Transcript Interview Talk - University of North Texas

Unnatural Protein Engineering:
Biochemical and Medicinal Applications
Youngha Ryu
Department of Chemistry
Texas Christian University
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
Protein biosynthesis machinery
DNA
tRNA
aa~AMP
Aminoacyl-tRNA
synthetase
(aaRS)
mRNA
R
NH2
peptide chain
R
O
NH2
O
O
H2N
O
EF-Tu
acylated tRNA
mRNA
ribosome
“Standard” Genetic Codes
PNAS 48, 1086 (1962)
Suppressor tRNAs
Nonsense Suppressors in E. coli
Suppressor
Type
Anticodon change
tRNA
Gene
Efficiency
supE
Amber
CUG --> CUA
tRNAGln
glnV
0.8-20%
supP
Amber
CAA --> CUA
tRNALeu
leuX
30-100%
supD
Amber
CGA --> CUA
tRNASer
serU
6-54%
supU
Amber
CCA --> CUA
tRNATrp
trpT
supF
Amber
GUA --> CUA
tRNATyr
tyrT
supZ
Amber
GUA --> CUA
tRNATyr
tyrU
supB
Ochre
UUG --> UUA
tRNAGln
glnU
supL
Ochre
UUU --> UUA
tRNALys
lysT
supN
Ochre
UUU --> UUA
tRNALys
lysV
supC
Ochre
GUA --> UUA
tRNATyr
tyrT
supM
Ochre
GUA --> UUA
tRNATyr
tyrU
glyT
Opal
UCC --> UCA
tRNAGly
glyT
trpT
Opal
CCA --> UCA
tRNATrp
trpT
11-100%
0.1-30%
In vitro System
P. G. Schultz et al. Science 1989, 244, 182
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
An “Orthogonal” pair from M. jannaschii
5'
pC
C
G
G
C
G
G
U
G
A
C
CU U G A
A
G
G
GA A C
G
G U A
G
C
G
G
A
C
U
C
3'
AOH
C
C
A
G
G
C
C
G
C
C
TA A
C G G C C
A
G C U G G
U U C
C
G
CA
G
G U
U
C
C
U
A
G
A
• 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
Directed Evolution of Mj TyrRS
Selection strategy
Wang, Brock, Herberich & Schultz Science 292, 498, (2001)
Synthetases for Unnatural amino acids
O
OH
H 2N
O
OH
H2 N
O
Turner, Graziano, Spraggon & Schultz J. Am. Chem. Soc., 127, 14976 (2005)
Proc. Natl. Acad. Sci., 103, 6483 (2006)
Expanded Genetic Code in E. coli
O
O
O
OH
H2 N
OH
H 2N
O
OH
H2 N
OH
H 2N
O
O
OH
H2 N
O
OH
N3
O
OH
OH
H 2N
OH
H2 N
O
O
I
NH2
OH
H2 N
O
O
OH
H2 N
O
N
C
NO2
OH
H2 N
O
N
O
O
OS
O
O
N
O
O
OH
NO 2
OH
H2 N
OH
H 2N
O
OH
H 2N
O
O
OH
H 2N
OH
H2 N
O
O
N N
CO2 H
N
B(OH)2
CF3
N
OH
H2 N
OH
H 2N
O
O
OH
H 2N
OH
H 2N
O
O
OH
H2 N
O
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
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
3' U U U U U U C G G A C G A G C
RNAse T, PH etc
5'
U
U
RNAse P
C
C
G
G
C
G
G
U
C G A CU U G A
A
G
G
A GA A C G
G
G
C
C
G
G
A
C
A
AAUUCGAAAAGCCUGCUC A
A
C
Terminator
C
RNAse III
A RNAse E
G
G
C
C
G
C
C
U A A
CGGCC
A
GCUGG
C
U
C
U
G
CA G
G U
C
C
U
A
U
A
C U A
JYTRN
Mutant glnS promoter enhances
the synthetase expression
-35 region
-10 region
+1
WT
AAAAAACTAACAGTTGTCAGCCTGTCCCGCTTATAAGATCATACGCCGTTATACGTT
Mutant
AAAAAACTAACAGTTGTCAGCCTGTCCCGCTT-TAATATCATACGCCGTTATACGTT
WT
BpaRS
Mutant
Asp286Arg (D286R) substitution enhances
tRNA(CUA) binding affinity
TyrRS (WT) – tRNATyr (WT)
TyrRS (WT) – tRNATyr (CUA)
TyrRS (D286R) – tRNATyr (CUA)
His283
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
Kobayashi et. al. Nat. Struct. Biol. 10, 425 (2003)
Km= 0.68 mM
kcat= 0.079 s-1
kcat/Km (relative) = 0.22
