Nickel nitrilotriacetic acid resin

Protein production was performed using standard protocols previously described (30 (link)), with the exception that FAH and GSH transferase ζ1 (GSTZ1) were grown at 37 °C for 16 h, whereas homogentisic acid dioxygenase (HGD) was grown at 25 °C until an A₆₀₀ of 0.6 to prevent the formation of inclusion bodies. Protein purification was obtained by nickel–nitrilotriacetic acid resin (Invitrogen) followed by size exclusion chromatography (Hi-Load 26/60 Superdex 75; GE Healthcare) and eluted in 20 mm Tris, 300 mm NaCl, pH 8, for FAH; 20 mm Tris, 500 mm NaCl, pH 7.0, for HGD; and 5 mm HEPES, pH 7.0, with 5% (v/v) glycerol for GSTZ1. Protein quantification was performed by spectrophotometrical measurement (E_FAH(280 nm) = 55,550 m⁻¹ cm⁻¹; E_HGD(280 nm) = 69,280 m⁻¹ cm⁻¹; and E_GSTZ1(280 nm) = 19,300 m⁻¹ cm⁻¹).

For the production of polyclonal antibodies, EmACT was expressed in the bacterial pBAD/TOPO ThioFusion Expression Kit (Invitrogen). To maximize the recognition of EmACT after processing by the generated antibodies, the full Emact (without stop codon) amplified using the primer pair Emact_Dw/Emact_Up coding for the preproprotein EmACT was chosen for immunization and subcloned in pBAD/TOPO ThioFusion expression vector (Invitrogen). The His-tagged Thioredoxin- fusion protein (Thio-EmACT) following arabinose induction (2 g/L; 4 h), was purified on a nickel-nitrilotriacetic acid resin (Invitrogen). The purified protein was diafiltered on against sterile PBS (1×) on Centrifugal Filter Units (Millipore) before quantification. NMRI mice were immunized with the purified Thio-EmACT resuspended in 100 μl of Freund Incomplete adjuvant (Sigma) by two subsequent sub-cutaneous injections initially and repletion 4 weeks later for boosting. Ten days after the second round of injections, animals were terminally bled from the heart and the serum was collected and stored at −20°C. To generate pre-immune serum, naïve NMRI mice were similarly bled by cardiac bleeding and serum collected.

The FN9-10_ELP fusion protein was produced as previously described by Lee et al [40 (link)]. Briefly, the ELP sequence was synthesized (Genotech, Daejeon, Korea) and cloned into the pBAD-His-FN9-10 expression vector using the restriction enzymes SacⅠ and HindⅢ. The FN9-10_ELP construct was transformed into E. coli TOP10, as the expression host.
To express FN9-10_ELP, the transformed TOP10 cells were incubated overnight at 37°C in lysogeny broth (LB) medium containing 100 μg·mL⁻¹ ampicillin (LB-Amp⁺). When the culture medium reached OD₆₀₀ = 0.6, induction was performed for 6 h by adding 0.1% (w/v) L-arabinose to induce protein expression. The bacteria were harvested and pelleted by centrifugation at 6000 g for 10 min, lysed in NaCl-Tris-EDTA (STE) buffer and sonicated. The soluble extract was centrifuged at 13,000 g for 2 x 10 min. The collected supernatant was purified by passing through a chromatography column containing a nickel-nitrilotriacetic acid resin (Invitrogen, Carlsbad, CA).
The recombinant FN9-10_ELP purity was evaluated using coomassie blue staining on 12% (v/v) SDS-PAGE gels. In addition, the molecular weight and expression of recombinant FN9-10_ELP were confirmed by western blotting using the horseradish peroxidase (HRP) conjugate of monoclonal anti-polyhistidine antibody (His antibody, sc-8036 HRP, Santa Cruz Biotechnology, Santa Cruz, CA, USA).