Establishment and characterization of a CHO cell line producing a secreted form of human ciliary neurotrophic factor: neuroprotective effects of the recombinant protein.
Human ciliary neurotrophic factor (CNTF) was inserted into a mammalian expression vector linked to the prepro sequence of human nerve growth factor. A Chinese hamster ovary cell line was established by resistance to neomycin and the plasmid integrated DNA was amplified using the metallothionein gene. This cell line contained several hundred copies of the human CNTF gene and produced an NH2 terminal truncated form of human CNTF (22 kDa) which was secreted into the medium. Although the copy number of the human CNTF gene was high and its mRNA was actively transcribed, the recombinant protein secreted into the medium constituted only 35-40% of the total amount of human CNTF synthesized by these cells. Both wild-type human CNTF produced in bacterial cells and the human CNTF obtained by forced secretion were effective in protecting hippocampal pyramidal neurons from injury induced by glucose deprivation, a form of excitotoxic neurodegeneration.
Negro
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A
Fidia Research Laboratories, Abano Terme, Italy.
Tavella
A
A
Soranzo
C
C
Petrelli
L
L
Skaper
S D
SD
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D015139:Blotting, Southern; D016466:CHO Cells; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D020934:Ciliary Neurotrophic Factor; D006224:Cricetinae; D004247:DNA / Q000032:analysis / Q000378:metabolism; D006624:Hippocampus / Q000166:cytology; D006801:Humans; D008668:Metallothionein / Q000235:genetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009414:Nerve Growth Factors / Q000096:biosynthesis*; D009419:Nerve Tissue Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000494:pharmacology; D009474:Neurons / Q000166:cytology* / Q000187:drug effects; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000096:biosynthesis / Q000378:metabolism; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000494:pharmacology; D017384:Sequence Deletion; D014158:Transcription, Genetic; D014162:Transfection
D020934:Ciliary Neurotrophic Factor; D009414:Nerve Growth Factors; D009419:Nerve Tissue Proteins; D012333:RNA, Messenger; D011994:Recombinant Proteins; D004247:DNA; D008668:Metallothionein