Polycistronic expression of MjtRNA
aaRS promoter
glnS
tRNA promoter
-
lpp
tRNA copy #
0
1
JYTRN
glnS glnS
proK
1
glnS’
glnS’
glnS’
proK
proK
proK
3
6
1
b-Galactosidase assay for suppression efficiency
TAG
araBAD promoter
leader
lacZ
proK
proK + D286R
proK + glnS’
proK + glnS’ + D286R
+ 3TRN
+ 6TRN
Efficient incorporation
of many different unnatural amino acids
O
O
N3
H2N
CO2H
Bpa
H2N
CO2H
pAcPhe
H2N
CO2H
pAzPhe
I
H2N
CO2H
pIPhe
Efficiency and fidelity
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)
Site-selective modification of proteins
O
H2N
N
O PEG
O
O
NH2
O PEG
O
N
O
HN
PEG
PEG
O
N
H
O
pAcPhe
N
H
N
H
O
O
Lys
N
H
O
http://www.ambrx.com
Photocaged Tyrosine
Tyr503
lactose
Deiters, Groff, Ryu, Xie & Schultz Angew. Chem. Int. Ed. 45, 2728 (2006)
Incorporation of a distance probe into proteins
Tsao, Summerer, Ryu & Schultz J. Am. Chem. Soc. 128, 4572 (2006)
Incorporation of an IR probe into proteins
Relative Absorbance
1.0
pCNPhe
A
2236
0.8
0.6
FeIII pCNPhe64 Mb
FeIII (CN) pCNPhe64 Mb
0.4
0.2
0.0
2180
2200
CO
[2239 cm-1]
Azide
[2234 cm-1]
NO
[2230 cm-1]
Cyanide
[2236 cm-1]
O2
[2230 cm-1]
__
1.0
Relative Absorbance
Met-ferric
[2248 cm-1]
2220
2240
2260
2280
Wavenumber (cm-1)
Ferrous (Fe2+) Ferric (Fe3+)
adducts
adducts
Deoxyferrous
[2233 cm-1]
2248
B
2230
0.8
FeII(CO) pCNPhe64 Mb
FeII(NO) pCNPhe64 Mb
0.6
FeII(O2) pCNPhe64 Mb
2239
0.4
0.2
0.0
2160
2180
2200
2220
2240
2260
Wavenumber (cm-1)
Schultz, Supekova, Ryu, Xie, Perera & Schultz J. Am. Chem. Soc. 128, 13984 (2006)
Co-translational protein modification
OH
OH
H2N
O
Tyrosine
Thrombin
Desulfo-hirudine
Ki = 307 fM
Thrombin
Sulfo-hirudine
Ki = 26 fM
Liu & Schultz, Nat. Biotech. 24, 1436 (2006)
Incorporation of an NMR probe
TE domain of the human FAS
Cellitti et. al. J. Am. Chem. Soc. 130, 9268 (2008)
Unnatural amino acids incorporated by
the mutant TyrRS in E. coli
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
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
N-Terminal acetylation of the Z-domain
is context-dependent
Position
Amino acid
Observed
M.W.
Bpa
7968.3
S3
Tyrosine
7886.5
Bpa
7913.9
V4
Tyrosine
7827.4
Bpa
7898.2
D5
Tyrosine
7811
- Met1
Na-Acetylation
Yes
Yes
Yes
No
Yes
No
Photo-crosslinking and proteomics analysis
kDa Marker
220 80 50 -
Control S3Bpa
2
3
1
V4Bpa
D5Bpa
2
1
1
30 20 15 10 -
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
Strategy to identify the YebF transporter
Unnatural protein medicinal chemistry
Directed evolution of the archaea LeuRS system
Next round of positive selection
Selection with a single reporter plasmid
Positive selection
+ Unnatural amino acid
+ Cm + Uracil
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
Deletion of the upp and pyrF genes
by the recombinase-based gene replacement
GeneHog D upp D pyrF
Minimal media
+ Uracil
Minimal media
+ Uracil
+ Uridine
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
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)