BL21 Rosetta pLacI-Rare cells were transformed with pIJ196-CDPS23 and grown in 2xTY medium containing 50 µg/mL ampicillin and 34 µg/mL chloramphenicol at 37°C until OD600nm = 1. Protein expression was then induced by addition of isopropyl-β-D-thiogalactopyranoside at a final concentration of 0.5 mM at 20°C during 18 h. Cells were resuspended in lysis buffer (10 mM HEPES pH = 7.5, 500 mM NaCl, 3 mM β-mercaptoethanol) and disintegrated by sonication. Cglo-CDPS was purified via Talon Metal affinity chromatography (Clontech) using standard protocols followed by anion exchange chromatography (Q sepharose Fast Flow, GE Healthcare). Enzyme concentration was calculated using a computed extinction coefficient of 33140 M−1cm−1. About 1 mg of Cglo-CDPS was obtained from 1 L of culture.
Talon metal affinity chromatography
Manufactured by Takara Bio
Sourced in France, United States, Spain
TALON metal affinity chromatography is a purification system that uses a cobalt-based resin to selectively bind and purify proteins containing a histidine-tag. The system provides a simple and efficient way to isolate recombinant proteins from complex mixtures.
Automatically generated - may contain errors
Lab products found in correlation
E coli bl21 de3, by Thermo Fisher Scientific (2 mentions)
Superdex 75, by GE Healthcare (2 mentions)
Glutathione sepharose 4b, by Bioworld Technology (2 mentions)
Pe sumo vector, by LifeSensors (2 mentions)
Superdex 200 10 300 gl column, by GE Healthcare (2 mentions)
Q sepharose fast flow, by GE Healthcare (1 mentions)
Pgex 6p 1, by GE Healthcare (1 mentions)
Hiload 16 600 superdex 75 pg column, by GE Healthcare (1 mentions)
Hitrap q hp anion exchange column, by GE Healthcare (1 mentions)
Superdex 200 increase 10 300 gl column, by GE Healthcare (1 mentions)
Superdex 75 10 300 gl column, by GE Healthcare (1 mentions)
Hitrap, by Cytiva (1 mentions)
Chemidoc xrs system, by Bio-Rad (1 mentions)
Quikchange site directed mutagenesis, by Agilent Technologies (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
X tremegene hp transfection reagent, by Roche (1 mentions)
12 protocols using talon metal affinity chromatography
1
Purification of Cglo-CDPS Protein
2
Purification of DksA and Associated Proteins
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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.
3
Purification and Expression of Truncated rGlEB1 Polypeptides
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Full-length rGlEB1 was expressed in Escherichia coli and purified as described previously [13] (link). In addition, the DNA fragment containing the GlEB1 ORF was dissected into two parts. The 5′-region of eb1 (552 bp) was amplified from the genomic DNA of Giardia WB by PCR using the primers, EB1-full-ERI-F and EB1-CHD-R (Table 2 ), and the 3′-region of eb1 (474 bp) was amplified with another set of primers, EB1-EBD-F and EB1-full-XhoI-R (Table 2 ). The resultant eb1 DNA fragments were cloned into pET21b (Novagen) to generate overexpression plasmids, pET21b-EB1-CHD and pET21b-EB1-EBD, for the truncated rGlEB1 polypeptides. These rEB1 polypeptides were expressed in E. coli BL21 (DE3) with 0.5 mM IPTG at 30°C for 3 h, and purified using a TALON metal affinity chromatography as described by the manufacturer (Clontech).
