Recombinant Human and Rat Ciliary Neurotrophic Factors

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.

Trophic Support of Motoneurons: Physiological, Pathophysiological, and Therapeutic Implications

“…The results obtained with this enriched preparation could later be confirmed by CNTF purified to homogeneity and recombinant CNTF (3,59,93,94).…”

“…That CNTF has a direct action on motoneurons could be deduced from the fact that the survival effect in single motoneuron cultures was identical to that in 80% pure low-density cultures (94) . The cloning of CNTF was the prerequisite for producing large quantities of recombinant CNTF (49,59,100) and for the determination of its developmental expression and cellular localization (20,27,86,94,97,100,101). These investigations demonstrated that CNTF is expressed postnatally in myelinating Schwann cells (27,86,97,100,101) and a subpopulation of type I astrocytes (20,101) in the CNS, that CNTF is a cytosolic molecule, and that the biological activity, e.g., in the adult sciatic nerve, is more than 10,000 _ times higher than that of (retrogradely) transported NGF in the rat sciatic nerve (35,97,98).…”

Glycinergic and GABAergic Synaptic Activity Differentially Regulate Motoneuron Survival and Skeletal Muscle Innervation

“…Cells were grown with 10% horse serum in Neurobasal medium supplemented with B27 and 500 mM glutamax I (Invitrogen, Eggensten, Germany) at 37°C in a humidified atmosphere with 5% CO 2 . After 1 h, the Neurobasal medium was supplemented with one of the following neurotrophic factors at a concentration of 1 ng/ml: glia-derived neurotrophic factor (GDNF) (PreProtech, Frankfurt, Germany); brain-derived neurotrophic factor (BDNF) (Amgen, Thousand Oaks, CA); and ciliary neurotrophic factor (CNTF) (rat and human expressed and purified as described by Masiakowski et al, 1991). The initial cell number per well was determined 3 h after plating by phase-contrast microscopy (magnification, 125ϫ).…”

Hepatocyte Growth Factor Promotes Motor Neuron Survival and Synergizes with Ciliary Neurotrophic Factor

“…Trophic Factors and Vincristine-Recombinant rat CNTF was prepared as described (26). HGF and vincristine were purchased from R&D Systems and Sigma, respectively.…”