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Abstract
Expression of human granulocyte macrophage colony stimulating factor (hGMCSF), a cytokine of therapeutic importance, as a thioredoxin (TRX) fusion has been investigated in Escherichia coli BL21 (DE3) codon plus cells. The expression of this protein was low when cloned under the T7 promoter without any fusion tags. High yield of GMCSF was achieved (∼88 mg/L of fermentation broth) in the shake flask when the gene was fused to the E. coli TRX gene. The protein was purified using a single step Ni2+-NTA affinity chromatography and the column bound fusion tag was removed by on-column cleavage with enterokinase. The recombinant hGMCSF was expressed as a soluble and biologically active protein in E. coli, and upon purification, the final yield was ∼44 mg/L in shake flask with a specific activity of 2.3 × 108 U/mg. The results of Western blot and RP-HPLC analyses, along with biological activity using the TF-1 cell line, established the identity of the purified hGMCSF. In this paper, we report the highest yield of hGMCSF expressed in E. coli. The bioreactor study shows that the yield of hGMCSF could be easily scalable with a yield of ∼400 mg/L, opening up new opportunities for large scale production hGMCSF in E. coli.
Highlights
Human granulocyte macrophage colony stimulating factor is a cytokine, secreted by macrophages, T cells, mast cells, endothelial cells and fibroblasts in response to immune and inflammatory stimuli
We describe the overexpression of human granulocyte macrophage colony stimulating factor (hGMCSF) as a soluble thioredoxin (TRX)-fusion and purification to homogeneity with very high yield after removal of the fusion tag by enterokinase digestion
As the poor expression of GMCSF was unsuitable for any further experimentation, the gene was cloned as a TRX fusion and the expression was carried out in the same cell line as described before
Summary
Human granulocyte macrophage colony stimulating factor (hGMCSF) is a cytokine, secreted by macrophages, T cells, mast cells, endothelial cells and fibroblasts in response to immune and inflammatory stimuli. Many attempts have been undertaken to synthesize biologically active recombinant hGMCSF; transfected mammalian cells are not preferred as an expression system for producing GMCSF for biological and structural studies due to low expression levels and the presence of contaminating CSFs secreted by the mammalian cells themselves [3]. This problem can be handled using an Escherichia coli expression system to produce large quantities of recombinant protein. We describe the overexpression of hGMCSF as a soluble thioredoxin (TRX)-fusion and purification to homogeneity with very high yield after removal of the fusion tag by enterokinase digestion
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