Download "non-natural" amino acids - RIKEN Systems and Structural Biology

Biosynthesis of proteins containing
"non-natural" amino acids
Takahito Mukai, Kensaku Sakamoto and Shigeyuki Yokoyama
RIKEN Systems and Structural Biology Center (SSBC)
All living organisms on earth are largely composed of proteins that are produced by decoding the information stored in DNA. Proteins are made up of the building
blocks called amino acids. It is known that there exist hundreds of amino acids in nature, however, only 20 of them serve as the protein constituents. Expanding
repertoire of amino acids beyond the standard 20 could impart novel biological functions not found in nature, and the use of non-natural amino acid can provide
one such technology. RIKEN Systems and Structural Biology Center (SSBC) conducts research in expanding the genetic code, the set of rules that translate
information encoded in DNA into proteins, to incorporate non-natural amino acids into proteins site-specifically. This technology can provide powerful tools for
exploring and illuminating the mechanism as well as mystery of life. Proteins substituted with non-natural amino acids, which we advocate to use the term
alloprotein , have immense potential in varied fields including drug development.
Brief overview
2) Site-specific Incorporation technology
of non-natural amino acid
1) Why non-natural amino acid?
◆ Difficult to attach novel functions
to natural amino acids
◆ What s the merit of using
non-natural amino acids?
Example of non-natural amino acids
Natural amino acid (Lysine)
＊ Enable synthesis of proteins with novel
functions
＊ Enable site-specific incorporation of
novel functions into proteins
＊ Enable the rational design of proteins
that can be used in variety of fields
Non-natural
amino acid
Attaching novel functions
Modified Lysine
Non-natural amino acid(AzZlys)
Mammalian
S.cerevisiae
AUC
Cell-free
Modified tRNA
Incorporation to
the targeted protein
AzZLys having novel functions
Modified aaRS
Novel functions
Alloprotein
E.Coli
Drosophila
melanogaster S2
Drug development
・Antibody drug
・DDS
・Optimization of commercial drug products
Val
Sys
Ala
AUC
Biopolymers
・Surgical thread
・Biopolymer scaffolds, etc
Researech tool ・Fluorescent labeling
・Photo-crosslinker ・Heavy atoms for structural analysis
Val
Sys
Ala
Ser
CGA
AUC
GCU
UAG
Ribosome
mRNA
Site-specific incorporation of non-natural amino acid -Host cell engineering1) E. coli RFzero strain hyperproducing Alloproteins
-100% incorporation of non-natural amino acids
at specific multiple sitesConventional (E.coli)
Non-natural
amino acid
5'
Ribosome
×
mRNA
mRNA
5'
P
E.coli
Non-natural amino acid
Tyrosine
A U C
U A G
U A G
P
A
Protein synthesis
the code of a non-natural
amino acid
(Incorporation rate:100%).
- Enable incorporation of nonnatural amino acids at as
3' many sites as desired .
A
archaebacteria
-derived TyrRS
Conventional
Knock out of inherent TyrRS and tRNATyr genes
2) Production of posttranslationally modified protein
Non-natural
amino acid
H2A
H3.1
H2AX
Toxin
H2AZ
Optimal site
antibody
Possible heterogeneous products of
undefined stoichiometry
CENP-A
H4
・Enables conjugation of cytotoxic drugs at the optimum site.
・Enables control over the number of conjugations to
cytotoxic drugs.
・Enables homogeneous conjugates.
