Unnatural Protein Engineering: Biochemical and Medicinal Applications

1
Unnatural Protein EngineeringBiochemical and
Medicinal Applications

• Youngha Ryu

Department of Chemistry Texas Christian
University
2
Methods of Protein Modification

• Chemical modification of the reactive side chains
• Total chemical synthesis
• Ligation of synthetic peptide to a truncated
  protein
• In vitro transcription/translation
• Expression in living organisms

3
Protein biosynthesis machinery
DNA
Aminoacyl-tRNA synthetase (aaRS)
mRNA
transcription
aminoacylation
peptide chain
acylated tRNA
ribosome
mRNA
translation
4
Standard Genetic Codes
5
PNAS 48, 1086 (1962)
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Suppressor tRNAs
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Nonsense Suppressors in E. coli
Suppressor Type Anticodon change tRNA Gene Efficiency
supE Amber CUG --gt CUA tRNAGln glnV 0.8-20
supP Amber CAA --gt CUA tRNALeu leuX 30-100
supD Amber CGA --gt CUA tRNASer serU 6-54
supU Amber CCA --gt CUA tRNATrp trpT
supF Amber GUA --gt CUA tRNATyr tyrT 11-100
supZ Amber GUA --gt CUA tRNATyr tyrU
supB Ochre UUG --gt UUA tRNAGln glnU
supL Ochre UUU --gt UUA tRNALys lysT
supN Ochre UUU --gt UUA tRNALys lysV
supC Ochre GUA --gt UUA tRNATyr tyrT
supM Ochre GUA --gt UUA tRNATyr tyrU
glyT Opal UCC --gt UCA tRNAGly glyT
trpT Opal CCA --gt UCA tRNATrp trpT 0.1-30
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In vitro System
P. G. Schultz et al. Science 1989, 244, 182
9
Incorporation of unnatural amino acids into
proteins in living organisms

• Efficient transport or biosynthesis of unnatural
  amino acids
• Unique codons (nonsense, four base, etc)
• tRNA/aminoacyl-tRNA synthetase pair that is
  orthogonal to the endogenous system
• Directed evolution of the aminoacyl-tRNA
  synthetase to selectively charge the orthogonal
  tRNA with an unnatural amino acid

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An Orthogonal pair from M. jannaschii
3'

• M. jannaschii tRNATyr is orthogonal to E. coli
  synthetases
• M. jannaschii TyrRS is orthogonal to E. coli
  tRNAs
• M. jannaschii TyrRS has minimal interaction with
  anticodon
• M. jannaschii TyrRS has no proofreading activity

5'
A
T
A
U
C
C G G C C
G
A
A
U U G
A
C
A
G
G C U G G
C
U
U
C
G
A A C
G
G
G
A
G
U
A
G
U
A
C
U
G
C
A
U
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Directed Evolution of Mj TyrRS
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Selection strategy
Wang, Brock, Herberich Schultz Science 292,
498, (2001)
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Synthetases for Unnatural amino acids
Turner, Graziano, Spraggon Schultz J. Am. Chem.
Soc., 127, 14976 (2005)
Proc. Natl.
Acad. Sci., 103, 6483 (2006)
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Expanded Genetic Code in E. coli
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Sub-optimal yields of proteins in E. coli

• Non-versatile two plasmid system? Integration
  of the synthetase and tRNA genes into a single
  plasmid, which is compatible with most E. coli
  expression vectors and strains
• Intrinsic low efficiency due to the competition
  with termination? High suppression efficiency
  is achieved by naturally occuring non-sense
  suppressors (e.g. XL1-Blue)
• Inefficient transcription and processing of tRNA?
  New promoter and flanking sequence
• Inefficient expression of aminoacyl-tRNA
  synthetase? New promoter

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New Mj tRNA expression cassette

• E. coli prolyl tRNAs have C1-G72 pair, which is
  major identity determinant of MjtRNA Important
  context for the precise tRNA processing
• proK tRNA is most frequently used in E. coli
• FIS enhances tRNA transcription

Terminator
JYTRN
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Mutant glnS promoter enhances the synthetase
expression
-35 region
-10 region
1
AAAAAACTAACAGTTGTCAGCCTGTCCCGCTTATAAGATCATACGCCGTT
ATACGTT
WT
AAAAAACTAACAGTTGTCAGCCTGTCCCGCTT-TAATATCATACGCCGTT
ATACGTT
Mutant
WT
Mutant
BpaRS
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Asp286Arg (D286R) substitution enhances tRNA(CUA)
binding affinity
TyrRS (WT) tRNATyr (WT)
TyrRS (WT) tRNATyr (CUA)
TyrRS (D286R) tRNATyr (CUA)
His283
Asp286
Asp286
Asp286
Arg286
G34
G34
C34
C34
Phe261
Km 0.35 mM kcat 0.19 s-1 kcat/Km (relative) 1
Km 39 mM kcat 0.070 s-1 kcat/Km (relative)
0.0033
Km 0.68 mM kcat 0.079 s-1 kcat/Km (relative)
0.22
Kobayashi et. al. Nat. Struct. Biol. 10, 425
(2003)
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Polycistronic expression of MjtRNA
aaRS promoter
glnS
glnS
glnS
glnS
glnS
glnS
tRNA promoter
lpp
proK
proK
proK
proK
-
tRNA copy
0
1
1
1
3
6
JYTRN
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b-Galactosidase assay for suppression efficiency
TAG
araBAD promoter
leader
lacZ
proK
proK D286R
proK glnS
proK glnS D286R
3TRN
6TRN
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Efficient incorporation of many different
unnatural amino acids
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Efficiency and fidelity
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Optimizing protein yields in E. coli

