2.4. Expression and purification of recombinant wild type and mutant PfAOP in Escherichia coli

Recombinant proteins were expressed and purified by affinity-chromatography as described previously [10], [11], [16]. Briefly, E. coli XL1-Blue cells were transformed with plasmid PFAOP/pQE30, PFAOP^C117S/pQE30, PFAOP^C143S/pQE30 or PFAOP^L109M/pQE30. Expression was induced with 0.5 mM isopropyl-β-D-1-thiogalactopyranoside for 4 h at 37 °C. Liquid cultures were harvested by centrifugation for 15 min at 4000×g and 4 °C. The bacteria were resuspended in buffer containing 20 mM imidazole, 300 mM NaCl, 50 mM Na_xH_yPO₄, pH 8.0, incubated with lysozyme and disrupted by sonication. Proteins were affinity-purified on Ni-NTA agarose columns and eluted in buffer containing 200 mM imidazol, 300 mM NaCl, 50 mM Na_xH_yPO₄, pH 8.0. Subsequently, samples were treated with 5 mM DTT for 30 min at 4 °C to fully reduce the protein. Remaining imidazole and DTT were removed using HiTrap desalting columns that were equilibrated with buffer containing 100 mM Na_xH_yPO₄, 0.1 mM DTPA, pH 7.4. Protein elution was monitored at 280 nm using an Äkta FPLC system. The protein concentration was determined spectrophotometrically using the molar extinction coefficient ε_280 nm = 21.43 mM⁻¹ cm⁻¹ as calculated for the primary sequence of the protein using the ProtParam ExPASy tool (http://web.expasy.org/protparam/). The thiol content of the proteins was analyzed with 5,5′-dithiobis-(2-nitrobenzoic acid) [17] revealing that 97% of the protein thiols were in a reduced state (data not shown).