123
22. 20 mM coenzyme A trilithium salt stock solution dissolved in
water, and stored at −20 °C.
23. For the determination of enzyme kinetics, use a multiwell
luminometer such as AB-2350 equipped with a sample injector
(ATTO, Tokyo, Japan).
24. For the calculation of enzyme kinetics, use a software that can
perform nonlinear curve fitting, such as Kaleida Graph 4.1
(Synergy Software, Reading, PA).
25. For FlimPIA with faster kinetics, use a tube-based luminometer such as NU-2600 equipped with a sample injector and a
stirrer (Microtech Nichion, Chiba, Japan).
26. Optional: For the measurement of LH 2 -AMP concentration,
use a fluorescence spectrometer such as FP-8500 (Jasco, Tokyo,
Japan).
3 Methods
1. Amplify the DNA fragment encoding FLuc derived of Photinus
pyralis (Ppy) by PCR using pGEX-Ppy [4] or pGEM-luc vector
as a template, and primers NotG4SBack (with G 4 S linker and a
NotI site) and XhoFor (with an XhoI site).
2. Digest the amplified fragment with restriction enzymes NotI
and XhoI, and subclone into the multicloning site of pET32b
between NotI and XhoI sites.
3. Prepare a pair of DNA fragments each encoding a binding
domain (BD) of your interest, which are appended with restriction sites NcoI and NotI by PCR. Digest the product with the
same enzymes, and ligate each with the plasmid prepared in 2
that was digested with NcoI and NotI (see Notes 3 and 4).
Here, the procedure is exemplified with FKBP12 and FRB that
are denoted as BD1 and BD2, respectively (see Note 5).
4. Transform JM109 competent E. coli cells with the ligation
mixture.
5. Culture the cells on LBA agar plates and incubate overnight at
37 °C.
6. Pick a colony and culture it in 4 mL LBA medium and incubate overnight at 37 °C.
7. Extract the plasmid, and confirm the nucleotide sequence of
the entire open reading frame.
1. To make H245D/K443A/L530R/E354K mutants as the
Donor, conduct a series of site-directed mutagenesis with
QuikChange Site-directed mutagenesis kit as per manufacturer’s
instructions. Use pET32/BD1/Fluc as the template and primers
H245D, K443A, L530R, and E354K (Table 1) in combination
with their complementary strand primers (see Notes 6 and 7).
3.1 Construction
of the Binding
Domain-Fused
Expression Vectors
(Fig. 2)
3.2 Construction
of BD1/Donor
Expression Vector
(Fig. 2a)
Sensitive Protein-Protein Interaction Assay
22. 20 mM coenzyme A trilithium salt stock solution dissolved in
water, and stored at −20 °C.
23. For the determination of enzyme kinetics, use a multiwell
luminometer such as AB-2350 equipped with a sample injector
(ATTO, Tokyo, Japan).
24. For the calculation of enzyme kinetics, use a software that can
perform nonlinear curve fitting, such as Kaleida Graph 4.1
(Synergy Software, Reading, PA).
25. For FlimPIA with faster kinetics, use a tube-based luminometer such as NU-2600 equipped with a sample injector and a
stirrer (Microtech Nichion, Chiba, Japan).
26. Optional: For the measurement of LH 2 -AMP concentration,
use a fluorescence spectrometer such as FP-8500 (Jasco, Tokyo,
Japan).
3 Methods
1. Amplify the DNA fragment encoding FLuc derived of Photinus
pyralis (Ppy) by PCR using pGEX-Ppy [4] or pGEM-luc vector
as a template, and primers NotG4SBack (with G 4 S linker and a
NotI site) and XhoFor (with an XhoI site).
2. Digest the amplified fragment with restriction enzymes NotI
and XhoI, and subclone into the multicloning site of pET32b
between NotI and XhoI sites.
3. Prepare a pair of DNA fragments each encoding a binding
domain (BD) of your interest, which are appended with restriction sites NcoI and NotI by PCR. Digest the product with the
same enzymes, and ligate each with the plasmid prepared in 2
that was digested with NcoI and NotI (see Notes 3 and 4).
Here, the procedure is exemplified with FKBP12 and FRB that
are denoted as BD1 and BD2, respectively (see Note 5).
4. Transform JM109 competent E. coli cells with the ligation
mixture.
5. Culture the cells on LBA agar plates and incubate overnight at
37 °C.
6. Pick a colony and culture it in 4 mL LBA medium and incubate overnight at 37 °C.
7. Extract the plasmid, and confirm the nucleotide sequence of
the entire open reading frame.
1. To make H245D/K443A/L530R/E354K mutants as the
Donor, conduct a series of site-directed mutagenesis with
QuikChange Site-directed mutagenesis kit as per manufacturer’s
instructions. Use pET32/BD1/Fluc as the template and primers
H245D, K443A, L530R, and E354K (Table 1) in combination
with their complementary strand primers (see Notes 6 and 7).
3.1 Construction
of the Binding
Domain-Fused
Expression Vectors
(Fig. 2)
3.2 Construction
of BD1/Donor
Expression Vector
(Fig. 2a)
Sensitive Protein-Protein Interaction Assay