1. Manufacture of Human Interleukin 13 Protein Using a Prokaryotic Expression System Ryan Rupp, York College of Pennsylvania, Department of Biological Sciences Abstract Previous research has shown the ability to mutate the human interleukin 13 (hIL13) protein at the 13 th position of  - helix A without degradation in structure. The mutated protein diminishes binding affinities for normal cell IL4/IL13 receptors, but increases binding affinities for cancer cell IL13 receptors (Thompson and Debinski 1999). Amplification of the intron-less DNA sequence from a eukaryotic gene was completed using a polymerase chain reaction. The resultant target sequence was then purified and ligated into a pQE-30UA vector system for placement into a prokaryotic system. Transformation of E. coli cells was completed to introduce the recombinant plasmid. Presence of an ampicillin resistance gene in the plasmid allowed for transformant cell isolation on ampicillin antibiotic plates. The plasmids in the E. coli were extracted and a polymerase chain reaction utilizing internal primers was completed to determine proper directionality of the target DNA sequence insert and overall success of the reaction. SDS-PAGE after induction of cells with IPTG was used to determine if the target protein was present. A Western blot antibody binding assay showed the presence of hIL13 at approximately 13 kDa, which is what was expected. Introduction Over expression of receptors for hIL13 is known to occur in high-grade gliomas (Debinski and Thompson 1999). Mutated proteins that interact with these receptors could be a possible treatment method (Thompson and Debinski 1999). Prior to manufacture of a protein for mutagenesis and possible cancer therapies a genetic clone needs to be manufactured. The pQE-30UA vector method has not previously been used to make a plasmid containing the hIL13 gene. The pQE-30UA method allows for easy ligation of PCR products, easy expression of recombinant proteins, and easy purification of the proteins using His-tag affinity. Objective Manufacture an expression clone containing the eukaryotic sequence for hIL13 and place it into a prokaryotic (E. coli) system for recombinant protein protein production. Methods 1.Primer design 2.PCR amplification 3.DNA extraction from the Gel 4.PCR product purification and gel 5.Ligation Reaction into pQE30-UA cloning vector 6.Transformation of Top Ten F’ E. coli 7.Plating of E. coli onto ampicillin plates 8.Selection of clones 9.Extraction of Plasmid DNA from clones. 10.PCR amplification of target sequence and internal Ampicillin resistance gene 11.Regrowth of potential clone 12.Induction with IPTG to produce hIL13 protein 13.Harvesting and lysis of E. coli expressing hIL13 protein 14.SDS-PAGE and Coomassie stain of total extracted E. coli proteins 15.Western blot using indirect detection of hIL13 proteins isolated from clones Results Figure 1. Initial amplification gel showing amplified bands at ~344 base pairs (hIL13). Figure 2. PCR purification gel showing bands at ~344 base pairs with no extraneous DNA fragments. Figure 3. Extraction of Plasmids following E. coli culturing on ampicillin plates. Figure 4. Gel assay using internal AmpR primers and target sequence primers to determine directionality of insert. Bands seen are at ~800 base pairs (Amp R ) and ~344 base pairs (hIL13). Figure 5. Following the SDS-PAGE a Coomassie blue stain was used to visualize the target protein. The dark band shows a protein at ~13 kDa (hIL13). Figure 6. Western blot assay for hIL13 protein. The bands seen at about 13 kDa represent the hIL13protein. Discussion Initial PCR amplification yielded bright bands at approximately 344 base pairs in size (Figure 1). The banding shows that the proper target sequence was amplified. The bands were extracted from the gel using a scalpel and the DNA was separated from the gel matrix. The separation yields a pure sample as seen in Figure 2. The pure hIL13 was then extracted again and separated from the gel matrix so it could be ligated into the pQE-30UA vector. The vector was then utilized in the transformation of the E.coli. The vector contained the gene for ampiciliin resistance so by plating the the cells on selection agar a visual inspection would show if transformation of the cells was achieved. Resultant transformed colonies were selected and the cells were lysed to liberate the plasmids. Gel electrophoresis was used to determine if the plasmids were present (Figure 3). PCR amplification with internal primers shows that the PCR worked and that at least some of the clones had the proper directionality of the insert (Figure 4). Following culturing of the suspect cells and inoculation with IPTG the the cells were lysed and SDS-PAGE was run to determine presence of the hIL13 (Figure 5). The proteins in the SDS-PAGE were transferred to a membrane and Western blot antibody binding analysis was completed to be sure of the hIL13 being present (Figure 6). Future work 1.Mutate the hIl13 at the 13 th position from a glutamic acid to a cysteine. 2. Use a linking agent to tether either fluorescent tags or chemotherapeutic drugs to the protein. 3. Complete signaling assays with the cancerous and normal cells. Literature Cited Debinski, Waldemar and Jeffrey P. Thompson. 1999. Retargeting Interleukin 13 for Radioimmunodetection and Radioimmmunotherapy of High-grade Human Gliomas. Clinical Cancer Research. 5:3143- 3147. Thompson, Jeffrey P. and Waldemar Debinski. 1999. Mutants of Interleukin 13 with Altered Reactivity toward Interleukin 13 Receptors. The Journal of Biological Chemistry. 274:29944-29950. Acknowledgements Dr. Jeffrey Thompson PhD Dr. Ronald Kaltreider PhD 506 bp~344 bp 506 bp ~344 bp ~4000 bp, but running faster due to plasmid supercoiling 789101112 1018 bp 506 bp ~800 bp ~344 bp ~13 kDA 111087 control ~13 kDa 18.3 kDa 7.5 kDa 15 kDa 10 kDa control 7810