl concentration was linearly increased to 1 M. The elution procedures were performed in a 10-column-volume gradient. Chromatographic fractions were analyzed by LC/MS/MS as previously described^{51 (link)}.