Q sepharose anion exchange chromatography

DNA corresponding to deletion constructs of PFA0660w and PfHsp70-x were PCR amplified, and cloned in pET-28a(+) bacterial expression vector before expression in E. coli BL21 (DE3) cells. Recombinant proteins were purified using Ni-NTA affinity chromatography followed by gel permeation chromatography. Gel filtration chromatography was performed using AKTA prime plus on Superdex 200 10/300 GL column (GE Healthcare Life Sciences, Chicago, IL, USA). Purified PFA0660w-S was used for raising polyclonal antibodies in rabbits commercially (Merck, Bangalore, India).
To confirm protein identity, purified recombinant protein constructs of PFA0660w and PfHsp70-x were resolved on SDS-PAGE and transferred on NC membrane. NC membrane was blocked overnight at 4 °C in 5% BSA. Following washing, the blot was probed with horseradish peroxidise (HRP) linked monoclonal anti-hexahistidine antibody (1:2000; Sigma) for 2 hours, and developed with diamino benzidine/H₂O₂ substrate (Sigma).
Recombinant ATS domain of PF08_0141 was purified for experiments as previously described^{36 ,37 (link)}. Briefly, protein was purified using Ni-NTA affinity chromatography followed by Q-Sepharose anion exchange chromatography (GE Healthcare) and then size exclusion on Superdex 200 10/300 GL column (GE Healthcare).

Recombinant TcdB was expressed and purified as previously described (48 (link)). Briefly, a plasmid encoding His-tagged TcdB (pBL377) was transformed into Bacillus megaterium according to the manufacturer’s protocol (MoBiTec). Six liters of Luria-Bertani medium supplemented with tetracycline (10 mg/liter) were inoculated with an overnight culture to an OD₆₀₀ of ∼0.1. Cells were grown at 37°C and 220 rpm. Expression was induced with 5 g/liter of d-xylose once cells reached an OD₆₀₀ of 0.3 to 0.5. After 4 hours, the cells were centrifuged and resuspended in 20 mM tris (pH 8.0), 500 mM NaCl, and protease inhibitors. An EmulsiFlex C3 microfluidizer (Avestin) was used at 15,000 lb/in² twice to generate lysates. Lysates were then centrifuged at 40,000g for 20 min. Supernatant containing toxin was passed through a Ni-affinity column (HisTrap FastFlow Crude, GE Healthcare) initially. Further purification was performed using Q-Sepharose anion-exchange chromatography (GE Healthcare) and gel filtration chromatography in 20 mM Hepes (pH 6.9)–50 mM NaCl.

The wt NA and mutant NA (I223R/H275Y) genes derived from A/H1N1 2009 pandemic influenza virus were amplified by PCR. The PCR products were cloned into the pAcGP67A vector (BD Biosciences) and transfected into the Sf9 insect cell line to produce recombinant proteins. The culture medium was collected, clarified by centrifugation at 110 × g, and subjected to further viral propagation. The cultured Sf9 cells were inoculated with recombinant baculovirus at a multiplicity of infection of 10 and incubated at 27 °C for 96 h. The cells were collected after centrifugation, and the cell pellet was resuspended in cell lysis buffer (50 mM Tris-HCl, pH 8.5, 5 mM 2-mercaptoethanol, 100 mM KCl, 1 mM phenylmethylsulfonyl fluoride), sonicated, and centrifuged at 16,000 × g for 15 min at 4 °C. After the cell lysate was sonicated to reduce its viscosity, the cell debris was removed by centrifugation for 1 h at 16,000 × g. The soluble protein from the cell supernatant was applied to Ni-nitrilotriacetic acid agarose resin (Qiagen), washed, and eluted with buffer (50 mM Tris-HCl, 0.5 M NaCl, 0.5 M imidazole, pH 8.0). The purified proteins were dialyzed against phosphate-buffered saline (PBS) and further purified by Q-Sepharose anion-exchange chromatography (GE Healthcare).