Optimization of Interleukin-5 Protein Expression in BL21 Strain of Escherichia Coli

Document Type : Original Article (s)

Authors

1 PhD in Genetics, Department of Biology, School of Basic Sciences, Science and Research Branch, Islamic Azad University, Tehran, Iran

2 Associate Professor, Department of Genetics and Molecular Biology, School of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran

3 Associate Professor, Department of Biology, School of Basic Sciences, Science and Research Branch, Islamic Azad University, Tehran, Iran

4 Associate Professor, Department of Genetics, School of Medicine, Ilam University of Medical Sciences, Ilam, Iran

Abstract

Background: In asthma, the relationship between number of eosinophils and severity of this disease supports the hypothesis that eosinophil is the major effector cell in inflammation of airway. Evolution of eosinophils is regulated by interlukin-5 (IL-5). Therefore, by blocking IL-5, at least one major reason of asthma would be prevented. To produce antagonists against IL-5 (like aptamer), it is necessary to have this protein in large scale and high purity. This study aimed to optimize IL-5 protein expression of BL21 strain of Escherichia coli (E. coli) to be used instead of antibody.Methods: At first, complementary DNA (cDNA) construct encoding IL-5 was designed, and was ordered to be produced in pET28a vector. Expression vector was transformed into competent E. coli Bl21 (DE3) origami. Then, protein expression was optimized by altering temperature, incubation time, and the amount of isopropyl β-d-1-thiogalactopyranoside (IPTG). Protein expression was assessed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and western blot in different levels of the test.Findings: The optimum conditions for protein expression were gained when the density of bacteria at the OD600 reached to 0.6 to 0.8, and culturing was done at 29 °C for 18 hours and 150 rpm, andinduction with 1mM IPTG. There was a 13-kDa protein band on SDS-PAGE and western blot that confirmed the expression of IL-5 protein.Conclusion: This protein can be used for producing aptamers against IL-5 and enzyme-linked immunosorbent assay (ELISA) kit for measuring IL-5. In all these process, there is no need to perfect folding of the protein. Therefore, the expression can be done in prokaryotic system, as it has high efficiency.

Keywords

References

  1. Haldar P, Brightling CE, Hargadon B, Gupta S, Monteiro W, Sousa A, et al. Mepolizumab and exacerbations of refractory eosinophilic asthma. N Engl J Med 2009; 360(10): 973-84.
  2. Greenfeder S, Umland SP, Cuss FM, Chapman RW, Egan RW. Th2 cytokines and asthma. The role of interleukin-5 in allergic eosinophilic disease. Respir Res 2001; 2(2): 71-9.
  3. Miranda C, Busacker A, Balzar S, Trudeau J, Wenzel SE. Distinguishing severe asthma phenotypes: role of age at onset and eosinophilic inflammation. J Allergy Clin Immunol 2004; 113(1): 101-8.
  4. Flood-Page PT, Menzies-Gow AN, Kay AB, Robinson DS. Eosinophil's role remains uncertain as anti-interleukin-5 only partially depletes numbers in asthmatic airway. Am J Respir Crit Care Med 2003; 167(2): 199-204.
  5. Rothenberg ME, Klion AD, Roufosse FE, Kahn JE, Weller PF, Simon HU, et al. Treatment of patients with the hypereosinophilic syndrome with mepolizumab. N Engl J Med 2008; 358(12): 1215-28.
  6. Menzella F, Lusuardi M, Galeone C, Taddei S, Facciolongo N, Zucchi L. Mepolizumab for severe refractory eosinophilic asthma: evidence to date and clinical potential. Ther Adv Chronic Dis 2016; 7(6): 260-77.
  7. Song KM, Lee S, Ban C. Aptamers and their biological applications. Sensors (Basel) 2012; 12(1): 612-31.
  8. Ruigrok VJ, Levisson M, Hekelaar J, Smidt H, Dijkstra BW, van der Oost J. Characterization of aptamer-protein complexes by X-ray crystallography and alternative approaches. Int J Mol Sci 2012; 13(8): 10537-52.
  9. Sambrook J, Russell DW. Gel retardation assays for dna-binding proteins. Cold Spring Harb Protoc 2006; 2006(1): prot3948.
  10. Sadeghian-Rizi T, Ebrahimi A, Moazzen F, Yousefian H, Jahanian-Najafabadi A. Improvement of solubility and yield of recombinant protein expression in E. coli using a two-step system. Res Pharm Sci 2019; 14(5): 400-7.
  11. Baeshen MN, Al-Hejin AM, Bora RS, Ahmed MM, Ramadan HA, Saini KS, et al. Production of biopharmaceuticals in E. coli: Current scenario and future perspectives. J Microbiol Biotechnol 2015; 25(7): 953-62.
  12. Dower WJ, Miller JF, Ragsdale CW. High efficiency transformation of E. coli by high voltage electroporation. Nucleic Acids Res 1988; 16(13): 6127-45.
  13. Sheykhi S, Amininasab M, Saffari B, Abdi S. DNA Cloning and expression of alpha-Synuclein Protein in E. coli. Modares Journal of Biotechnology 2018; 9(1): 145-52. [In Persian].
  14. Boshtam M, Khanahmad SH, Feizollahzadeh S, Rahimmanesh I, Asgary S. Expression and purification of biologically active recombinant rabbit monocyte chemoattractant protein1 in Escherichia coli. FEMS Microbiol Lett 2018; 365(9): fny070.
  15. Crowl R, Seamans C, Lomedico P, McAndrew S. Versatile expression vectors for high-level synthesis of cloned gene products in Escherichia coli. Gene 1985; 38(1-3): 31-8.
  16. Zarei Jaliani H, Farajnia S, Safdari Y, Mohammadi SA, Barzegar A, Talebi S. Optimized condition for enhanced soluble-expression of recombinant mutant anabaena variabilis phenylalanine ammonia lyase. Adv Pharm Bull 2014; 4(3): 261-6.
  17. Hu Y, An Y, Fang N, Li Y, Jin H, Nazarali A, et al. The Optimization of Soluble PTEN Expression in Escherichia coli. Open Biochem J 2015; 9: 42-8.
  18. Malik A, Alsenaidy AM, Elrobh M, Khan W, Alanazi MS, Bazzi MD. Optimization of expression and purification of HSPA6 protein from Camelus dromedarius in E. coli. Saudi J Biol Sci 2016; 23(3): 410-9.
  19. Lee SE, Kwon SH, Huh JW. Soluble expression and purification of human monocyte chemoattractant protein-1 in escherichia coli via fusion with a small ubiquitin-like modifier. Int J Recent Sci 2015; 6(7): 4999-5003.
  20. Hutchison CA, III, Chuang RY, Noskov VN, Assad-Garcia N, Deerinck TJ, Ellisman MH, et al. Design and synthesis of a minimal bacterial genome. Science 2016; 351(6280): aad6253.
Journal of Isfahan Medical School
Volume 38, Issue 592 - Serial Number 592
July and August 2020
Pages 694-700

Files

History

  • Receive Date: 30 April 2020
  • Revise Date: 25 April 2024
  • Accept Date: 06 April 2022

Share

How to cite

Statistics