High recovery refolding of rhG-CSF from Escherichia coli, using urea gradient size exclusion chromatography.
Protein folding liquid chromatography (PFLC) is a powerful tool for simultaneous refolding and purification of recombinant proteins in inclusion bodies. Urea gradient size exclusion chromatography (SEC) is a recently developed protein refolding method based on the SEC refolding principle. In the presented work, recombinant human granulocyte colony-stimulating factor (rhG-CSF) expressed in Escheriachia coli (E. coli) in the form of inclusion bodies was refolded with high yields by this method. Denatured/reduced rhG-CSF in 8.0 mol.L(-1) urea was directly injected into a Superdex 75 column, and with the running of the linear urea concentration program, urea concentration in the mobile phase and around the denatured rhG-CSF molecules was decreased linearly, and the denatured rhG-CSF was gradually refolded into its native state. Aggregates were greatly suppressed and rhG-CSF was also partially purified during the refolding process. Effects of the length and the final urea concentration of the urea gradient on the refolding yield of rhG-CSF by using urea gradient SEC were investigated respectively. Compared with dilution refolding and normal SEC with a fixed urea concentration in the mobile phase, urea gradient SEC was more efficient for rhG-CSF refolding--in terms of specific bioactivity and mass recovery, the denatured rhG-CSF could be refolded at a larger loading volume, and the aggregates could be suppressed more efficiently. When 500 microL of solubilized and denatured rhG-CSF in 8.0 mol.L(-1) urea solution with a total protein concentration of 2.3 mg.mL(-1) was loaded onto the SEC column, rhG-CSF with a specific bioactivity of 1.0 x 10(8) IU.mg(-1) was obtained, and the mass recovery was 46.1%.
Wang
Chaozhan
C
Key Laboratory of Synthetic and Natural Functional Molecule Chemistry of Ministry of Education, Institute of Modern Separation Science, Northwest University, Xi'an 710069, China. czwang@nwu.edu.cn
Wang
Lili
L
Geng
Xindu
X
D002850:Chromatography, Gel / Q000379:methods*; D004926:Escherichia coli / Q000235:genetics*; D016179:Granulocyte Colony-Stimulating Factor / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry* / Q000378:metabolism; D015203:Reproducibility of Results; D014508:Urea / Q000737:chemistry*
D011994:Recombinant Proteins; D016179:Granulocyte Colony-Stimulating Factor; D014508:Urea