4.2 Expression and purification of full-length hIGFBP2
A 15N-labeled sample of full-length h IGFBP2 was
prepared as described previously. Briefly, the h IGFBP2 construct
was transformed into E. coli BL21(DE3) StarTMcompetent cells. The transformed colony was inoculated in 10 mL primary
culture of LB medium containing 200 µg/ml ampicillin and incubated at 37
°C for 16 h, 200 rpm. The cells were then diluted 100-fold into fresh LB
(amp) and grown up to a cell density corresponding to
OD600~ 0.6. The cells were centrifuged
and the cell pellet transferred to 1 L minimal media followed by growth
at 37 °C up to a cell density corresponding to OD600~ 0.8 before inducing the protein expression with 0.5 mM
IPTG at 25°C for 6 h. Cells were harvested by centrifugation,
solubilized in Buffer-I [Phosphate-buffer (50 mM sodium phosphate, 0.3
M NaCl, pH 8.0) containing 8 M urea, 5 mM DTT, and 1 mM PMSF] and then
sonicated on ice. The supernatant was incubated at 4 °C for 2 h with 1
mL of pre-equilibrated His-Select™ Nickel affinity agarose
(Sigma–Aldrich) followed by washing and elution of the protein with 200
mM imidazole in Buffer-I. The eluate was dialyzed in Buffer-I to remove
urea, followed by removal of DTT. The dialyzed protein was exchanged
with phosphate buffer (50 mM sodium phosphate, 50 mM NaCl, pH 6.0) and
concentrated to 500 µL for NMR experiments.