Recombinant Human and Rat Ciliary Neurotrophic Factors

Recombinant Human and Rat Ciliary Neurotrophic Factors The human ciliary neurotrophic factor (CNTF) gene was identified and cloned, based on homology with the recently cloned rat cDNA. The gene encodes a protein of 200 amino acids, which shares about 80% sequence identity with rat and rabbit CNTF and, like these homologues, lacks an apparent secretion signal sequence. The human CNTF gene, like the rat gene, appears to contain a single intron separating two protein coding exons. An intronless human CNTF gene was constructed by the use of polymerase chain reactions and introduced into vectors designed for expression of foreign proteins in E. coli. The rat CNTF gene was also introduced into similar vectors. Both the human and rat proteins were expressed at exceptionally high levels, at 20–40% and 60–70% of total protein, respectively. Extraction of the recombinant proteins from inclusion bodies by guanidinium chloride, followed by two column chromatography steps, produced high yields of pure CNTF that supported survival and neurite outgrowth from embryonic chick ciliary neurons in culture. The biological activity of both recombinant proteins was comparable to that of native rat CNTF. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Neurochemistry Wiley

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Publisher
Wiley
Copyright
Copyright © 1991 Wiley Subscription Services, Inc., A Wiley Company
ISSN
0022-3042
eISSN
1471-4159
DOI
10.1111/j.1471-4159.1991.tb08250.x
Publisher site
See Article on Publisher Site

Abstract

The human ciliary neurotrophic factor (CNTF) gene was identified and cloned, based on homology with the recently cloned rat cDNA. The gene encodes a protein of 200 amino acids, which shares about 80% sequence identity with rat and rabbit CNTF and, like these homologues, lacks an apparent secretion signal sequence. The human CNTF gene, like the rat gene, appears to contain a single intron separating two protein coding exons. An intronless human CNTF gene was constructed by the use of polymerase chain reactions and introduced into vectors designed for expression of foreign proteins in E. coli. The rat CNTF gene was also introduced into similar vectors. Both the human and rat proteins were expressed at exceptionally high levels, at 20–40% and 60–70% of total protein, respectively. Extraction of the recombinant proteins from inclusion bodies by guanidinium chloride, followed by two column chromatography steps, produced high yields of pure CNTF that supported survival and neurite outgrowth from embryonic chick ciliary neurons in culture. The biological activity of both recombinant proteins was comparable to that of native rat CNTF.

Journal

Journal of NeurochemistryWiley

Published: Sep 1, 1991

References

  • Purification of the chick eye ciliary neuronotrophic factor
    Barbin, Barbin; Manthorpe, Manthorpe; Varon, Varon
  • The nerve growth factor: biochemistry, synthesis, and mechanism of action
    Greene, Greene; Shooter, Shooter
  • Ciliary neurotrophic factor induces cholinergic differentiation of rat sympathetic neurons in culture
    Saadat, Saadat; Sendtner, Sendtner; Rohrer, Rohrer
  • Neurotrophic molecules
    Snider, Snider; Johnson, Johnson

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