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
 
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