454
Cell-free Translation Systems
steps. In the case of the elongation process,
Elongation Factor (EF)-Tu was ±rst found
to be a factor that stimulates poly U-
dependent
polyphenylalanine
synthesis
and
thereafter
it
appeared
to
convey
aminoacylated tRNA to the ribosome as
a GTP-binding form. EF-Ts were shown
to catalyze the exchange of GTP to GDP
on EF-Tu. EF-G appeared to catalyze the
hydrolysis of the triphosphate of GTP
depending on the ribosome and appeared
to be involved in the translocation of tRNA
from the A-site to the P-site.
In the case of the initiation process, ini-
tiation factor-2 (IF-2) was shown to carry
initiator tRNA attached by formymethio-
nine onto a 30S subunit of ribosome.
Initiation factor-3 (IF-3) was indicated to
dissociate 70S ribosome resulting from the
termination reaction. Initiation factor-1
(IF-1) was found to enhance the disas-
sembling activity of IF3 by entering an
inter-subunit space. Regarding the termi-
nation process, release factor-1(RF-1) and
release factor-2 (RF-2) enter the A-site of
the ribosome depending on the termina-
tion codons to catalyze the hydrolysis of
peptidyl-tRNA on the P-site of ribosome.
RF-1 recognizes UAG and UAA codons,
while RF-2 is responsible for UGA and
UAA. While the function of these two RFs
was veri±ed at an early stage of the study
on translation, RF-3 and the ribosome re-
cycling factor (RRF) had remained less
understood. Very recently RF-3 was shown
to be involved in the release of RF-1 and
RF-2 from the ribosome. RRF was ±rst dis-
covered as a factor enhancing the activity of
the cell-free translation system and shown
to promote the dissociation of mRNA and
tRNA from the ribosome.
Similarly, eukaryotic translational fac-
tors have been studied by the cell-free
translation system. Although most essen-
tial factors coincide with bacterial factors,
±nding a number of additional proteins
enhancing the cell-free translation system,
especially in the initiation process, sug-
gests that the eukaryotic system appeared
to be much more complicated than the
bacterial system.
2
Applications
2.1
Protein Production
Although having long served as a useful
system for the elucidation of the transla-
tion mechanism, the cell-free translation
system had been paid only slight attention
as a tool for the preparation of pro-
tein. Most protein productions at present
are based upon the expression system of
cloned genes in living cells, in which we
sometimes undergo failure, particularly in
cases of toxic or unstable proteins. There-
fore, it was desired that an
in vitro
gene
expression system using the cell-free trans-
lation system would compensate for the
in vivo
expression system. However, the
low yield of protein product had hindered
the utilization of the cell-free translation
system. The conventional cell-free trans-
lation system using crude extracts, such
as
E. coli
S30, rabbit reticulocyte lysate
and wheat germ extracts, produces trans-
lation reactions for no longer than 1 h
in the batch system and synthesized pro-
tein products to such a small degree as to
be detected only by labeling with radioac-
tive amino acids. To improve productivity,
transcription/translation, coupled with us-
ing phage RNA polymerases such as T7
or SP6 RNA polymerases, and energy
regenerating systems for ATP recycling
such as creatine phosphate/creatine kinase
or phosphoenolpyruvate/pyurvate kinase
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