Volume 7, Issue 1 (Feb 2019)                   Res Mol Med (RMM) 2019, 7(1): 16-25 | Back to browse issues page

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1- ImmunoBiochemistry lab, Immunology Research Center, Mashhad University of medical Sciences, Mashhad, Iran
2- Immunology Department, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran , sm-mousavi@razi.tums.ac.ir
Abstract:   (1959 Views)
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-2 recombinant.
Materials and methods: cDNA synthesis was performed after RNA extraction of mouse splenocytes. PCR product purification carried out after IL-2 coding sequence amplification and was ligated into the pET-21b (+) vector and transformed into the competent BL21 E.coli. Expression and purification of recombinant mouse IL-2 were done using IPTG inducer and metal affinity chromatography respectively.
Results: DNA sequencing confirmed the accuracy of the insertion process. A 23 kDa exogenous protein was observed on the SDS-PAGE. Specificity and concentration of produced mouse recombinant IL-2 protein were confirmed by western blotting and BCA methods.
Conclusion: Recombinant IL-2 was produced in BL21 and pET-21b (+) expression system at 24°C in the soluble form.
Full-Text [PDF 588 kb]   (653 Downloads)    
Type of Study: Research | Subject: Immunology
Received: 2019/05/24 | Accepted: 2019/07/7 | Published: 2019/02/15