Abstract
Human granulocyte-macrophage colony stimulating factor (hGMCSF) is an important therapeutic cytokine. As a novel attempt to purify hGMCSF protein, without the enzymatic cleavage of the affinity tag, an intein-based system was used. The gene was fused by overlap extension PCR to the intein sequence at its N-terminal in pTYB11 vector. The hGMCSF was expressed as a fusion protein in E. coli BL21(DE3), and E. coli GJ1158. In the former, the protein was expressed as inclusion bodies and upon purification the yield was 7 mg/l with a specific activity of 0.5 × 107 IU/mg. In salt-inducible E. coli GJ1158, hGMCSF was expressed in a soluble form at 20 mg/l and a specific activity of 0.9 × 107 IU/mg. The intein-hGMCSF was purified on a chitin affinity column by cleaving intein with 50 mM DTT resulting in a highly pure 14.7 kDa hGMCSF.
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Acknowledgement
We gratefully thank All India Council for Technical Education, Govt. of India for providing fellowship (NDF) to Mr. K Srinivasa Babu and Council of Scientific and Industrial Research for providing fellowship (SRF) to Mr. T Muthukumaran. Also we would like to thank the company, M/S Virchow Biotech Private Ltd, Hyderabad, India for assistance in determining the bioactivity of recombinant proteins.
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Srinivasa Babu, K., Muthukumaran, T., Antony, A. et al. Single step intein-mediated purification of hGMCSF expressed in salt-inducible E. coli . Biotechnol Lett 31, 659–664 (2009). https://doi.org/10.1007/s10529-009-9921-8
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DOI: https://doi.org/10.1007/s10529-009-9921-8