163
2. Culture supernatant (2 mL) can be precipitated with 10%
trichloroacetic acid, and washed twice with cold acetone, and
resuspended in 20 μL of PBS and used for SDS-PAGE.
3. The supernatant including soluble protein should be kept for
SDS-PAGE.
4. Before adding to the solubilized protein, TALON metal affinity
resin should be washed twice with TALON extraction buffer
containing 6 M Gdn-HCl.
5. Keep stirring the buffer using a magnetic stirrer.
6. The purified enzyme in the buffer containing 15% glycerol can
be stored for several months at −80 °C. The concentration of
the proteins in stock solution was typically 1 μM in our hands.
Acknowledgment
We thank Tatsuya Shinoda of Kyowa Medex Co. for generously
allowing the use of the genes derived from KTM-219 IgG. This
study was supported by Grant-in-Aid for Scientific Research
(Grants 24360336 and 15H04191 to H.U. and Grant 46420793
to J.D.) from JSPS, Japan, and partly by Strategic International
Collaborative Research Program, Japan Science and Technology
Agency (JST).
Fig. 4 S-V plots for BLA and Cbody-cpBLA. (a) 2 nM BLA or (b) 70 nM Cbody-cpBLA was added with nitrocefin
substrate in the presence or absence of antigen 1 μM BGP-C10, in PBS containing 1.2 M urea, incubated at
30 °C, and read for absorbance. Background absorbance of the samples without BLA or Cbody-cpBLA was
subtracted for each condition. Initial velocities are plotted against the substrate concentration. Averages of
three samples with an error bar for 1 SD are shown. Fitted curves based on the Michaelis-Menten equation are
shown for each condition. Statistical analysis was conducted using the two-tailed unpaired Student’s t-test.
(∗∗) p < 0.01. Reproduced from Iwai et al. [10] with permission from American Chemical Society
Regulation of β-Lactamase Activity by Clampbody
2. Culture supernatant (2 mL) can be precipitated with 10%
trichloroacetic acid, and washed twice with cold acetone, and
resuspended in 20 μL of PBS and used for SDS-PAGE.
3. The supernatant including soluble protein should be kept for
SDS-PAGE.
4. Before adding to the solubilized protein, TALON metal affinity
resin should be washed twice with TALON extraction buffer
containing 6 M Gdn-HCl.
5. Keep stirring the buffer using a magnetic stirrer.
6. The purified enzyme in the buffer containing 15% glycerol can
be stored for several months at −80 °C. The concentration of
the proteins in stock solution was typically 1 μM in our hands.
Acknowledgment
We thank Tatsuya Shinoda of Kyowa Medex Co. for generously
allowing the use of the genes derived from KTM-219 IgG. This
study was supported by Grant-in-Aid for Scientific Research
(Grants 24360336 and 15H04191 to H.U. and Grant 46420793
to J.D.) from JSPS, Japan, and partly by Strategic International
Collaborative Research Program, Japan Science and Technology
Agency (JST).
Fig. 4 S-V plots for BLA and Cbody-cpBLA. (a) 2 nM BLA or (b) 70 nM Cbody-cpBLA was added with nitrocefin
substrate in the presence or absence of antigen 1 μM BGP-C10, in PBS containing 1.2 M urea, incubated at
30 °C, and read for absorbance. Background absorbance of the samples without BLA or Cbody-cpBLA was
subtracted for each condition. Initial velocities are plotted against the substrate concentration. Averages of
three samples with an error bar for 1 SD are shown. Fitted curves based on the Michaelis-Menten equation are
shown for each condition. Statistical analysis was conducted using the two-tailed unpaired Student’s t-test.
(∗∗) p < 0.01. Reproduced from Iwai et al. [10] with permission from American Chemical Society
Regulation of β-Lactamase Activity by Clampbody