155
3 Methods
To create a Clampbody as an antigen-dependent switch, any antibody for small molecule antigen, whose variable region
(Fv = V H + V L ) is stabilized by the bound antigen, will suffice.
Here, we employ the V H and V L domains of antibody KTM219
[22], which recognizes the C-terminal peptide of the human bone
Gla protein (BGP) as a model. First, clone the V H and V L genes by
the following procedure.
1. Amplify the DNA fragment encoding V H fragment of the antibody by PCR using the primers NcoVHrev and VHSpefor,
pET26/Fv-cpBLA as a template, and KOD FX DNA polymerase and the DNA fragment encoding V L fragment of the
antibody by PCR with AgeVLrev and VLNotfor as primers.
2. Purify the resulting DNA fragments by Wizard
®
SV Gel and
PCR Clean-Up System as per manufacturer’s instructions.
3. Ligate these fragments into pCR4Blunt-TOPO as per manufacturer’s instructions.
Make a construct for a circularly permuted V H (cpV H ), whose new
termini are generated between Pro41 and Gly42 (as described
according to Kabat numbering) while the native N- and C-termini
are connected via a flexible linker (G 4 S) 3 . Considering the possible
lesser stability of V H fragment, two Cys residues are added at the
N- and C-termini of cpV H to promote the correct folding and
increased stability through the disulfide bridge.
1. Amplify the DNA fragment encoding Gly42 to Ser113 of V H
with a linker peptide (G 4 S) 3 at the C-terminus by PCR using
the primers NcoCcpVHrev and VHG4Sfor, pET26/Fv-cpBLA
as a template, and KOD FX DNA polymerase.
2. Amplify the DNA fragment encoding Glu1 to Pro41, described
according to Kabat numbering, with the linker at the
N- terminus by PCR using G4S3VHrev and cpVHCSpefor as
primers.
3. Purify these fragments by Wizard
®
SV Gel and PCR Clean-Up
System.
4. Perform overlap-extension PCR with the gel-purified PCR
products (20 ng each) and KOD FX for 15 cycles without
primers and 35 cycles with NcoCcpVHrev and cpVHCSpefor
primers.
5. Purify these fragments by Wizard
®
SV Gel and PCR Clean-Up
System.
6. Ligate into pCR4Blunt-TOPO.
3.1 Construction
of the V H and V L genes
for Anti- BGP
C-Terminal Peptide
Antibody
3.2 Construction
of the cpV H Gene
Regulation of β-Lactamase Activity by Clampbody
3 Methods
To create a Clampbody as an antigen-dependent switch, any antibody for small molecule antigen, whose variable region
(Fv = V H + V L ) is stabilized by the bound antigen, will suffice.
Here, we employ the V H and V L domains of antibody KTM219
[22], which recognizes the C-terminal peptide of the human bone
Gla protein (BGP) as a model. First, clone the V H and V L genes by
the following procedure.
1. Amplify the DNA fragment encoding V H fragment of the antibody by PCR using the primers NcoVHrev and VHSpefor,
pET26/Fv-cpBLA as a template, and KOD FX DNA polymerase and the DNA fragment encoding V L fragment of the
antibody by PCR with AgeVLrev and VLNotfor as primers.
2. Purify the resulting DNA fragments by Wizard
®
SV Gel and
PCR Clean-Up System as per manufacturer’s instructions.
3. Ligate these fragments into pCR4Blunt-TOPO as per manufacturer’s instructions.
Make a construct for a circularly permuted V H (cpV H ), whose new
termini are generated between Pro41 and Gly42 (as described
according to Kabat numbering) while the native N- and C-termini
are connected via a flexible linker (G 4 S) 3 . Considering the possible
lesser stability of V H fragment, two Cys residues are added at the
N- and C-termini of cpV H to promote the correct folding and
increased stability through the disulfide bridge.
1. Amplify the DNA fragment encoding Gly42 to Ser113 of V H
with a linker peptide (G 4 S) 3 at the C-terminus by PCR using
the primers NcoCcpVHrev and VHG4Sfor, pET26/Fv-cpBLA
as a template, and KOD FX DNA polymerase.
2. Amplify the DNA fragment encoding Glu1 to Pro41, described
according to Kabat numbering, with the linker at the
N- terminus by PCR using G4S3VHrev and cpVHCSpefor as
primers.
3. Purify these fragments by Wizard
®
SV Gel and PCR Clean-Up
System.
4. Perform overlap-extension PCR with the gel-purified PCR
products (20 ng each) and KOD FX for 15 cycles without
primers and 35 cycles with NcoCcpVHrev and cpVHCSpefor
primers.
5. Purify these fragments by Wizard
®
SV Gel and PCR Clean-Up
System.
6. Ligate into pCR4Blunt-TOPO.
3.1 Construction
of the V H and V L genes
for Anti- BGP
C-Terminal Peptide
Antibody
3.2 Construction
of the cpV H Gene
Regulation of β-Lactamase Activity by Clampbody