Journal of Isfahan Medical School

Journal of Isfahan Medical School

Cloning and Expression of Recombinant Human Interleukin 1 Receptor Antagonist in Escherichia Coli Strains, BL21 (DE3), Rosetta (DE3), and Origami (DE3)

Document Type : Original Article (s)

Authors
Hanieh Fardgoli 1
Seyed Nezameddin Hoseini 2
Setareh Haghighat 3
1 MSc Student, Department of Microbiology, School of Advanced Science and Technology, Tehran Medical Sciences Branch, Islamic Azad University, Tehran, Iran
2 Associate Professor, Department of Recombinant Hepatitis B Vaccine, Production and Research Complex, Pasteur Institute of Iran, Iran
3 Assistant Professor, Department of Microbiology, School of Advanced Science and Technology, Tehran Medical Sciences Branch, Islamic Azad University, Tehran, Iran
10.22122/jims.v37i539.12229
Abstract
Background: Recombinant human interleukin-1 receptor antagonist (IL-1RA) is a protein with 153 amino acids and molecular weight of 16.83 kDa. This drug protein is known as Anakinra, and has an effective application in the treatment of rheumatoid arthritis. This study was conducted to examine the produce of the recombinant IL-1RA protein in Escherichia coli (E. coli) strains.Methods: Codon optimization of IL-1RA gene was done using GenScript, and the gene was cloned in the pUC18 as cloning vector. Then, plasmid was cut by two restriction enzymes including NdeI and BamH1 enzymes. IL-1RA gene was purified from the agarose gel. IL-1RA gene was ligated into expression vector. The constructed expression cassette was transformed into E. coli BL21 (DE3) Origami (DE3) and Rosetta (DE3) using CaCl2 and heat shock method.Findings: Identification and confirmation of transformed colonies was performed using colony polymerase chain reaction (PCR). Induction of this gene was done with isopropyl β- d-1-thiogalactopyranoside (IPTG). The protein expression was analyzed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and western blotting techniques, and it purified by Ni nickel resin. Expression analysis of transformed E. coli strains confirmed that gene integrated into expression host. Molecular weight of expressed protein was estimated to be 16.83 kDa.Conclusion: In this study, Human IL-1RA was successfully produced in E. coli Origami with high quantity other than the rest of E. coli strains. Therefore, E. coli BL21 Origami (DE3) can be used as the suitable host for production of recombinant IL-1RA, and this technology has a potential for localization.
Keywords
Interleukin 1 receptor antagonist protein
Recombination
Escherichia coli
  1. Wood DD, Ihrie EJ, Dinarello CA, Cohen PL. Isolation of an interleukin-1-like factor from human joint effusions. Arthritis Rheum 1983; 26(8): 975-83.
  2. Schulz MF, Buell G, Schmid E, Movva R, Selzer G. Increased expression in Escherichia coli of a synthetic gene encoding human somatomedin C after gene duplication and fusion. J Bacteriol 1987; 169(12): 5385-92.
  3. Carter DB, Deibel MR, Jr., Dunn CJ, Tomich CS, Laborde AL, Slightom JL, et al. Purification, cloning, expression and biological characterization of an interleukin-1 receptor antagonist protein. Nature 1990; 344(6267): 633-8.
  4. Perrier S, Darakhshan F, Hajduch E. IL-1 receptor antagonist in metabolic diseases: Dr Jekyll or Mr Hyde? FEBS Lett 2006; 580(27): 6289-94.
  5. Arend WP. Cytokine imbalance in the pathogenesis of rheumatoid arthritis: the role of interleukin-1 receptor antagonist. Semin Arthritis Rheum 2001; 30(5 Suppl 2): 1-6.
  6. Chen Z, Chen H, Wang X, Ma X, Huang B. Expression, purification, and characterization of secreted recombinant human insulin-like growth factor-binding protein-6 in methylotrophic yeast Pichia pastoris. Protein Expr Purif 2007; 52(2): 239-48.
  7. Kim SO, Lee YI. High-level expression and simple purification of recombinant human insulin-like growth factor I. J Biotechnol 1996; 48(1-2): 97-105.
  8. Khuzaima I, Iftikhar K, Tabarruk U, Sadia H, Akhtar H, Shami K. Studying the pharmacokinetics of interleukin-1RA mutants. BioScientific Review 2019; 1(1): 17-23.
  9. Brinkmann U, Mattes RE, Buckel P. High-level expression of recombinant genes in Escherichia coli is dependent on the availability of the dnaY gene product. Gene 1989; 85(1): 109-14.
  10. Al-Hejin A, Bora R, Ahmed M. Plasmids for optimizing expression of recombinant proteins in E. coli. In: Gull M, editor. Plasmid. London, UK: IntechOpen; 2019.
  11. Studier FW. Improved cloning and expression vectors and systems (WO2017139412A1) [Google Patents]. 2017.
  12. Miller MG, Chng C, Alvizo O, Mayo MA, Riggins JN, Yi X, et al. T7 rna polymerase variants. Redwood City, CA; US20190002851. 2019.
  13. Ranjbari J. Engineered recombinant protein production systems originated from Escherichia coli. Trends in Peptide and Protein Sciences 2018; 3: e2.
  14. Buryskova M, Pospisek M, Grothey A, Simmet T, Burysek L. Intracellular interleukin-1alpha functionally interacts with histone acetyltransferase complexes. J Biol Chem 2004; 279(6): 4017-26.
  15. Wang YX, Yang ZX, Zhu HQ, Zhou XW, Huang PT. Construction, expression and preliminary pharmacokinetic analysis of IL-1ra mutants. Sheng Wu Gong Cheng Xue Bao 2006; 22(3): 472-6. [In Chinese].
  16. Birikh KR, Lebedenko EN, Boni IV, Berlin YA. A high-level prokaryotic expression system: Synthesis of human interleukin 1 alpha and its receptor antagonist. Gene 1995; 164(2): 341-5.
  17. Zanette D, Dundon W, Soffientini A, Sottani C, Marinelli F, Akeson A, et al. Human IL-1 receptor antagonist from Escherichia coli: Large-scale microbial growth and protein purification. J Biotechnol 1998; 64(2-3): 187-96.
Journal of Isfahan Medical School
Volume 37, Issue 539 - Serial Number 539
July and August 2019
Pages 965-972

  • Receive Date 03 September 2019
  • Accept Date 06 April 2022