460
Cell-free Translation Systems
against the presence of RFs in the cell-
extract of the cell-free translation sys-
tem. RFs in cell-free extracts hydrolyze
polypeptidyl-tRNA on the P-site, respond-
ing to the occupation of the termination
codon at the A-site, causing a problem-
atical release of the complex. To prevent
this instability, mRNA lacking the termi-
nation codon should be designed. The
degradation of mRNA during cell-free
translation sometimes reduces recovery of
the objective mRNA. Thus, nuclease-free
cell-free translation systems are eagerly
anticipated.
Despite these problems, these two meth-
ods allow the mRNA (genotype) to be
±shed out depending upon the protein
function (phenotype)
in vitro
, relatively
more conveniently and expeditiously than
an
in vivo
system. Moreover,
in vitro
sys-
tems are capable of dealing with a huge
genetic library comprising 10
13
molecules,
which is an advantage over
in vivo
sys-
tems. Particularly, RNA fragments coding
for polypeptides with binding properties
toward certain ligands, such as antibodies,
are enriched in a cloning-independent way
by af±nity selection from a variety of ge-
netic pools. The multiple cycles of
in vitro
selection and ampli±cation of functional
proteins and genes, starting from a ran-
domized pool, can be designated as an
in
vitro
evolution system.
3
Perspectives
At present, most protein production pro-
cesses are carried out utilizing an expres-
sion system in living cells. However, we
are now able to amplify DNA molecules
in a test tube by PCR technology using a
DNA polymerase and to synthesize RNA
by
in vitro
transcription reaction using an
RNA polymerase. Considering that these
cell-free systems de±nitely accelerated an
advance in molecular biology, the devel-
opment of the cell-free translation system
will also greatly contribute to studies on
the structures and/or functions of pro-
teins. Because of simplicity compared to
an
in vivo
expression system, the cell-
free translation system is well suited for
high-throughput protein production in a
postgenome era. Moreover, modi±cation
of the translation process is so easily
achieved that we will be able to develop
an arti±cial system in addition to synthesis
of protein with unnatural amino acids, the
in vitro
virus method and ribosome-display
method.
See
also
DNA
Replication
and
Transcription; Ribosome Structure
and Function, High Resolution.
Bibliography
Books and Reviews
Jermutus,
L.,
Ryabova,
L.A.,
Pluckthun, A.
(1998) Recent advances in producing and
selecting functional proteins by using cell-
free translation,
Curr.
Opin.
Biotechnol.
9
,
534–548.
Primary Literature
Hirao, I., Ohtsuki, T., Fujiwara, T., Mitsui, T.,
Yokogawa, T.,
Okuni, T.,
Nakayama, H.,
Takio, K., Yabuki, T., Kigawa, T., Kodama, K.,
Nishikawa, K.,
Yokoyama, S.
(2002)
An
unnatural base pair for incorporating amino
acid analogs into proteins,
Nat. Biotechnol.
20
,
177–182.
Noren, C.J., Anthony-Cahill, S.J., Grif±th, M.C.,
Schultz, P.G. (1989) A general method for
site-speci±c incorporation of unnatural amino
acids into proteins,
Science
244
, 182–188.
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