Structure and activity of granulocyte colony-stimulating factor derived from CHO cells containing cDNA coding for alternatively spliced sequences.
Two different cDNAs have been isolated, coding for two forms of granulocyte colony-stimulating factor (G-CSF): one for a polypeptide of 174 amino acids and the other for a polypeptide of 177 amino acids. In this paper, we have expressed these two forms in Chinese hamster ovary cells and characterized the purified proteins for activity and conformation. In vitro mitogenic assay showed a 50-fold lower activity for the 177 form than for the 174 form. In vitro receptor binding assay showed that binding of the 177 form to the purified extracellular domain of G-CSF receptor was also diminished, while the 174 form complexed with the receptor. Circular dichroic spectra showed that both forms are similar in the secondary structure, but are slightly different in the tertiary structure. Infrared spectra also showed a slight difference between the two forms. Both techniques also demonstrated differences in stability; i.e., the 174 form is more stable than the 177 form during storage or against heat denaturation.
Arakawa
T
T
Amgen Inc., Amgen Center, Thousand Oaks, California 91320-1789.
Horan
T P
TP
Leong
K
K
Prestrelski
S J
SJ
Narhi
L O
LO
Hu
S
S
D017398:Alternative Splicing; D000818:Animals; D001681:Biological Assay; D016466:CHO Cells; D002942:Circular Dichroism; D006224:Cricetinae; D018076:DNA, Complementary / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D016179:Granulocyte Colony-Stimulating Factor / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism / Q000494:pharmacology; D007525:Isoelectric Focusing; D011489:Protein Denaturation; D016188:Receptors, Granulocyte Colony-Stimulating Factor / Q000378:metabolism; D011994:Recombinant Proteins / Q000494:pharmacology; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet; D017550:Spectroscopy, Fourier Transform Infrared
D018076:DNA, Complementary; D016188:Receptors, Granulocyte Colony-Stimulating Factor; D011994:Recombinant Proteins; D016179:Granulocyte Colony-Stimulating Factor