157
1. Amplify the DNA fragment encoding NcoI, SpeI, AgeI, and
NotI sites by PCR using the primers MCScpBLArev and
cpBLAMCSfor, pET26/Fv-cpBLA as a template, and KOD
FX DNA polymerase.
2. Purify these fragments by Wizard
®
SV Gel and PCR Clean-Up
System.
3. Digest the gel-purified PCR product (2 μg) with NcoI HF and
NotI HF.
4. Purify the resulting DNA fragments using the Wizard
®
SV Gel
and PCR Clean-Up System and insert into pIT2(13CG2)
digested with the same, resulting in pIT2/MCS.
5. Digest the cpV H gene with Nco I and Spe I.
6. Purify these fragments by Wizard
®
SV Gel and PCR Clean-Up
System and inserted into pIT2/MCS digested with the same.
7. After the cpV H insertion, digest the cpV L gene with Age I and
Not I.
8. Purify these fragments using Wizard
®
SV Gel and PCR
Clean- Up System and inserted into cpV H -inserted pIT2/
MCS.
9. Digest these plasmids with NcoI and NotI.
10. Purify these fragments by Wizard
®
SV Gel and PCR Clean-Up
System and insert into pET30b(+) digested with the same,
resulting in pET30b/cpVH-cpVL.
11. Insert the cpBLA(RG13) gene into pET30b/cpVH-cpVL
with In-Fusion
®
HD Cloning Kit according to the manufacturer, resulting in pClamp-cpBLA.
Prepare a single-chain protein construct where cpV H and cpV L are
connected by a short linker G 4 S (single-chain Clampbody, scCb)
to evaluate the antigen-binding activity of the Clampbody.
1. Anneal TSG4TGrev and TSG4TGfor in Tris-EDTA buffer to
prepare the DNA fragment encoding the SG 4 linker.
2. Insert the fragment into pClamp-cpBLA digested with AgeI
and SpeI, resulting in pClamp-SG4.
When Clampbody and Cbody-cpBLA are expressed in E. coli, the
majority of the expressed proteins will accumulate in inclusion
body even if cultured at lower temperature. In such cases, perform
the following procedures to solubilize the insoluble Clampbodies.
1. Transform SHuffle T7 Express lysY competent cells with
expression vectors (see Note 1).
3.6 Construction
of scCb Expression
Vectors
3.7 Expression
and Purification
of Clampbodies [6, 25]
Regulation of β-Lactamase Activity by Clampbody
1. Amplify the DNA fragment encoding NcoI, SpeI, AgeI, and
NotI sites by PCR using the primers MCScpBLArev and
cpBLAMCSfor, pET26/Fv-cpBLA as a template, and KOD
FX DNA polymerase.
2. Purify these fragments by Wizard
®
SV Gel and PCR Clean-Up
System.
3. Digest the gel-purified PCR product (2 μg) with NcoI HF and
NotI HF.
4. Purify the resulting DNA fragments using the Wizard
®
SV Gel
and PCR Clean-Up System and insert into pIT2(13CG2)
digested with the same, resulting in pIT2/MCS.
5. Digest the cpV H gene with Nco I and Spe I.
6. Purify these fragments by Wizard
®
SV Gel and PCR Clean-Up
System and inserted into pIT2/MCS digested with the same.
7. After the cpV H insertion, digest the cpV L gene with Age I and
Not I.
8. Purify these fragments using Wizard
®
SV Gel and PCR
Clean- Up System and inserted into cpV H -inserted pIT2/
MCS.
9. Digest these plasmids with NcoI and NotI.
10. Purify these fragments by Wizard
®
SV Gel and PCR Clean-Up
System and insert into pET30b(+) digested with the same,
resulting in pET30b/cpVH-cpVL.
11. Insert the cpBLA(RG13) gene into pET30b/cpVH-cpVL
with In-Fusion
®
HD Cloning Kit according to the manufacturer, resulting in pClamp-cpBLA.
Prepare a single-chain protein construct where cpV H and cpV L are
connected by a short linker G 4 S (single-chain Clampbody, scCb)
to evaluate the antigen-binding activity of the Clampbody.
1. Anneal TSG4TGrev and TSG4TGfor in Tris-EDTA buffer to
prepare the DNA fragment encoding the SG 4 linker.
2. Insert the fragment into pClamp-cpBLA digested with AgeI
and SpeI, resulting in pClamp-SG4.
When Clampbody and Cbody-cpBLA are expressed in E. coli, the
majority of the expressed proteins will accumulate in inclusion
body even if cultured at lower temperature. In such cases, perform
the following procedures to solubilize the insoluble Clampbodies.
1. Transform SHuffle T7 Express lysY competent cells with
expression vectors (see Note 1).
3.6 Construction
of scCb Expression
Vectors
3.7 Expression
and Purification
of Clampbodies [6, 25]
Regulation of β-Lactamase Activity by Clampbody