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).
