The recombinant Nef-Vpr-Gp160-P24 polypeptide was expressed in theE. coli Rosetta strain transformed with pET-24a(+)-nef-vpr-gp160-p24 and purified by an affinity chromatography technique using Ni-NTA agarose column (Macherey-Nagel) under denaturing conditions as previously reported by our group (Davoodi et al.2019a (link),b (link)). To produce Nef protein,Escherichia coli Rosetta strain was transformed with pET-23a (+) harboring thenef gene. Nef protein was expressed and purified under native conditions as previously reported by our group (Davoodi et al.2019a (link),b (link)). In this study, the Nef-Vpr-Gp160-P24 polypeptide and Nef protein were produced in a large scale for mice immunization.
Ni nta agarose column
Manufactured by Macherey-Nagel
The Ni-NTA agarose column is a chromatographic resin used for the purification of recombinant proteins containing a histidine-tag. The column utilizes the high-affinity interaction between nickel-nitrilotriacetic acid (Ni-NTA) and the histidine residues to selectively capture and purify the target protein from complex mixtures.
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Lab products found in correlation
Nanodrop spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Gsh agarose beads, by Merck Group (1 mentions)
Resourceq 6 ml column, by GE Healthcare (1 mentions)
Bradford protein assay kit, by Merck Group (1 mentions)
10 kda dialysis membrane, by Thermo Fisher Scientific (1 mentions)
Qcl 1000, by Lonza (1 mentions)
3 protocols using ni nta agarose column
1
Production of HIV-1 Fusion Proteins
2
Purification of Recombinant Human ASF1
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Recombinant human ASF1 (hASF1A(1-156)) was purified as already described from expression in E. coli of a (His)6-GST-Tev site-Asf1 fusion protein using the pETM30 plasmid.(37) Briefly, soluble (His)6-tagged GST fusion protein was purified on reduced glutathione (GSH) agarose beads (Sigma). After cleavage with recombinant (His)6-TEV protease at room temperature overnight, the (His)6-GST tag and the protease were trapped in a Ni-NTA agarose column (Macherey Nagel). The flow-through fraction containing ASF1 protein was further purified by anion exchange chromatography using a Resource Q 6mL column (GE Healthcare). ASF1 was then concentrated using an Amicon device (Millipore) and the buffer was replaced with a 50mM Tris-HCl (pH7.5). Unlabled ASF1 used for ITC experiments was purified from pellets of bacteria grown in LB medium, and uniformly labeled ASF1 from bacteria gown in M9 minimal media supplemented with ( 15 NH4)Cl (Eurisotop, 0.5 g/L) as the sole nitrogen source and or 13 C glucose (Eurisotop, 2 g/L).
3
Purification and Characterization of rNef-Vpr-Gp160-P24 Peptide
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The rNef-Vpr-Gp160-P24 peptide was purified by an affinity chromatography technique using Ni-NTA agarose column (Macherey-Nagel) under denaturing conditions (i.e., 8 M urea buffer and pH 4.5) and Tris-HCl lysis buffer. Then, the recombinant peptide was dialyzed in phosphate buffered saline (PBS) solution using a 10 kDa dialysis membrane (Thermo Scientific). Finally, its concentration and purity were measured using the Bradford protein assay kit (Sigma, Germany) and NanoDrop spectrophotometer (Thermo Fisher Scientific). According to LAL assay, contamination with LPS was \ 0.5 EU/mg (QCL-1000, Lonza).
Preparation of the MPG/DNA and HR9/DNA complexes and gel retardation assay To form the MPG/pEGFP-N1-nef-vpr-gp160-p24 and HR9/pEGFP-N1-nef-vpr-gp160-p24 complexes, the MPG and HR9 peptides were mixed with an equal amount (2 lg) of pEGFP-N1-nef-vpr-gp160-p24 at different nitrogen to phosphate ratios (N/P: 0.5, 1, 2, 5 and 10) and incubated at room temperature for 45 min. The formation of the complexes was investigated using gel retardation assay. For this purpose, 1% agarose gel was used to evaluate the electrophoretic mobility of the complexes.
Preparation of the MPG/DNA and HR9/DNA complexes and gel retardation assay To form the MPG/pEGFP-N1-nef-vpr-gp160-p24 and HR9/pEGFP-N1-nef-vpr-gp160-p24 complexes, the MPG and HR9 peptides were mixed with an equal amount (2 lg) of pEGFP-N1-nef-vpr-gp160-p24 at different nitrogen to phosphate ratios (N/P: 0.5, 1, 2, 5 and 10) and incubated at room temperature for 45 min. The formation of the complexes was investigated using gel retardation assay. For this purpose, 1% agarose gel was used to evaluate the electrophoretic mobility of the complexes.
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