The cloning, expression and purification of wild type R17 and its variants has been described in Lubman et al. (Lubman et al., 2014 (link)). For crystallization, we employed an alternate version of the published protocol developed to minimize the amount of N-linked carbohydrate (Chang et al., 2007 (link)). This involved expression of both R17 and R17GAG2 mutant in medium containing 1mM of glycosylation processing inhibitor kifunensine. The culture medium was collected 10 days after transfection and was purified using Ni-Agarose beads (Qiagen, Valencia, CA). The eluted protein was buffer exchanged into 50mM Hepes pH7.5, 600mM NaCl and incubated at room temperature overnight with Endoglycosidase Hf (3000U of EndoH for 1μg of protein) (New England Biolabs). The digested material passed over an amylose column to remove the EndoHf/maltose-binding protein fusion, followed by size exclusion chromatography (SEC) on a HiLoaD 26/60 Superdex 200pg column (G.E. Healthcare). For purification of the wild type R17, the NaCl concentration was maintained at 600mM throughout purification and crystallization. For the R17GAG2 variant, the NaCl concentration was maintained at 150mM for subsequent co-purification with CCL3 (see below).
Ni agarose beads
Manufactured by Qiagen
Ni-Agarose beads are a type of affinity chromatography media used for the purification of histidine-tagged recombinant proteins. These beads consist of nickel-chelated agarose particles that bind to the histidine tag, allowing the target protein to be isolated from complex mixtures.
Automatically generated - may contain errors
Lab products found in correlation
Hiload 26 60 superdex 200 pg column, by GE Healthcare (2 mentions)
Gst agarose beads, by Qiagen (2 mentions)
Endoglycosidase hf, by New England Biolabs (1 mentions)
Escherichia coli strain, by Thermo Fisher Scientific (1 mentions)
Hrp conjugated streptavidin, by Thermo Fisher Scientific (1 mentions)
Escherichia coli strain bl21, by Thermo Fisher Scientific (1 mentions)
6 protocols using ni agarose beads
1
Cloning, Expression, and Purification of R17 and Variants
2
Purification of Human PKM2 Protein
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Human cDNA for PKM2 was cloned into pET28a+ with a N-terminal His tag and purified from Escherichia coli strain BL21 (Invitrogen) using Ni-Agarose beads (Qiagen) as described previously25 .
3
Expression and Purification of Key Metabolic Enzymes
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Human cDNA for PKM2, PKM1, and VDAC3 were separately cloned into a pET28a (+) and pGEX-4T-1 vector with an N-terminal His-tag and N-terminal GST-tag and purified from the Escherichia coli strain BL21 (Invitrogen) using Ni–agarose beads and GST agarose beads (Qiagen) as described previously7 (link). PKL was purchased from Sino Biological Inc. in China.
4
Purification of His6-tagged Ubiquitin Conjugates
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His6-tagged ubiquitin (his6-Ub) or ubiquitin (Ub) was overproduced from plasmids with the inducible nmt1 promoter in WT or Δpqr1 cells in which the endogenous pho84+ gene was replaced with a pho84+-GFP fusion gene. These cells were cultured in EMM2 without thiamin for 20 h to induce expression of his6-Ub or Ub, and then cells were harvested. Harvested cells (8 × 108 cells) were suspended in 400 μl of denaturing lysis buffer containing 10 mM imidazole (pH 8.0), and proteins were extracted as described above. Protein extracts were incubated with 40 μl of Ni-agarose beads (QIAGEN) at 4 °C for 120 min to purify proteins covalently bonded to His6-Ub. Then Ni-agarose beads were washed 4× with denaturing lysis buffer containing 20 mM imidazole. After washing, Ni-agarose beads were suspended in 1× LDS sample buffer with 2-mercaptoethanol and were boiled at 70 °C for 10 min to solubilize bead-bound proteins.
5
Production and Characterization of Biotinylated Tetrameric SCT
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Biotinylated soluble pQa-1-SCT or pQa-1-dtSCT monomer was produced by transient transfection of 293 F cells with the corresponding plasmid DNA as previously described (Nelson et al., 2014 (link)). The secreted protein containing a C-terminal 6His tag was purified from culture medium using Ni-agarose beads (Qiagen) followed by size-exclusion chromatography on a HiLoaD 26/60 Superdex 200 pg column (GE Healthcare). The proteins were biotin labeled on an included BirA-peptide-tag with enzyme in vitro following the manufacture's protocol (Avidity). The labeled protein was tested for biotinylation by ELISA using HRP conjugated streptavidin (Invitrogen), and then tetramerized through addition of phycoerythrin (PE)-labeled streptavidin (SA) (BD Biosciences) at a molar ratio of 4 molecules of SCT monomers to 1 molecule of PE-SA.
6
OTUD3 Protein Purification Protocols
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Human cDNA for OTUD3 were respectively cloned into a pGEX-4T-1 and pET28a (+) vector with a N-terminal GST-tag and purified from the Escherichia coli strain BL21 (Invitrogen) using GST Agarose beads and Ni-Agarose beads (Qiagen) separately.
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