N-terminally His-tagged recombinant proteins of CrPV-1A, DCV-1A and FHV-B2 were expressed in Escherichia coli Rosetta2 strain (Merck Millipore). Typically, the cells in 1-litre cultures with 100 μg/ml ampicillin were cultivated to an OD600 of 0.4–0.5 at 37°C and then grown at 15°C overnight with 1 mM isopropyl-β-d -thiogalactoside (IPTG). The cell pellets were resuspended in His A buffer (30 mM HEPES–KOH pH 7.4, 200 mM KOAc, 2 mM Mg(OAc)2, 5% glycerol, 20 mM imidazole–HCl (pH 7.5), 0.2 mM TCEP) containing 1 × EDTA-free protease inhibitor cocktail (Roche), sonicated and centrifuged at 10 000 × g for 20 min. The supernatant was loaded onto HisTrap FF crude 5 ml (GE Healthcare Life Sciences) and eluted with a linear gradient from His A buffer to His B buffer (His A buffer containing 400 mM imidazole) using AKTA purifier (GE Healthcare Life Sciences). The peak fractions were collected and buffer-exchanged to 30 mM HEPES–KOH (pH 7.4), 100 mM KOAc, 2 mM Mg(OAc)2, 10% glycerol, 1 mM DTT with PD-10 (GE Healthcare Life Sciences). All purified proteins were flash-frozen in liquid nitrogen and stored at –80°C. The concentrations of the proteins were measured by SDS-PAGE with defined dilutions of BSA as concentration standards after CBB staining of the gel.
Histrap ff crude 5 ml
Manufactured by GE Healthcare
HisTrap FF crude 5 ml is a laboratory equipment product designed for protein purification. It is a prepacked column that utilizes Ni Sepharose for the purification of histidine-tagged proteins. The core function of this product is to enable efficient and convenient affinity chromatography-based purification of recombinant proteins.
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4 protocols using histrap ff crude 5 ml
1
Purification of Recombinant Viral Proteins
2
Purification of His-tagged and Twin-Strep-tagged Proteins
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Expression and purification of His-tagged proteins were performed as described23 (link),24 (link),26 (link). E. Coli BL21-Gold (DE3) Competent Cells (Agilent Technologies) were transformed with the plasmid coding for the different proteins. Cells were grown at 37 °C in 2YT medium containing 200 µg.L−1 ampicillin until the OD600nm reached 0.6. Protein expression was induced by addition of 1 mM IPTG and the cells were further incubated for 5 h at 37 °C. The cells were harvested, suspended in 50 mM sodium phosphate pH7.5, 150 mM NaCl (PBS), submitted to three freezing/thawing cycles, treated with DNase I for 30 min and sonicated. The His-tagged proteins were purified using nickel-affinity chromatography (Histrap™ FF crude 5 mL, GE Healthcare). The His-tag A3-A3 and A3-bGFPD was cleaved by TEV protease for some SPR experiments.
Twin-Strep®-tagged proteins (bA3-2, eGFP and BFP) were also purified using a StrepTactin Sepharose affinity chromatography (StrepTrap™ HP 5 mL, GE Healthcare); both samples obtained from this first step purification were then submitted to a size exclusion chromatography.
Twin-Strep®-tagged proteins (bA3-2, eGFP and BFP) were also purified using a StrepTactin Sepharose affinity chromatography (StrepTrap™ HP 5 mL, GE Healthcare); both samples obtained from this first step purification were then submitted to a size exclusion chromatography.
3
Specificity of Cheytin Binders
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Two different genes encoding fusion proteins were constructed to test the specificity of Cheytin binders selected against Kaz-AlphaRep. First the CheytinWT-Kaz-alphaRep fusion protein (the CheytinWT-Kα), corresponds to the fusion between the CheytinWT and the Kα. Second the fusion bF5K-Kα, corresponds to the fusion between the binder F5 of Kaz, selected from the Lib-Cheytins 2.1 and the Kα. These two genes cloned in PQE81L-vector were obtained by the Gibson assembly approach (data not shown). CheytinWT-Kα and bF5K-Kα were purified using an affinity chromatography (Histrap™ FF crude 5 mL GE Healthcare); samples obtained from this first step purification were then submitted to a size-exclusion chromatography (Hiload 16/60 SuperdexTM 75) equilibrated in Cheytin-buffer Tris 20 mM NaCl 150 mM MgCl2 5 mM pH 7,4.
4
Recombinant AgraGH45-1 Production in P. pastoris
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A X-33 P. pastoris colony transformed with pGAPZα-B-AgraGH45-1 ΔSP was inoculated in 100 mL of YPG/Zeocine 100 µg/mL and kept at 28 °C and 225 rpm for 3 days. After that, 10 mL of the culture was used to inoculate 1 L of YPG/Zeocine 100 µg/mL. The culture was kept at 28 °C and 225 rpm for 4 days. The cells were harvested by centrifugation, the supernatant filtered through 0.2 µm and then diluted in sodium phosphate buffer pH 7.4 (1:1) to a final concentration of 20 mM. Sodium chloride was added to a final concentration of 0.5 M. The final volume was kept circulating through a HisTrap FF crude 5 mL (G.E. Healthcare) column previously equilibrated with binding buffer (20 mM sodium phosphate pH 7.4, 0.5 M NaCl). The column was washed with 6 volumes of binding buffer containing 20 mM imidazole, and the recombinant AgraGH45-1 was eluted with binding buffer containing 0.5 M imidazole. Eluted fractions were dialyzed against 0.25 mM sodium bicarbonate and freeze-dried. Protein samples were quantified and used in electrophoretic assays to molecular weight determination. AgraH45-1 was detected by Western Blotting with 6 × -His Tag Monoclonal Antibody (Thermo Fisher Scientific, USA).
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