Hemin

Leishmania tarentolae cell-free lysate was produced as described by Johnston & Alexandrov29 (link)30 (link)83 (link). Briefly, Leishmania tarentolae Parrot strain was obtained as LEXSY host P10 from Jena Bioscience GmbH, Jena, Germany and cultured in TBGG medium containing 0.2% v/v Penicillin/Streptomycin (Life Technologies) and 0.05% w/v Hemin (MP Biomedical). Cells were harvested by centrifugation at 2500 x g, washed twice by resuspension in 45 mM HEPES, pH 7.6, containing 250 mM Sucrose, 100 mM Potassium Acetate and 3 mM Magnesium Acetate and resuspended to 0.25 g cells/g suspension. Cells were placed in a cell disruption vessel (Parr Instruments, USA) and incubated under 7000 KPa nitrogen for 45 minutes, then lysed by rapid release of pressure. The lysate was clarified by sequential centrifugation at 10 000 x g and 30 000 x g and anti-splice leader DNA leader oligonucleotide was added to 10 μM. The lysate was then desalted into 45 mM HEPES, pH 7.6, containing, 100 mM Potassium Acetate and 3 mM Magnesium Acetate, supplemented with a coupled translation/transcription feeding solution and snap-frozen until required.

Bacteroides strains were grown in liquid TYG (tryptone yeast glucose) medium, in liquid minimal medium (14 (link)), or on brain heart infusion (BHI; Becton Dickinson) agar supplemented with 50 mg/liter hemin (MP Biomedicals) and 0.5 mg/liter vitamin K₃ (MP Biomedicals) in an anaerobic chamber (Coy Laboratory Products) filled with 70% N₂, 20% CO₂, and 10% H₂. Escherichia coli S17-1 lambda pir and BL21 Rosetta strains were grown in LB medium and incubated aerobically at 37°C with shaking at 300 rpm. Antibiotics were added when required at the following concentrations: anhydrotetracycline (aTC), 2 ng/ml; ampicillin, 100 μg/ml; erythromycin, 25 μg/ml; gentamicin, 200 μg/ml; kanamycin, 50 μg/ml; tetracycline, 2 μg/ml; and 5-fluoro-2′-deoxyuridine (FUdR), 200 μg/ml.

Leishmania tarentolae extracts were prepared in house using the protocol described previously [36 (link)]. Briefly, Leishmania tarentolae Parrot strain was obtained as LEXSY host P10 from Jena Bioscience GmbH, Jena, Germany and cultured in TBGG medium containing 0.2% v/v Penicillin/Streptomycin (Life Technologies) and 0.05% w/v Hemin (MP Biomedical). Cells were harvested by centrifugation at 2500 × g, washed twice by resuspension in 45 mM HEPES, pH 7.6, containing 250 mM Sucrose, 100 mM Potassium Acetate and 3 mM Magnesium Acetate and resuspended to 0.25 g cells/g suspension. Cells were placed in a cell disruption vessel (Parr Instruments, USA) and incubated under 7000 KPa nitrogen for 45 min, then lysed by rapid release of pressure. The lysate was clarified by sequential centrifugation at 10 000 × g and 30 000 × g and anti-splice leader DNA leader oligonucleotide was added to 10 μM. The lysate was then desalted into 45 mM HEPES, pH 7.6, containing, 100 mM Potassium Acetate and 3 mM Magnesium Acetate, supplemented with a coupled translation/transcription feeding solution and snap-frozen until required. We verified that the expression patterns and cleavage of the proteins in this study was independent of the batch of LTE used.

The Leishmania tarentolae Parrot strain was obtained as LEXSY host P10 from Jena Bioscience GmbH, Jena, Germany, and cultured in TBGG media (12 g/L tryptone, 24 g/L yeast extract, 0.8% glycerol, 5.55 mM glucose, 17 mM KH₂PO₄, 72 mM K₂HPO₄) containing 0.2% v/v penicillin/streptomycin (Life Technologies) and 0.05% w/v hemin (MP Biomedical)^{117 (link)}. Cells were collected by centrifugation at 2500 × g, washed twice by resuspension in 45 mM HEPES pH 7.6, containing 250 mM sucrose, 100 mM potassium acetate and 3 mM magnesium acetate, and resuspended to 0.25 g cells/g suspension. Cells were placed in a cell disruption vessel (Parr Instruments, USA) and incubated under 7000 kPa nitrogen for 45 min, then lysed by a rapid release of pressure. The lysate was clarified by sequential centrifugation at 10,000 × g and 30,000 × g and anti-splice leader oligonucleotide was added to 10 mM. The lysate was then desalted into 45 mM HEPES pH 7.6, containing 100 mM potassium acetate and 3 mM magnesium acetate, and snap-frozen until required.

Cell-free lysate was collected from Leishmania tarentolae (LT) as per Johnston & Alexandrov [22 (link), 43 (link), 44 (link)]. Leishmania tarentolae Parrot strain was acquired as LEXSY host P10 from Jena Bioscience GmbH, Jena, Germany, and cultured in the TBGG medium containing 0.2% v/v penicillin/streptomycin (Life Technologies) and 0.05% w/v hemin (MP Biomedical). LT cells were harvested through centrifugation at 2500×g, washed twice by resuspension in 45 mM HEPES (pH 7.6) containing 3 mM magnesium acetate, 100 mM potassium acetate, and 250 mM sucrose. Cells were resuspended to 0.25 g cells/g suspension and incubated under 7000 kPa nitrogen for 45 min, then lysed by rapid release of pressure in a cell disruption vessel (Parr Instruments, USA). Through sequential centrifugation at 10,000×g and 30,000×g, the cell-free lysate was clarified and 10 μM anti-splice leader DNA leader oligonucleotide was added. The cell-free lysate was then desalted into 45 mM HEPES (pH 7.6) containing 100 mM potassium acetate and 3 mM magnesium acetate. The LTE was supplemented with a coupled translation/transcription feeding solution and snap-frozen until required for further experimentation.

Leishmania tarentolae cell-free lysate was produced as described by Johnston and Alexandrov [42 (link),43 (link),44 (link)]. Briefly, Leishmania tarentolae Parrot strain was obtained as a LEXSY host P10 from Jena Bioscience GmbH, Jena, Germany and cultured in TBGG medium containing 0.2% v/v Penicillin/Streptomycin (Life Technologies, Carlsbad, CA, USA) and 0.05% w/v Hemin (MP Biomedicals, Seven Hills, NSW, Australia). Cells were harvested by centrifugation at 2500× g, washed twice by resuspension in 45 mM HEPES buffer, pH 7.6, containing 250 mM Sucrose, 100 mM Potassium Acetate and 3 mM Magnesium Acetate and resuspended to 0.25 g cells/g suspension. Cells were placed in a cell disruption vessel (Parr Instruments, Moline, IL, USA) and incubated under 7000 KPa nitrogen for 45 min, and then lysed by rapid release of pressure. The lysate was clarified by sequential centrifugation at 10,000× g and 30,000× g and anti-splice leader DNA leader oligonucleotide was added to 10 µM. The lysate was then desalted into 45 mM HEPES, pH 7.6, containing, 100 mM Potassium Acetate and 3 mM Magnesium Acetate, supplemented with a coupled translation/transcription feeding solution and snap-frozen until required.