High-throughput protein purification by nickel affinity chromatography

12× DW24 (corresponding to the over-expression of 24 venom peptides) were thawed, in one go, for 10 min at 37 °C in a water-bath followed by 15 min shaking at 20 °C in a Microtron shaking incubator (INFORS-HT, Switzerland) (800 rpm). During this time the cultures were lysed by lysozyme and the solution became viscous. After the addition of 10 µg/mL DNAse and 20 mM MgSO₄ in each well and another 10 min shaking at 20 °C, the 12 × 0.5 mL lysed bacteria for each peptide were pooled and transferred to a new DW24 to have 24 distinct 6 mL lysates of DsbC-His-peptide fusions per DW24. To ensure complete cell lysis, the DW24 was sonicated in a plate sonicator (Ultrasonic processor XL, Misonix Inc., USA) for 5 min (power 5, 30 s ON/OFF cycles). Purification was performed by immobilized metal affinity chromatography (IMAC) as described in the accompanying paper [13] where all the steps were automated on a Tecan Freedom EVO 200 robot (Switzerland) containing a vacuum manifold. Briefly, the 6 mL of crude cell lysates were incubated with 4 × 200 µL Ni²⁺ Sepharose 6 Fast flow resin (GE Healthcare, 17-5318-02) with bound Nickel and then transferred (4 wells per peptide) into 96-well filter plates (20 µm) (Macherey–Nagel). The wells were washed twice with 800 µL buffer A (50 mM Tris, 300 mM NaCl, 10 mM Imidazole, pH 8) followed by three washes with 800 µL of 50 mM Tris, 300 mM NaCl, 50 mM Imidazole, pH 8 buffer. The recombinant fusion proteins were eluted from the resin beads with 1 mL of elution buffer (50 mM Tris, 300 mM NaCl, 250 mM Imidazole, pH 8). Rather than collecting the elution in a 96 deep-well plate (DW96) like in the standard procedure, this DW plate was replaced by a DW24, pooling the 4 × 1 mL of elution of a single peptide into a single well of the DW24. This procedure was reproduced four times in one day in order to purify the 96 fusion proteins. The total time taken for a single round of this process was around 4 h.