Presentation on theme: "Construction, Transformation, and Prokaryote Expression of a Fused GFP and Mutant Human IL-13 Gene Sequence Lindsay Venditti, Department of Biological."— Presentation transcript:
Construction, Transformation, and Prokaryote Expression of a Fused GFP and Mutant Human IL-13 Gene Sequence Lindsay Venditti, Department of Biological Sciences Introduction The word tumor is a universal term for a new growth of tissue (Sloan-Kettering, 2002). Glioblastoma multiforme, a brain tumor, is a high-grade glioma that originates in the supportive cells of the brain. They are the most common malignant brain tumors in adults and most resistant to treatment. Large amounts of IL-13R alpha 2 receptors are found on the surface of Glioblastoma multiforme cells (Debinski and Thompson 1999). Normal tissue has an alternative form of human interleukin 13 receptor on the cell surface. A mutant called hIL-13.E13C lost its ability to interact with the interleukin 13/4 receptor of normal tissue and it had enhanced ability to interact with the interleukin 13 receptor on the brain tumor tissue. Using a mutant that interacts with IL-13 R alpha 2 receptors would allow for specific cancer treatments using a tumor targeted approach. This mutant is not naturally made in our bodies and would have to be manufactured in a laboratory setting by having a culture of cells produce the protein with subsequent extraction and purification from the culture. The gene sequence of the mutant hIL-13.E13C was generated (Thompson and Debinski 1999) with hopes to accomplish two goals. 1. Create a GFP/hIL-13.E13C vector and clone into a pBAD plasmid. Then insert into Top-10 E. coli. 2. Force expression and extraction of this protein from E. coli broth. Methods 1. Design primers for PCR amplification of hIL-13 gene 2. PCR amplification of DNA with primers 3. Ligation with pBAD vector containing GFP 4. Purify sample 5. Transform into Top Ten E. coli 6. Plate on Amp + plate and L- Arabinose 7. Choose colonies that glow green under UV light 8. Grow colonies in LB broth and induce expression of protein 9. Lyse and digest cell cultures with 8M urea 10. All purified through Ni agarose beads 11. Digestion or purification with: a. Glycerin b. NB-40 c. Factor Xa enzyme 12.SDS-PAGE and coomassie stain of proteins 13. Western blot for detection of hIL-13 proteins Acknowledgement Dr. Thompson Results Figure 3. SDS PAGE Gel showing standards with lanes of enzyme(3), cut(4) and uncut(5) protein and GFP (6)(control) Figure 4. SDS PAGE Gel showing initial, flow through and elute of NB-40(2,3,4) Glycerin (5,6,7) unpurified (8) Figure 1. PCR amplification of hIL-13 Figure 2. Digestion of clones after transformation 1. PCR amplification of hIL-13/GFP DNA segment accurately done 2. Ligation into pBAD vector made with cut sites 3.Plasmid was successful in being transferred into Top Ten E. coli and expressed by L- arabinose 4.Popping cells and purification of protein sample was shown successful through SDS PAGE gel 5. Purification of hIL-13/GFP for western blot using indirect detection was not successful Discussion The GFP/hIL-13 was able to be amplified with the primers by PCR (Figure 1). This sample was then able to be ligated into a pBAD vector to create a plasmid that was transformed in to E. coli. The colonies that grew and glowed were selected and grown in LB broth. The pBAD promoter was induced by L-arabinose and this allowed the GFP/hIL-13 to be transcribed and translated. The cultures were screened for GFP/hIL-13 presence by glowing green under UV light. The clones were also popped and digested for extraction of the hIL- 13 (Figure 2). The protein had a HIS tag preceding the GFP/hIL-13 that had a high affinity for Ni agarose beads. This was used for purification. The purified protein was run on a SDS PAGE gel and stained for the presence of protein ~42 kDa. Confirming tests were by cutting the protein with factor Xa which is an protease that cleaves the hIl-13 from the GFP (Figure 3). This worked and the samples were then purified with either NB-40 (lanes 2,3,4) or Glycerin (lanes 5,6,7) to remove residual proteins. Glycerin yielded less residual bands than NB-40 in a SDS PAGE gel (Figure 4). The sample did not appear at ~42 kDa when using a western blot by indirect detection. Future work 1.Purification of GFP/hIL-13 2. Western blot to confirm presence of GFP/hIL-13 3. Induce correct folding pattern of GFP/hIL-13 Abstract Tumors affect many people and Glioblastoma multiform is one that affects the brain. Large amounts of IL-13 receptor alpha 2 protein cover the surface of these cells while normal cells do not possess this protein in great amounts. A tumor targeted approach of killing these cells using modified hIL-13 as a vehicle has been effective. IL-13 combined with GFP was cloned into a prokaryote system for the expression of this protein. GFP was used in combination with the mutated hIL-13 because it will fluoresce under UV light and it could be used as a potential diagnostic fluorescent tumor marker. The protein was then expressed, extracted, and purified for GFP/hIL-13. SDS –PAGE gels of this protein and a diagnostic protease digestion proved the protein was present. A western blot however did not confirm the presence of GFP/hIL-13. Lit Cited Debinski W and Thompson JP. Retargeting interleukin 13 for radioimmunodetection and radioimmunotherapy of human high-grade gliomas. Clin Cancer Res 5: 3143s-3147s,1999. Thompson JP and Debinski W. Mutants of interleukin 13 with altered reactivity towards interleukin 13 receptors. J Biol Chem 274: 29944- 29950, 1999. 2002. Sloan-Kettering – Brain and Spinal Cord Cancers: Overview. Available from: http://www.mskcc.ord/mskcc/html/268.cfmhttp://www.mskcc.ord/mskcc/html/268.cfm 1. 2. 3. 4. 5. 6. 1. 2. 3. 4. 5. 6. 7. 8.