Stable Expression of Recombinant Saxiphilin in
Insect and Yeast Cells for Large Scale Production
and Purification
Niall Mangan,1 Manoj Krishnan2 and Edward Moczydlowski3
Department of Biology, Clarkson University
This research project explores two methods of production of saxiphilin. One method uses
insect cells transfected with the gene for saxiphilin while the other uses transfected yeast cells. In
both methods the protein can be purified using chromatography and tested for through gel
electrophoresis. One of these methods may become a cost effective way of producing saxiphilin.
Researchers have identified saxiphilin as a non-membrane bound binding site for the
toxin saxitoxin and are interested in its applications. Cyanobacteria and dinoflagelletes produce
certain toxins, including saxitoxin which causes paralytic shellfish poisoning in
humans(Landsberg 2002). Humans are at risk for paralytic shellfish poisoning when they
consume organisms such as bivalves which have accumulated large amounts of paralytic toxins.
saxitoxin blocks the sodium channels in neurons, which regulate the electrical charge used to
transmit signals by allowing sodium ions to flow in and out of the cells. Besides causing paralysis
and potentially death by blocking these channels, saxitoxin also presents itself as a useful probe
for scientists exploring the structure and functionality of the channels. During such an experiment
researchers discovered a non-membrane bound binding site for saxitoxin in certain frogs, snakes,
newts and toads, including Rana catesbeina (bullfrog) (Yi et al., 1993). This protein, latter called
saxiphilin for its affinity for saxitoxin, was differentiated from the protein channel binding sites
by comparing its binding affinity with tetrodotoxin, another neurotoxin which did not bind to
saxiphilin (Moczydlowski et al., 1988b). saxiphilin was determined to be a member of the
transferrin family of plasma proteins because of high sequence similarity. Relating saxiphilin to
transferrin gave some insight into its function, perhaps as transport or detoxification protein
(Morabito et al., 1994).
Because it binds to saxitoxin with a high affinity, saxiphilin is a potentially very useful
protein. It could be used to explore saxitoxin and sodium channels further, create an antidote or
1
2008, University studies, Clarkson University, Honors Program, Edward Moczydlowski, oral presentation
Research Associate Department of Biology Clarkson University
3
Professor and Chairman of Department of Biology Clarkson University
2
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toxicity test for saxitoxin. Before any of its applications can be realized an efficient method of
mass producing the protein needs to be created.
The first method for producing saxiphilin using insect cells was chosen because
saxiphilin requires a eukaryotic cellular environment to fold. Prokaryotic cells such as E. coli
have a reductive environment which would have broken the bonds necessary for saxiphilin’s
structure (Morabito, M. A., Llewellyn et al., 1995). Using the InsectSelect System, Sf9 High Five
insect cells were transfected with a pIZ/V5-His vector containing the gene for saxiphilin. Cells
transfected with the gene for saxiphilin are grown as a monolayer. Serum free medium is used to
avoid contamination with serum proteins during purification. They are passaged once every three
days, at which point the supernatant containing the protein was collected and stored.
After harvesting the medium containing saxiphilin, the protein was purified using
chromatography. Agarose beads with nickel binding sites were used as the resin for the first
purification process. The saxiphilin was tagged with a His 6 sequence creating an affinity for
nickel. The saxiphilin bound to the nickel via the His 6 tag and low gradients of Imidazole were
used to wash the column. A final gradient of 150 mM Imidazole displaced the nickel saxiphilin
bonds and the purified saxiphilin was collected. Because other substances besides saxiphilin bind
with the same affinity to Nickel, the sample required further purification. This time Heparin
Sepharose was used as the resin for the column. Increasing levels of NaCl were used to wash the
column. The sample collected at 50 mM NaCl was run through gel electrophoresis produced a
well defined band at about 91 kDa indicating saxiphilin’s presence. There were also faint bands
of contamination at other point on the gel which could have been caused by the high
concentration used to run the gel. To see if further purification can be reached the procedure will
be repeated using a protease inhibitor. This may reduce excess substances in the samples.
Potentially, a second method of production of saxiphilin transfects the saxiphilin gene
into yeast cells. Yeast cells like insect cells have the same eukaryotic slightly oxidative
environment while also reproducing faster than insect cells and costing less. The protein may then
be collected and purified in a similar process as the insect cell produced protein. Gels will be run
to determine the presence and purity of saxiphilin. Success will be measured by the presence of a
band at 91 kDa and lack of other bands indicating purified saxiphilin.
Although the second method using yeast is only in the planning stages of development if
it works it will be a highly efficient system of production. Yeast would be a inexpensive and fast
method of production compared to stable expression in insect cells. If either of these methods
proves to be cost- effective the next project would be to create a saxitoxin test using the saxiphiln.
The risk of paralytic shellfish poisoning, as well as the human desire for shellfish, creates a need
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for some cost-effective method of testing for high levels of saxitoxin. If a fluorescent tag
activated by saxitoxin binding could be attached to saxiphilin and a low cost procedure could be
developed for the manufacture of fluorescently tagged saxiphilin, the result would be very useful
for testing.
References
Landsberg, Jan H. (2002) Effects of Algal Blooms on Aquatic Organisms. Reviews in Fisheries
Science. Ed. Robert R. Stickney. Vol. 10. Issue #2. CRC Press. 191-204.
Li, Y., Llewellyn, L.E., and Moczydlowski, E. (1994) Mol. Pharmacol. 44, 742-748.
Moczydlowski, E., Mahar, J., Ravindran, A. (1988b) Mol. Pharmacol. 33:202-211
Morabito, M.A., and Moczydlowski, E. (1995) Proc. Natl. Acad. Sci. U.S.A. 92, 6651.
Moravito, M.A., Llewellyn, L.E., Mocztdlowski, E.G., 1995. Biochemistry 34, 13027-13033.
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