High pH solubilization and chromatography-based renaturation and purification of recombinant human granulocyte colony-stimulating factor from inclusion bodies.
Recombinant human granulocyte colony-stimulating factor (rhG-CSF) is a very efficient therapeutic protein drug which has been widely used in human clinics to treat cancer patients suffering from chemotherapy-induced neutropenia. In this study, rhG-CSF was solubilized from inclusion bodies by using a high-pH solution containing low concentration of urea. It was found that solubilization of the rhG-CSF inclusion bodies greatly depended on the buffer pH employed; alkalic pH significantly favored the solubilization. In addition, when small amount of urea was added to the solution at high pH, the solubilization was further enhanced. After solubilization, the rhG-CSF was renatured with simultaneous purification by using weak anion exchange, strong anion exchange, and hydrophobic interaction chromatography, separately. The results indicated that the rhG-CSF solubilized by the high-pH solution containing low concentration of urea had much higher mass recovery than the one solubilized by 8 M urea when using anyone of the three refolding methods employed in this work. In the case of weak anion exchange chromatography, the high pH solubilized rhG-CSF could get a mass recovery of 73%. The strategy of combining solubilization of inclusion bodies at high pH with refolding of protein using liquid chromatography may become a routine method for protein production from inclusion bodies.
Key Laboratory of Synthetic and Natural Functional Molecule Chemistry of Ministry of Education, College of Chemistry and Materials Science, Northwest University, Xi'an, People's Republic of China.
Ming
M
Li
Hua
H
Fan
Jiahua
J
Liu
Minhong
M
Wang
Lili
L
Wang
Chaozhan
C
Wang
D002852:Chromatography, Ion Exchange / Q000379:methods*; D004926:Escherichia coli / Q000166:cytology; D016179:Granulocyte Colony-Stimulating Factor / Q000737:chemistry* / Q000302:isolation & purification*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D057927:Hydrophobic and Hydrophilic Interactions; D002479:Inclusion Bodies / Q000737:chemistry* / Q000187:drug effects; D020673:Protein Renaturation / Q000187:drug effects; D011994:Recombinant Proteins / Q000737:chemistry* / Q000302:isolation & purification*; D012995:Solubility; D012997:Solvents / Q000494:pharmacology; D014508:Urea / Q000494:pharmacology
D011994:Recombinant Proteins; D012997:Solvents; D016179:Granulocyte Colony-Stimulating Factor; D014508:Urea