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| Bacterial Expression | Bacteria from 50 mL induced culture were pelleted by centrifugation at 5000 rpm for 10 minutes at 4?C. The bacterial pellet was resuspended in 50 mL PBS, then lysed by three cycles of freeze-thawing in liquid nitrogen and cold water (4?C), sonicated for 20 cycles of 20 sec, and centrifuged at 12000 rpm for 15 minutes at 4?C. The pellet was resuspended in buffer B (100 mM NaH2PO4, 10 mM Tris-HCl, 8 M urea; pH 8.0) containing cocktail of protease inhibitor (Roche, Germany) and incubated for 1h at room temperature with shaking to solubilize the inclusion body proteins. The suspension was centrifuged at 12000 rpm for 15 minutes at 4?C. The clear supernatant was collected and loaded on l ml of Ni-NTA resin (Genscript, USA) pre equilibrated with buffer B. The resin-sample suspension was gently agitated at room temperature for 1 h to allow protein to bind to resin and then centrifuged at 1000 rpm for 5 minutes. The supernatant was removed and the resin was washed with 2 ml of buffer B and buffer C (100 mM NaH2PO4, 10 mM Tris-HCl, 8 M urea; pH 6.3), respectively. The fusion protein bound with Ni-NTA resin was eluted for five times by adding 1 ml of buffer E (100 mM NaH2PO4, 10 mM Tris-HCl, 8 M urea; pH 4.5). Fractions corresponding to the fusion protein, as identified by SDS-PAGE, were pooled and refolded by dialysis against decreasing concentrations of urea in 100 bed volumes of 25 mM Tris-HCl buffer containing150 mM NaCl, 14 mM β-mercaptoethanol, 10% glycerol, 0.2 M Arginine, at 4?C for 24 h. After dialysis, Protein concentrations were estimated by using the Bradford assay reagent. | Get A Quote |
Interleukin-24 (IL-24) is a novel tumor-suppressor gene that has different alternative splice isoforms. It has been shown that new smallest isoform of human IL-24 gene, lacking three exons, induces higher levels of cytotoxicity than all the isoforms, indicating shortest isoform of IL-24 may be a new promising anti-cancer agent. In this study, we aimed to provide a reproducible method for recombinant production of the smallest isoform of IL-24 (sIL-24). The Structure of sIL-24 was analyzed using bioinformatics tools (I-TASSER, Prosa, RAMPAGE and SPDBV version 4.1). The DNA sequence encoding sIL-24 was chemically synthesized and sub-cloned into the pET-32a (+) vector for further protein expression in Escherichia ... More
