Superdex 200 gel filtration column

DNA sequences encoding human truncated CD27 (extracellular domain of CD27), CD70 extracellular domain, B7-H3 extracellular domain, CD70 specific scFv (derived from the CD70-16D_cc-scFv sequence, Patent number: WO2017021354A1) and B7-H3 specific scFv (derived from mAb-J42) were synthesized by GENEWIZ. And all the cDNA was sub-cloned into a pVAX1 based expression vector with human or murine IgG1 Fc and (His)₆ tag fusion at the C-terminus, respectively. Transient expression in the HEK293T cell line was performed by using expression vectors and optimal DNA to PEI ratio was determined with 1:3. The cells were cultured in FreeStyle™ 293 Expression Medium (Thermo Fisher Scientific) for 4~5 days. Then the culture supernatants were harvested and centrifuged for 30 min at 10 000 × g, 4 °C. The recombinant proteins were initially purified by Ni-NTA column chromatography, and eluted with elution buffer (25mM Tris, pH 8.0, 250mM NaCl, 250mM Imidazole, 5% (v/v) glycerol and 1mM PMSF). The eluted recombinant proteins were then loaded to a Superdex200 gel filtration column (GE Healthcare) with gel filtration buffer (25mM Tris, pH 8.0, 250 mM NaCl, and 5% (v/v) glycerol, 1mM PMSF) followed by analysis of recombinant proteins purity through SDS-PAGE. Finally, recombinant proteins were concentrated and stored at -80 °C for later studies.

Nucleosomes were reconstituted as previously described (Dyer et al., 2004 (link)). Recombinant Xenopus histones were expressed in E. coli BL21(DE3) pLysS and purified from inclusion bodies via Sephacryl S-200 gel filtration (GE Healthcare). Stoichiometric amounts of each core histone were incubated together under high salt conditions (2 M NaCl) and the resulting histone octamer purified using a Superdex 200 gel filtration column (GE Healthcare). 216 bp DNA carrying the nucleosome-positioning 601 sequence was PCR amplified from the pGEM-3Z 601 plasmid and purified using a Resource Q anion exchange column (GE Healthcare). Purified DNA, in slight excess to octamers, was mixed together in 2 M NaCl and diluted stepwise with 10 mM Tris (pH 7.5) to reach a final concentration of 100 mM NaCl. The reconstituted nucleosomes were then analysed on a 0.8% Tris-borate agarose gel, and concentrated using a 5000 MWCO spin concentrator (GE Healthcare).

Unless otherwise stated, oligonucleotides were from Eurofins Operon. KOD polymerase and Escherichia coli DE3 pLysS Rosetta cells were from Novogen. RNase inhibitor murine and T7 RNA polymerase were from New England Biolabs. Glycogen was from Roche, Proteinase K from Invitrogen, and Heparin Sepharose from Sigma.
The rapid DNA ligation kit was from Thermo Scientific. QIAquick PCR purification, QIAquick Gel elution, and QIAquick nucleotide removal kits were from Qiagen. The QuikChange site-directed mutagenesis kit was from Agilent. A 2 ml HiTrap heparin column and Superdex™ 200 gel filtration column were from GE Healthcare Life Sciences.

Briefly, proteins were overexpressed in E. coli BL21 (DE3) cells by growing at 37°C in LB media containing 50 μg/mL kanamycin to an OD₆₀₀ of 0.4–0.6. Expression was induced by the addition of isopropyl-β-D-thiogalactopyranoside (IPTG) to a final concentration of 0.5 mM. After induction, the cultures were grown at 16°C for an additional 16 hr. The cells were pelleted and lysed via sonication in lysis buffer [50 mM Tris-HCl pH 8.0, 500 mM NaCl, 15 mM Imidazole, 10% Glycerol, 0.1% Triton X- 100, 10 mM β-mercaptoethanol (BME)]. The lysate was cleared by centrifugation, and the supernatant was initially purified using Ni-NTA (Qiagen) beads and eluted with elution buffer (50 mM Tris-HCl pH 8.0, 500 mM NaCl, 200 mM Imidazole, and 10 mM BME). The protein was dialyzed against gel filtration buffer (20 mM Tris-HCl pH 8.0, 50 mM NaCl, 10 mM BME for GST-yCTD and 20 mM Tris-HCl pH 7.5, 200 mM NaCl, 10 mM BME). Finally, proteins were concentrated and ran on a Superdex 200 gel filtration column (GE). Erk2 was purified using a previously published protocol (Khokhlatchev et al., 1997 (link)). Homogeneity of the eluted fractions was determined via Coomassie Brilliant Blue stained SDS-PAGE. Samples were concentrated in vivaspin columns (Sartorius).

Nanosota-1A, 1B and 1C were each purified from the periplasm of ss320 E. coli after the cells were induced by 1 mM IPTG. The cells were collected and re-suspended in 15 ml TES buffer (0.2 M Tris pH 8, 0.5 mM EDTA, 0.5 M sucrose), shaken on ice for 1 hour and then incubated with 40 ml TES buffer followed by shaking on ice for another hour. The protein in the supernatant was sequentially purified using a Ni-NTA column and a Superdex200 gel filtration column (GE Healthcare) as previously described (20 (link)). Nanosota-1C-Fc was purified from the cytoplasm of Shuffle T7 E. coli. The induction of protein expression was the same as above. After induction, the cells were collected, re-suspected in PBS and disrupted using Branson Digital Sonifier (Thermofisher). The protein in the supernatant was sequentially purified on protein A column and Superdex200 gel filtration column as previously described (20 (link)).