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Updated: Jun 18, 2026
Expression, Isolation, and Purification of Soluble and Insoluble Biotinylated Proteins for Nerve Tissue Regeneration
Published on: January 22, 2014
Expression of recombinant rat ciliary neurotrophic factor in Escherichia coli and its synergy with basic fibroblast
1Department of Cell, Molecular and Structural Biology, Northwestern University Medical and Dental Schools, 303 East Chicago Avenue, Chicago, Illinois 60611, USA.
Abstract:
Rat CNTF was expressed in Escherichia coli using a T7 RNA polymerase vector system. rCNTF was obtained as an insoluble aggregate at levels approaching 70% of total E. coli protein. After extraction, renaturation, and purification by anion-exchange chromatography and gel filtration the yield of biologically active rCNTF was nearly 0.5 mg/ml of E. coli culture broth. The effects of rCNTF and bFGF on cultured chick ciliary neurons were compared. bFGF was primarily neuritogenic and did not support ciliary neuron survival beyond 2 days in culture. rCNTF induced neuritogenesis more slowly and supported ciliary neuron survival in culture for a longer period. bFGF strongly potentiated the effect of rCNTF. Without bFGF, the ED(50) for rCNTF was 102 +/- 25 pg/ml. With bFGF, the potency of rCNTF increased to an ED(50) of 42 +/- 6 pg/ml. Chick nodose neurons also responded to rCNTF and their response was potentiated by bFGF. Thus, bFGF modulates the response of ciliary and nodose neurons to CNTF in vitro and may have a similar effect in vivo.
