151
designed a novel antibody Fv domain format whose termini were
located at these sites, and named this doubly circularly permuted
antibody Fv as Clampbody. In addition, we also designed a fusion
protein comprising Clampbody and cpBLA by connecting cpV H
and cpV L to the N- and C-termini of cpBLA [7] with a minimum
number of linker residues between them (Clampbody-cpBLA,
Cbody-cpBLA) (Fig. 1b).
We demonstrated the specific antigen binding of Clampbody,
and more importantly, the antigen-dependent catalytic activity of
novel molecular switch Cbody-cpBLA. Interestingly, we also found
that the antigen-dependency of Cbody-cpBLA became more significant in reaction buffers containing some denaturant or detergent such as urea and Triton X-100. From these results, we
reasoned that Cbody-cpBLA was stabilized by the binding of the
antigen peptide, and therefore showed antigen-dependent catalytic
activity. This would be a good example of the allosteric regulation
driven by ligand-induced stabilization or thermodynamic shift,
which is collectively called “ensemble” model [12–15]. By optimizing the reaction condition, Cbody-cpBLA could detect their
antigen peptide specifically at single nM level. Considering the
variety of available substrates, Cbody-cpBLA would be applied to
biosensors, prodrug therapies, and possibly to bacterial growth
regulation. We also propose Clampbody as a useful tool in antibodybased sensing proteins such as protein complementation assay
[16], fluorescence resonance energy transfer-based assay [17, 18],
and transduction assay of reaction intermediate [19, 20]. Taking
advantage of the proximity of their termini, Clampbody might be
fused with many protein domains and fragments, and therefore be
able to control the distance and orientation of fused protein(s) in
an antigen-dependent manner.
2 Materials
Prepare all solutions using ultrapure water (made by purifying
deionized water to attain resistivity of 18 MΩ cm at 25 °C). For all
the reagents, use them of the highest grade available. The chemicals and other reagents, unless otherwise indicated, were obtained
from Sigma (St. Lois, MO), Nacalai Tesque (Kyoto, Japan), or
Wako Pure Chemical Industries (Osaka, Japan).
1. Oligonucleotides (see Table 1) (e.g., Greiner Japan, Tokyo,
Japan; Eurofins genomics, Tokyo, Japan).
2. High-fidelity thermostable DNA polymerase (e.g., KOD FX,
Toyobo, Osaka, Japan; PrimeSTAR
®
Max DNA Polymerase,
Takara-Bio, Shiga, Japan).
2.1 Construction
of Clampbody Genes
Regulation of β-Lactamase Activity by Clampbody
designed a novel antibody Fv domain format whose termini were
located at these sites, and named this doubly circularly permuted
antibody Fv as Clampbody. In addition, we also designed a fusion
protein comprising Clampbody and cpBLA by connecting cpV H
and cpV L to the N- and C-termini of cpBLA [7] with a minimum
number of linker residues between them (Clampbody-cpBLA,
Cbody-cpBLA) (Fig. 1b).
We demonstrated the specific antigen binding of Clampbody,
and more importantly, the antigen-dependent catalytic activity of
novel molecular switch Cbody-cpBLA. Interestingly, we also found
that the antigen-dependency of Cbody-cpBLA became more significant in reaction buffers containing some denaturant or detergent such as urea and Triton X-100. From these results, we
reasoned that Cbody-cpBLA was stabilized by the binding of the
antigen peptide, and therefore showed antigen-dependent catalytic
activity. This would be a good example of the allosteric regulation
driven by ligand-induced stabilization or thermodynamic shift,
which is collectively called “ensemble” model [12–15]. By optimizing the reaction condition, Cbody-cpBLA could detect their
antigen peptide specifically at single nM level. Considering the
variety of available substrates, Cbody-cpBLA would be applied to
biosensors, prodrug therapies, and possibly to bacterial growth
regulation. We also propose Clampbody as a useful tool in antibodybased sensing proteins such as protein complementation assay
[16], fluorescence resonance energy transfer-based assay [17, 18],
and transduction assay of reaction intermediate [19, 20]. Taking
advantage of the proximity of their termini, Clampbody might be
fused with many protein domains and fragments, and therefore be
able to control the distance and orientation of fused protein(s) in
an antigen-dependent manner.
2 Materials
Prepare all solutions using ultrapure water (made by purifying
deionized water to attain resistivity of 18 MΩ cm at 25 °C). For all
the reagents, use them of the highest grade available. The chemicals and other reagents, unless otherwise indicated, were obtained
from Sigma (St. Lois, MO), Nacalai Tesque (Kyoto, Japan), or
Wako Pure Chemical Industries (Osaka, Japan).
1. Oligonucleotides (see Table 1) (e.g., Greiner Japan, Tokyo,
Japan; Eurofins genomics, Tokyo, Japan).
2. High-fidelity thermostable DNA polymerase (e.g., KOD FX,
Toyobo, Osaka, Japan; PrimeSTAR
®
Max DNA Polymerase,
Takara-Bio, Shiga, Japan).
2.1 Construction
of Clampbody Genes
Regulation of β-Lactamase Activity by Clampbody