1. A Study of a Peptide Secretion Signal Addition to Human Interleukin 13 Receptor Alpha 2 and Green Fluorescent Protein Jacqueline Freebery and Jeffrey P. Thompson, Ph.D. Department of Biology, York College of Pennsylvania, York, PA 17405 INTRODUCTION METHODS RESULTS DISCUSSION CONCLUSION LITERATURE CITED ACKNOWLEDGEMENTS OBJECTIVES HYPOTHESES Eukaryotic cells transcribe their DNA into mRNA in the nucleus; the mRNA translation to amino acids is accomplished in the cytoplasm at the ribosome. Protein trafficking is determined in some eukaryotes by peptide signals contained within proteins that target their end locations. The majority of secreted proteins are secreted by a secretory pathway that is directed by secretory signal peptides (Barash et. al. 2002). A Hidden Markov Model (HMM) has been computer generated to determine artificial signal peptide sequences; using known human signal peptides as a training set, it functions to predict, identify, and generate signal peptides that direct strong protein secretion and expression (Barash et. al. 2002). Human Interleukin 13 Receptor Alpha 2 (IL13Rα2) is significant in tumor biology because it binds and internalizes the Interleukin 13 Ligand with higher affinity than the IL13Rα1 without mediating signal transduction (Kawakami et. al. 2003). Green Fluorescent Protein (GFP) is a useful tool for protein targeting in living cells. Engineering of GFP into chimeric proteins is valuable in biochemical research. The Ligase Chain Reaction was successful in creating the primer coding for the secretion signal. All four plasmids were successfully created. Initial transfections of U-87MG cells were successful. Cell death occurred in U-87MG transfectants with plasmids containing GFP containing. U-87MG transfectants with IL13Rα2-sec produce IL13Rα2. U-87MG transfectants with IL13Rα2-sec secrete IL13Rα2. However, the CRP10 cell line produces and secretes a greater amount of IL13Rα2. Molecular weights of all positive results in the comparative western blot are heavier than the weight of the protein portion of IL13Rα2, indicating that post- translational events have occurred. Obtain IL13Rα2 in a secretable form so that it is manufactured by the cell and emitted into surrounding media instead of staying attached to the cell membrane or trapped in the cytoplasm. Determine whether the addition of SP-HMM34 peptide secretion sequence to IL13Rα2 and GFP will enhance their secretion into the media. H 1 : The addition of SP-HMM34 peptide secretion signal to IL13Rα2 and GFP will enhance their secretion into their surrounding media. H 0 : The addition of SP-HMM34 peptide secretion signal to IL13Rα2 and GFP will not enhance their secretion into their surrounding media. I would like to thank Dr. Thompson for his continued guidance and support throughout this study. Barash, S., Wang, W., Shi, Y. Human secretory signal peptide description by hidden Markov model and generation of a strong artificial peptide for secreted protein expression. Biochemical and Biophysical Research Communications 2002. 294: 835-842. Kahlon, K.S., Brown, C., Cooper, L.J., Raubitschek, A., Forman, S.J., and Jensen, M.C. 2004. Specific recognition and killing of glioblastoma multiforme by interleukin 13-zetakine redirected cytolytic T cells. Cancer Res. 24:9160-9166. Kawakami, K., Kawakami, M., Husain, S.R., Puri, R.K. 2003. Potent antitumor activity of IL-13 cytotoxin in human pancreatic tumors engineered to express IL- 13 receptor 2 chain in vivo. Gene therapy [serial online] 10:1116-1128. Available from: http://www.nature.com/gt/journal/v10/n13/full/3301956a.htmlhttp://www.nature.com/gt/journal/v10/n13/full/3301956a.html Tsien, R.Y. 1998. The Green Fluorescent Protein. Annual Review of Biochemistry. 67: 509-544. Primer Design LCR PCR Plasmid Restriction Digestion Ligation Reaction Transformation in E. coli Plasmid Purification Nucleotide Sequencing Stable Transfection of U-87MG Cell Line Plasmid with IL13Rα2-sec Plasmid with GFP-sec Plasmid with GFP alone Empty Plasmid Media and Cell Analysis Affinity Chromatography SDS-PAGE Western Blot Analysis The addition of the SP-HMM34 peptide secretion signal to IL13Rα2 does not enhance secretion into surrounding media. Therefore, the initial hypothesis, H 1, is rejected, and H 0 is accepted. The effect of the SP-HMM34 peptide secretion signal to GFP is unknown due to the unforeseeable occurrence of cell death in the U-87MG transfectants with GFP. A repeat study with the addition of SP-HMM34 to GFP is needed to determine a definite conclusion. HMM Secretion Sequence Selection Figure 5. Western Blot Analysis showing positive results for positive control (2), IL13Rα2-sec cell pellet (6), CRP5 cell pellet (8), CRP10 media (9), and CRP10 cell pellet (10). Negative results are shown for blasticidin resistant media (3) and cell pellet (4), IL13Rα2-sec media (5), and CRP5 media (7). Figure 3. Gel Electrophoresis showing 100 BP ladder (1) and 500 BP Ladder (2), followed by plasmid digestion with endonucleases AgeI and BspHI to show SPHMM34 secretion fragment (3). Figure 4. Western Blot Analysis showing positive results for positive control (6), and IL13Rα2-sec transfectant media (8) and cell pellet (10). Negative results are shown for blasticidin resistant transfect media (7) and cell pellet (9). Figure 1. SP-HMM34 peptide sequence and back translation for encoding cDNA sequence MRPTWAWWLFLVLLLALWAPARG ATG CGC CCC ACC TGG GCC TGG TGG CTG TTC CGT GTG CTG CTG CTG GCC CTG TGG GCC CCC GCC CGC GGC http://cancerres.aacrjournals.org/content/vol64/issue24/cover.shtml http://expasy.org/spotlight/back_issues/2001/06/the_greenest_of.shtmlGFP Figure 2. Plasmid used to create proteins containing SP-HMM34 Figure 6. Shows standard curve to determine the molecular weights of standards. Sample Migration Distance (mm) LOG of the Molecular Weight Molecular Weight (kD) IL13Rα2-sec Cell Pellet5.31.6040.01 CRP5 Cell Pellet4.81.7049.67 CRP10 Media4.71.7151.86 CRP10 Cell Pellet4.81.7049.67 Figure 7. Shows data for measured migration distances (mm) of positive results from comparative western blot along with molecular weight calculations from linear regression equation. 1 2 3 4 5 6 7 8 9 10 1 2 3 l 10 9 8 7 6 5