Evaluation of soluble expression of recombinant granulocyte macrophage stimulating factor (rGM-CSF) by three different E. coli strains

Sina Soheili , Ali Jahanian-Najafabadi, Vajihe Akbari


Background and purpose: Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a cytokine with a wide range of therapeutic applications although expression of GM-CSF in Escherichia coli (E. coli) usually leads to formation of insoluble aggregates mostly lack biological activity. The aim of this study was                                   to compare the soluble expression level of GM-CSF in three E. coli strains, BL21 (DE3), SHuffle® T7 and OrigamiTM 2 (DE3).

Experimental approach: The effect of different temperatures and inducer concentrations on soluble expression of GM-CSF was evaluated. The soluble GM-CSF was subjected to endotoxin removal and purification using nickel-nitrilotriacetic acid (Ni-NTA) affinity chromatography, ultrafiltration. The biological activity of produced GM-CSF was evaluated based on its growth promotion effect on TF-1 cell lines by MTT assay method.

Findings / Results: A significant improvement of the soluble yield of GM-CSF (about 30% of GM-CSF was expressed as soluble proteins) was observed when protein expression was induced at 30 °C with 0.5 mM isopropyl β- d-1-thiogalactopyranoside (IPTG) in E. coli Shuffle T7. The soluble GM-CSF with a high purity up to 95 % and specific activity of 1.25 × 104 IU/μg was obtained.

Conclusion and implications: The proposed strategy here can be used to improve the soluble expression of other hard-to-express proteins with similar structural properties (i.e., containing disulfide binds or cysteine).




Disulfide bond isomerase; Expression; GM-CSF; Shuffle T7; Soluble.

Full Text:



Metcalf D. The colony-stimulating factors and cancer. Cancer Immunol Res. 2013;1(6):351-356.

DOI: 10.1158/2326-6066.CIR-13-0151.

Martin-Christin F. Granulocyte colony stimulating factors: how different are they? How to make a decision? Anticancer Drugs. 2001;12(3):185-191.

DOI: 10.1097/00001813-200103000-00002.

Conti L, Gessani S. GM-CSF in the generation of dendritic cells from human blood monocyte precursors: recent advances. Immunobiology. 2008;213(9-10):859-870.

DOI: 10.1016/j.imbio.2008.07.017.

Rini B, Wadhwa M, Bird C, Small E, Gaines-Das R, Thorpe R. Kinetics of development and characteristics of antibodies induced in cancer patients against yeast expressed rDNA derived granulocyte macrophage colony stimulating factor (GM-CSF). Cytokine. 2005;29(2):56-66.

DOI: 10.1016/j.cyto.2004.09.009.

Sezonov G, Joseleau-Petit D, D'Ari R. Escherichia coli physiology in Luria-Bertani broth. J Bacteriol. 2007;189(23):8746-8749.

DOI: 10.1128/JB.01368-07.

Shiloach J, Fass R. Growing E. coli to high cell density-a historical perspective on method development. Biotechnol Adv. 2005;23(5):345-357.

DOI: 10.1016/j.biotechadv.2005.04.004.

Green MR, Sambrook J. Easy Transformation of Escherichia coli: nanoparticle-mediated trans-formation. Cold Spring Harb Protoc. 2019;2019 (12). pdb. prot101204.

DOI: 10.1101/pdb.prot101204.

Singh SM, Panda AK. Solubilization and refolding of bacterial inclusion body proteins. J Biosci Bioeng. 2005;99(4):303-310.

DOI: 10.1263/jbb.99.303.

Zhuo XF, Zhang YY, Guan YX, Yao SJ. Co-expression of disulfide oxidoreductases DsbA/DsbC markedly enhanced soluble and functional expression of reteplase in Escherichia coli. J Biotechnol. 2014;192 Pt A:197-203.

DOI: 10.1016/j.jbiotec.2014.10.028.

Rosano GL, Ceccarelli EA. Recombinant protein expression in Escherichia coli: advances and challenges. Front Microbiol. 2014;5:172-188.

DOI: 10.3389/fmicb.2014.00172.

Long X, Gou Y, Luo M, Zhang S, Zhang H, Bai L, et al. Soluble expression, purification, and characterization of active recombinant human tissue plasminogen activator by auto-induction in E. coli. BMC Biotechnol. 2015;15(1): 1-9.

DOI: 10.1186/s12896-015-0127-y.

Lobstein J, Emrich CA, Jeans C, Faulkner M, Riggs P, Berkmen M. SHuffle, a novel Escherichia coli protein expression strain capable of correctly folding disulfide bonded proteins in its cytoplasm. Microb Cell Fact. 2012;11:56-71.

DOI: 10.1186/1475-2859-11-56.

Malekian R, Jahanian-Najafabadi A, Moazen F, Ghavimi R, Mohammadi E, Akbari V. High-yield production of granulocyte-macrophage colony-stimulating factor in E. coli BL21 (DE3) by an auto-induction strategy. Iran J Pharm Res. 2019;18(1): 469-478.

