EXPANDING THE GENETIC CODE WITH UNNATURAL AMINO

ACIDSKevin CravediBiochemistry,

B.S.Class of 2010

Department of Chemistry

Senior Seminar

March 11, 2010

I. Thesis

II. Background of Method

III. Application of Method

IV. Example 1: AzoPhe

V. Example 2: Sulfotyrosine (Sulfohirudin)

VI.Conclusion

Outline

THESIS That the incorporation of unnatural amino acids can be expressed in E. Coli with efficiency using the method of Peter G. Schultz, his students and collaborators.

BACKGROUND OF UNNATURAL AMINO ACID INCORPORATION

Incorporation of

unnatural amino acid

Suppression of Stop

Codon

Use of orthogonal

tRNAUse of

orthogonal tRNA

synthetase

Incorporation of unnatural amino acid into protein is a pre-translational process.

Reddi, O. S. Recombinant DNA Technology: A Laboratory Manual; Allied Publishers: Mayapuri, New Dehli, 2000; pp 1.

WHAT THE NOVEL METHOD LOOKS LIKE.

Wang, L. Wang Lab. Salk Institute for biological studies. http://wang.salk.edu/research.php (accessed Jan 17, 2010).

APPLICATION OF METHOD

Mj•Methanoccocus Jannaschii (Mj): tRNA and Aminoacyl tRNA synthetase (aaRS).

TAG•Suppression of a

stop (amber) codon such as TAG

aaRS •Change in the active site of (aaRS)

Ligase

•Acylation of tRNA and ligation using T-4 ligase.

Pontrello, J. K. 3rd year seminar requirement. http://www.chem.wisc.edu/areas/organic/studsemin/pontrello/pontrello-abs.pdf (accessed Jan 27, 2010)

1

23

1. METHANOCCOCUS JANNASCHII (MJ)

Tyrosyl tRNA and synthetase

Similar Recognition Elements as E. coli

No cross reaction with E. coli machinery

TAG

Least used

codon in E. coli

Sufficiently

translated

No disruption

or terminatio

n

2. WHY USE TAG (UAG CODON)

3. MODIFICATION OF SYNTHETASE TO ACCOMMODATE UNNATURAL AMINO ACID

Direct evolution method was implemented in order to rearrange the active site to accomodate the unnatural amino acid.

1. A library of 109 possible synthetase active sites were randomized for one example.2. Result: Active site specific to unnatural amino acid.

3. CONTINUATION

Positive Selectio

n

Negative

Selection

Proper incorporati

on of unnatural

amino acid into

protein

Wang, Q.; Parrish, A. R.;Wang, L. Chembiol. 2009, 16(3), 323.

EXAMPLE (1) INCORPORATION OF PHOTO-ISOMERIZABLE UNNATURAL AMINO ACID PHENYLALANINE-4-AZOBENZENE

CisLeast Stable Irradiation using 334 nm

lightTrans

Most Stable Irradiation using 420 nm light

PHENYLALANINE-4-AZOBENZENE (AzoPhe)

Bose, M.; Groff, D.; Xie, J.; Eric, B.; Schultz, P. G. J. Am. Chem. Soc. 2005, 128, 388.

Tyrosyl tRNA synthetase(MjTyrRS)

Tyrosyl mutant amber suppressor tRNA

(MjtRNATyrCUA)

Orthogonal Pair

THE ACTIVE SITE OF AZOPHE SYNTHETASEDirect evolution and positive and negative selections were made.

Tyr-32, Leu-65, Phe-108, Gln-109, Asp-158, and Leu-162

Tyr32Gly, Leu65Glu, Phe108Ala, Gln109Glu, Asp158Gly, and Leu162His.

Determination of Efficiency

Whale sperm myoglobin (residue 75)

In the presence

of (AzoPheR

S)MjtRNATyr

CUA

1mM AzoPh

e

Negative (Absenc

e of AzoPhe)

EXAMPLE 2: SULFOTYROSINE (TYS) (1) BUILDING SPECIFICITY OF SYNTHETASE AND (2)

INCORPORATION INTO PROTEIN Tyr32 Leu65 Asp158 Ile159 Leu162

Tyr32Leu, Leu65Pro, Asp158Gly, Ile159Cys, and Leu162Lys

Kehoe, J. W.; Bertozzi, C. R. Chem. Biol. 2000, 7, R57.

Result: Peak of 7,876 Da only sulfotyrosine.

Incorporation of sulfo-Tyr-protein

into E. coli.

Plasmid 1: Z-domain

(residue 7), tRNA, and 2

mM sulfotyrosine

Plasmid 2: Containing

StyrRS

SDS-PAGE confirms this incorporation.

Direct evolution and positive and negative selections were made.

1) SULFOTYROSINE-(MALDI-TOF)

Kehoe, J. W.; Bertozzi, C. R. Chem. Biol. 2000, 7, R57.

SULFOTYROSINE APPLICATION Recombinant hirudin

(desulfo-hirudin) is a less potent thrombin inhibitor than native protein as an anticoagulant. This early experiment was done to create natural sulfo-hirudin by incorporation of sulfotyrosine into hirudin.

SULFO-HIRUDIN IN THROMBIN COMPLEX

Liu, C. C.; Brustad, E.; Liu, W.; Schultz, P. G. J. Am. Chem. Soc. 2007, 129, 10648.

CONCLUSION Placement of unnatural amino acids into proteins with high

efficiency has been effectively implemented.

New innovations are being

implemented

Upwards of 1-10 unnatural amino

acid incorporation in

one protein

4 and 5 codon pairs

Therapuetic drug for cancer

and HIV

THE END

SPECIAL THANKS Dr. Ildliko Kovach

Peter G. Schultz and Co.

The Chemistry Department

REFERENCES Pontrello, J. K. 3rd year seminar requirement.

http://www.chem.wisc.edu/areas/organic/studsemin/pontrello/pontrello-abs.pdf (accessed Jan 27, 2010)

Reddi, O. S. Recombinant DNA Technology: A Laboratory Manual; Allied Publishers: Mayapuri, New Dehli, 2000; pp 1.

Kehoe, J. W.; Bertozzi, C. R. Chem. Biol. 2000, 7, R57. Bose, M.; Groff, D.; Xie, J.; Eric, B.; Schultz, P. G. J. Am. Chem. Soc. 2005, 128,

388. Wang, L. Wang Lab. Salk Institute for biological studies.

http://wang.salk.edu/research.php (accessed Jan 17, 2010). Wang, Q.; Parrish, A. R.;Wang, L. Chembiol. 2009, 16(3), 323. Liu, C. C.; Brustad, E.; Liu, W.; Schultz, P. G. J. Am. Chem. Soc. 2007, 129,

10648.