A simplified purification procedure for recombinant human granulocyte macrophage-colony stimulating factor from periplasmic space of Escherichia coli.
Cytoplasmic expression is commonly used for production of recombinant human granulocyte macrophage-colony stimulating factor (rhGM-CSF) which most often comes with inclusion body formation. We expressed rhGM-CSF in periplasmic space of Escherichia coli and optimized its extraction by osmotic shock and purification by anion exchange chromatography. Our works show that MgCl2 at 2 mM in osmotic shock buffer improves extraction of the protein and reduces contamination with other proteins. To achieve a simplified purification procedure for rhGM-CSF, efforts were focused on the adjustment of pH of the buffers and application of proper concentration of salt. Following to measurement of the pI of 5.4 for rhGM-CSF by isoelectric focusing, the pH of dialysis buffer and buffers used in anion exchange chromatography were adjusted to 6.5 for optimal binding of the protein to the column and removal of proteins with higher pIs during washing of the column. In addition, it was found that appliance of NaCl at a concentration of 20 mM in dialysis and column washing buffers prior to elution with elution buffer containing 120 mM NaCl significantly improves purification of the protein. Starting with specific amount of total proteins obtained by osmotic shock, it was possible to recover 95% of which following to purification with a purification yield of 72% for rhGM-CSF along with appropriate biological activity.
Rastgar Jazii
F
F
National Institute of Genetic Engineering and Biotechnology, Tehran, Iran. rastgar_jazii@yahoo.com
Karkhane
A A
AA
Yakhchali
B
B
Fatemi
S S
SS
Deezagi
A
A
D002852:Chromatography, Ion Exchange / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000235:genetics / Q000302:isolation & purification*; D006863:Hydrogen-Ion Concentration; D008274:Magnesium / Q000737:chemistry; D019897:Periplasm / Q000378:metabolism; D011994:Recombinant Proteins
D011994:Recombinant Proteins; D016178:Granulocyte-Macrophage Colony-Stimulating Factor; D008274:Magnesium