Genes encoding wild-type or dksA point mutants were cloned into the GST fusion plasmid pGEX-6P-1 (GE) (10 (link)). Full-length or truncated dnaJ, tig, and rpoC genes were directionally cloned as C-terminal 6His fusions into the NdeI and XhoI sites of the pET-22b(+) plasmid (Novagen). All constructs were confirmed by sequence analysis. Plasmids were expressed in E. coli BL21 (DE3) (Invitrogen) or E. coli Origami B (DE3) pLysS (Novagen) (SI Appendix, Table S1 ). Cells grown in LB broth at 37 °C to an OD600 of 0.5–0.7 were then treated with 0.1 mM isopropyl-β-d -thiogalactopyranoside (IPTG). After 3 h, the cells were harvested, disrupted by sonication, and centrifuged to obtain cell-free supernatants. GST and 6His-tagged fusion proteins were purified using Glutathione-Sepharose 4B (bioWORLD) and TALON metal-affinity chromatography (Clontech), respectively. To remove the GST tag from recombinant GST-DksA protein, PreScission protease (PSP), prepared in PBS buffer supplemented 10 mM DTT, was added. After an overnight incubation at 4 °C, protein was eluted and further purified by size-exclusion chromatography on Superdex 75 (GE Healthcare Life Sciences). Purified DksA proteins were aliquoted inside a BACTRON anaerobic chamber (Shel Lab). The purity and mass of the recombinant proteins were assessed by SDS/PAGE.
Glutathione sepharose 4b
Manufactured by Bioworld Technology
Sourced in United States
Glutathione-Sepharose 4B is a chromatography resin designed for the purification of glutathione-binding proteins. It consists of glutathione, a tripeptide, covalently coupled to Sepharose 4B, a cross-linked agarose matrix. This resin can be used to capture and isolate proteins that have an affinity for glutathione, a common ligand for many cellular proteins.
Automatically generated - may contain errors
Lab products found in correlation
Superdex 75, by GE Healthcare (3 mentions)
E coli bl21 de3, by Thermo Fisher Scientific (2 mentions)
Talon metal affinity chromatography, by Takara Bio (2 mentions)
Pgex 6p 1, by GE Healthcare (1 mentions)
E coli bl21 de3 plyss, by Thermo Fisher Scientific (1 mentions)
Ni nta affinity chromatography, by Qiagen (1 mentions)
3 protocols using glutathione sepharose 4b
1
Purification of DksA and Associated Proteins
2
DksA Protein Expression and Purification
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Protein expression and purification were performed as previously described (35 ). Briefly, E. coli BL21 (DE3) (Thermo Fisher Scientific) (Table S1 ) grown in LB broth at 37 °C to an absorbance of 0.5 to 0.8 at 600 nm were treated with 0.1 mM isopropyl-β-d -thiogalactopyranoside. After 3 h, the cells were harvested, disrupted by sonication, and centrifuged to obtain cell-free extracts. Glutathione-S-transferase (GST) and 6His-tagged fusion proteins were purified using Glutathione-Sepharose 4B (bioWORLD) and TALON metal-affinity chromatography (Clontech), respectively, according to manufacturer’s protocols. To purify the DksA proteins, the GST tags were removed from GST-DksA proteins bound to a Glutathione-Sepharose 4B resin. PreScission protease was added to recombinant GST-DksA proteins in PBS containing 10 mM DTT. After overnight incubation at 4 °C, untagged proteins were eluted with PBS. For further purification of DksA protein, size-exclusion chromatography on Superdex 75 (GE Healthcare Life Sciences) was used. Purified DksA proteins were aliquoted inside a BACTRON anaerobic chamber (Shel Lab). The purity and mass of the recombinant proteins were assessed by SDS-PAGE.
3
Recombinant Protein Purification of GreA, GreB, and DksA
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Recombinant 6XHis-tag GreA or 6XHis-tag GreB were produced by cloning greA and greB genes into NdeI and BamHI sites of the pET14B vector (Novagen) using greA F and greA R or greB F and greB R primers, respectively (Tables b and c in S1 Text ). All constructs were confirmed by sequencing. Plasmids were expressed in E. coli BL21 (DE3) pLysS (Invitrogen). Cells grown in LB broth at 37°C to an OD600 of 0.5 were treated with 1 mM isopropyl β-D-1-thiogalactopyranoside. After 3 h, the cells were harvested, disrupted by sonication, and centrifuged to obtain cell-free supernatants. 6XHis-tag fusion proteins were purified using Ni-NTA affinity chromatography (Qiagen) as per manufacturer’s instructions. DksA protein was purified as described previously [56 (link)]. A GST-DksA fusion protein was purified using Glutathione-Sepharose 4B (bioWORLD, Dublin, Ohio, USA) according to manufacturer’s protocols. To remove the GST tag, PreScission protease was added to recombinant GST-DksA protein in phosphate-buffered saline (PBS) containing 10 mM DTT. After overnight incubation at 4°C, proteins were eluted and further purified by size-exclusion chromatography on Superdex 75 (GE Healthcare Life Sciences). Purified DksA proteins were aliquoted inside a BACTRON anaerobic chamber (Shel Lab, Cornelius, Oregon, USA). The purity and mass of the recombinant proteins were assessed by SDS/PAGE.
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