Transcript Boehmler - York College of Pennsylvania

Cloning and Expression of a Novel GFP:Botox Fusion Protein from a Prokaryotic Expression System
Jessica M. Boehmler* and Jeffrey P. Thompson
Department of Biological Sciences, York College of Pennsylvania
ABSTRACT
OBJECTIVE
Botulinum neurotoxin (botox), found on the list of
potential biological weapons, is the most lethal protein
yet discovered. Research into the protein has shown
that its poisoning domain is structurally and
functionally distinct from its cell-binding domain.
Although binding of botox to neurons is a crucial step
in its poisoning pathway, its binding mechanism
remains unclear. To address these questions, a safe,
useful form of botox was developed. This novel
recombinant protein combines the binding domain of
botox with green fluorescent protein, eliminating the
poisoning component of the toxin. The DNA
sequence for the binding domain of botox serotype A
was fused to the 3’ end of the GFP gene. This
construct was subsequently cloned into a prokaryotic
expression vector, allowing the GFP:Botox A fusion
protein to be expressed in and purified from E. coli.
Characterization of the binding properties of the
purified GFP:BotoxA protein are underway.
To clone the GFP:Botox gene and express the
protein in a prokaryotic expression vector for
characterization of the binding properties of
GFP:Botox
INTRODUCTION
The botulinum toxin is a nerve-toxin produced
by the anaerobic, Gram-positive, spore-forming
bacteria Clostridium botulinum. In the United States
approximately 110 cases of botulism are reported
every year. These cases are caused by ingestion of
contaminated food, inhalation of spores or wound
infection by the bacteria (Botulism 2001). More
recently, however, the botulinum toxin has become
widely feared for its potential use as a biological
weapon. The toxin is a polypeptide that is composed
of a heavy chain and a light chained linked by a
disulfide bond (Davis 1993) (Figure 1). The
carboxyterminus of the heavy chain contains the
receptor-binding site, which allows for the attachment
of the entire polypeptide to its target cell. The
aminoterminus portion of the heavy chain consists of
a domain responsible for channel formation that
allows the polypeptide to enter the target cell (Davis
1993). The toxic portion is in the light chain. Since
the toxin survives digestion and translocates from the
digestive system into the bloodstream intact, previous
research has shown that an inactive form of the toxin
may be used as an oral vaccine to raise antibodies
against the botulinum toxin (Kiyatkin et al 1997). By
using a fusion protein containing the carboxyterminus
of the heavy chain and different antigenic proteins, it
may be possible to elicit an immune response to a
variety of antigens through oral administration of this
fusion protein.
Figure 1.
METHODS
2. Ligated the phosphorylated PCR product into a
linearized, dephosphorylated pQE30 vector, a
prokaryotic expression vector.
2
3
2.0 kbp
1.6 kbp
3. Transformed prokaryotic vector containing
GFP:Botox gene into Top 10 F’ E. coli cells (Figure
3).
Figure 4. Product from PCR performed to verify presence of the
GFP:Botox gene in the transformed glowing cells. Lane 1 contains
1 kbp ladder. Lane 2 contains the empty pQE30 vector used as a
control for size. Lane 3 contains PCR product. Bright band of
GFP:Botox running at about 2kbp.
4. Isolated plasmid DNA containing the GFP:Botox
gene from broth culture.
Figure 5.
1
5. Used PCR to verify the presence and size of the
GFP:Botox gene (Figure 4).
3
4
The fusion protein GFP:Botox, was successfully
cloned and expressed in E. coli cells. After isolation
of the plasmid DNA from a sample of the E. coli
culture, the GFP:Botox gene appeared to be in the
plasmid and intact. Both PCR and a restriction digest
verified the presence of the gene and its proper size.
However, after running the GFP:Botox protein on an
SDS-PAGE, the size of the molecule was
approximately 20 kDa smaller than expected. The
DNA sample was then sent out to Elim
Biopharmaceutical Inc. for automated DNA
sequencing to determine if a frame-shift mutation had
resulted in the occurrence of a stop codon within the
gene. Analysis showed the DNA sequence to be
correct and free of mutations. The inconsistency in
running size of the protein could be the result of
residual tertiary structure prior to electrophoresis,
making it appear to be of smaller molecular weight.
