Metal affinity chromatography

The full-length coding sequences of gTrxR and gEF1Bγ (GiardiaDB accession number GL50803_9827 and GL50803_12102, respectively) were PCR amplified from the Giardia WB-C6 genomic DNA using primers reported in Supplemental Table S1. PCRs were performed on a T-Personal Thermocycler (Biometra, Göttingen, Germany) using 100 ng of gDNA, 10 units of high fidelity Pfu turbo DNA polymerase (Agilent Technologies, Santa Clara, CA, USA), 50 μM dNTP, 20 pmol of each primer in 50 μl of reaction mixture. Amplification conditions were: 1 cycle at 95 °C for 2 min; 30 cycles at 95 °C for 30 s, 56 °C for 30 s and 72 °C for 30 s; and 1 cycle at 72 °C for 7 min. The coding sequence of g14-3-3 was excised from p14-X vector3. For the expression of N-terminal 6xHIS-tagged fusion protein in Escherichia coli, the BamHI/NotI digested fragments were cloned in BamHI/NotI linearized pQ30 vector (Qiagen, Germany) and transformed in M15 strain. Expression of recombinant proteins and purification under native conditions by metal affinity chromatography (Qiagen, Germany) were performed as described (Lalle et al., 2015 (link)).

Overnight pre-culture was grown in LB supplemented with 200 μg/mL of ampicillin. Then, 200 mL of LB supplemented with 200 μg/mL of ampicillin with constant vigorous shaking at 37 °C until an OD₆₀₀ of 0.5 was reached. 1 mM of IPTG was added to the culture and kept in vigorous shaking at 37 °C for 18 hrs. Protein expression and maturation was verified by withdrawing 1 ml of cell culture and spinning down cells by centrifugation (2500 x g for 5 min at 4 °C) to confirm color.
Cells were collected by centrifugation (2500 x g for 5 min at 4 °C) and lysed on ice using 10 μg/ml lysozyme in PBS plus 5% glycerol for 30 min., and then two freeze-thaw (between -80 °C and 4 °C) cycles were used to complete the lysis. After lysis is completed, cell debris were removed by centrifugation at 10,000 RPMs for 10 min. The protein obtained is pink (aeBlue-pink).
For the blue form of the protein, an equally prepared lysate was incubated at 25 °C (aeBlue-blue) for 60 min, reaching a total change to the blue form.
Both supernatants were subjected to metal affinity chromatography (Qiagen) following the manufacturer's protocol for soluble protein purification, but aeBlue-pink and aeBlue-blue purification was carried at 4 °C to prevent protein degradation.
Protein purity was verified by SDS-PAGE. Protein was quantified using the DC Protein Assay (Bio-Rad).