Senior Seminar Department of Chemistry EXPANDING THE GENETIC CODE WITH UNNATURAL AMINO ACIDS Kevin Cravedi Biochemistry, B.S.

Class of 2010 March 11, 2010

Outline

I. Thesis II. Background of Method III. Application of Method IV. Example 1: AzoPhe V. Example 2: Sulfotyrosine (Sulfohirudin) VI. Conclusion

THESIS 

That the incorporation of unnatural amino acids can be expressed in collaborators.

E. Coli

with efficiency using the method of Peter G. Schultz, his students and

BACKGROUND OF UNNATURAL AMINO ACID INCORPORATION Incorporation of unnatural amino acid into protein is a pre translational process.

Suppression of Stop Codon Use of orthogonal tRNA Use of orthogonal tRNA synthetase Incorporation of unnatural amino acid

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).

Mj

APPLICATION OF METHOD •

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. 3 rd year seminar requirement. http://www.chem.wisc.edu/areas/organic/studsemin/pontrello/pontrello-abs.pdf

(accessed Jan 27, 2010)

1. METHANOCCOCUS JANNASCHII (MJ) Tyrosyl tRNA and synthetase Similar Recognition Elements as E. coli No cross reaction with E. coli machinery

2. WHY USE TAG (UAG CODON) Least used codon in E. coli No disruption or termination TAG Sufficiently translated

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 10 9 possible synthetase active sites were randomized for one example.

2. Result: Active site specific to unnatural amino acid.

Positive Selection 3. CONTINUATION Proper incorporation of unnatural amino acid into protein Negative Selection

Wang, Q.; Parrish, A. R.;Wang, L.

Chembiol.

2009, 16(3), 323.

EXAMPLE (1) INCORPORATION OF PHOTO ISOMERIZABLE UNNATURAL AMINO ACID PHENYLALANINE-4-AZOBENZENE Bose, M.; Groff, D.; Xie, J.; Eric, B.; Schultz, P. G.

J. Am. Chem. Soc

. 2005

, 128,

388.

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

 Tyr-32, Leu-65, Phe-108, Gln-109, Asp-158, and Leu-162  Tyr32 Gly , Leu65 Glu , Phe108 Ala , Gln109 Glu , Asp158 Gly , and Leu162 His .

Determination of Efficiency Whale sperm myoglobin (residue 75) In the presence of (AzoPheRS) MjtRNA Tyr CUA 1mM AzoPhe Negative (Absence of AzoPhe)

EXAMPLE 2: SULFOTYROSINE (TYS) (1) BUILDING SPECIFICITY OF SYNTHETASE AND (2) INCORPORATION INTO PROTEIN Direct evolution and positive and negative selections were made.

Tyr32 Leu65 Asp158 Ile159 Leu162 Tyr32 Leu , Leu65 Pro , Asp158 Gly , Ile159 Cys , and Leu162 Lys SDS-PAGE confirms this incorporation.

Plasmid 1: Z domain (residue 7), tRNA, and 2 mM sulfotyrosine Plasmid 2: Containing StyrRS Incorporation of sulfo-Tyr protein into E. coli.

Result: Peak of 7,876 Da only sulfotyrosine.

Kehoe, J. W.; Bertozzi, C. R.

Chem. Biol.

2000,

7,

R57.

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. 3 rd year seminar requirement. http://www.chem.wisc.edu/areas/organic/studsemin/pontrello/pontrello-abs.pdf

Jan 27, 2010) (accessed 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.