• E. coli prolyl-tRNA
  promoter and terminator for the amber suppressor
  tRNA
• Mutated form of the glnS promoter for the
  synthetase
• D286R substitution in the synthetase gene
• Multiple copies of the suppressor tRNA gene
• Yield of adiponectin (Glu123Bpa) mutant 0.4g/L

Ryu Schultz Nat. Methods 3, 263 (2006)
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Site-selective modification of proteins
http//www.ambrx.com
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Photocaged Tyrosine
Tyr503
lactose
Deiters, Groff, Ryu, Xie Schultz Angew. Chem.
Int. Ed. 45, 2728 (2006)
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Incorporation of a distance probe into proteins
Tsao, Summerer, Ryu Schultz J. Am. Chem. Soc.
128, 4572 (2006)
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Incorporation of an IR probe into proteins
pCNPhe
Ferric (Fe3) adducts
Ferrous (Fe2) adducts
Met-ferric 2248 cm-1
Deoxyferrous 2233 cm-1
Azide 2234 cm-1
CO 2239 cm-1
Cyanide 2236 cm-1
NO 2230 cm-1

• __

O2 2230 cm-1
Schultz, Supekova, Ryu, Xie, Perera Schultz J.
Am. Chem. Soc. 128, 13984 (2006)
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Co-translational protein modification
Thrombin
Thrombin
Desulfo-hirudine
Sulfo-hirudine
Ki 307 fM
Ki 26 fM
Liu Schultz, Nat. Biotech. 24, 1436 (2006)
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Incorporation of an NMR probe
TE domain of the human FAS
Cellitti et. al. J. Am. Chem. Soc. 130, 9268
(2008)
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Unnatural amino acids incorporated by the mutant
TyrRS in E. coli
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Identification of the protein modification and
secretion pathways by photo-crosslinking in E.
coli

• N-Acetylation of recombinant proteins in E. coli
• Na-Acetylation (e.g. Z-domain etc)
• Ne-Acetylation of lysine side chains
  (e.g. Porcine and bovine
  somatotropins)
• Secretion pathway of the YebF protein in E. coli

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N-Terminal acetylation of the Z-domain depends
on E. coli strains and expression plasmids
E. coli Strain Deleted Gene Plasmid - Met1 N-Acetylation
BL21(DE3) pET Yes/No Yes
BL21(DE3) pBAD Yes No
AD494(DE3) pET Yes No
JW4335 rimI pBAD Yes No
JW1053 rimJ pBAD Yes No
JW1423 rimL pBAD Yes No
JW2293 ack pBAD Yes No
JW2294 pta pBAD Yes No
JW4030 acs pBAD Yes No
LCB90 ack pBAD Yes No
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N-Terminal acetylation of the Z-domainis
context-dependent
Position Amino acid Observed M.W. - Met1 Na-Acetylation
S3 Bpa 7968.3 Yes Yes
S3 Tyrosine 7886.5 Yes Yes
V4 Bpa 7913.9 Yes No
V4 Tyrosine 7827.4 Yes No
D5 Bpa 7898.2 Yes No
D5 Tyrosine 7811 Yes No
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Photo-crosslinking and proteomics analysis
Band S3Bpa V4Bpa D5Bpa
1 Protein A Protein A Protein A EF-Ts
2 Protein A Hsp70 PBP EF-Tu Protein F
3 EF-Tu Protein A
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Strategy to identify the YebF transporter
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Unnatural protein medicinal chemistry
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Directed evolution of the archaea LeuRS system
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Selection with a single reporter plasmid
Unnatural amino acid
Positive selection
Cm Uracil
Next round of positive selection
Survivors containing aaRS capable of charging any
natural or unnatural aa on the orthogonal tRNA
- Unnatural amino acid
Negative selection
5-FU
Cells that incorporate natural amino acids make
toxic product from 5-FU and die
Cells that incorporate unnatural amino acid only
survive on 5-FU
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Deletion of the upp and pyrF genes by the
recombinase-based gene replacement
GeneHog D upp D pyrF
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Summary

• A single plasmid system for the high yield
  expression of proteins containing unnatural amino
  acids
• Broad applications depending on the
  physicochemical properties of unnatural amino
  acids chemical and photochemical reactions,
  spectroscopic probes, novel therapeutics etc.
• Ongoing projects
• Identification of the protein acetylation and
  secretion pathways
• Unnatural protein medicinal chemistry
• Directed evolution of the leucyl-tRNA synthetase

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Acknowledgments
Graduate Students

• Minoro Aoshima
• Lina Bernal-Perez
• Pradeep Budhathoki
• Aery Lee

Undergraduate Students

• Kiran Butt
• Michael Foster
• Brett McKnight
• Fatima Sahyouni
• Diana Tran

Collaborators

• Dr. Laszlo Prokai (UNTHSC)
• Dr. Peter Schultz (TSRI)

Financial Support

• TCU (Start-up, RCAF, SERC)