Functional phage display of ciliary neurotrophic factor.
We report the display of human ciliary neurotrophic factor (hCNTF), a survival factor for neuronal cells belonging to the alpha-helical cytokine superfamily, on the surface of the filamentous bacteriophage fd. The hCNTF cDNA was fused to a DNA sequence encoding the C-terminal domain of pIII, a minor coat protein exposed at one end of fd. Gene fusions were cloned into a plasmid containing the ColE1 plasmid and fd origins of replication, and were packaged into phagemid particles upon superinfection with M13KO7 helper phage. The resulting fusion phage bound specifically to anti-CNTF antibodies and to the recombinant soluble CNTF alpha-receptor. Moreover, phage-displayed hCNTF was found to possess biological activity at concentrations comparable to those of the soluble cytokine. These results demonstrate that CNTF can be displayed on phage in a correctly folded and functionally active form. Binding of fusion phage to immobilized CNTF alpha-receptor and subsequent elution at low pH resulted in affinity purification of CNTF-displaying virions. Utilization of this technology should enable the selection of high-affinity variants from libraries of CNTF mutants displayed on phage.
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Istituto di Ricerche di Biologia Molecolare P. Angeletti (IRBM), Pomezia (Roma), Italy.
Gloaguen
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Laufer
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D000595:Amino Acid Sequence; D001483:Base Sequence; D001681:Biological Assay; D020934:Ciliary Neurotrophic Factor; D004305:Dose-Response Relationship, Drug; D004797:Enzyme-Linked Immunosorbent Assay; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D017912:Inovirus / Q000235:genetics; D008969:Molecular Sequence Data; D009414:Nerve Growth Factors / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D009419:Nerve Tissue Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D009447:Neuroblastoma; D011485:Protein Binding; D020801:Receptor, Ciliary Neurotrophic Factor; D017475:Receptors, Nerve Growth Factor / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism / Q000494:pharmacology; D014407:Tumor Cells, Cultured
D020934:Ciliary Neurotrophic Factor; D009414:Nerve Growth Factors; D009419:Nerve Tissue Proteins; D020801:Receptor, Ciliary Neurotrophic Factor; D017475:Receptors, Nerve Growth Factor; D011994:Recombinant Proteins