456
Cell-free Translation Systems
extensions of reaction and high yields
of proteins. More recently, a reconstruc-
tion of the cell-free translation system
using puriFed components was success-
fully achieved. Three initiation factors,
three elongation factors, three release fac-
tors, RR±s, 20 individual aminoacyl tRNA
synthetases and methionyl-tRNA trans-
f
o
rm
y
l
a
s
ew
e
r
ec
l
on
e
df
r
omt
h
e
E. coli
genome for efFcient expression in
E. coli
cells and puriFed. This reconstituted sys-
tem, designated as the Protein synthesis
using recombinant elements (PURE) sys-
tem, is capable of synthesizing approxi-
mately 0.1 mg of proteins in 1-mL volume
per hour without any supplemental system
such as C±C± and CEC±.
2.2
Synthesis of ArtiFcial Protein
One application of cell-free translation
is the incorporation of unnatural amino
acids
into
polypeptides.
The
amino
acid–comprising proteins are canonically
conFned to 20 kinds, barring exceptions
such as selenocystein, and therefore
in vivo
systems are unable to synthesize proteins
with other amino acids. In 1989, Shultz’s
group developed a method to incorporate
unnatural amino acids at a desired po-
sition in polypeptides using the cell-free
translation system. In this system, as il-
lustrated in ±ig. 2, synthesized suppressor
tRNA are Frst attached by a chemical or en-
zymatic method to unnatural amino acids
and subjected to cell-free translation for
DNA templates possessing amber codons
at the desired position. The attachment of
unnatural amino acids is mostly carried
out as follows. An unnatural amino acid
is Frst attached to 3
0
OH of adenosine of
pCpA dinucleotide by a chemical method.
Later, the resultant aminoacylated pCpA
is ligated to an amber suppressor tRNA
lacking a 3
0
-terminal CA sequence using
UAG
Unnatural
amino acid
A
C
C
3
C
pCpA-Unnatural amino acid
T4 RNA ligase
Suppressor tRNA (-CA)
mRNA
TAG
DNA
In vitro
transcription
Cell-free
translation
Protein with
unnatural amino acid
at a specific site
AUC
Amber
CUA
±ig. 2
Strategy for incorporation of unnatural amino acid into polypeptide using the cell-free
translation system.
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