期刊
BIOCHEMICAL ENGINEERING JOURNAL
卷 43, 期 2, 页码 197-202出版社
ELSEVIER SCIENCE SA
DOI: 10.1016/j.bej.2008.09.018
关键词
Recombinant human granulocyte colony-stimulating factor; Inclusion bodies; Protein refolding and purification; Immobilized metal ion affinity chromatography; Protein folding liquid chromatography
资金
- National Natural Science Foundation in China [20705028]
- Foundation of Key Laboratory of Modern Separation Science in Shaanxi Province [4005JS61]
Immobilized metal ion affinity chromatography (IMAC) is a new technique for protein refolding, but it is limited to the refolding of fusion proteins with histidine affinity tags. In the present work, a non-fusion recombinant protein, recombinant human granulocyte colony-stimulating factor (rhG-CSF) expressed in Escheriachia coli (E. coli) in the form of inclusion bodies was successfully refolded with simultaneous purification by IMAC. rhG-CSF inclusion bodies solubilized in 8.0 mol/L urea was injected into a CU(II)-iminodiacetic acid (IDA)-IMAC column, the soluble and active form of rhG-CSF in aqueous solution was obtained after desorbed from the column by linear increase of imidazole concentration. Several factors in the refolding process, including urea concentration and pH of mobile phases, type of buffer, glycerol concentration and loading sample volume, were investigated, respectively. When 200 mu L of denatured/reduced rhG-CSF solution at a total protein concentration of 2.8 mg/mL was loaded on the IMAC column, rhG-CSF with a specific activity of 2.3 x 10(8) IU/mg and a mass recovery of 39% was obtained after IMAC refolding, and rhG-CSF was also purified during this chromatographic process, its purity was determined to be 97%. (C) 2008 Elsevier B.V. All rights reserved.
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