1. Design and Production of a GFP and Human IL-13 Linked Chimeric Protein in E. coli Using pQE-30 Vector Stephen R. Suknaic Department of Biology, York College of Pennsylvania Introduction Interleukin 13 (IL-13) is an immuno- regulatory cytokine that has been a useful tool in breast and brain cancer research (Kawakami 2004). This protein is a ligand for the surface receptors IL- 13R α 1 and IL-13R α 2. These receptors are effective targets for cancer research due to their over-expression on the surface of certain cancer cell types. IL-13 ligand has been experimentally attached to other proteins to produce a new fusion or ‘chimera’ protein. By successfully developing a fused protein consisting of more than one active part, researchers have been able to successfully develop a single protein with multiple functions and potential uses. A prime example of this is a fusion of IL-13 and Pseudomonas exotoxin A, which can carry a toxic molecule to a cell expressing the IL-13 receptor. The primary objective of this experiment is to see if IL-13 could be successfully fused with Green Fluorescent protein to create a green glowing chimera protein containing the IL-13 ligand. Such a protein would have many theoretical uses:  Competition assays with drugs targeting the IL-13R α 2 Receptor  Quantification of the number of IL-13 receptors on a cell membrane  Luminescence can be used to identify tumor locations or sizes Primary Objectives I. Integrate modified DNA sequences for GFP and IL-13 into pQE-30 vector II. Transform said vector into competent E. coli and induce protein production with IPTG III. Isolate chimera and analyze it for proper biological activity Methods Results The following amino acid sequence was inferred from the final pQE-30 DNA sequence obtained from Elim Biopharmaceuticals:  Histidine Tag presence and function was verified by testing the chimera for affinity to Nickel- ion beads using IMAC  Enterokinase Cut Site presence was confirmed by treatment with Enterokinase enzyme to release the protein from the beads  GFP presence was confirmed by the characteristic bright green glow  Linker Region presence was confirmed by the DNA sequencing  Factor Xa Cut Site presence was confirmed by treatment of the chimera with Factor Xa enzyme  IL-13 presence was supported by the polyacrylamide gel run after the Factor Xa enzyme treatment Conclusions  All the amino acid codons for GFP and IL-13 were properly integrated in sequence into the pQE-30 vector  Modified pQE-30 vector was successfully created and used to produce chimera  The proper structure of this protein was confirmed by protease digests, Ion- affinity chromatography, and presence of the characteristic glow of Green Fluorescent Protein Take Home Message  The GFP and IL-13 chimera was successfully made using the pQE-30 vector as a means to transform E. coli  The components of this protein were tested to confirm their presence beyond just their amino acid sequence  Future studies could test to see if the chimera will bind effectively to the IL-13 α 1 and/or α 2 receptor Works Cited Kawakami K, Kawakami M, Puri RK. Specifically targeted killing of interleukin-13 (IL-13) receptor-expressing breast cancer by IL-13 fusion cytotoxin in animal model of human disease. Molecular Cancer Theory. 2004 Feb;3(2):137-47. Kawakami K, Kawakami M, Puri RK. IL-13 receptor- targeted cytotoxin cancer therapy leads to complete eradication of tumors with the aid of phagocytic cells in nude mice model of human cancer. Journal of Immuonology. 2002 Dec 15;169(12):7119-26 http://www.ncbi.nlm.nih.gov/sites/entrez?db=gene&cmd=S howDetailView&TermToSearch=3596 http://www.conncoll.edu/ccacad/zimmer/GFP- ww/structure.html http://www.rcsb.org/pdb/explore/images.do?structureId=1I JZ http://www1.qiagen.com/Products/Protein/Expression/QIAe xpressExpressionSystem/pQE-30XaVector.aspx?ShowInfo=1 Acknowledgements I would like to thank Dr. Thompson for his guidance, wisdom and support throughout the duration of this project. I would also like to thank the entire York College Biology Faculty Staff for any help or suggestions that they have made with regards to this project. His-Tag and Entero-kinase Cut Site IL-13GFP Linker Region and Factor Xa Cut Site This project began with the design of a protein with the following structure: The following timeline is a summary of the protocols involved with this experiment: Figure 1: Structure of the Quiagen pQE-30 Vector Structure of GFP Structure of IL-13 mrgshhhhhhgsacddddkaskgeelftgvvpilveldgdvnghkfsvs gegegdatygkltlkficttgklpvpwptlvttfsygvqcfsrypdhmkrhdf fksampegyvqertisfkddgnyktraevkfegdtlvnrielkgidfkedg nilghkleynynshnvyitadkqkngikanfkirhniedgsvqladhyqqn tpigdgpvllpdnhylstqsalskdpnekrdhmvllefvtaagithgmdel ykgtggsgggiegrasspgpvppstalrelieelvnitqnqkaplcngsm vwsinltagmycaaleslinvsgcsaiektqrmlsgfcphkvsagqfsslhv rdtkievaqfvkdlllhlkklfregrfn Histidine TagEnterokinase Cut Site GFPLinker Region Factor Xa Cut SiteIL-13 Figure 2: Polyacrylamide gel showing the intact chimera as well as after digestion with Factor Xa enzyme. The IL-13 that was digested is running at ~12 kD. Lane 1 contained standard kaleidoscope ladder. Lane 2 contained only the purified chimera with a mass of ~42kD. Lane 3 contained the chimera digested with Factor Xa enzyme. Lane 4 contained only the Factor Xa enzyme. 1 2 3 4 IL-13 ~12 kd Factor Xa enzyme kD 50 37 25 20 15 10 Intact Chimera ~42 kD GFP and Linker ~30 kd