FUTURE DIRECTIONS
1.0 kbp
Further characterization of the GFP:Botox
protein is needed to identify its accurate size. The
binding characteristics of this fusion protein also need
to be examined. Using this protein, the binding
mechanism of the botulinum toxin can be better
understood with the presence of the GFP tag allowing
for facilitated visualization of the absorbance of the
protein and its location within the cell. Oral
administration of this protein to mice will allow for the
determination of whether this fusion protein is able to
escape digestion as is possible with the holotoxin. By
escaping digestion this form of the botulinum toxin
should theoretically allow for an immune response to
the GFP attached to it, resulting in the formation of
anti-GFP antibodies. With the further development
and characterization of this protein, its use as a
carrier molecule for oral vaccines to various antigens
can be achieved. This will allow a less painful and
more widespread distribution of medical care.
0.5 kbp
LITERATURE CITED
6. Restriction digested the isolated plasmid with
HindIII to verify presence of gene (Figure 5).
7. Extracted the protein from the E. coli and used
affinity chromatography to isolate the GFP:Botox
protein using its aminoterminus 6XHis tag.
8. Ran protein sample on SDS-PAGE to verify the
size of the protein (Figure 6).
RESULTS
Figure 2.
1
3.0 kbp
2.0 kbp
1.6 kbp
2
4.0 kbp
3.0 kbp
2.0 kbp
1.6 kbp
Figure 5. Product from restriction digest of pQE30 vector
containing GFP:Botox gene with HindIII. Botox gene itself has 2
internal HindIII sites at 297 bp and and 2347bp. Lane 1 contains 1
kb ladder. Lane 3 contains plasmid digested with HindIII. There are
2 bands of digested plasmid running at about 2.0 and 3.6 kbp
which was consistent with expected band size. Lane 4 contains
undigested plasmid.
Figure 6.
GFP:Botox
Figure 2. Lane 1 contains 1 kbp ladder. Lane 2 contains PCR
product run on 1% agarose electrophoresis gel. Band running at
about 2 kbp which was the predicted size for the GFP-Botox gene.
1
75 kDa
2
3
4
GFP:
Botox
50 kDa
Figure 3.
2001. Botulism. Available from:
http://www.cdc.gov/ncidod/dbmd/diseaseinfo/botulism_g.htm#What
%20is%20botulism. Accessed 31 March 2003.
Davis, Larry E. Botulinum toxin: from poison to medicine. The
Western Journal of Medicine 158:25-30.
Kiyatkin, Nikita, Maksymowych, Andrew B., and Simpson, Lance L.
1997. Induction of an immune response by oral administration of
recombinant botulinum toxin. Infection and Immunity 65:45864591.
S
S
Figure 1. Schematic diagram of botulinum toxin structure.
1
1. Used PCR to amplify the GFP:Botox gene out of a
mammalian expression vector (Figure 2).
Light chain
N-terminus
C-terminus
of heavy chain
DISCUSSION
Figure 4.
Figure 3. E. coli cells expressing the GFP:Botox protein after
successful transformation of the pQE30 vector containing the
GFP:Botox insert. Photograph taken using fluorescence
microscopy using a FITC filter at 100X magnification.
Figure 6. SDS-PAGE of GFP:Botox fusion protein to verify correct
size of the protein. Gel stained with Coomassie blue to visualize
bands of protein. Lane 1 contains standard ladder. Lane 2
contains the protein from a prokaryotic expression vector
containing an unrelated gene as a negative control. Lane 3
contains the extracted soluble protein. Lane 4 contains the eluted
GFP:Botox protein. Size of GFP:Botox protein was about 65 kDa.
Predicted size of the fusion protein was approximately 83 kDa.
Artwork:
http://qiagen.com/literature/pqesequences/pQE-30_UA.pdf
http://www.biochemtech.uni-halle.de/PPS2/projects/jonda/structur.htm
ACKNOWLEDGEMENTS
This research was funded by a grant from the Pennsylvania
Academy of Science.