Instrument
Bruker Avance - 800 MHz
Sample
State isotropic, Solvent system 90% H2O/10% D2O, Pressure 1 atm, Temperature 298 K, pH 6.4, Details Uniformly 15N-labelled (u-15N UVI31+) and uniformly 13C/15N
doubly-labelled (u-13C/15N UVI31+) samples were produced in minimal (M9)
media. E. coli strain BL21 (DE3) harboring the vector pET was grown in the
appropriate minimal (M9) medium containing ampicillin (100 mg/ml) to an
absorbance A600 of 0.5 at 33 C. The cells were harvested and resuspended in
fresh minimal (M9) medium without ampicillin, followed by induction with 1
mM IPTG at 25 C for overnight. Cells were collected by centrifugation,
resuspended in lysis buffer [50 mM sodium phosphate (pH 7.6), 50 mM NaCl, 1 mM
PMSF, 5 mM Imidazole, 2% Tween 20, and 1 mg/ml lysozyme] and incubated
on ice for 30 min. Cells were disrupted by ultrasonication. The cell
debris was removed by centrifugation (15,000 rpm for 20 min at 4 C). The
UVI31+ protein was purified from the resulting supernatant using Ni NTA
(Ni2+-nitrilotriacetate) agarose (Qiagen, Hilden, Germany). His6-tagged UVI31+
was eluted with 250 mM imidazole in 50 mM sodium phosphate (pH 7.6), 50 mM
NaCl. The eluted fractions were dialyzed overnight against 50 mM
sodium phosphate (pH 6.4), 50 mM NaCl at 4 C. The protein was further purified
by gel filtration using a Sephadex G75 column (GE healthcare, USA)
equilibrated with 50 mM sodium phosphate (pH 6.4), 50 mM NaCl. The
recombinant protein eluted at a volume corresponding to a monomer of
approximately 13 kDa. N-terminal amino acid sequence analysis was performed by
the Proteomics International Pty Ltd, Australia. The UVI31+ was quantitated
by Bradfords with bovine serum albumin as a standard, and by measuring
absorbance at 280 nm (Bradford 1976). Typical yields were 25 30 mg/l of
culture.
| # | Name | Isotope labeling | Type | Concentration |
|---|
| 1 | S114A mutant of UVI31+ | [U-100% 13C; U-100% 15N] | | 0.8 (±0.03) mM |
|---|
| 2 | H2O | natural abundance | | 90 % |
|---|
| 3 | D2O | [U-2H] | | 10 % |
|---|
