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.
Keywords: human granulocyte macrophage colony stimulating factor; on-column cleavage; TRX fusion; IMAC; enterokinase
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Das, K.M.; Banerjee, S.; Shekhar, N.; Damodaran, K.; Nair, R.; Somani, S.; Raiker, V.P.; Jain, S.; Padmanabhan, S. Cloning, Soluble Expression and Purification of High Yield Recombinant hGMCSF in Escherichia coli. Int. J. Mol. Sci. 2011, 12, 2064-2076.
Das KM, Banerjee S, Shekhar N, Damodaran K, Nair R, Somani S, Raiker VP, Jain S, Padmanabhan S. Cloning, Soluble Expression and Purification of High Yield Recombinant hGMCSF in Escherichia coli. International Journal of Molecular Sciences. 2011; 12(3):2064-2076.
Das, Krishna M.P.; Banerjee, Sampali; Shekhar, Nivedita; Damodaran, Karpagavalli; Nair, Rahul; Somani, Sandeep; Raiker, Veena P.; Jain, Shweta; Padmanabhan, Sriram. 2011. "Cloning, Soluble Expression and Purification of High Yield Recombinant hGMCSF in Escherichia coli." Int. J. Mol. Sci. 12, no. 3: 2064-2076.