Synthesis, purification, and characterization of human ciliary neuronotrophic factor from E. coli.
The cDNA for human ciliary neuronotrophic factor (CNTF) has been cloned into an expression vector under the control of the T7 promoter. The BL21 strain of E. coli was transformed with this vector. Human CNTF accounted for about 30% of the total bacterial protein after induction with isopropyl-B-D-thiogalactopyranoside. This human CNTF was purified to homogeneity from inclusion bodies by a combination of ion exchange chromatography and reverse-phase high performance liquid chromatography. The amino-terminal amino acid sequence of the purified protein was identical to the deduced amino acid sequence; however, the methionyl residue has been removed. On SDS-PAGE gels, human CNTF displayed a molecular weight of about 24 kDa, in accord with its deduced molecular mass; a pI of 5.8 indicates the acidic nature of the molecule. A proposed structure for human CNTF includes major alpha helical regions. The ED50 of purified human CNTF was approximately 30 pM, using cultured embryonic day 10 chicken dorsal root ganglion neurons; no activity was observed with neurons from embryonic day 8 ganglia. Polyclonal antibodies prepared against both a synthetic peptide of CNTF and the entire human CNTF protein recognized a single 24 kDa band on Western blots, corresponding to human CNTF. However, only the antibodies against intact CNTF blocked its biological activity. This represents the first molecular expression and purification of human CNTF.
Negro
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Advanced Technology Division, Fidia S.p.A., Abano Terme, Italy.
Corona
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Bigon
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Martini
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Grandi
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Skaper
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SD
Callegaro
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D001483:Base Sequence; D020934:Ciliary Neurotrophic Factor; D002942:Circular Dichroism; D004926:Escherichia coli / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D009414:Nerve Growth Factors / Q000302:isolation & purification; D009419:Nerve Tissue Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D009474:Neurons / Q000187:drug effects; D012995:Solubility
D020934:Ciliary Neurotrophic Factor; D009414:Nerve Growth Factors; D009419:Nerve Tissue Proteins