Determining if the fused product of Botox A and GFP can be used to
observe the binding patterns of Botulinum toxin A.
Felicia Yothers
Department of Biological Sciences, York College of Pennsylvania
www.3dchem.com
www.3dchem.com
Introduction
•Known as the most toxic naturally occurring protein on
earth
•Botulinum toxins are used for a variety of therapeutic
uses ranging from headaches to spastic conditions
and cosmetic purposes (Dolly 2003).
Methods
Results
•Had to culture two colonies to determine if the
fluorescent glow could be identified and then sent
out for sequencing
PCR copy of the Botox-gene from a mammalian
vector that has been on ice for quite awhile in
Biochemistry Lab (Thompson 2009)
•Assumed since there was no glow that the
protein did not fold correctly
Run PCR on that gene and checked reaction with
an electrophoresis gel looking for 1800 base pair
line
•Affects peripheral and autonomic nervous systems by
preventing the release of acetylcholine.
•Disrupts neurotransmission and muscle paralysis.
•Discovery of potent small molecule inhibitors of the
botulinum toxin helped scientists learn how the heavy
and light chain worked together and led to the question
of how binding patterns worked (Burnett, J.C. et.al.
2007)
•The question at hand is where does the Botox bind to
the cell; is it glyco-proteins or glyco-lipids or some
other small molecular structure found on the cell
Discussion
•The plate was left out in the cooler room air
•Glow appeared
Purified and combined with an E. coli vector
sequence
•Conclusion was that the product that we made
was temperature sensitive and did not like the 37
degree Celsius incubator
Transformation of that sample and then plated
on ampicillin agar where colonies are grown to
observe green fluorescence
Protein product was extracted and sent out for
DNA sequencing
Figures 1. Electrophoresis gel showing the
ladder and GFP-Botox gene at about 1800 base
pairs to determine the product was isolated.
Ran an SDS- PAGE
•Also we conclude that the PCR copy was ligated
correctly into the plasmid and can be expressed
•The protein can be made and GFP does fold
correctly
•Because the product is stable if it can be purified
from the E. coli culture it can in fact be used to
observe the binding patterns of Botox within
neuronal cells
Ran a Western Blot using an anti-GFP antibody.
Future Research
•Attempting to try the SDS-page and western blot
again to detect the GFP-botox band
Purify from the E. coli using the histidine-tag
through affinity chromatography
•Purify the product using a histidine tag through
column chromatography.
Figure 2. A western blot gel that was overexposed to show a GFP-Botox band, but
unfortunately the SDS-page did not come out
correctly and nothing was detected.
•Taking this new fused product and applying it to
real life situations where botox A binding patterns
need to be understood for possibly cancer or
disease research
•Hopefully in the future we can identified if the
binding of Botox is specific to glyco-lipids or
glyco-proteins or some other unknown complex
Works Cited
Burnett, J.C. et.al. 2007. Inhibition of Metalloprotease
Botulinum Serotype from a Pseudo-peptide Binding Mode to
a Small Molecule that is Active in Primary Neurons. Journal
of Biochemistry. 282: 7: 5004-5014
www.derm.net
Objectives
Determine if the heavy chain of Botox A
and GFP protein can be fused together
Then determine if this fusion product can
be used to observe the binding patterns of
botulinum toxin
MSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPT
LVTTFSYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDGNYKTRAEVKFEG
DTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYITADKQKNGIKANFKIRHNIEDGSV
QLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITHG
MDELYKSGSGPVLAVPSSDPLVQCGGIALGYELYKMRLLSTFTEYIKNIINTSILNLRYESN
HLIDLSRYASKINIGSKVNFDPIDKNQIQLFNLESSKIEVILKNAIVYNSMYENFSTSFWI
RIPKYFNSISLNNEYTIINCMENNSGWKVSLNYGEIIWTLQDTQEIKQRVVFKYSQMIN
ISDYINRWIFVTITNNRLNNSKIYINGRLIDQKPISNLGNIHASNNIMFKLDGCRDTHRY
IWIKYFNLFDKELNEKEIKDLYDNQSNSGILKDFWGDYLQYDKPYYMLNLYDPNKYVD
VNNV
Above figure shows the amino acid sequence of the fused gene of botox and
GFP; showing that the gene was correctly ligated together and did in fact
exhibit a green fluorescent glow. (Green indicates the GFP sequencing and
Red indicates the botox portion with a linker between the two)
Dolly, O. 2003. Synaptic Transmission: Inhibition of
Neurotransmitter Release by Botulinum Toxins. Journal of
Headache. 43: 16-24.
Figure 3. A template of what the SDS-page
should look like once completed correctly and
then subjected to a western blot to focus on
the GFP-Botox gene.
Acknowledgments
I would like to thank Dr. Thompson for his help and
guidance throughout my senior thesis project.