153
3. DNA purification kits (e.g., Wizard
®
SV Gel and PCR
Clean- Up System, Promega, WI).
4. Restriction enzymes (e.g., New England Biolabs, Beverly, MA).
5. DNA ligation kit (e.g., Ligation high ver. 2, Toyobo).
6. BGP C-terminal peptides (e.g., Genscript, Piscataway, NJ).
7. pET26/Fv-cpBLA (previously described [6]).
8. pIT2(13CG2) (previously described [21]).
9. pET30b(+) (Novagen, Darmstadt, Germany).
10. pCR4Blunt-TOPO (Zero Blunt TOPO PCR Cloning Kit for
Sequencing, Thermo Fisher Scientific, Waltham, MA).
11. In-Fusion
®
HD cloning kit (Clontech, CA).
12. Tris-EDTA buffer: 10 mM Tris–HCl, 1 mM EDTA, pH 7.4.
1. SHuffle T7 express lysY competent cells (New England Biolabs).
2. LB medium: 1% tryptone, 0.5% yeast extract, 0.5% NaCl, pH 7.0.
3. LBK medium: LB containing antibiotics (50 μg/mL kanamycin).
4. Agar.
5. 1 M Isopropyl-β-d-thiogalactopyranoside (IPTG).
6. Supersonic homogenizer (e.g., VP-15S Taitec, Saitama, Japan).
7. 2-mercaptoethanol.
8. 0.5 M EDTA, pH 8.0.
9. Guanidium hydrochloride (Gdn-HCl).
10. TALON extraction buffer: 300 mM NaCl, 50 mM sodium
phosphate, adjusted to pH 7.0.
11. TALON metal affinity resin and TALON disposable gravity
column (Clontech, CA).
12. TALON elution buffer: TALON extraction buffer supplement
with 200 mM imidazole.
13. Pierce™ BCA protein assay kit for determining protein concentrations (Thermo Fisher Scientific, Waltham, MA).
1. Slide-A-Lyzer Dialysis Cassettes, 3.5 K MWCO, 3 mL
(Thermo Fisher Scientific).
2. Dialysis buffer: 50 mM Tris–HCl, 200 mM NaCl, 1 mM
EDTA, pH 8.0.
1. SDS-PAGE gradient gels (5 ~ 20%) (e.g., e-PAGEL, ATTO,
Tokyo, Japan).
2. SDS running buffer: 25 mM Tris–HCl, 192 mM l-glycine,
1 g/L sodium dodecyl sulfate.
3. PVDF membrane (e.g., Clear Blot Membrane –P plus, ATTO).
2.2 Protein
Expression
2.3 Refolding
of Clampbodies
2.4 Western Blot
of Clampbodies
Regulation of β-Lactamase Activity by Clampbody
3. DNA purification kits (e.g., Wizard
®
SV Gel and PCR
Clean- Up System, Promega, WI).
4. Restriction enzymes (e.g., New England Biolabs, Beverly, MA).
5. DNA ligation kit (e.g., Ligation high ver. 2, Toyobo).
6. BGP C-terminal peptides (e.g., Genscript, Piscataway, NJ).
7. pET26/Fv-cpBLA (previously described [6]).
8. pIT2(13CG2) (previously described [21]).
9. pET30b(+) (Novagen, Darmstadt, Germany).
10. pCR4Blunt-TOPO (Zero Blunt TOPO PCR Cloning Kit for
Sequencing, Thermo Fisher Scientific, Waltham, MA).
11. In-Fusion
®
HD cloning kit (Clontech, CA).
12. Tris-EDTA buffer: 10 mM Tris–HCl, 1 mM EDTA, pH 7.4.
1. SHuffle T7 express lysY competent cells (New England Biolabs).
2. LB medium: 1% tryptone, 0.5% yeast extract, 0.5% NaCl, pH 7.0.
3. LBK medium: LB containing antibiotics (50 μg/mL kanamycin).
4. Agar.
5. 1 M Isopropyl-β-d-thiogalactopyranoside (IPTG).
6. Supersonic homogenizer (e.g., VP-15S Taitec, Saitama, Japan).
7. 2-mercaptoethanol.
8. 0.5 M EDTA, pH 8.0.
9. Guanidium hydrochloride (Gdn-HCl).
10. TALON extraction buffer: 300 mM NaCl, 50 mM sodium
phosphate, adjusted to pH 7.0.
11. TALON metal affinity resin and TALON disposable gravity
column (Clontech, CA).
12. TALON elution buffer: TALON extraction buffer supplement
with 200 mM imidazole.
13. Pierce™ BCA protein assay kit for determining protein concentrations (Thermo Fisher Scientific, Waltham, MA).
1. Slide-A-Lyzer Dialysis Cassettes, 3.5 K MWCO, 3 mL
(Thermo Fisher Scientific).
2. Dialysis buffer: 50 mM Tris–HCl, 200 mM NaCl, 1 mM
EDTA, pH 8.0.
1. SDS-PAGE gradient gels (5 ~ 20%) (e.g., e-PAGEL, ATTO,
Tokyo, Japan).
2. SDS running buffer: 25 mM Tris–HCl, 192 mM l-glycine,
1 g/L sodium dodecyl sulfate.
3. PVDF membrane (e.g., Clear Blot Membrane –P plus, ATTO).
2.2 Protein
Expression
2.3 Refolding
of Clampbodies
2.4 Western Blot
of Clampbodies
Regulation of β-Lactamase Activity by Clampbody