Cell-free Translation Systems
455
systems, had been developed, though both
showed limited results.
However, the short lifetime of the cell-
free translation system was overcome by
the development of the continuous-flow
cell-free (CFCF) system by A Spirin’s
group in 1988. In CFCF, prolongation of
the reaction was achieved by the contin-
uous feeding of amino acids and energy
components, and continuous removal of
reaction products. The reaction was carried
out in a chamber connected to a reservoir
to supply the substrates, ATP, GTP, and
amino acids, by pumping, and the reactor
was separated at the outlet by a membrane
facilitating permeation of the synthesized
protein and a low molecular weight re-
action product (Fig. 1a). By this system,
it appeared that the reaction using wheat
germ extract proceeds for up to 100 h and
was able to synthesize 120 to 240
µ
gofpro-
tein in a 1-mL reaction chamber. The most
plausible explanation for this long sustain-
ability is that the shortage of an energy
source such as ATP and GTP during the
reaction is compensated by the supple-
ment of a feeding solution, considering
that in the batch system using cell extracts,
the ATPase inevitably dwelling in the ex-
tract rapidly consumes ATP. The simpler
system without the aid of pumping was de-
veloped on the basis of the dialysis method
of low molecular weight compounds and
designated as the continuous-exchange
cell-free (CECF) system (Fig. 1b).
The improvement of productivity and
reproducibility of cell-free translation has
also been achieved by examining the reac-
tion condition and preparation procedures
of crude cell-extract. Preparation of crude
extract from polished wheat germ ensures
constant production of proteins and pro-
longation of reaction in the CFCF system.
By washing the embryo, endogenous in-
hibitors of translation, including ribosome
inactivating proteins (RIP) such as ricin
toxin, are successfully eliminated from ex-
tracts, which may result in such drastic
Cell-free translation
Feeding
solution
Ultrafiltration
membrane
Amino acids
ATP, GTP
Ultrafiltration
membrane
Pump
Amino acids
ATP, GTP
Cell-free translation
Feeding
solution
Protein product
Reactor
(a)
(b)
Fig. 1
Schematic illustrations of (a) CFCF and (b) CECF translation systems. A feeding
solution containing ATP, GTP, and amino acids is supplied to the reaction chamber in which
cell-free translation proceeds, by pumping (CFCF) or diffusion (CECF).
previous page 1129 Encyclopedia of Molecular Cell Biology and Molecular Medicine read online next page 1131 Encyclopedia of Molecular Cell Biology and Molecular Medicine read online Home Toggle text on/off