Kta purifier system

To generate antibody specific to ZmMADS47, cDNA fragment containing the first 400 bp from 5’ ORF was cloned into the pGEX-4T-1 GST-tagged vector by BamHI/XhoI sites. Recombinant protein were expressed in bacterial strain BL21 (DE3) (Novagen) by adding 500 μM IPTG (Dingguo) to a final concentration of 0.4 mM under 25°C, and purified using the ÄKTA purifier system (GE Healthcare). The antibody was prepared by Shanghai ImmunoGen Biological Technology in rabbit according to standard protocol. To generate antibodies specific to O2, O2 ORF was amplified and cloned into NotI/NcoI sites of pET-32a (Novagen). His-O2 fused protein was induced in E. coli strain Rosetta (DE3), and purified by the ÄKTA purifier system (Amersham Biosciences). The antibodies were prepared by Shanghai ImmunoGen Biological Technology according to standard protocol. The 19-kD, 22-kD α-zein, 16-kD, 27-kD, 50-kD γ-zein, 14-kD β-zein, 10-kD δ-zein specific antibodies were produced according to the protocol published before [4 (link)].

Bacterial cells were grown to mid-log phase (OD_{600 nm} = ∼0.8) in LB media at 37°C in the presence of 50 μg/mL Kanamycin and 100 μg/mL chloroamphenicol. Induction of the culture was then carried out with 0.3 mM isopropyl-1-thio-β-D-galactopyranoside (IPTG) at 20°C. Cells were pelleted after 20 h by centrifugation at 8, 000 rpm for 10 min at 4°C. The cell pellet was resuspended in buffer A [20 mM Tris, 300 mM NaCl, 5% (v/v) glycerol, and 1 mM PMSF, pH 8.0] and lysed by ultrasonification on ice. The cell debris and membranes were pelleted by centrifugation at 15,000 rpm (R20A2 rotor, Hitachi high-speed refrigerated centrifuge R21GIII) for 45 min at 4°C. The soluble C-terminally hexahistidine-tagged PaHigA was purified by affinity chromatography with nickel-nitrilotriacetic acid resin (Bio-Rad). Untagged proteins were removed with buffer A containing 50 mM imidazole. Recombinant PaHigA was then eluted with buffer A containing 250 mM imidazole. The protein was further purified by gel filtration (Superdex 200, GE Healthcare) equilibrated in buffer B [20 mM Tris, 300 mM NaCl, 5% (v/v) glycerol, pH 8.0] using an ÄKTA Purifier System (Amersham).

V. fluvialis LMGT 4216 was cultivated in 1 L of BHI broth at 30°C for 24 h. Cells were removed by centrifugation (10,000 × g for 30 min at 4°C), and the bacteriocin was precipitated from the culture supernatant with ammonium sulfate (60% saturation at 4°C overnight). The precipitate was harvested by centrifugation (15,000 × g for 40 min at 4°C), redissolved in 700 mL of distilled water, and adjusted to pH 3.5 with 1 M hydrochloric acid. The sample was applied to a Hi-Prep 16/10 SP-XL column (GE Healthcare, Chicago, IL, USA). Unbound material was washed from the column with 150 mL of 25 mM sodium citrate-phosphate buffer (pH 3.5). The bacteriocin was eluted with 100 mL of 0.5 M sodium chloride, and the eluate was then applied to a 1-mL Resource RPC column (GE Healthcare) connected to an Äkta purifier system (Amersham Pharmacia Biotech, Amersham, UK). The column was previously equilibrated with 0.1% (vol/vol) trifluoroacetic acid (TFA), and the bacteriocin was eluted from the column using a linear gradient (40 column volumes [CV]) of isopropanol containing 0.1% (vol/vol) TFA at 1 mL/min.

All size exclusion chromatography (SEC) experiments were carried out using an Äkta purifier system (Cytiva) with indicated buffers and columns. 10/300 columns were loaded with max. 8 mg protein with 0.5 ml/min flow rate at 4 °C. 16/600 columns were loaded with max. 80 mg protein with 1 ml/min flow rate at 4 °C.

Size exclusion chromatography (SEC) experiments were carried out using an Äkta purifier system (Cytiva) and a Superdex 75 10/300 column (Cytiva). The column was loaded with max. 10 mg protein with a flow rate of 0.5 ml/min at 4 °C using the indicated buffers.

SEC was carried out at 12 °C, using an Äkta purifier system (Cytiva, Munich, Germany) and Increase Superose 6 5/150 or 3.2/300 columns (Cytiva, Munich, Germany). The columns were equilibrated in sterile-filtrated, degased and pre-cooled buffer (100 mM NaCl, 20 mM HEPES, pH 7.4) before injecting the affinity purified protein samples. Flow rate was 0.15 mL/min for Superose 6 5/150 and 0.05 mL/min for Superose 6 3.2/300. UV absorbance was recorded at 280 nm and data were plotted using GraphPad Prism (v.9.5).