FimCH variant complexes were purified from periplasm preparations, as previously described (14 (link)). FimGNteH complexes were assembled by a spontaneous in vitro DSE reaction, in which FimGNte peptide (FimG residues 1 to 15; EZBiolab) was mixed in ~10× molar excess with FimCH variant complexes in 15 mM MES (pH 5.6) and 50 mM NaCl and incubated at 37°C for 16 hours. FimGNte displaces FimC in this reaction, and resultant FimGNteH variant complexes were purified away from excess FimGNte peptide and free FimC using a SOURCE 15S column (GE) in 15 mM MES (pH 5.6) with a gradient of 0 to 400 mM NaCl. Pooled fractions containing FimGNteH variant complexes were dialyzed against 15 mM MES (pH 5.6) and 50 mM NaCl, concentrated to 1 to 5 mg/ml, and stored stably at 4°C for use in biophysical assays.
Source 15s column
Manufactured by GE Healthcare
Sourced in United States
The SOURCE 15S column is a chromatography column designed for protein purification. It features a stable, high-quality resin that provides efficient separation and recovery of target proteins. The column's core function is to facilitate the purification process, allowing researchers to isolate and concentrate specific proteins from complex mixtures.
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Lab products found in correlation
Source 15q column, by GE Healthcare (2 mentions)
Sephadex g 25 medium, by GE Healthcare (1 mentions)
Shuffle t7 express e coli cells, by New England Biolabs (1 mentions)
Quickchange lightning site directed mutagenesis kit, by Agilent Technologies (1 mentions)
Arctic express de3 ril, by Agilent Technologies (1 mentions)
Superdex 75 column, by GE Healthcare (1 mentions)
5 protocols using source 15s column
1
Purification of FimCH variant complexes
2
Purification of MgGH51 Enzyme
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Culture broth containing secreted MgGH51 (Table 1 ▸ ) was produced as described by Sørensen et al. (2006 ▸ ). Filtrated broth was applied onto a Sephadex G-25 medium (GE Healthcare, Piscataway, New Jersey, USA) column equilibrated with 25 mM sodium acetate pH 4 and applied onto a SOURCE 15S column (GE Healthcare, Piscataway, New Jersey, USA) equilibrated with the same buffer. Bound proteins were eluted with a linear gradient from 0 to 1000 mM sodium chloride over ten column volumes. Fractions were collected and analysed by SDS–PAGE. MgGH51-bearing fractions were pooled and the pH was adjusted to 5.5.
3
Purification of Yeast Translational Factors
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Yeast eEF1A and eEF2 preparations were adapted from the eEF2 preparation method described previously (1 (link), 54 (link)). Briefly, clarified lysate (150 mL) from fresh yeast cake in Buffer 1 (20 mM Hepes-KOH, pH 7.2, 10% glycerol, 5 mM MgCl2, and 1 mM DTT), supplemented with 300 mM KCl, 1 mM PMSF, and 10 protease inhibitor mini tablets, was dialyzed against Buffer 1 overnight at 4 °C. Following filtration, the resulting solution (∼150 mL) was loaded onto a 10-mL SOURCE 15S-column (GE Life Sciences) pre-equilibrated with Buffer 1 supplemented with 20 mM KCl. Using a linear gradient of 20 mM to 150 mM KCl in Buffer 1 resulted in separation of eEF2 and eEF1A, which eluted at 20 mM KCl and 90 mM KCl, respectively. eEF1A was used without further purification. The pooled eEF2 fraction was loaded onto an 8-mL SOURCE 15Q-column (GE Life Sciences) from which eEF2 was eluted using a linear gradient of 40 mM to 500 mM KCl in 20 mM Tris⋅HCl, pH 7.6, 10% glycerol, 5 mM MgCl2, and 1 mM DTT.
4
Recombinant Expression and Purification of C5a
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Recombinant hC5a (mutant Cys704Arg), mC5a and mC5a-desArg were expressed recombinantly in bacteria23 (link). All proteins were expressed as fusions with an N-terminal thioredoxin tag (Trx-A) followed by a hexahistidine tag and a TEV protease cleavage site (Trx-His6-TEV-C5a), in Shuffle T7 Express E. coli cells (New England Biolabs). The proteins were purified using a two-step Ni-column affinity chromatography, including removal of the affinity-tag by overnight incubation with TEV protease, followed by a cation exchange chromatography on a Source 15S column (GE Healthcare Life Sciences). The final protein buffer was adjusted to 20 mM HEPES, pH 7.5, 150 mM NaCl before flash freezing in liquid nitrogen and storage at −80 °C until use. Mutants of hC5a and mC5a were generated using the Quick-Change Lightning Site Directed Mutagenesis Kit from Agilent Technologies and all mutants were expressed and purified using the same protocol as for native proteins. Human C3a was expressed recombinantly following the same protocol as for the C5a proteins50 (link).
5
Sortase-Mediated Ligation of WRC Complex
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To create WRC:Abi2-(MBP)2, the sortase ligation sequence, LPGTG, was genetically fused to the C-terminus of MBP-Abi2 (1-158). Meanwhile, a TEV site was added to the N-terminus of an (MBP)2 tag, which after Tev cleavage would expose a Gly required for sortase ligation. MBP-Abi2 (1-158)-LPGTG was expressed, purified, and incorporated into the WRC as described above to create WRC-LPGTG. GG-2MBP was expressed in Arctic Express (DE3) RIL (Agilent) cells after induction with 0.75 mM IPTG at 10°C for 16 hr, purified on amylose resin, and subjected to TEV cleavage overnight, followed by anion exchange chromatography using a Source 15Q column (GE Healthcare). Sortase5M (sortase A pentamutant) was a gift from David Liu (Addgene plasmid # 75144), expressed in Arctic Express (DE3) RIL (Agilent) cells, purified over Ni-NTA agarose resin, followed by cation exchange using a Source 15S column (GE Healthcare) and size exclusion chromatography using a Superdex 75 column (GE Healthcare) (Chen et al., 2011 (link)). WRC-LPGTG (1 µM) was mixed with GG-MBP (25 µM) and sortase (10 µM) in 50 mM Tris pH 7.5, 150 mM NaCl, and 10 mM CaCl2 and left at RT for 2 hr. The reaction was quenched by adding 25 mM EGTA, and the WRC-(MBP)2 was purified over a Superdex 200 column to separate the WRC-(MBP)2 from unligated molecules and sortase.
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