rCI, rNTD and His-σA (primary sigma factor of S. aureus with a histidine tag) were purified from E. coli strains SAU1304, SAU1315, and SAU1283, respectively, using standard methods [7] (link), [37] (link). Briefly, IPTG-induced cells were ruptured in buffer B [20 mM Tris-HCl (pH 8.0), 300 mM NaCl, 10 mM imidazole, 5% glycerol and 10 µg/ml PMSF], followed by purification of the polyhistidine-tagged recombinant protein (rCI or rNTD or His-σA) from the crude extract by Ni-NTA column chromatography (Qiagen). The eluted proteins were dialyzed against buffer C [10 mM Tris-HCl (pH 8.0), 200 mM NaCl, 1 mM EDTA, and 5% glycerol] for 12–16 h at 4°C. All proteins in buffer C were stored on ice until use. The molar concentrations of the recombinant proteins were determined using the molecular masses of their respective monomeric forms.
Ni nta column chromatography
Manufactured by Qiagen
Ni-NTA column chromatography is a laboratory equipment used for protein purification. It utilizes a nickel-nitrilotriacetic acid (Ni-NTA) resin to selectively bind and purify proteins with a histidine tag. The Ni-NTA resin interacts with the histidine tag, allowing the target protein to be separated from other components in the sample.
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Lab products found in correlation
4 protocols using ni nta column chromatography
1
Purification of Recombinant Proteins from E. coli
2
Cloning, Expression, and Purification of Glycosyltransferase BLC
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Glycosyltransferase BLC was cloned, expressed, and purified from the genomic DNA of B. licheniformis DSM13, as reported previously (Wu et al. 2012 (link)). Briefly, the amplified PCR products were purified, digested with BamHI and XhoI, and cloned into the same sites of the pET28a vector. The recombinant expression vectors were transformed into E. coli BL21(DE3). The E. coli BL21(DE3) culture transformed with the recombinant expression vector was induced with isopropyl-1-thio-β-d -galactopyranoside (IPTG) (at a final concentration of 0.5 mM). After centrifugation of the cell lysate, BLC enzyme was purified by nickel–nitriloriacetic acid (Ni–NTA) column chromatography (Qiagen). The fractions eluted with 50 and 100 mM imidazole were pooled and concentrated with an Amicon Ultra-15 instrument (Millipore). The concentrated enzyme was dialyzed overnight at 4 °C using 100 mM Tris–HCl (pH 8.0) containing 20% (vol/vol) glycerol and stored at 4 °C until use.
3
Recombinant Protein Expression and Purification
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After designing and ordering the chimeric IpaD-StxB-TolC gene in pET28a (ShineGene, China), this structure was transferred to E. coli BL21 (DE3) by the heat shock method. Then, by adding 1 mM isopropyl-β-d-thiogalactoside (IPTG; Sigma, USA) to the bacterial culture medium, recombinant protein production was induced and incubated at 37°C for 5 hr. After that, the culture medium was centrifuged (5000×g at 25°C for 10 min) and the bacterial precipitate was mixed with the lysis buffer (100 mM NaH2PO4, 10 mM Tris-HCl, 8 M urea, pH=8.0). In the next step, after sonication (6 times for 10 s with high power), the lysed cell was centrifuged at 10000 g at 4°C for 15 min, the precipitate was discarded, and the supernatant was loaded in the Ni-NTA chromatography column (Qiagen). Protein elution was achieved using the elution buffer containing 100 mM NaH2PO4, 10 mM Tris-base, and 8 M urea (pH=4.2). SDS PAGE 10% and Western blot analysis using anti-His tag antibody were performed to confirm the recombinant protein expression.
4
Optimization of Recombinant Protein Expression
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All recombinant plasmids were transformed into Escherichia coli BL21(DE3) competent cells, selected by ampicillin. To obtain the maximum yield, the bacterial solution was added with isopropyl‐β‐d ‐thiogalactopyranoside (IPTG) at a final concentration of 0.1, 0.2, 0.5 and 1.0 mM, and shaken for 12 h at 20, 25, 30 and 37°C. The cells were harvested, resuspended in the lysis buffer and sonicated for 20 min. After centrifugation, the supernatant was retained and filtered (0.45 μm) to be applied to a Ni‐NTA chromatography column (Qiagen). Bound proteins were eluted in a gradient with different concentrations of imidazole buffer and the fractions were dialysed against PBS overnight at 4°C. Each purified protein was concentrated with a 10–30 kDa MWCO ultrafiltration tube (Millipore) according to the molecular weight.
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