4
Purification of p19 Protein Constructs
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The PFO-based endosome-disrupting agent (C225.2/PFOT490A,L491V) was prepared as previously described (17 (link)). The p19, p19-E6, p19-E18 clones and SUMO-E18 were expressed from the pE-SUMO vector (LifeSensors) in Rosetta 2 (DE3) Escherichia coli (Novagen) and purified by Talon metal affinity chromatography (Clontech) following previously described methods (17 (link)). Following cleavage of the SUMO tag, the p19 constructs were purified by anion exchange chromatography (AEX) and size-exclusion chromatography (SEC). AEX was performed using a HiTrap Q HP anion exchange column (GE Healthcare Life Sciences) with an increasing salt gradient (10–500 mM NaCl) in 20 mM Bis–Tris, pH 6.5. SEC was performed using a HiLoad 16/600 Superdex 75 pg column (GE Healthcare Life Sciences) in PBS. Analytical SEC was performed using a Superdex 75 10/300 GL column (GE Healthcare Life Sciences) or Superdex 200 Increase 10/300 GL column (GE Healthcare Life Sciences) in PBS. Detailed methods for the expression and purification of p19 are provided in Supplementary Methods .
5
Recombinant Fibronectin Fragment Production
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We used the human cDNA encoding the FNIII7-10 fragment and subcloned in the expression vector pET-15b (Takahashi et al., 2007 (link)). To generate the FNIII7-10syn we mutated by site-directed mutagenesis the two arginines in the synergy sequence: DRVPHSRN>DAVPHSAN. We performed two rounds of PCR using the following primers: 5´-GATGCGGTGCCCCACTCTCGGAAT-3´ (forward) and 5´-GATGCGGTGCCCCACTCTGCGAAT-3´ (forward) and the complementary reverse primers. The expression of recombinant FN fragments was done in the E. coli strain Rosetta T1R. Purification was performed using TALON Metal Affinity chromatography (Clontech, Saint Germain en Laye, France). Finally the protein was obtained by gel filtration chromatography using Superdex 200 10/300 GL columns (GE Healthcare) and Superdex Size Exclusion Media (GE Healthcare, Valencia, Spain) and eluted in PBS.
6
FlgA-FlgI Complex Formation Assay
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Pull-down assays of FlgA-FlgI complex formation with TALON metal affinity chromatography were carried out following the manufacturer’s instructions (Clontech, Mountain View, CA). Cell extracts prepared from E. coli Origami2 (DE3), harboring the N-terminally His-tagged FlgI (His-FlgI, pHMK385) co-expressed without FlgA (pHMK14) or FlgA proteins (pHMK720, pHMK844 or pHMK869) were loaded onto a TALON spin column. Following extensive washing with a buffer containing 10 mM imidazole, bound proteins were eluted with 300 mM imidazole and separated by SDS-PAGE, transferred to a PVDF membrane, and probed with custom antibodies raised against FlgA and FlgI (MBL, Nagoya, Japan). Membrane images were acquired with a ChemiDoc XRS+ system (Bio-Rad, Hercules, CA).
7
Recombinant FNIII7-10 Protein Expression
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We subcloned the human cDNA encoding FNIII7-10 in the expression vector pET-15b (Takahashi et al., 2007 (link)). The substitution of D to E or deletion of the entire RGD motif was achieved by site-directed mutagenesis and allowed expression of FNIII7-10RGE or FNIII7-10ΔRGD fragments, respectively, in the Escherichia coli strain Rosetta T1R. The fragments were purified using TALON Metal Affinity chromatography (Clontech) and gel filtration chromatography using Superdex 200 10/300 GL columns (GE Healthcare) and eluted in PBS.
8
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.
9
Recombinant Protein Expression and Purification
10
Cloning and Purification of GlMBP1
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A 1,338 bp DNA fragment encoding the GlMBP1 open reading frame (ORF) was amplified by PCR from the genomic DNA of G. lamblia, using 2 primers, 8405F and 8405R (Table 1 ), and cloned into pET21b (Novagen, Darmatadt, Germany), resulting in pETGlMBP1. The rGlMBP1 was expressed in Escherichia coli BL21 (DE3), with 0.5 mM ITPG at 30˚C for 3 hr, and purified using TALON Metal affinity chromatography, as described by the manufacturer (Clontech, Mountain View, California, USA).
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