The full sequence of TrGDH was amplified by PCR with specific primers (Supplemental Table 1 ), and the PCR products were digested with BamHI and HindIII and then inserted into a pCold-TF vector with the same sites to yield the recombinant prokaryotic expression vector pCold-TF-TrGDH with a trigger factor (TF) and a His-tag sequence at the 5′-end (Takara, Japan). In addition, the rice homogenous NADP(H)-GDH gene OsGDH4 was also cloned and inserted into the same prokaryotic expression vector, which served as a control. The pCold-TF-TrGDH vector was then transformed into E. coli BL21 (DE3), and positive clones were isolated for His6-TF-TrGDH expression. The recombinant protein His6-TF-TrGDH was affinity purified with nickel-nitrilotriacetic acid (Ni-NTA) resin (Invitrogen, USA) according to previously described methods (Zhou et al. 2015a ). To verify the His6-TF-TrGDH, a western blot was used to examine the purified fusion proteins using anti-His monoclonal antibodies (1:5,000; Abmart, China) as described previously (Zhou et al. 2015b (link)).
Nickel nitrilotriacetic acid ni nta resin
Manufactured by Thermo Fisher Scientific
Sourced in United States
Nickel-nitrilotriacetic acid (Ni-NTA) resin is a versatile affinity chromatography matrix used for the purification of recombinant proteins containing polyhistidine (His) tags. The resin consists of nickel ions immobilized on a cross-linked agarose support, which can selectively bind to and retain His-tagged proteins. This allows for efficient capture, washing, and elution of the target protein from the resin.
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2 protocols using nickel nitrilotriacetic acid ni nta resin
1
Cloning and Expression of TrGDH
2
Recombinant Asc l 5 Protein Expression
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The cDNA sequence coding for Asc l 5 protein was subcloned without signal peptide into the expression vector pET-45b+ (GenScript, USA) and E. coli Origami (DE3) competent cells were transformed by electroporation. The E. coli cultures were grown overnight in Luria–Bertani medium containing 100 mg/L ampicillin at 37 °C. Expression of the recombinant protein was induced by adding IPTG to a final concentration of 1 mM at an OD600 of 0.5. After cultivation for additional 5 h at 37 °C, E. coli cultures were centrifuged (30 min, 3500 rpm, 4 °C). Induced cultures were re-suspended in native buffer (50 mM NaH2PO4, 300 mM NaCl), incubated with lysozyme (1 mg/mL, 30 min on ice) and sonicated. Lysates were incubated with Nickel-Nitrilotriacetic Acid (Ni-NTA) resin (Invitrogen) for one hour, washed with native buffer plus 20 mM imidazole, and eluted with native buffer plus 250 mM imidazole as a 6xHis-tagged protein [8 (link)]. The purified recombinant was directly subjected to SDS-PAGE analysis.
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