Cloning, Soluble Expression and Purification of High Yield Recombinant hGMCSF in Escherichia coli

International Journal of Molecular Sciences

Krishna M.P. Das⁵  Sampali Banerjee⁵  Nivedita Shekhar⁴  Karpagavalli Damodaran¹  Rahul Nair²  Sandeep Somani³  Veena P. Raiker¹  Shweta Jain¹ 

[1] Analytical development Team, Lupin Limited, Biotechnology R & D, Gat #1156, Ghotawade Village, Mulshi Taluka, Pune-411042, India; E-Mails:;Upstream Development Team, Lupin Limited, Biotechnology R & D, Gat #1156, Ghotawade Village, Mulshi Taluka, Pune-411042, India; E-Mail:;Downstream Development Team, Lupin Limited, Biotechnology R & D, Gat #1156, Ghotawade Village, Mulshi Taluka, Pune-411042, India; E-Mail:;Mammalian Bioassay Team, Lupin Limited, Biotechnology R & D, Gat #1156, Ghotawade Village, Mulshi Taluka, Pune-411042, India; E-Mail:;Clone Development Team, Lupin Limited, Biotechnology R & D, Gat #1156, Ghotawade Village, Mulshi Taluka, Pune-411042, India; E-Mails:

关键词: human granulocyte macrophage colony stimulating factor;    on-column cleavage;    TRX fusion;    IMAC;    enterokinase;   

DOI  :  10.3390/ijms12032064

来源: mdpi

【 摘 要 】

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.

【 授权许可】

CC BY   
© 2011 by the authors; licensee MDPI, Basel, Switzerland.