Optimization of Cloning Conditions for High-Level Production of Recombinant Mouse Interleukin-۲ in Escherichia coli

Backgrounds Many proteins have been expressed so far in bacterial host. Due to its simple culture conditions, having a short life cycle, and easily genetic manipulation, E.coli have been regarded as a preferable host to produce recombinant proteins, but protein cloning in bacterial host have many challenges. Therefore, we aimed to review some of these problems by an experience from mice IL-۲ recombinant. Materials and methods: cDNA synthesis was performed after RNA extraction of mouse splenocytes. PCR product purification carried out after IL-۲ coding sequence amplification and was ligated into the pET-۲۱b (+) vector and transformed into the competent BL۲۱ E.coli. Expression and purification of recombinant mouse IL-۲ were done using IPTG inducer and metal affinity chromatography respectively. Results: DNA sequencing confirmed the accuracy of the insertion process. A ۲۳ kDa exogenous protein was observed on the SDS-PAGE. Specificity and concentration of produced mouse recombinant IL-۲ protein were confirmed by western blotting and BCA methods. Conclusion: Recombinant IL-۲ was produced in BL۲۱ and pET-۲۱b (+) expression system at ۲۴°C in the soluble form.

کلیدواژه ها:

Recombinant protein ، pET-۲۱b (+) ، IL-۲ protein ، Cloning