Malekian R, Sima S, Jahanian-Najafabadi A, Moazen F, Akbari V. Improvement of soluble expression of GM-CSF in the cytoplasm of Escherichia coli using chemical and molecular chaperones. Protein Expr Purif. 2019;160:66-72.

DOI: 10.1016/j.pep.2019.04.002.

Akbari V, Sadeghi HMM, Jafrian-Dehkordi A, Abedi D, Chou CP. Functional expression of a single-chain antibody fragment against human epidermal growth factor receptor 2 (HER2) in Escherichia coli. J Ind Microbiol Biotechnol. 2014;41(6):947-956.

DOI: 10.1007/s10295-014-1437-0.

Akbari V, Sadeghi HM, Jafarian-Dehkordi A, Chou CP, Abedi D. Optimization of a single-chain antibody fragment overexpression in Escherichia coli using response surface methodology. Res Pharm Sci. 2015;10(1):75-83. PMCID: PMC4578215.

Kosobokova EN, Skrypnik KA, Pinyugina MV, Shcherbakov AI, Kosorukov VS. Optimization of the refolding of recombinant human granulocyte-macrophage colony-stimulating factor immobilized on affinity sorbent. Appl Biochem Biotechnol. 2014;50(8):773-779.

DOI: 10.1134/S0003683814080031.

Oloomi M, Bouzari S, Rechinsky V. Purification and characterization of the cloned human GM-CSF gene expressed in Escherichia coli. Med J Islam Rep Iran. 1999;12(4):353-357.

Hansen G, Hercus TR, McClure BJ, Stomski FC, Dottore M, Powell J, et al. The structure of the GM-CSF receptor complex reveals a distinct mode of cytokine receptor activation. Cell. 2008;134(3):496-507.

DOI: 10.1016/j.cell.2008.05.053.

de Groot NS, Ventura S. Protein aggregation profile of the bacterial cytosol. PLoS One. 2010;5(2):e9383,1-17.

DOI: 10.1371/journal.pone.0009383.

Thomson CA, Olson M, Jackson LM, Schrader JW. A simplified method for the efficient refolding and purification of recombinant human GM-CSF. PLoS One. 2012;7(11): e49891, 1-6

DOI: 10.1371/journal.pone.0049891.

Das KMP, Banerjee S, Shekhar N, Damodaran K, Nair R, Somani S, et al. Padmanabhan, cloning, soluble expression and purification of high yield recombinant hGMCSF in Escherichia coli. Int J Mol Sci. 2011;12(3):2064-2076.

DOI: 10.3390/ijms12032064.

Lebendiker M, Danieli T. Production of prone-to-aggregate proteins. FEBS Lett. 2014;588(2):236-246.

DOI: 10.1016/j.febslet.2013.10.044.

Berkmen M. Production of disulfide-bonded proteins in Escherichia coli. Protein Expr Purif. 2012;82(1):240-251.

DOI: 10.1016/j.pep.2011.10.009.

Ahmadzadeh M, Farshdari F, Nematollahi L, Behdani M, Mohit E. Anti-HER2 scFv Expression in Escherichia coli SHuffle®T7 Express Cells: Effects on Solubility and Biological Activity. Mol Biotechnol. 2020;62,18-30.


Rasooli F, Hashemi A. Efficient expression of EpEX in the cytoplasm of Escherichia coli using thioredoxin fusion protein. Res Pharm Sci. 2019;14(6),554-565.

DOI: 10.4103/1735-5362.272564

Safarpour H, Banadkoki SB, Keshavarzi Z, Morowvat MH, Soleimanpour M, Pourmolaei S, et al. Expression analysis and ATR-FTIR characterization of the secondary structure of recombinant human TNF-alpha from Escherichia coli SHuffle(R) T7 Express and BL21 (DE3) cells. Int J Biol Macromol. 2017;99:173-178.

DOI: 10.1016/j.ijbiomac.2017.02.052.

Nasiri M, Babaie J, Amiri S, Azimi E, Shamshiri S, Khalaj V, et al. SHuffleTM T7 strain is capable of producing high amount of recombinant human fibroblast growth factor-1 (rhFGF-1) with proper physicochemical and biological properties. J Biotechnol. 2017;259:30-38.

DOI: 10.1016/j.jbiotec.2017.08.015.

Ratelade J, Miot MC, Johnson E, Betton JM, Mazodier P, Benaroudj N. Production of recombinant proteins in the lon-deficient BL21(DE3) strain of Escherichia coli in the absence of the DnaK chaperone. Appl Environ Microbiol. 2009;75(11):3803-3807.

DOI: 10.1128/AEM.00255-09.

Fathi-Roudsari M, Akhavian-Tehrani A, Maghsoudi N. Comparison of three Escherichia coli strains in recombinant production of reteplase. Avicenna J Med Biotechnol. 2016;8(1):16-22.

Ferrer M, Chernikova TN, Yakimov MM, Golyshin PN, Timmis KN. Chaperonins govern growth of Escherichia coli at low temperatures. Nat Biotechnol. 2003;21(11):1266-1267.

DOI: 10.1038/nbt1103-1266.


  • There are currently no refbacks.

Creative Commons LicenseThis work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly.