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

XML Print


Download citation:
BibTeX | RIS | EndNote | Medlars | ProCite | Reference Manager | RefWorks
Send citation to:

Abdi A, Hosseinpour M, Mashayekhi K, Mousavi M J, Badiee Kheirabadi S E, Sankian M. Optimization of Cloning Conditions for High-Level Production of Recombinant Mouse Interleukin-2 in Escherichia coli. Res Mol Med (RMM). 2019; 7 (1) :16-25
URL: http://rmm.mazums.ac.ir/article-1-309-en.html
Abstract:   (600 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]   (68 Downloads)    
Type of Study: Research | Subject: Immunology
Received: 2019/05/24 | Accepted: 2019/07/7

Add your comments about this article : Your username or Email:
CAPTCHA

Send email to the article author


© 2019 All Rights Reserved | Research in Molecular Medicine

Designed & Developed by : Yektaweb