128
C-terminal domain rotates as the reaction proceeds from the
adenylation to the oxidative luminescent steps. As a result,
when the BD is fused to the C-terminus, its rotation might be
hampered.
4. Thioredoxin (Trx), which is originally encoded in the pET32
vector, is fused to the N-terminus of BD. Trx is used to increase
the protein solubility in the bacterial expression process.
5. FRB associates with FKBP12 in a rapamycin-dependent manner [8–10]. Rapamycin is known to act as immunosuppressant drug, anticancer agent, and accelerating agent for
autophagy.
6. H245 and K443 are essential for the oxidative reaction of Fluc,
and the mutations H245D and K443A selectively suppress the
oxidative activity [5, 11, 12]. The mutation L530R stabilizes
bound ATP in Fluc to accelerate adenylation activity [13]. E354K
is a mutation that confers additional thermostability [14].
7. QuikChange Multi Site-Directed Mutagenesis kit is also
useful.
8. K529 is a residue that stabilizes phosphates of bound ATP, and
the K529Q mutation selectively suppresses the adenylation
activity [15]. The hinge region residue R437 is located close
to the active site of Fluc in the adenylation conformation.
The mutation R437K further decreases the distance between
the residue and LH 2 , and thus suppresses the adenylation
activity [2].
9. SHuffle T7 Express lysY is an engineered E. coli B strain in
which DsbC is constitutively expressed. DsbC works as a chaperone to promote the folding of protein.
10. To remove secreted β-lactamase from the precultured E. coli,
the medium of the preculture should not be added to the main
culture.
11. From this step the samples must be kept on ice or at 4 °C.
12. The purified enzyme can be stored at −80 °C in the buffer
containing 15% glycerol for several months.
13. The synthesis and use of LH 2 -AMP is optional. It is used to measure LH 2 -AMP kinetics of BD1/Donor and BD2/Acceptor.
14. The color of the solution turns yellow when the reaction is
completed.
15. LH 2 -AMP is stable in an acidic condition.
16. The solution for elution should be cooled because LH 2 -AMP
is unstable.
17. The peak of the fluorescent spectrum of LH 2 -AMP is at
approximately 535 nm, and the peak of the spectrum of LH 2 is
Yuki Ohmuro-Matsuyama and Hiroshi Ueda
C-terminal domain rotates as the reaction proceeds from the
adenylation to the oxidative luminescent steps. As a result,
when the BD is fused to the C-terminus, its rotation might be
hampered.
4. Thioredoxin (Trx), which is originally encoded in the pET32
vector, is fused to the N-terminus of BD. Trx is used to increase
the protein solubility in the bacterial expression process.
5. FRB associates with FKBP12 in a rapamycin-dependent manner [8–10]. Rapamycin is known to act as immunosuppressant drug, anticancer agent, and accelerating agent for
autophagy.
6. H245 and K443 are essential for the oxidative reaction of Fluc,
and the mutations H245D and K443A selectively suppress the
oxidative activity [5, 11, 12]. The mutation L530R stabilizes
bound ATP in Fluc to accelerate adenylation activity [13]. E354K
is a mutation that confers additional thermostability [14].
7. QuikChange Multi Site-Directed Mutagenesis kit is also
useful.
8. K529 is a residue that stabilizes phosphates of bound ATP, and
the K529Q mutation selectively suppresses the adenylation
activity [15]. The hinge region residue R437 is located close
to the active site of Fluc in the adenylation conformation.
The mutation R437K further decreases the distance between
the residue and LH 2 , and thus suppresses the adenylation
activity [2].
9. SHuffle T7 Express lysY is an engineered E. coli B strain in
which DsbC is constitutively expressed. DsbC works as a chaperone to promote the folding of protein.
10. To remove secreted β-lactamase from the precultured E. coli,
the medium of the preculture should not be added to the main
culture.
11. From this step the samples must be kept on ice or at 4 °C.
12. The purified enzyme can be stored at −80 °C in the buffer
containing 15% glycerol for several months.
13. The synthesis and use of LH 2 -AMP is optional. It is used to measure LH 2 -AMP kinetics of BD1/Donor and BD2/Acceptor.
14. The color of the solution turns yellow when the reaction is
completed.
15. LH 2 -AMP is stable in an acidic condition.
16. The solution for elution should be cooled because LH 2 -AMP
is unstable.
17. The peak of the fluorescent spectrum of LH 2 -AMP is at
approximately 535 nm, and the peak of the spectrum of LH 2 is
Yuki Ohmuro-Matsuyama and Hiroshi Ueda