Genetic Incorporation of Unnatural Amino Acids into Proteins

Monica Amin Yang Song Yan Liu Harbani Malik Vipul Madahar Jiayu Liao

Department of Chemical and Biological Engineering, Rensselaer Polytechnic Institute, New York Department of Bioengineering, University of California, Riverside

GOALS

Use an unnatural amino acid as a chemical handle for site- specific immobilization Current Method

Different reaction sites on the protein Protein Protein

Our Method

Azide-alkyne Huisgen cycloaddition reaction: Specific binding, no change in conformation of protein

SUMOylation Pathway

Cascade event involving multiple protein-protein interactions SUMO: Small – Ubiquitin – like MOdifiers 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 adenocarcinoma 3 . Figure 1. Yang Song

FRET – Based Analysis

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) Binding assays based on steady state and time resolved FRET can be used to monitor interactions in the SUMOylation Pathway

Site- Specific Incorporation of the unnatural amino acid

Site-specific incorporation of unnatural amino acid,

p

propargyloxy phenylalanine (

p

Ppa) [Figure 3] , into Cypet-SUMO1 in

Escherichia coli.

Mutated

M. Janaschii

tyrosyl-tRNA synthetase created to selectively charge an amber suppressor tRNA with

p

Ppa.

Figure 3. Nature Methods

(2007 ) UAG

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 Start Codon: ATG Ser HisTag

PCR Mutation

SUMO Stop Codon: TAA Start Codon: ATG TAG HisTag SUMO Stop Codon: TAA

Immobilization on Glass Plate

Azide-Alkyne Huisgen Cycloaddition

Figure 5 .

Bioorganic & Medicinal Chemistry Letters

(2005) 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.

METHODS : CLONING

cDNA Cloning

Cloning Region 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

pCR2.0- TOPO (3.9kb)

Transformation using TOP 10 cells DNA Extraction Characterization: Digestion Check, Sequencing

METHODS : CLONING

Gene Cloning

1 2 Digestion of SUMO1-pCR2.0 and pET-28B vector (specific digestion enzymes) NcoI NdeI NotI Ligation of SUMO1 gene to pET-28B vector TAG-His Cypet SUMO1 pET- 28B (5368bp) Transformation using TOP10 cells DNA Extraction & Sequencing Clone TAG- SUMO1 into SUMO1 pET-28B vector [1] Clone Cypet gene into TAG-SUMO1- pET-28B vector [2] Restriction Sites for : 1. TAG-SUMO  NotI, NcoI 2. TAG-SUMO-Cypet  NotI, NdeI

METHODS: PROTEIN EXPRESSION & PURIFICATION

Protein Expression: Protein Purification:

Use column chromatography (Nickel-NTA Agarose column) and dialysis 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

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 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. A 6 7 8 9 10 (a) (b)

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

Denotes the specific interaction between SUMO1 and Ubc9. We determined the interaction between Cypet-SUMO1 and Ypet- Ubc9 using FRET [Figure on next slide].

Kept constant Cypet SUMO1 concentration and gradually increased Ypet Ubc9 concentration

Cypet- SUMO1 Ypet-Ubc9

5 uL 0 uL 5 uL 5 uL 5 uL 1 uL 2 uL 3 uL

Dialysis Buffer

15 uL 14 uL 13 uL 12 uL 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 and Ypet- Ubc9 Proof of Concept

2000000 1800000 1600000 Decrease in Cypet – SUMO1 emission 0 uL/ug 1 uL/ug 2 uL/ug 3 uL/ug 1400000 1200000 1000000 800000 600000 400000 200000 0 450 470 Increase in Ypet-Ubc9 absorption 490 510 Emission Wavelength in nm 530 550

SUMMARY

• 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

p

Ppa into interested proteins.

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

Acknowledgments THANK YOU Jun Wang Dr. Rodgers BRITE Program Dr. Liao Dr. Liao’s Lab National Science Foundation

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