Cell-free Translation Systems
459
In order to link polypeptide and mRNA,
two approaches using cell-free transla-
tion are now available: (1) the ‘‘
in vitro
virus’’ method and (2) the ribosome (or
polysome)-display method.
In the ‘‘
in
vitro
virus
’’ method, cre-
ation of a covalent linkage of bonding
between the mRNA and the protein is ex-
ecuted by cell-free translation depending
on mRNA tagged with puromycin, an an-
tibiotic with a structure resembling the 3
0
end of aminoacyl tRNA (Fig. 4a). Emerg-
ing nascent polypeptide at the P-site is
transferred to mRNA-linked puromycin
at the A-site, giving rise to a covalent-
bonded molecule of the protein product
and responsible mRNA. Consequently,
through the selection toward a polypeptide
portion, mRNA is concurrently isolated,
followed by an RT-PCR reaction of the
mRNA fragment.
Meanwhile, the ribosome (or polysome)-
display method is based upon a non-
covalent complex formation of mRNA,
ribosome, and polypeptide (Fig. 4b). The
mRNA libraries are translated in the
cell-free translation system and the re-
action is hampered by antibiotics, for
instance, chloramphenicol for the sys-
tem derived from
E. coli
,o
rs
im
p
l
yb
y
cooling on ice. The stalling ribosomes
display nascent polypeptides emerging
from translating mRNA, and the selection
is directed toward arresting the mRNA-
ribosome-polypeptide complex. The iso-
lated complex is dissociated with EDTA
by splitting the ribosomal subunit, and the
eluted mRNA is subjected to the RT-PCR
reaction.
To ensure an ef±cient ribosome display,
the stability of the mRNA–ribosome–poly-
peptide
complex
should
be
sustained
RT-PCR
Selection
Dissociation of
the complex
Transcription
DNA
Ribosome
Ligand
Cell-free translation
(stalling of translation)
mRNA
Protein
RT-PCR
Selection
Transcription
DNA
Cell-free
translation
mRNA
Pu
Ligand
Pu
Synthesis of
puromycin
linked
mRNA
mRNA
Pu
Pu
Pu
Ribosome
Protein
Pu
Pu
(a)
(b)
Fig. 4
Schematic drawing of two-selection
method of mRNA by protein function. Ribosome
display (a) is based on the selection toward
attached polypeptide on stalling ribosome. The
RNA-ribosome-polypeptide complex is screened
by immobilized ligand and RNA derived from a
selected complex was ampliFed by RT-PCR.
In
vitro
virus (b) is synthesized puromicin-linked
mRNA, which attacks nascent polypeptides
attached to tRNA on the ribosome. The covalent
complex of mRNA and polypeptide is selected by
the ligand and subjected to RT-PCR.
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