Protein expression and purification were performed as described previously (35 (link)). To clone the L. enzymogenes rpfB1 and rpfB2 genes, genomic DNA extracted from L. enzymogenes was used for PCR amplification using Pfu DNA polymerase, and the primers are listed in Table 3 . The PCR products were inserted into pET-28b (+) to produce the plasmids pET-rpfB1 and pET-rpfB1. The L. enzymogenes rpfB1 and rpfB2 genes were verified by nucleotide sequencing by Genscript (Nanjing, Jiangsu, China). rpfB1 and rpfB2 with a vector-encoded His6-tagged N terminus were expressed in E. coli BL21(DE3) and purified with Ni-nitrilotriacetic acid (NTA) agarose (Qiagen, Chatsworth, CA, USA) using a nickel-ion affinity column (Qiagen). The protein purity was monitored by SDS-PAGE and matrix-assisted laser desorption ionization–time of flight (MALDI-TOF) mass spectrometry.
Nickel ion affinity column
Manufactured by Qiagen
Sourced in United States
The Nickel-ion affinity column is a laboratory equipment used for the purification and isolation of recombinant proteins containing a histidine tag. It consists of a matrix with immobilized nickel ions that selectively bind to the histidine tag on the target protein, allowing it to be separated from other components in the sample.
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Lab products found in correlation
Ni nta agarose, by Qiagen (4 mentions)
Coomassie brilliant blue r 250, by Merck Group (2 mentions)
Ni nitrilotriacetic acid nta agarose, by Qiagen (1 mentions)
E coli strain bl21 de3 competent cells, by Transgene (1 mentions)
Takara mutanbest kit, by Takara Bio (1 mentions)
Superdex 200 10 300 column, by GE Healthcare (1 mentions)
Bovine serum albumin (bsa), by Thermo Fisher Scientific (1 mentions)
9 protocols using nickel ion affinity column
1
Protein Expression and Purification of L. enzymogenes RpfB1 and RpfB2
2
Site-directed mutagenesis of ILVC enzyme
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Site-directed mutagenesis was performed with TaKaRa MutanBest kit (TaKaRa, Beijing, China). Briefly, the ilvC sequence was amplified by PCR from the cDNA template and PCR products were inserted into pOT2 plasmid. pOT2-Ilvc plasmid was amplified with designed mutant primers by PCR, and blunted using Blunting Kination Enzyme Mix, then ligated using ligation solution I in the kit. Plasmids were transformed into Escherichia coli Migula (Enterobacterales: Enterobacteriaceas) DH5α (TransGen, Beijing, China) and verified by DNA sequencing. Five amino acid residues contacting both NADP(H) and Mg2+ are conserved among bacteria, fungi, and plants; thus, their 5 active-site residue mutageneses were performed as mentioned above, respectively [15 (link)].
The recombinant ILVC protein was expressed in E. coli [16 (link)]. Briefly, ilvC with a 6× His-tag sequence at the C-terminus was amplified by PCR from the cDNA template or pOT2-Ilvc, and PCR products were inserted into pET-28b (+) vector (Novagen, Beijing, China), then transformed into E. coli strain BL21 (DE3)-competent cells (TransGen, Beijing, China). ILVC protein expression was induced by the addition of isopropyl β-D-thiogalactoside (IPTG) to a final concentration of 0.5 mM and purified with Ni-NTA agarose (Qiagen, Chatsworth, CA, USA) using a nickel-ion affinity column (Qiagen). Protein purity was monitored by SDS-PAGE.
The recombinant ILVC protein was expressed in E. coli [16 (link)]. Briefly, ilvC with a 6× His-tag sequence at the C-terminus was amplified by PCR from the cDNA template or pOT2-Ilvc, and PCR products were inserted into pET-28b (+) vector (Novagen, Beijing, China), then transformed into E. coli strain BL21 (DE3)-competent cells (TransGen, Beijing, China). ILVC protein expression was induced by the addition of isopropyl β-D-thiogalactoside (IPTG) to a final concentration of 0.5 mM and purified with Ni-NTA agarose (Qiagen, Chatsworth, CA, USA) using a nickel-ion affinity column (Qiagen). Protein purity was monitored by SDS-PAGE.
3
Expression and Purification of FadR Proteins
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In addition to the FadR protein with origins of both E. coli and V. cholerae, the S. oneidensis FadR protein was produced in solubility via the induced expression with 0.2 mmol/L isopropyl β-D-1-thiogalactopyranoside (IPTG) at 30°C for 3.5 h. The bacterial lysis by two rounds of sonication treatment was clarified by centrifugation, and the resultant supernatant was loaded onto a nickel-ion affinity column (Qiagen). The contaminant proteins were removed with wash buffer containing 50 mmol/L imidazole, and subsequently the 6× His-tagged FadR proteins in three versions (FadR_she, FadR_ec and FadR_vc) were eluted in elution buffer containing 100 mmol/L imidazole. The protein was concentrated by ultra-filtration (30 kDa cutoff) and exchanged into 1× PBS buffer (pH 7.4) containing 10% glycerol. The purified proteins were visualized by 15% SDS-PAGE followed by staining with Coomassie Brilliant Blue R250 (Sigma, St. Louis, MO).
