Preparation and a structure-function analysis of human ciliary neurotrophic factor.
Ciliary neurotrophic factor (CNTF) is a trophic protein that promotes survival and/or differentiation of a variety of neuronal cell types including sensory, sympathetic, and motor neurons. CNTF, leukemia inhibitory factor (LIF), interleukin-6 (IL-6) and oncostatin M (OSM) share a predicted common helical framework and partially identical receptor components. In this study, we present the preparation and structure--functional analysis of recombinant human CNTF. The human CNTF gene was expressed under the control of the PL promoter in Escherichia coli, and the mutants were constructed by insertion, deletion and site-directed mutagenesis. The recombinant proteins were purified from bacteria via DEAE A-50 and Sephacryl S-200 chromatography, and their survival promoting activities were determined using cultures of embryonic chick dorsal root ganglion (DRG) neurons. Insertion at position 23 with APGL, or at position 79 with PRGA, or substitution of 162L163Q for PIDG resulted in proteins with no neurotrophic activity. However, insertion at position 186 with PRGI did not alter human CNTF activity. Deletion of the carboxy-terminal amino acid 186-200 did not reduce the biological activity, but elimination of the amino acid 162-186 abolished the activity. The mutant substituting of 17 Cys for Ser was found to display a biological activity equivalent to that of the wild type. Our data provided experimental confirmation for the structural prediction of CNTF.
He
C
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Department of Neurobiology, Second Military Medical University, Shanghai, China.
Chen
J
J
Ao
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S
Lu
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D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002642:Chick Embryo; D020934:Ciliary Neurotrophic Factor; D003001:Cloning, Molecular; D003545:Cysteine / Q000235:genetics; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D005727:Ganglia, Spinal / Q000187:drug effects / Q000502:physiology; D015964:Gene Expression Regulation, Bacterial / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009414:Nerve Growth Factors / Q000235:genetics*; D009419:Nerve Tissue Proteins / Q000235:genetics*; D011401:Promoter Regions, Genetic / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000494:pharmacology
D020934:Ciliary Neurotrophic Factor; D009414:Nerve Growth Factors; D009419:Nerve Tissue Proteins; D011994:Recombinant Proteins; D003545:Cysteine