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.