Renaturation with simultaneous purification of rhG-CSF from Escherichia coli by ion exchange chromatography.
Protein refolding is a key step for the production of recombinant proteins, especially at large scales, and usually their yields are very low. Application of liquid chromatography to protein refolding is an exciting step forward for this field. In this work, recombinant human granulocyte colony-stimulating factor (rhG-CSF) expressed in Escherichia coli was renatured with simultaneous purification by ion exchange chromatography (IEC) with a Q Sepharose FF column. Several chromatographic parameters affecting the refolding yield of the denatured/reduced rhG-CSF, such as the urea concentration, pH value, concentration and ratio of reduced/oxidized glutathione in the mobile phase, as well as the flow rate of the mobile phase, were investigated in detail and indicated that the urea concentration and the pH value were of great importance. At the optimal conditions, the renatured and purified rhG-CSF was found to have a specific bioactivity of 3.0 x 10(8) IU/mg, a purity of 96%, and a mass recovery of 49%. Compared with the usual dilution method, the IEC method developed here is more effective for rhG-CSF refolding in terms of specific bioactivity and mass recovery.
Wang
Chaozhan
C
Institute of Modern Separation Science, Key Laboratory of Separation Science in Shaanxi Province, Department of Chemistry, Northwest University, Xi'an, PR China.
Wang
Lili
L
Geng
Xindu
X
D000595:Amino Acid Sequence; D002852:Chromatography, Ion Exchange / Q000379:methods; D004926:Escherichia coli / Q000737:chemistry*; D016179:Granulocyte Colony-Stimulating Factor / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D011994:Recombinant Proteins; D013997:Time Factors; D014508:Urea / Q000737:chemistry
D010446:Peptide Fragments; D011994:Recombinant Proteins; D016179:Granulocyte Colony-Stimulating Factor; D014508:Urea