genetic incorporation of unnatural amino acids into proteins monica amin yang song yan liu harbani...
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Genetic Incorporation of Unnatural Amino Acids into
ProteinsMonica Amin Yang Song
Yan Liu Harbani Malik Vipul Madahar
Jiayu LiaoDepartment of Chemical and Biological Engineering, Rensselaer Polytechnic Institute,
New York
Department of Bioengineering, University of California, Riverside
GOALS
ProteinAzide-alkyne Huisgen cycloaddition reaction: Specific binding, no change in conformation of protein
Use an unnatural amino acid as a chemical handle for site- specific immobilization
Current Method
Protein
Our Method
Different reaction sites on the protein
SUMOylation Pathway
Several proteins catalyze covalent conjugation between SUMO and cellular target proteins that are involved in regulation of various cellular processes.
Disregulation of the pathway is linked to diseases like ovarian carcinoma, melanoma, and lung adenocarcinoma3.
Cascade event involving multiple protein-protein interactions
SUMO: Small – Ubiquitin – like MOdifiers
Figure 1. Yang Song
FRET – Based Analysis
Binding assays based on steady state and time resolved FRET can be used to monitor interactions in the SUMOylation Pathway
Forster (Fluorescence) Resonance Energy Transfer
• Non- radiative process
• Donor (Cypet) and Acceptor (Ypet) (Fluorophores)
• Donor transfers energy to ground state acceptor
• Proximity of 1-10nm
• Dipole- dipole interactions
Figure 2. FRET principle . Angewandte Chemie (2006)
Site- Specific Incorporation of the unnatural amino acid
UAG
Site-specific incorporation of unnatural amino acid, ppropargyloxy- phenylalanine (pPpa) [Figure 3] , into Cypet-SUMO1 in Escherichia coli.
Mutated M. Janaschii tyrosyl-tRNA synthetase created to selectively charge an amber suppressor tRNA with pPpa.
Figure 3. Nature Methods (2007 )
Figure 4. ChemCommun (2002)
Site- Specific Incorporation of the Unnatural Amino Acid
Once we have our DNA construct with the TAG mutation
DNA gets transcribed to mRNA [TAG UAG]
In response to this unique codon the tRNA with the unnatural amino acid attaches to the mRNA
After the translation, the unnatural amino acid is incorporated into the peptide sequence
PCR Mutation
SUMOSer HisTag
StartCodon:
ATG
StopCodon:
TAA
SUMOTAG HisTag
StartCodon:
ATG
StopCodon:
TAA
Immobilization on Glass PlateAzide-Alkyne Huisgen Cycloaddition
Achieve site-specific immobilization of a fluorescent tag protein (Cypet- SUMO1) on azide modified glass surface under mild conditions
Detect the resonance energy transfer with Ypet tagged enzymes in SUMOylation pathway, like ubc9, AOS1/Ubo2, SENPs, and PIASs.
Figure 5 . Bioorganic & Medicinal Chemistry Letters (2005)
METHODS : CLONING
A+
pCR2.0- TOPO(3.9kb)
K+
Cloning Region
METHODS : CLONING
TAG-His SUMO1Cypet
NcoI NotINdeI
2
1
pET- 28B (5368bp)
Restriction Sites for :1. TAG-SUMO NotI, NcoI2. TAG-SUMO-Cypet NotI, NdeI
METHODS: PROTEIN EXPRESSION & PURIFICATION
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
RESULTS
A 1 2 3 4 5
A 6 7 8 9 10
(a)
(b)
Figure (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.
RESULTS
Incorporated Cypet fluorescence gene using cloning procedures mentioned in methods
We grew the cells on a Kanamycin resistant agar plate; got colonies
Sent for sequencing.
Proof of Concept
We determined the interaction between Cypet-SUMO1 and Ypet- Ubc9 using FRET [Figure on next slide].
Cypet-SUMO1 is excited at 414nm
Emission from Cypet- SUMO1 slowly decreases as the absorption of Ypet- Ubc9 gradually increases do to the increasing concentration of Ypet- Ubc9.
Cypet- SUMO1 Ypet-Ubc9 Dialysis Buffer
5 uL 0 uL 15 uL
5 uL 1 uL 14 uL
5 uL 2 uL 13 uL
5 uL 3 uL 12 uL
Kept constant Cypet-SUMO1 concentration and gradually increased Ypet-Ubc9 concentration
Denotes the specific interaction between SUMO1 and Ubc9.
RF
U
Emission Wavelength in nm
Cypet-SUMO1 and Ypet- Ubc9 Proof of Concept
•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.
•The TAG- Cypet-SUMO1/pET-28B will allow us to site-specifically incorporate pPpa into interested proteins.
SUMMARY
FUTURE DIRECTIONS• Use FRET- based assays to monitor protein-protein interaction within the SUMOylation Pathway
• Use protein micro array [protein immobilization on glass plate] to find the inhibitors in the Sumoylation pathway
•Incorporate unnatural amino acids in proteins in the mammalian system
AcknowledgmentsTHANK YOU
Jun WangDr. Rodgers
BRITE Program Dr. Liao
Dr. Liao’s LabNational Science Foundation
REFERENCES1. 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.