Imidazole

Recombinant His₆-tagged 4E-BP1 protein was produced by transforming Escherichia coli BL21 RP electrocompetent bacteria with the pETM-11-4E-BP1 vector described earlier according to standard procedures. Briefly, protein expression was induced with isopropyl-β-d-thiogalactopyranoside for 3 h at 30 °C, and His₆–4E-BP1 was purified using Ni-NTA agarose (cat. no. 1018244, Qiagen) and eluted with 250 mM imidazole (A1073, Applichem).

In this experiment, plasmid pET22 b (+) (5493 bp, Novagene, USA) was used as the expression vector. Also, E. coli strains DH5 α and BL21 (DE3) (Iranian Biological Resources Center) were used as hosts for cloning (amplification) and expression of recombinant peptide, respectively. E. coli BL21 (DE3) has the λ prophage carrying the T7 polymerase that for protein over-expression needed T7 promoters. The recombinant protein expression was induced by IPTG (Sigma, Country) under the control of a strong T7 promoter in E. coli BL21 (DE3). Ampicillin was used to select the transformed colonies on the Luria–Bertani (LB) agar (Miller). The Luria–Bertani (LB) medium (Miller) was used for bacterial culture. A pre-equilibrated Ni–NTA affinity column (Bio-Rad) and imidazole (AppliChem, Spain) were used to Purification of recombinant protein. 1 kb DNA Ladder and 245 kDa protein Ladder, and NcoI and XhoI Restriction enzymes were purchased from Roche (Germany) and Thermo Fisher Scientific (USA) companies, respectively.