Purification of recombinant human granulocyte-macrophage colony stimulating factor expressed in Escherichia coli

Abstract

Recombinant human granulocyte-macrophage colony stimulating factor (rhGM-CSF) was expressed as inclusion bodies (IB) in E. coli. A simple and effective protocol has been worked out for the purification. IB collected after the breakage of bacteria through sonication were subjected to repeated washing followed by solubilization in TE buffer (50 mmol/L of Tris.HCl, 1 mmol/L of EDTA, pH 8.3) containing 8 mol/L urea and 10 mmol/L DL-dithiothreitol. By means of Sephacryl-200 HR, refolding, and Q Sepharose Fast Flow, rhGM-CSF was obtained with a purity of 99%. The total protein recovery was 10% and specific activity of rhGM-CSF was 1 x 10(7) u/mg. The sequence of N-terminal 16 amino acid residues of purified rhGM-CSF was determined and found to be identical to the native protein. This study provided useful parameters for mass production of rhGM-CSF.