452
Cell-free Translation Systems
Unnatural Amino Acid
Amino acids that are not used by living organisms.
In vitro Virus
The selection method by RNA molecules covalently bound to the protein product.
Ribosome Display
The selection method for mRNA by the function of the polypeptide displayed on
ribosome stalling during the translation process.
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A disrupted cell is capable of synthesizing protein from amino acids depending upon
the template RNA. This discovery, half a century ago, set the stage for biochemical
studies on the gene expression process occurring in cells. The cell-free translation
system successfully revealed the function of a number of key molecules participating
in the translation system, such as ribosome, mRNA, tRNA, and protein factors
and, moreover, elucidated the very complicated mechanism of protein synthesis.
Although still frequently utilized in these experimental studies, cell-free translation
is beginning to make a mark as an attractive tool for protein production as a potential
alternative to an
in vivo
expression system. Moreover, the cell-free translation system
is prospective as a method for the synthesis of protein with unnatural amino acids
and for the selection of genotypes from peptide libraries.
1
History
1.1
Beginning
Proteins are translated on ribosomes from
mRNAs transcribed from DNA. The out-
line of how translation proceeds had been
elucidated by both
in vitro
and
in vivo
approaches for two decades after the dis-
covery of the double-helix structure of
DNA. In the 1950s, most studies focusing
on the gene expression process were car-
ried out on the basis of a genetic approach
using bacteria and phages. The hypothesis
of the central dogma, DNA makes the RNA
that makes protein, had been evidenced by
various observations obtained from
in vivo
experiments. However, the precise mech-
anism of translation, such as deciphering
information on mRNA, had to await a
series of veriFcations by the
in vitro
trans-
lation system using disrupted cells. The
Frst demonstration of protein synthesis
using cell-extract was described by Zamec-
nik’s group in 1952. The Fnding that rat
liver extract was capable of incorporating
amino acids into polypeptides allowed us
to elucidate the involvement of individual
molecular players in the gene expression
process. Thereafter, it was shown in 1955
that the ribonucleoprotein complex, which
wasdes
ignatedasr
ibosome
,cata
lyzesthe
peptide bond formation. Up to 1960, the
construction of bacterial cell-free transla-
tion systems had been achieved by several
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