Biomedical Engineering Reference
In-Depth Information
NH
2
O
-
N
-
Codon for residue of interest
O
PO
NHAlloc
O
N
O
O
A
ll
UUC
TAG
N
O
1
AllO
PO
NH
2
O
N
O
O
3
N
O
N
-
O
PO
NHAlloc
Plasmid
Plasmid
O
N
O
N
N
O
N
AllO
PO
O
N
N
O
O
O
R
Plasmid DNA containing
the gene of interest
H
OH
OH
O
2
pdCpA
pdCpA-uAA
4
2
C
UAG
mRNA
O
R
CUA
NH
2
A
C
C
Truncated tRNA
d
5
CUA
tRNA loaded with an
unnatural amino acid
Xenopus
oocyte
7
Mutant protein evaluated
with electrophysiology
Xenopus
oocyte
expressing mutant protein
FIGURE 4.8
Example of site-specii c incorporation of unnatural amino acids into proteins expressed in
Xenopus
oocytes.
Chemical synthesis and aminoacylation of the dinucleotide, pdCpA;
Ligation of pdCpA-
uAA to a truncated tRNA bearing the amber stop anticodon;
Mutation of the codon encoding the residue
of interest into TAG amber stop codon by using site-directed mutagenesis;
Generation of mRNA through
in vitro
transcription;
Expression of mut a nt protei n i n
Xenopus
oocyte after coinjection of mRNA and tRNA,
and the evaluation of the mutant protein with electrophysiology (two-electrode voltage-clamp recordings).
Similarly, the technology has been applied to evaluate the importance of a proline residue for
opening and closure of 5-HT
3
receptors. It was hypothesized that
cis-trans
isomerism of the proline
residue in the ion channel domain was important for the opening and closure of 5-HT
3
receptors.
This was investigated by the incorporation of proline analogs with either increased or decreased
probability of
cis-trans
isomerism and there was a distinct correlation between this ability and the
ability of the ion channel to open.
Although the technology has obvious and wide-ranging potential, it also has substantial limi-
tations. Firstly, the generation of aa-tRNAs requires highly skilled persons in both chemistry
and molecular biology. Secondly, the amount of protein generated is very low, thus exceptionally