Porcine brain tubulin was purified as described previously (Castoldi and Popov, 2003 (link)). The pET30a(+):IPGA1 (full length coding sequence) and pET30a(+):IPGA1 (1–220 aa) constructs were generated and transformed into the Escherichia coli strain BL21 (DE3). The recombinant protein, His–IPGA1, was purified with a nickel-nitrilotriacetic acid column (Qiagen) equilibrated with the elution buffer (50 mM NaH2PO4, 300 mM NaCl, and 250 mM imidazol, pH 8.0). Recombinant protein samples were analysed by SDS-PAGE and western blotting with an anti-His antibody. The microtubule polymerization and co-sedimentation assay was performed as described previously (Mao et al., 2005 (link)). For the microtubule co-sedimentation assay of IPGA1 proteins, 2.5 μM His–IPGA1 proteins were added to taxol-stabilized microtubules (5 μM porcine brain tubulin) in PEMT buffer (100 mM PIPES, 1 mM EGTA, 1 mM MgCl2, and 20 µM taxol, pH 6.9). After incubation at 25 °C for 30 min, samples were centrifuged at 100 000 g at 25 °C for 20 min. Pellets were analysed by 12% SDS-PAGE, and then visualized by staining the protein gels with Coomassie Brilliant Blue R-250. His-MAP65-1 and BSA were used as positive and negative controls, respectively.
Nickel nitrilotriacetic acid column
Manufactured by Qiagen
Sourced in Germany
The Nickel-nitrilotriacetic acid column is a type of chromatography column used for the purification of proteins containing a histidine tag. It functions by leveraging the strong interaction between nickel ions and histidine residues, allowing for the selective capture and elution of the target protein.
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Lab products found in correlation
Amylose column, by New England Biolabs (2 mentions)
Escherichia coli strain bl21, by Merck Group (2 mentions)
Qiaexpressionist, by Qiagen (2 mentions)
Imidazole, by Merck Group (1 mentions)
Bradford protein quantification kit, by Tiangen Biotech (1 mentions)
Hek293ft, by Thermo Fisher Scientific (1 mentions)
Escherichia coli bl21 de3 cells, by Thermo Fisher Scientific (1 mentions)
7900ht fast real time pcr system, by Thermo Fisher Scientific (1 mentions)
Protein thermal shift dye kit, by Thermo Fisher Scientific (1 mentions)
Polyethyleneimine, by Merck Group (1 mentions)
Endotoxin removal system, by GenScript (1 mentions)
Octyl sepharose 4 fast flow column, by GE Healthcare (1 mentions)
Escherichia coli bl21 de3, by Thermo Fisher Scientific (1 mentions)
Superdex 200, by GE Healthcare (1 mentions)
Flexstation 3, by Molecular Devices (1 mentions)
Bead beater, by Biospec (1 mentions)
Bamhi, by New England Biolabs (1 mentions)
Ecori, by New England Biolabs (1 mentions)
Protein assay kit, by Bio-Rad (1 mentions)
14 protocols using nickel nitrilotriacetic acid column
1
Purification and Characterization of IPGA1
2
Expression and Purification of LOX-1 C-Terminal Domain
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The C-terminal domain of LOX-1 was expressed by using the protocols reported earlier12 (link). As this domain contains several disulfide bridges, it was co-expressed with DsbC, a Chaperone disulfide isomerase. The genes were custom synthesized by Genscript (Piscataway, NJ) using gene map described in Park el al.12 (link). It encodes for 147 amino acids for the LOX-1 fragment (residues 136–273) and 236 amino acids for DsbC. The gene was incorporated into a pET15b vectors by Genscript. The vectors were then transformed into Dh5α cells for amplification and BL21 (DE3) E. coli (Novagen) cells for expression. The proteins were expressed by adding 0.4 mM isopropyl β-D-thiogalactoside in E. coli cells grown in LB medium containing kanamycin, tetracycline, and carbenicillin at 23 °C, with 250 rpm agitation, for 20 h. The protein was purified using a nickel-nitrilotriacetic acid column (Qiagen, Valencia, CA). And then, the protein samples were concentrated to 10 mg/ml.
3
Recombinant 1,2-α-L-Fucosidase Expression
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The plasmid encoding the 1,2-α-L-fucosidase active domain was a gift from Takane Katayama (University of Kyoto, Japan). The enzyme was bacterially expressed in BL21 (DE3) delta-lacZ Escherichia coli following induction with 0.2 mM isopropyl β-D − 1-thiogalactopyranoside diluted in LB broth for 2 days at room temperature (Katayama et al., 2004 (link)). Soluble protein was extracted following sonication in ice water. Cell disruption was finalized by passing the cell lysate through a 5/8 in. gauge needle. Disrupted cells were then centrifuged at 10,000 g for 30 min at 4°C. Cleared supernatant was collected and expressed protein was purified on a nickel nitrilotriacetic acid column following manufacturer’s recommendation (Qiagen, France). Recombinant protein was eluted with 500 mM imidazole (Sigma, France) and was dialyzed overnight against 100 mM Na2HPO4 pH 6.5 containing 10% glycerol. Purified recombinant protein was aliquoted at 4 mg/ml, flash-frozen, and stored at −40°C until further use.
