Isolation and characterization of ciliary neurotrophic factor from rabbit sciatic nerves.
Ciliary neurotrophic factor (CNTF) has been purified 35,000-fold to homogeneity from rabbit sciatic nerves using its ability to promote the survival of chick embryo ciliary ganglion neurons as the bioassay. The purification involved a combination of acid treatment, ammonium sulfate fractionation, hydrophobic interaction chromatography, chromatofocusing, preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and reversed-phase high performance liquid chromatography. Overlapping peptide sequences were obtained which accounted for 49% of the primary structure of the molecule. This information was used to prepare synthetic peptides in order to elicit antibodies. Purified CNTF exhibited two major and several minor bands between 24 and 22 kDa on silver-stained sodium dodecyl sulfate-polyacrylamide electrophoresis gels. All of the molecular forms were immunostained in Western blots by antiserum to synthetic peptides. The peptide sequences also provided a basis for cloning and expression of the rabbit CNTF gene (Lin, L-F. H., Mismer, D., Lile, J. D., Armes, L. G., Butler, E. T., III, Vannic, J. L., and Collins, F. (1989) Science 246, 1023-1025) confirming that the protein purified as reported here is CNTF.
Lin
L F
LF
Synergen, Inc., Boulder, Colorado 80301.
Armes
L G
LG
Sommer
A
A
Smith
D J
DJ
Collins
F
F
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies; D002478:Cells, Cultured; D002642:Chick Embryo; D002851:Chromatography, High Pressure Liquid; D020934:Ciliary Neurotrophic Factor; D004591:Electrophoresis, Polyacrylamide Gel; D005346:Fibroblast Growth Factors / Q000494:pharmacology; D015151:Immunoblotting; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009414:Nerve Growth Factors / Q000302:isolation & purification* / Q000494:pharmacology; D009419:Nerve Tissue Proteins / Q000302:isolation & purification* / Q000494:pharmacology; D009474:Neurons / Q000166:cytology / Q000187:drug effects; D010455:Peptides / Q000138:chemical synthesis; D012584:Sciatic Nerve / Q000502:physiology*
D000906:Antibodies; D020934:Ciliary Neurotrophic Factor; D009414:Nerve Growth Factors; D009419:Nerve Tissue Proteins; D010455:Peptides; D005346:Fibroblast Growth Factors