4
Cloning and Purification of Xcc ilvC
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To clone the Xcc ilvC gene, genomic DNA extracted from strain Xcc Xc1 was used for PCR amplification with Pfu DNA polymerase, using primers in Supplementary Table S2 . PCR products were inserted into pET-28b (+) to produce plasmids pKH2. The ilvC gene was confirmed by nucleotide sequencing by Shanghai Sangon Inc. (Shanghai, China). Xcc ilvC with a vector-encoded His6-tagged N-terminus was expressed in E. coli BL21 (DE3), and purified with Ni-NTA agarose (Qiagen, Chatsworth, CA, United States) using a nickel-ion affinity column (Qiagen). Protein purity was monitored by SDS-PAGE.
5
Overexpression and Purification of BioR1/BioR2
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Both BioR1 and BioR2 of P. denitriifcans were overexpressed using prokaryotic expression system with induction of 0.3 mmol/L isopropyl β-d -1-thiogalactopyranoside (IPTG) at 30°C for 3 h. The clarified supernatant of bacterial lysates was loaded onto a nickel-ion affinity column (Qiagen, Hilden, Germany). After removal of the contaminant proteins with wash buffer containing 50 mmol/L imidazole, the 6x His-tagged protein of interest was eluted in elution buffer containing 150 mmol/L imidazole. The purified proteins were exchanged into 1X PBS buffer (pH 7.4) containing 10% glycerol, and visualized by 15% SDS-PAGE followed by staining with Coomassie Brilliant Blue R250 (Sigma, St. Louis, MO). Of note, the BioR1 is somewhat a weird protein, in that it easily precipitates during the process of purification, which is almost similar to scenarios seen with FabR proteins (Feng and Cronan 2011 (link)).
6
Cloning and Purification of Xcc FabH
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The Xcc fabH gene was cloned into pET-28b to yield plasmid pYYH-2. Xcc FabH with a vector-encoded His6-tagged N-terminus was expressed in E. coli BL21 (DE3), and purified with Ni-NTA agarose (Qiagen) using a nickel-ion affinity column (Qiagen). The purities of proteins were monitored by SDS-PAGE. The E. coli FabD, FabH, FabG, FabZ, and FabI, and E. coli holo-ACP proteins were purified as described previously43 (link).
7
Purification of Vibrio FadR Proteins
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Since all the other three FadR proteins of Vibrio origins were tagged with hexa-histidine on the N-terminus, similar to the paradigm E. coli FadR protein stocked in our lab. The recombinant proteins were routinely prepared as we described before (Zhang et al., 2014 (link)). Briefly, when the optical density at wavelength of 600 nm (OD600) reached 0.6, the bacterial culture was induced with 0.2 mM isopropyl-β-D-1-thiogalactopyranoside (IPTG) at 30°C overnight. Following lysis by sonication, the cell lysate was clarified by centrifugation and loaded onto a nickel-ion affinity column (Qiagen). After removal of contaminant proteins with washing buffer containing 50 mM imidazole, the 6xHis-tagged FadR protein was eluted in elution buffer containing 150 mM imidazole, concentrated by ultra-filtration (30-kDa cutoff) and exchanged into 20 mM Tris-HCl (pH 7.5) containing 100 mM NaCl. The soluble fractions were further purified by gel filtration with a Superdex 200 10/300 column (GL, GE Healthcare) and the resultant FadR proteins were judged with 12% SDS-PAGE. Protein concentrations were measured using the BCA method with BSA as internal reference (Thermo Scientific).
8
Cloning and Purification of PhoP Transcription Factor
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Protein expression and purification were performed according to Li et al. (2017) (link). To clone XAC phoP gene, the genomic DNA extracted from strain X. citri was used for PCR amplification with Pfu DNA polymerase and primers (Supplementary Table 2 ). PCR products were inserted into pET-28b (+) to produce plasmids pET-phoP. The phoP gene was confirmed through nucleotide sequencing by Genscript (Nanjing, Jiangsu, China). phoP with a vector-encoded His6-tagged N-terminus was expressed in E. coli BL21 (DE3), and purified with Ni-NTA agarose (Qiagen, Chatsworth, CA, United States) using a nickel-ion affinity column (Qiagen). Protein purity was monitored by SDS-PAGE.
9
Recombinant protein purification by affinity chromatography
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The target protein-coding regions were amplified with primers and inserted into the expression vector pQE-2 with an N-terminal hexahistidine tag to generate eight plasmids from pBHK466 to pBHK473. The constructed recombinant plasmids were transformed into the host strain E. coli Rosetta pLysS and grown at 37°C in LB medium in the presence of kanamycin (50 μg/ml) and chloramphenicol (30 μg/ml). The bacterial cultures were induced with 0.2 mM isopropyl-β-d -thio-d -galactoside at an OD600 of 0.8 and were grown at 37°C for an additional 3 hours before harvest. The cells were collected, resuspended in lysis buffer [50 mM sodium phosphate, 300 mM NaCl, 10 mM imidazole, and 1 mM dithiothreitol (pH 8.0)], lysed by sonication, and centrifuged. The clarified bacterial supernatant was loaded onto a nickel-ion affinity column (QIAGEN). The column was washed with wash buffer [50 mM sodium phosphate, 300 mM NaCl, 40 mM imidazole, and 1 mM dithiothreitol (pH 8.0)] and eluted in the same buffer (elution buffer) containing 200 mM imidazole. The protein was concentrated by ultrafiltration (10-kDa cutoff) and exchanged into sodium phosphate buffer [50 mM sodium phosphate, 200 mM NaCl, and 1 mM dithiothreitol (pH 8.0)]. The purity of the samples was monitored by SDS-PAGE. Strains, plasmids, and primers used for protein overexpression are listed in tables S3 and S4.
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