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

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

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

References

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Journal of Isfahan Medical School
Volume 37, Issue 539 - Serial Number 539
July and August 2019
Pages 965-972

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History

  • Receive Date: 03 September 2019
  • Revise Date: 01 November 2024
  • Accept Date: 06 April 2022

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