Users Online: 933
Home Print this page Email this page
Home About us Editorial board Search Browse articles Submit article Ahead of Print Instructions Subscribe Contacts Login 
ORIGINAL ARTICLE
Year : 2019  |  Volume : 8  |  Issue : 1  |  Page : 71

Cloning, optimization of periplasmic expression and purification of recombinant granulocyte macrophage-stimulating factor in Escherichia coli BL21 (DE3)


Department of Pharmaceutical Biotechnology, Isfahan Pharmaceutical Research Center, Faculty of Pharmacy, Isfahan University of Medical Sciences, Isfahan, Iran

Correspondence Address:
Dr. Vajihe Akbari
Department of Pharmaceutical Biotechnology, Isfahan University of Medical Sciences, Isfahan
Iran
Login to access the Email id

Source of Support: None, Conflict of Interest: None


DOI: 10.4103/abr.abr_166_19

Rights and Permissions

Background: Molgramostim, a nonglycosylated version of recombinant granulocyte-macrophage colony-stimulating factor (GM-CSF), can be produced in a high level by Escherichia coli. However, overexpression of GM-CSF in bacterial cells usually leads to formation of inclusion bodies and insoluble protein aggregates which are not biologically active. The aim of the present study was to improve the expression of soluble and biologically active GM-CSF in periplasmic space of E. coli BL21 (DE3). Materials and Methods: The codon-optimized GM-CSF gene was subcloned into pET-22b expression vector, in frame with the pelB secretion signal peptide for periplasmic secretion. Cultivation conditions including as isopropyl β-D-1-thiogalactopyranoside (IPTG) concentration, incubation temperature, and presence of sucrose were optimized to improve periplasmic expression of GM-CSF. The expressed protein was purified using Ni-NTA affinity column. Biological activity of GM-CSF on HL-60 cells was evaluated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Results: The amount of soluble protein for periplasmic expression was more when compared with one of the cytoplasmic expressions. The optimum condition for periplasmic expression of GM-CSF was expression at 23°C, using 1 mM IPTG as inducer and in the presence of 0.4 M sucrose. The biological activity of purified GM-CSF on HL-60 cell line was assessed by MTT assay, and the specific activity of produced GM-CSF was determined as 1.2 × 104 IU/μg. Conclusion: The present work suggests that periplasmic expression and optimization of cultivation conditions could improve soluble expression of recombinant proteins by E. coli.


[FULL TEXT] [PDF]*
Print this article     Email this article
 Next article
 Previous article
 Table of Contents

 Similar in PUBMED
   Search Pubmed for
   Search in Google Scholar for
 Related articles
 Citation Manager
 Access Statistics
 Reader Comments
 Email Alert *
 Add to My List *
 * Requires registration (Free)
 

 Article Access Statistics
    Viewed204    
    Printed14    
    Emailed0    
    PDF Downloaded53    
    Comments [Add]    

Recommend this journal