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Recombinant human and rat ciliary neurotrophic factors
P Masiakowski1, H X Liu, C Radziejewski
1Regeneron Pharmaceuticals Inc., Tarrytown, New York.
Journal of Neurochemistry
|September 1, 1991
Summary
Researchers cloned the human ciliary neurotrophic factor (CNTF) gene and expressed it in E. coli. Recombinant human and rat CNTF proteins showed high expression and biological activity, supporting neuronal survival and growth.
Area of Science:
- Molecular Biology
- Neuroscience
- Biotechnology
Background:
- The human ciliary neurotrophic factor (CNTF) gene was identified and cloned through homology with rat cDNA.
- Human CNTF shares high sequence identity with rat and rabbit CNTF, lacking a secretion signal sequence.
- Both human and rat CNTF genes contain a single intron separating two protein-coding exons.
Purpose of the Study:
- To clone and express the human CNTF gene.
- To investigate the expression levels and biological activity of recombinant human and rat CNTF.
- To produce purified CNTF for potential therapeutic applications in neuronal regeneration.
Main Methods:
- Polymerase chain reaction (PCR) was used to construct an intronless human CNTF gene.
- Genes were inserted into E. coli expression vectors.
- Recombinant proteins were expressed, extracted from inclusion bodies, and purified using chromatography.
Main Results:
- High-level expression of both human (20-40%) and rat (60-70%) CNTF in E. coli.
- Purified recombinant CNTF supported survival and neurite outgrowth of embryonic chick ciliary neurons.
- Biological activity of recombinant CNTF was comparable to native rat CNTF.
Conclusions:
- The human CNTF gene can be successfully cloned and expressed in E. coli.
- Recombinant human and rat CNTF possess significant biological activity.
- This study provides a method for producing pure, biologically active CNTF for further research and therapeutic development.