Synthesis and purification of biologically active rat brain-derived neurotrophic factor from Escherichia coli.
The cDNA for rat brain-derived neurotrophic factor was cloned as the prepro and mature sequences into two independent expression vectors under control of the T7 promoter. When these vectors were transfected into Escherichia coli the prepro and mature forms of brain-derived neurotrophic factor accounted for about 20% and 25% of total E. coli proteins, and displayed molecular sizes of 26 kDa and 15 kDa, respectively. Mature brain-derived neurotrophic factor was extracted from E. coli inclusion bodies, refolded in the presence of CuCl2 and purified. The resulting protein had an ED50 of 3 ng/ml in supporting survival of cultured embryonic dorsal root ganglion neurons.
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Advanced Technology Division, Fidia S.p.A., Abano Terme, Italy.
Corsa
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Moretto
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Skaper
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Callegaro
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D000818:Animals; D001483:Base Sequence; D019208:Brain-Derived Neurotrophic Factor; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D002642:Chick Embryo; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D005727:Ganglia, Spinal / Q000166:cytology; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009414:Nerve Growth Factors / Q000096:biosynthesis*; D009419:Nerve Tissue Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D009474:Neurons / Q000166:cytology / Q000187:drug effects; D009838:Oligodeoxyribonucleotides; D010957:Plasmids; D011487:Protein Conformation; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000302:isolation & purification / Q000494:pharmacology; D015183:Restriction Mapping; D014158:Transcription, Genetic
D019208:Brain-Derived Neurotrophic Factor; D009414:Nerve Growth Factors; D009419:Nerve Tissue Proteins; D009838:Oligodeoxyribonucleotides; D012333:RNA, Messenger; D011994:Recombinant Proteins; D004247:DNA