Synthesis of the biologically active beta-subunit of human nerve growth factor in Escherichia coli.
The gene (NGFB) encoding the beta subunit of mature human nerve growth factor (hNGFB) was subcloned into the pJLA503 expression vector under the control of bacteriophage promoters PR and PL, and expressed in Escherichia coli. The recombinant protein represented approximately 3% of the total cellular protein. Biologically active hNGFB was solubilized (0.2% total NGFB) and purified by cation-exchange chromatography and it yielded two bands on polyacrylamide-gel electrophoresis under nonreducing conditions, corresponding to the monomeric (14 kDa) and homodimeric (26.5 kDa) forms of the molecule. Both hNGFB forms were immunopositive on Western blots with rabbit anti-NGFB antibodies; however, following additional purification, only the species corresponding to the hNGFB homodimer was biologically active on cultured chicken dorsal root ganglion neurons. These results demonstrate the feasibility of synthesizing the biologically active form of hNGFB in E. coli.
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Advanced Technology Division, Abano Terme, Italy.
Martini
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Bigon
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Cazzola
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Minozzi
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Skaper
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Callegaro
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D001483:Base Sequence; D015153:Blotting, Western; D002478:Cells, Cultured; D002845:Chromatography; D003001:Cloning, Molecular; D004274:DNA, Recombinant / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D009414:Nerve Growth Factors / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification / Q000494:pharmacology; D010957:Plasmids / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000302:isolation & purification / Q000494:pharmacology
D004274:DNA, Recombinant; D046911:Macromolecular Substances; D009414:Nerve Growth Factors; D011993:Recombinant Fusion Proteins