13c glucose

BRD4 (Uniprot accession
number O60885) genes were cloned into pET28 expression vectors containing an N-terminal
His₆-tag followed by a tobacco etch virus (TEV) protease
site. Sequences for the individual constructs covered residues N44
to E168 for the N-terminal bromodomain, BD1; H341 to E460 for the
C-terminal bromodomain, BD2; and N44 to E460 for the tandem BRD4(1,2).
The TEV protease-digested BD1 and BRD4(1,2) constructs retained four
non-native residues (G40, S41, H42, M43) prior to the native N44,
whereas the BD2 construct retained a non-native Gly–Gly sequence
prior to its native H341. Expression and purification closely followed
the protocol described previously.^{21 (link)} Uniform
labeling with ¹⁵N and ¹³C isotopes was achieved
by expression in minimal M9 medium with ¹⁵NH₄Cl (Sigma-Aldrich) and ¹³C glucose (Cambridge Isotope
Laboratories) as the sole sources of nitrogen and carbon, respectively.
In addition, ¹⁵N and ¹³C labeled Celtone medium
(Cambridge Isotope Laboratories) was supplemented to the growth medium
at 5 g/L. For perdeuteration, either glucose-d₇ for U-[²H,¹⁵N] labeling or ¹³C-glucose-d₇ for [²H,¹³C,¹⁵N] labeling was used in an M9/D₂O medium with supplements of ²H variants of the Celtone
Base Powder.

Wild type and mutant evpP sequences were cloned between BamH I and EcoR I sites of the pET-M vector (derived from pET32-a, Novagen; Madison, WI) and then transformed into E. coli BL21 (DE3) competent cells. The cells were allowed to grow in LB with ampicillin at 37°C until the O.D. reached 0.6. IPTG (0.4 mM) was added into the media to induce protein expression and the cells were grown overnight at 20°C. The cells were then harvested in 20 mM Tris buffer, pH 7.0, sonicated and centrifuged. The supernatant was collected and passed through a Ni-NTA affinity column. Expression of ¹⁵N- or ¹³C-labeled protein was carried out in similar conditions as mentioned above, except that the cells were grown in M9 minimal medium supplemented with ¹⁵N-labeled ammonium chloride and/or ¹³C-labeled D-glucose. To obtain a single labeled sample (either ¹⁵N-labeled or ¹³C-labeled sample), cells were grown in 1×M9 minimal medium in the presence of ¹⁵N-ammonium chloride (Cambridge Isotope Laboratories; Andover, MA) or with ¹³C-glucose (Cambridge Isotope Laboratories) as the sole nitrogen or carbon source. For double labeled ¹⁵N- and ¹³C-sample, 1×M9 minimal medium with ¹⁵N-ammonium chloride and ¹³C-glucose (Cambridge Isotope Laboratories) was used as the sole nitrogen and carbon sources, respectively.