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 characterize...

Full description

Saved in:
Bibliographic Details
Published in:Archives of biochemistry and biophysics 1995-01, Vol.316 (1), p.285-289
Main Authors: Arakawa, T., Horan, T.P., Leong, K., Prestrelski, S.J., Narhi, L.O., Hu, S.
Format: Article
Language:English
Subjects:
Alternative Splicing
Animals
Biological Assay
CHO Cells
Circular Dichroism
Cricetinae
DNA, Complementary - genetics
Electrophoresis, Polyacrylamide Gel
Granulocyte Colony-Stimulating Factor - chemistry
Granulocyte Colony-Stimulating Factor - genetics
Granulocyte Colony-Stimulating Factor - metabolism
Granulocyte Colony-Stimulating Factor - pharmacology
Isoelectric Focusing
Protein Denaturation
Receptors, Granulocyte Colony-Stimulating Factor - metabolism
Recombinant Proteins - pharmacology
Spectrometry, Fluorescence
Spectrophotometry, Ultraviolet
Spectroscopy, Fourier Transform Infrared
Citations: Items that cite this one
Online Access:Get full text
Tags: Add Tag
No Tags, Be the first to tag this record!
Description
Summary: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.
ISSN:0003-9861
1096-0384
DOI:10.1006/abbi.1995.1039