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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.
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