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

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Fardgoli Hanieh; HOSEINI SEYED NEZAMEDDIN; HAGHIGHAT SETAREH

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Journal: JOURNAL OF ISFAHAN MEDICAL SCHOOL (I.U.M.S) Year:2019 | Volume:37 | Issue:539 Page(s): 965-972

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Title

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

Author(s)

Fardgoli Hanieh | HOSEINI SEYED NEZAMEDDIN | HAGHIGHAT SETAREH | Issue Writer Certificate

Keywords

Interleukin 1 receptor antagonist proteinQ1

RecombinationQ1

Escherichia coliQ1

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.

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APA: Copy

Fardgoli, Hanieh, HOSEINI, SEYED NEZAMEDDIN, & HAGHIGHAT, SETAREH. (2019). Cloning and Expression of Recombinant Human Interleukin 1 Receptor Antagonist in Escherichia Coli Strains, BL21 (DE3), Rosetta (DE3), and Origami (DE3). JOURNAL OF ISFAHAN MEDICAL SCHOOL (I.U.M.S), 37(539 ), 965-972. SID. https://sid.ir/paper/395979/en

Vancouver: Copy

Fardgoli Hanieh, HOSEINI SEYED NEZAMEDDIN, HAGHIGHAT SETAREH. Cloning and Expression of Recombinant Human Interleukin 1 Receptor Antagonist in Escherichia Coli Strains, BL21 (DE3), Rosetta (DE3), and Origami (DE3). JOURNAL OF ISFAHAN MEDICAL SCHOOL (I.U.M.S)[Internet]. 2019;37(539 ):965-972. Available from: https://sid.ir/paper/395979/en

IEEE: Copy

Hanieh Fardgoli, SEYED NEZAMEDDIN HOSEINI, and SETAREH HAGHIGHAT, “Cloning and Expression of Recombinant Human Interleukin 1 Receptor Antagonist in Escherichia Coli Strains, BL21 (DE3), Rosetta (DE3), and Origami (DE3),” JOURNAL OF ISFAHAN MEDICAL SCHOOL (I.U.M.S), vol. 37, no. 539 , pp. 965–972, 2019, [Online]. Available: https://sid.ir/paper/395979/en

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