E. coli derived human granulocyte-macrophage colony-stimulating factor (rh GM-CSF) available for clinical trials.
Recombinant human GM-CSF has been expressed as a fusion protein in E. coli in the form of inclusion bodies. Using denaturing agents, acid cleavage and sulfitolysis, the biologically inactive GM-CSF protein could be highly purified and additionally renaturated under suitable reoxidizing conditions. The thorough repair of the two disulfide bridges could be confirmed by sequencing fragments obtained by tryptic digestion. Refolding of the molecule has been studied by CD spectrometry and identity by Western blotting and SDS-PAGE analysis. As could be demonstrated, full biological activity (colony-forming assay with fresh human bone marrow cells) was restored during renaturation of the GM-CSF protein. Further proof of biological equivalence of the E. coli-derived protein with a yeast-derived biologically active rh GM-CSF has been published elsewhere.
Gronski
P
P
Research Laboratories of Behringwerke AG, Marburg/Lahn, W.-Germany.
Badziong
W
W
Habermann
P
P
List
W
W
Müllner
H
H
Neurohr
K J
KJ
Tripier
D
D
Seiler
F R
FR
D002942:Circular Dichroism; D002986:Clinical Trials as Topic; D003001:Cloning, Molecular; D003115:Colony-Stimulating Factors / Q000235:genetics / Q000302:isolation & purification / Q000627:therapeutic use*; D004926:Escherichia coli / Q000235:genetics; D005260:Female; D016179:Granulocyte Colony-Stimulating Factor; D006801:Humans; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000627:therapeutic use*
D003115:Colony-Stimulating Factors; D011994:Recombinant Proteins; D016179:Granulocyte Colony-Stimulating Factor