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Genetic Incorporation of Unnatural Amino Acids into Proteins
1
Monica Amin ,
Yang
2
Song ,
Yan
2
Liu ,
Harbani
2
Malik ,
Vipul
2
Madahar ,
Jiayu
2
Liao
Department of Chemical and Biological Engineering, Rensselaer Polytechnic Institute, New York
Department of Bioengineering, University of California, Riverside
Cloning
Region:
Introduction
Figure 7 (Right)
Digestion gel of the TAGSUMO1/pET-28B plasmid.
(a)TAG-pET-28B ~5kbp
(b)SUMO1 ~300bp
NcoI (296)
NdeI (238)
NotI (166)
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 adenocarcinoma3.
Analyzing the protein- protein binding is important to understand this
complex pathway.
1-8, 10 were positive and well
9 was negative.
pET-28B
(5368bp)
pCR2.0- TOPO
(3.9kb)
A
1
2
3
4
5
(a)
(b)
A
6
7
8
9
10
Samples 1 and 2 were sent for
sequencing and got the correct
sequence.
Figure 5. pCR 2.0 and pET-28B vectors and cloning regions
Protein Expression:
1. Transform TAG-CypetSUMO1 plasmid, orthogonal
tRNA and tRNA synthetase
plasmids into BL21 cells
Figure 4. FRET principle . Angewandte Chemie (2006)
Figure 1. SUMOylation Pathway. Yang Song
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 codons1.
We developed a new technology that
allows site-specific incorporation of
unnatural amino acid, ppropargyloxyphenylalanine (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.
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 interactions3.
Binding assays based on steady state and time resolved FRET can be
used to monitor interactions.
cDNA Cloning
SUMO1 gene- commercial
plasmid
PCR amplify SUMO1 and
TAG SUMO1 (specifically
designed primers)
FRET based Protein-Protein
Interaction:
Determine the interaction
between TAG-Cypet-SUMO1
and Ypet- Ubc9 and compare
to no mutation interaction
2. Grow Transformed cells
(step 1) in presence of
unnatural amino acid and
related antibiotics in the
medium
Methods
Protein Purification:
Use column chromatography
(Nickel-NTA Agarose
column) and dialysis
Results
Gene Cloning
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.
Digestion of SUMO1-pCR2.0
and pET-28B vector (specific
digestion enzymes)
Ligation of SUMO1 gene to
pET-28B vector
Cypet-SUMO1 and Ypet- Ubc9 Proof of Concept
Figure 2 . Nature Methods
(2007 )
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.
2000000
Ligation of SUMO1 and
TAG-SUMO1 using TOPO
cloning vector, pCR2.0
0 uL/ug
1800000
Transformation using TOP10
cells
1 uL/ug
Decrease in
Cypet –SUMO1
emission
1600000
2 uL/ug
RFU
Increase in
Ypet-Ubc9
absorption
1000000
800000
Figure 3 . Bioorganic & Medicinal Chemistry Letters (2005)
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).
Clone TAG- SUMO1 into
SUMO1- pET-28B vector
DNA Extraction
600000
400000
200000
Characterization: Digestion
Check , Sequencing
Clone Cypet gene into TAGSUMO1- pET-28B vector
Conclusion
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.
Future Directions
• 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
Acknowledgements
Steven Bach, Timothy Han Chen, Sylvia Chu, Richard Lauhead,
Randall Mello, Yongfeng Zhao. The National Science Foundation.
References
1200000
DNA Extraction & Sequencing
Colonies on TAG CypetSUMO1/ pET-28B plate.
(Kanamycin Resistance)
3 uL/ug
1400000
Transformation using TOP 10
cells
Figure 8 (Left):
0
450
470
490
510
530
Emission Wavelength in nm
Figure 6. Excitation of Cypet at 414nm
This research was supported by the National Science Foundation
550
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