+
P
Post-translational
modifications
Core histones
Optimal site
Me
3) Novel protein purification system
①Gene
H1
+
ＤＮＡ sequence
variation
and modification
TAG
sequence
H5
Normal cell
TAG
P
Ac
support TAG
Ac
1) Photo-crosslink for structural analysis 2) Successful determination of unknown
3) Applying to crystal structure analysis of
binders by utilizing the photo-crosslinking membrane protein site-specific photolabeling
Formation of stable complex
Incorporation of azide-containing
methodology comprehensively
amino acids into membrane protein
tag
Az
pBpa85 gankyrin
SH3
SH2
SH3
The appropriate positions for
introducing the crosslinker are defined
based on the structural information
S6C
No significant
Site-specific photostructural distortion
crosslinking.
by photo-crosslinking
pBpa85
Hydroxy acid
Target protein
②Expression of tagged fusion protein
Target protein
Solubilization of membrane protein and
fluorescent labeling
tag
Purification of membrane protein
g
followed by removal of tag
ta
E356
(Sato, S. et al. Biochemistry 50, 250 (2011))
Target protein
④Removal of tag by enzymatic cleavage
supportTAG
TAG
Target protein
supportTAG
Target protein
④Removal of tag under moderate conditions
Target protein
supportTAG
Target protein
Target protein
Target protein
High cost (Enzyme is expensive)
・Removal of tag may result in a final product
with extra residues
・Unwanted cleavage may occur within the
target protein
Site-specific incorporation of non-natural amino acid -Application 2-
GRB2
TAG
sequence
Target protein
②Expression of tagged fusion protein
The synergistic effects of these factors generate the epigenetic information
↓
Recreating the precise environment mimicking epigenetic regulatory mechanisms
↓
Useful for screening chemical compounds that modulate epigenetic regulation
Tumor cell
Cleavage
sequence
HMG1/2
Linker histones
Me
Selective attack on tumor cell expected to reduce
side-effects and enhance therapeutic efficacy.
Incorporation of
α-hydroxy acid
①Gene
③Purification：Tagged fusion protein captured
on a solid support
Antibody selectively binds to overexpressed
antigens on tumor cells
Release the drug into the intracellular
Corresponding aaRS-derived tRNATyr
required
Conventional
Ac
+
H3.3
H2B
archaebacteriaderived tRNATyr
Overexpresion of
archaebacteria-derived
TyrRS and tRNATyr genes
Site-specific incorporation of non-natural amino acid -Application 11) Drug-antibody conjugate
Non-natural amino acid
Encoding mutant E.coli
TyrRS genes and
suppressor tRNATyr genes
Protein containing
non-natural amino
acids at multiple sites - UAG is now redefined as
RF-1
A U C
ｔＲＮＡ
ＵＡＧ
2) E.coli engineered
for tyrosine analog
incorporation
E.coli
TyrRS mutants E.coli suppressor
tRNATyr
knockout
Release factor
（RF1）
ｔＲＮＡ
ＵＡＧ
3'
A
RFzero-iy
E. coli
Competition between the tRNA and RF-1 at
the UAG stop codon takes place, causing
inevitable inefficiency in incorporating nonnatural amino acids. (Incorporation rate: ≦
30%.）
A U C
U A G
P
Non-natural
amino acid
RFzero
Release factor
（RF1）
Amber suppressor tRNA
Compete
AUGGCTUAGGCUUGGUAGCCGUACGCUUAGGAUUAA
Non-natural Non-natural
amino acid amino acid
Non-natural
amino acid
Release
factor
RF-1
E.coli RFzero
AUGGCTUAGGCUUGGGAUCCGUACGCUUGGGAUUAA
Ribosome
E.coli
・Low cost
・Homogeneity of a final product
・Allows purification of target protein
without extra residues
4)Structural analysis in protein crystallography by the SAD method
-Site-specific incorporation of 3-iodotyrosineComparison of
anomalous signals →
Observation of membrane protein
complex in the mesophase
S
Se
I
CuKa
1.14
6.9
CrKa
0.56
2.28
12.6
(Se -peak, 0.98Å)
1.14
0.23
3.85
3.17
Easier visualization of protein molecules by X -Ray crystallography
ligand
The formation of a ligand-protein
complex. Ligands are labeled with
different fluorescent labels.
・The crystal structure of N-acetyltransferase with iodotyrosine was successfully determined at
1.8 and 2.2 Å resolutions by SAD phasing at CuKα and CrKα wavelengths, respectively
・A comparison with the native structure revealed no significant structural distortion caused by
the iodotyrosine incorporation（rmsd＝0.25Å）.
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