title: Recombinant human and rat ciliary neurotrophic factors.
abstract: The human ciliary neurotrophic factor (CNTF) gene was identified and cloned, based on homology with the recently cloned rat cDNA. The gene encodes a protein of 200 amino acids, which shares about 80% sequence identity with rat and rabbit CNTF and, like these homologues, lacks an apparent secretion signal sequence. The human CNTF gene, like the rat gene, appears to contain a single intron separating two protein coding exons. An intronless human CNTF gene was constructed by the use of polymerase chain reactions and introduced into vectors designed for expression of foreign proteins in E. coli. The rat CNTF gene was also introduced into similar vectors. Both the human and rat proteins were expressed at exceptionally high levels, at 20-40% and 60-70% of total protein, respectively. Extraction of the recombinant proteins from inclusion bodies by guanidinium chloride, followed by two column chromatography steps, produced high yields of pure CNTF that supported survival and neurite outgrowth from embryonic chick ciliary neurons in culture. The biological activity of both recombinant proteins was comparable to that of native rat CNTF.
authors.affiliation: Regeneron Pharmaceuticals Inc., Tarrytown, New York.
authors.forename: P
authors.initials: P
authors.lastname: Masiakowski
authors.forename: H X
authors.initials: HX
authors.lastname: Liu
authors.forename: C
authors.initials: C
authors.lastname: Radziejewski
authors.forename: F
authors.initials: F
authors.lastname: Lottspeich
authors.forename: W
authors.initials: W
authors.lastname: Oberthuer
authors.forename: V
authors.initials: V
authors.lastname: Wong
authors.forename: R M
authors.initials: RM
authors.lastname: Lindsay
authors.forename: M E
authors.initials: ME
authors.lastname: Furth
authors.forename: N
authors.initials: N
authors.lastname: Panayotatos
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002850:Chromatography, Gel; D020934:Ciliary Neurotrophic Factor; D003001:Cloning, Molecular; D004274:DNA, Recombinant / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D005796:Genes / Q000235:genetics; D005822:Genetic Vectors; D006801:Humans; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000235:genetics* / Q000378:metabolism; D010957:Plasmids; D051381:Rats; D011994:Recombinant Proteins / Q000235:genetics
chemical_list: D020934:Ciliary Neurotrophic Factor; D004274:DNA, Recombinant; D009419:Nerve Tissue Proteins; D011994:Recombinant Proteins