1. Genetic Incorporation of Unnatural Amino Acids into Proteins Monica Amin 1, Yang Song 2, Yan Liu 2, Harbani Malik 2, Vipul Madahar 2, Jiayu Liao 2 Department of Chemical and Biological Engineering, Rensselaer Polytechnic Institute, New York Department of Bioengineering, University of California, Riverside Introduction This research was supported by the National Science Foundation Conclusion Results 1.Deiters, Alexander, and Peter G. Schultz. "In vivo incorporation of an alkyne into proteins in Escherichia coli." Bioorganic & Medicinal Chemistry Letters (2005): 1521- 524. Print. 2.Liu, Wenshe, Ansgar Brock, Shuo Chen, and Peter G. Schultz. "Genetic Incorporation of Unnatural Amino Acids into Proteins in mammalian cells." Nature Methods 4.3 (2007): 239-44. Print. 3.Martin, Sarah F., Michael H. Tatham, Ronald T. Hay, and Iford D.W. Samuel. "Quantitative analysis of multi-protein interactions using FRET: Application to the SUMO pathway." Protein Science (2008): 777-84. Print. 4.Sapsford, Kim E., Lorenzo Berti, and Igor L. Medintz. "Materials for Fluorescence Resonance Energy Transfer Analysis: Beyond Traditional Donor- Acceptor Combinations." Angewandte Chemie (2006): 4562-588. Print. 5.Wang, Lei, and Peter G. Schultz. "Expanding the genetic code." ChemCommun (2002): 1-11. Print. 6.Zhang, Zhiwen, Brian A.C. Smith, Lei Wang, Ansgar Brock, Charles Cho, and Peter G. Schultz. "A New Strategy for the Site-Specific Modification of Proteins in Vivo." Biochemistry (2003): 6735-746. Print. Incorporate unnatural amino acids in proteins in the mammalian system Use protein micro array [protein immobilization on glass plate] to find the inhibitors in the Sumoylation pathway Methods SUMOylation pathway [Figure 1] is a cascade event involving multiple protein-protein interactions. Several proteins catalyze covalent conjugation between Small- Ubiquitin- like MOdifiers (SUMO) that are ubiquitin-related proteins and cellular target proteins that are involved in regulation of various cellular processes. Disregulation of the SUMO pathway has been linked to diseases including ovarian carcinoma, melanoma, and lung adenocarcinoma 3. Analyzing the protein- protein binding is important to understand this complex pathway. SUMO1 gene- commercial plasmid PCR amplify SUMO1 and TAG SUMO1 (specifically designed primers) Ligation of SUMO1 and TAG-SUMO1 using TOPO cloning vector, pCR2.0 Transformation using TOP 10 cells DNA Extraction Characterization: Digestion Check, Sequencing cDNA CloningGene Cloning Digestion of SUMO1-pCR2.0 and pET-28B vector (specific digestion enzymes) Ligation of SUMO1 gene to pET-28B vector Transformation using TOP10 cells DNA Extraction & Sequencing Clone TAG- SUMO1 into SUMO1- pET-28B vector Clone Cypet gene into TAG- SUMO1- pET-28B vector K+ Cloning Region: NcoI (296) NdeI (238) NotI (166) pET-28B (5368bp) A+ pCR2.0- TOPO (3.9kb) K+ We have determined the interaction between Cypet-SUMO1 and Ypet- Ubc9 using FRET [Figure 6]. With constant Cypet-sumo1 concentration and increasing Ypet-Ubc9 concentration, this figure shows when Cypet-SUMO1 is excited at 414nm, the emission from Cypet slowly decreases as the absorption of Ypet- Ubc9 gradually increases do to the increasing concentration of Ypet- Ubc9. Which denotes the specific interaction between SUMO1 and Ubc9. RFU Emission Wavelength in nm Cypet-SUMO1 and Ypet- Ubc9 Proof of Concept Genetic incorporation of unnatural amino acids site-specifically into proteins provides a way to manipulate the structures of proteins, monitor protein function and create proteins with novel properties. In previous studies, by creating orthogonal tRNA- synthetase pairs with specificity to unnatural amino acids, more than thirty unnatural amino acids have been incorporated selectively and efficiently into proteins in response to nonsense codons 1. Using azide-alkyne Huisgen cycloaddition [Figure 3], one of the most popular reactions within the click chemistry philosophy, we can achieve site-specific immobilization of the mutated protein on azide modified glass surface under mild conditions, and, thus detect protein-protein interaction using florescence resonance energy transfer (FRET). A 1 2 3 4 5 A 6 7 8 9 10 Figure 7 (Right) Digestion gel of the TAG- SUMO1/pET-28B plasmid. (a)TAG-pET-28B ~5kbp (b)SUMO1 ~300bp 1-8, 10 were positive and well 9 was negative. Samples 1 and 2 were sent for sequencing and got the correct sequence. Protein Expression: 1. Transform TAG-Cypet- SUMO1 plasmid, orthogonal tRNA and tRNA synthetase plasmids into BL21 cells 2. Grow Transformed cells (step 1) in presence of unnatural amino acid and related antibiotics in the medium Protein Purification: Use column chromatography (Nickel-NTA Agarose column) and dialysis FRET based Protein-Protein Interaction: Determine the interaction between TAG-Cypet-SUMO1 and Ypet- Ubc9 and compare to no mutation interaction Future Directions Acknowledgements Steven Bach, Timothy Han Chen, Sylvia Chu, Richard Lauhead, Randall Mello, Yongfeng Zhao. The National Science Foundation. References FRET [Figure 4] is a nonradiative process in which an excited state donor (usually a fluorophores) transfers energy to a ground state acceptor in proximity of 1-10nm, through dipole- dipole interactions 3. Binding assays based on steady state and time resolved FRET can be used to monitor interactions. This mutation along with a fluorescence tag on the protein can aid in developing microarrays by permanent immobilization of biological samples (such as the mutated protein) while maintaining bioactivity on a solid surface. We developed a new technology that allows site-specific incorporation of unnatural amino acid, ppropargyloxy- phenylalanine (pPpa) [Figure 2], into Cypet-SUMO1 in Escherichia coli. A mutated M. Janaschii tyrosyl-tRNA synthetase was created to selectively charge an amber suppressor tRNA with pPpa. Figure 1. SUMOylation Pathway. Yang Song Figure 2. Nature Methods (2007 ) Figure 3. Bioorganic & Medicinal Chemistry Letters (2005) Figure 4. FRET principle. Angewandte Chemie (2006) Figure 6. Excitation of Cypet at 414nm Figure 5. pCR 2.0 and pET-28B vectors and cloning regions Figure 8 (Left): Colonies on TAG Cypet- SUMO1/ pET-28B plate. (Kanamycin Resistance) (a) (b) Amber stop codon –TAG has been successfully incorporated into SUMO1/pET-28B plasmid to recognize unnatural amino acid. TAG incorporated Cypet-SUMO1/pET-28B construct is currently being studied. This will allow us to site-specifically incorporate pPpa into interested proteins.