Jessica M. Boehmler* and Jeffrey P. Thompson Department of Biological Sciences, York College of Pennsylvania ABSTRACT 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. 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. OBJECTIVE Figure 1. Figure 1. Schematic diagram of botulinum toxin structure. METHODS 1.Used PCR to amplify the GFP:Botox gene out of a mammalian expression vector (Figure 2). 2.Ligated the phosphorylated PCR product into a linearized, dephosphorylated pQE30 vector, a prokaryotic expression vector. 3.Transformed prokaryotic vector containing GFP:Botox gene into Top 10 F’ E. coli cells (Figure 3). 4.Isolated plasmid DNA containing the GFP:Botox gene from broth culture. 5.Used PCR to verify the presence and size of the GFP:Botox gene (Figure 4). 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). Artwork: Figure 2. 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. Figure 4. RESULTS Cloning and Expression of a Novel GFP:Botox Fusion Protein from a Prokaryotic Expression System To clone the GFP:Botox gene and express the protein in a prokaryotic expression vector for characterization of the binding properties of GFP:Botox Figure 3. 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. 1.6 kbp 0.5 kbp 1.0 kbp 2.0 kbp 3.0 kbp GFP:Botox 12 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. Figure 5. 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. DISCUSSION FUTURE DIRECTIONS Figure 6. 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. 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. 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. LITERATURE CITED kbp 2.0 kbp 1.6 kbp kbp 3.0 kbp 4.0 kbp 24 GFP: Botox 50 kDa 75 kDa Botulism. Available from: %20is%20botulism. Accessed 31 March Davis, Larry E. Botulinum toxin: from poison to medicine. The Western Journal of Medicine 158: Kiyatkin, Nikita, Maksymowych, Andrew B., and Simpson, Lance L Induction of an immune response by oral administration of recombinant botulinum toxin. Infection and Immunity 65: ACKNOWLEDGEMENTS This research was funded by a grant from the Pennsylvania Academy of Science Light chain N-terminus C-terminus of heavy chain S S