156
Similarly, make a construct for the circularly permuted V L (cpV L )
with new termini between Pro40 and Gly41. In this case, two Cys
residues at the N- and C-termini of cpV L are not added to avoid
unfavorable interdomain crosslinking at the later stage.
1. Amplify the DNA fragment encoding Gly41 to Arg108 of V L
with a linker peptide (G 4 S) 3 at the C-terminus by PCR using
the primers AgecpVLrev and VLG4Sfor, pET26/Fv-cpBLA as
a template, and KOD FX DNA polymerase.
2. Amplify the DNA fragment encoding Asp1 to Pro40 with the
linker at the N-terminus by PCR with G4S3VLrev and cpVLNotfor 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 DNA polymerase for
15 cycles without primers and 35 cycles with AgecpVLrev and
cpVLNotfor primers.
5. Purify these fragments by Wizard
®
SV Gel and PCR Clean-Up
System.
6. Ligate into pCR4Blunt-TOPO.
To make a fusion protein comprising Clampbody and cpBLA, first
make a construct for the cpBLA with known 3D structure that
Guntas et al. reported [23, 24].
1. Amplify the DNA fragment encoding Trp227 to Trp286 of
TEM-1 β-lactamase with a short peptide linker (GSGGS) at
the C-terminus by PCR using the primers InfVHSpeRG13rev
and RG13OL1for, pIT2/31IJ3 as a template, and PrimeSTAR
®
Max DNA Polymerase.
2. Amplify the fragment encoding His24 to Gly226 with the
linker at the N-terminus by PCR with RG13OL2rev and
RG13AgeVLInffor as primers.
3. Purify these fragments by Wizard
®
SV Gel and PCR Clean-Up
System.
4. With the gel-purified PCR products (20 ng each), perform
overlap-extension PCR with PrimeSTAR
®
Max DNA
Polymerase for 15 cycles without primers and 35 cycles with
InfVHSpeRG13rev and RG13AgeVLInffor primers.
5. Purify these fragments by Wizard
®
SV Gel and PCR Clean-Up
System.
Make a construct for 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 amino acid residues
(Clampbody-cpBLA, Cbody-cpBLA) (Fig. 1b, c).
3.3 Construction
of the cpV L Gene
3.4 Construction
of the cpBLA(RG13)
Gene
3.5 Construction
of Cbody-cpBLA
Fusion Protein
Expression Vectors
Hiroto Iwai et al.
Similarly, make a construct for the circularly permuted V L (cpV L )
with new termini between Pro40 and Gly41. In this case, two Cys
residues at the N- and C-termini of cpV L are not added to avoid
unfavorable interdomain crosslinking at the later stage.
1. Amplify the DNA fragment encoding Gly41 to Arg108 of V L
with a linker peptide (G 4 S) 3 at the C-terminus by PCR using
the primers AgecpVLrev and VLG4Sfor, pET26/Fv-cpBLA as
a template, and KOD FX DNA polymerase.
2. Amplify the DNA fragment encoding Asp1 to Pro40 with the
linker at the N-terminus by PCR with G4S3VLrev and cpVLNotfor 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 DNA polymerase for
15 cycles without primers and 35 cycles with AgecpVLrev and
cpVLNotfor primers.
5. Purify these fragments by Wizard
®
SV Gel and PCR Clean-Up
System.
6. Ligate into pCR4Blunt-TOPO.
To make a fusion protein comprising Clampbody and cpBLA, first
make a construct for the cpBLA with known 3D structure that
Guntas et al. reported [23, 24].
1. Amplify the DNA fragment encoding Trp227 to Trp286 of
TEM-1 β-lactamase with a short peptide linker (GSGGS) at
the C-terminus by PCR using the primers InfVHSpeRG13rev
and RG13OL1for, pIT2/31IJ3 as a template, and PrimeSTAR
®
Max DNA Polymerase.
2. Amplify the fragment encoding His24 to Gly226 with the
linker at the N-terminus by PCR with RG13OL2rev and
RG13AgeVLInffor as primers.
3. Purify these fragments by Wizard
®
SV Gel and PCR Clean-Up
System.
4. With the gel-purified PCR products (20 ng each), perform
overlap-extension PCR with PrimeSTAR
®
Max DNA
Polymerase for 15 cycles without primers and 35 cycles with
InfVHSpeRG13rev and RG13AgeVLInffor primers.
5. Purify these fragments by Wizard
®
SV Gel and PCR Clean-Up
System.
Make a construct for 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 amino acid residues
(Clampbody-cpBLA, Cbody-cpBLA) (Fig. 1b, c).
3.3 Construction
of the cpV L Gene
3.4 Construction
of the cpBLA(RG13)
Gene
3.5 Construction
of Cbody-cpBLA
Fusion Protein
Expression Vectors
Hiroto Iwai et al.