Mono s 10 100 gl column

The PGT128 Fab was expressed in FreeStyle™ 293 F cells (Invitrogen; cat. no. R790-07) by transfection at a 1:2 ratio of plasmids encoding light and heavy chain (truncated at Asp^H234), respectively^{21 (link)}. Supernatants harvested 6 days after transfection were passed over an anti-human lambda affinity matrix (CaptureSelect Fab λ; Life Technologies) equilibrated in Dulbecco’s PBS. Fab fragments were eluted with 0.1 M glycine, pH 2.7 and neutralized with 1 M Tris-Cl, pH 9.0 at a volume of 2 ml per 15 ml. Eluted fractions were pooled and buffer exchanged into 20 mM sodium acetate, pH 5.6, and loaded onto a Mono S 10/100 GL column (GE Healthcare). A 0–0.5 M KCl linear gradient was used for elution, and the Fab peak eluted at ≈16 mS cm⁻¹. PGT128 peak fractions were pooled, exchanged into 20 mM Tris-HCl, 150 mM NaCl, pH 8.0 buffer and concentrated to 29.5 mg ml⁻¹ with a 10 kDa ultrafiltration concentrator.

The fragment carrying 1348 was amplified from the genomic DNA of Pseudomonas sp. Os17 by PCR with the primers 25F1348 and 25R1348 (Supplementary Table S2). The PCR product was inserted into the NdeI/BamHI sites of the expression vector pET25b (+) (Novagen) with the In-Fusion HD Cloning system (Clontech). The resultant plasmid, pET25-1348, was introduced into E. coli BL21 (DE3) (Merck) and the strain was grown at 37°C. The expression of the recombinant Os1348 protein was induced by the addition of isopropyl-β-D-thiogalactopyranoside at a final concentration of 0.4 mM and growth continued at 16°C for 16 h. Harvested cells were resuspended in PBS (pH 7.4) and lysed by sonication. Debris was removed by centrifugation at 40,000 × g for 30 min. The lysate was applied to a 5-ml HiTrap SP HP column (GE Healthcare). The column was washed with 20 mM sodium acetate (pH 5.2) and eluted with a linear gradient of 0–500 mM NaCl. Os1348 fractions were purified further on HiLoad 26/60 Superdex75 prep grade (GE Healthcare) pre-equilibrated with buffer consisting of 10 mM Tris–HCl (pH 7.5) and 150 mM NaCl. After dialysis at 4°C for 16 h against 20 mM sodium acetate (pH 5.2), the supernatant was purified by a Mono S 10/100 GL column (GE Healthcare) and a 0–300 mM NaCl elution gradient.

Gene encoding MotA was synthesized and subcloned to pET21a by GENEWIZ, Inc. Escherichia coli strain BL21(DE3) (Invitrogen, Inc.) was transformed with plasmid pET21a-MotA (GENEWIZ, Inc.) encoding MotA under the control of the bacteriophage T7 gene 10 promoter. Single colonies of the resulting transformants were used to inoculate 1 L LB broth containing 50 μg/ml ampicillin, cultures were incubated at 37°C with shaking until OD₆₀₀ = 0.6, cultures were induced by the addition of IPTG to 1 mM, and cultures were incubated 3 h at 37°C. Then, cells were harvested by centrifugation (5000 rpm; 10 min at 4°C), resuspended in 20 ml buffer B (10 mM Tris-HCl, pH 7.5, 0.2 M NaCl, 5% glycerol, 1 mM EDTA and 1 mM dithiothreitol (DTT)) and lysed using a JN-02C cell disrupter (JNBIO, Inc.). The lysate was centrifuged (12 000 rpm; 45 min at 4°C), and the supernatant was loaded onto a 5 ml column of HiTrap Heparin HP (GE Healthcare, Inc.) equilibrated in buffer B and eluted with a 100 ml linear gradient of 0.2–1 M NaCl in buffer B. The sample was further purified by cation-exchange chromatography on a Mono S 10/100 GL column (GE Healthcare, Inc.; 160 ml linear gradient of 0.2–1 M NaCl in buffer B). Fractions containing MotA were pooled and stored at −80°C. Yields were ∼2 mg/l, and purities were >95%.