4
Cloning and Purification of VraR Protein
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A recombinant expression plasmid (pET28a-vraR) was constructed by inserting the vraR fragment amplified from SE1457 with the primers pET28a-vraR-F/pET28a-vraR-R (listed in Table 4 ) into the vector pET28a (+). The plasmid pET28a-vraR was transformed into E. coli BL21(DE3). When the transformant was grown to an OD600 value of 0.6 at 37°C, 0.8 mM isopropyl β-d -1-thiogalactopyranoside (IPTG) was added for overnight incubation at 22°C. The cells resuspended in lysis buffer (50 mM NaH2PO4, pH 8.0, 300 mM NaCl, 0.1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride [PMSF]) were sonicated and centrifuged at 15,000 × g for 30 min, and the supernatants were loaded onto a nickel-nitrilotriacetic acid column (Qiagen GmbH, Hilden, Germany). His-tagged VraR (6×His-VraR) was eluted using a linear gradient of 30 to 300 mM imidazole, and the protein concentration was determined using a Bradford protein quantification kit (Tiangen, Beijing, China).
5
CAPS Protein Purification by Affinity Chromatography
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For purification of CAPS-Myc-His and CAPS-mKate2-His, CAPS was transfected into HEK293-FT (Invitrogen) cells using a standard calcium phosphate protocol. Two days after transfection, cells were washed and removed with PBS. Cells were resuspended in 0.5% Triton X-100, 20 mm HEPES, 300 mm NaCl, 5 mm imidazole (IDA) with protease inhibitors and pelleted to remove cell debris. Supernatants were loaded onto a nickel-nitrilotriacetic acid column (Qiagen), washed, and eluted with 20 mm HEPES, 300 mm NaCl, 200 mm IDA. Eluates were diluted with 20 mm HEPES to 20 mm NaCl, 100 mm NaCl and concentrated to ∼10 μm CAPS.
6
Recombinant Porcine and Human IL-8 Expression
7
Purification and Thermal Stability of YARS2
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The wild-type and mutant human YARS2 cDNAs were amplified by PCR and cloned in-frame with a C-terminal His tag into the pET-28a vector. Recombinant wild-type and mutant YARS2 were produced as His tag fusion proteins in Escherichia coli Rosetta (DE3), as detailed elsewhere.31 (link) The proteins were purified using a nickel–nitrilotriacetic acid column (Qiagen). The stability of proteins was assessed using a Protein Thermal Shift dye kit (Life Technologies, Carlsbad, CA, USA), and unfolding temperature (melting temperature, Tm) tests were performed on a 7900HT Fast Real-Time PCR System (Thermo Fisher Scientific, Waltham, MA, USA), according to the modified manufacturer's instructions. The data were analyzed as detailed elsewhere.32 The Tm value was estimated from the transition midpoint of the fluorescence curve, which corresponds to the temperature at which half of the protein population unfolded.
8
Production and Assembly of PAT Protein
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The production of PAT was conducted in-house according to the previous protocol 12 . In brief, pET28a-His-pA-Tn5 was transformed into BL21 (DE3)-competent cells. The colonies obtained were then cultured in 500 ml LB medium. To induce PAT expression, 0.2 mM IPTG was added when the optical density of the bacterial culture reached 0.5-0.8. Incubation of the culture was carried out at 23 °C and 80 rpm for 5 h. The bacterial pellets were collected and lysed using HGX buffer, followed by sonication. Genomic DNA in the lysate was precipitated with polyethyleneimine (Sigma). The clear lysate was then applied to a nickel-nitrilotriacetic acid column (QIAGEN) and PAT was eluted from the column using 100 mM imidazole. The concentration of PAT was quantified by Coomassie blue staining after resolution in a 7.5% PAGE gel. Next, PAT was mixed with the annealed barcoded adaptor (Supplementary Table 1) at an equal molar ratio of 37.5 µM, and the mixture was incubated at 25 °C for 1 h. The assembled PAT was either stored at -20 °C or used directly for further experiments.
9
Cloning and Purification of CsGSTo Proteins
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The nucleotide sequences of CsGSTo1 (ANK78262) and 2 (ANK78263) were amplified from an adult C. sinensis cDNA library. The sequences were verified by sequencing, cloned into pET-28a(+) vector (Novagen, Madison, WI, USA) and transformed into Escherichia coli BL21 (DE3) (Thermo Fisher Scientific, Waltham, MA, USA). Bacterial cells were cultured with Luria–Bertani medium supplemented with kanamycin (50 µg/mL). Recombinant proteins, induced with 0.1 mM isopropyl-β-D-1-thiogalactopyranoside, were purified by a nickel–nitrilotriacetic acid column (Qiagen, Hilden, Germany), followed by thrombin cleavage. The proteins were delipidated using an Octyl-Sepharose 4 Fast Flow column (GE Healthcare, Little Chalfont, UK) and bacterial endotoxin was removed using the Endotoxin Removal System (GenScript, Piscataway, NJ, USA). The homogeneity of the recombinant proteins was monitored by 15% reducing SDS-PAGE. Enzyme activity was assayed using dehydroascorbate substrate [13 (link)].
10
Purification of MBP-L375-HIS10 and VACV MBP-D10
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Wild-type or mutated pMAL-c2x-malE-L375-his10 plasmids were transformed into Escherichia coli strain BL21 (EMD Millipore) for subsequent growth in LB broth supplemented with 50 μg/ml carbenicillin and 0.2% (w/v) glucose. MBP-L375-HIS10 expression was induced with 0.15 mM isopropyl β-D-1 thiogalactopyranoside (IPTG) followed by growth at 28°C. After 4 h of induction, cell lysates were produced using sonication. The recombinant protein was sequentially purified using an amylose column (New England Biolabs) followed by a nickel-nitrilotriacetic acid column (Qiagen) and then dialyzed against buffer comprised of 10 mM Tris-HCl pH 7.5, 100 mM NaCl, 10% glycerol, 1 mM DTT, and 2 mM Mg acetate [27 (link)]. Recombinant VACV MBP-D10 was produced and purified by affinity column chromatography as detailed in Parrish et al. [13 (link)].
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