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

Abstract


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).

 

 


Keywords


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

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