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An Enhanced Green Fluorescence Protein-based Assay for Studying Neurite Outgrowth in Primary Neurons
Published on: October 19, 2019
Molecular cloning, functional expression and characterization of p15, a novel fungal protein with potent
S Wakatsuki1, T Yokoyama, S Nakashima
1Department of Biotechnology, University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, 113-8657, Tokyo, Japan.
Abstract:
p15 is a novel fungal protein which induces neurite outgrowth and neuronal differentiation of PC12 cells. In the present study, we report molecular cloning, functional expression and characterization of the gene encoding p15. The deduced amino acid sequence suggested that p15 is synthesized as a precursor with 31 extra amino-terminal amino acids including a putative signal sequence, and 20 carboxy-terminal amino acids, in addition to the 118 amino acids-long mature region with neurite-inducing activity. From the poly(A)(+) RNA prepared from the producing fungal strain, a cDNA fragment encoding the mature region of p15 was amplified and His(6)-tagged recombinant p15 was produced in Escherichia coli. The recombinant protein purified by a single step on Ni(2+) agarose column chromatography exhibited comparable specific activity as native p15 in the PC12 neurite extension assay. The effect of His(6)-p15 was blocked by nicardipine, suggesting that Ca(2+) influx through the L-type Ca(2+) channels is essential for its neurite-inducing activity. In addition, mutational analysis of His(6)-p15 demonstrated that both intramolecular disulfide bonds are essential for its biological activity.
Insights
A novel fungal protein, p15, promotes neurite outgrowth and neuronal differentiation in PC12 cells. Its activity relies on calcium influx via L-type channels and essential intramolecular disulfide bonds.
Area of Science:
- Neuroscience
- Molecular Biology
- Protein Biochemistry
Background:
- p15 is a novel fungal protein.
- p15 induces neurite outgrowth and neuronal differentiation in PC12 cells.
Purpose of the Study:
- To report the molecular cloning, functional expression, and characterization of the gene encoding p15.
- To investigate the mechanism underlying p15's neurite-inducing activity.
Main Methods:
- Molecular cloning and functional expression of the p15 gene in E. coli.
- Purification of His(6)-tagged recombinant p15.
- PC12 neurite extension assay.
- Inhibition studies with nicardipine.
- Mutational analysis of p15.
Main Results:
- Recombinant p15 exhibited comparable activity to native p15.
- Nicardipine blocked p15's effect, indicating essential Ca(2+) influx through L-type channels.
- Intramolecular disulfide bonds were found to be essential for p15's biological activity.
Conclusions:
- The gene encoding p15 was successfully cloned and expressed.
- p15's neurite-inducing activity is dependent on calcium influx and disulfide bonds.
- p15 represents a potential therapeutic agent for neuronal regeneration.
