N dodecylphosphocholine dpc

Expression and
purification of ChiZ1-64 was performed as previously described.^{20 (link)13}C–¹⁵N labeled
ChiZ-FL containing a noncleavable N-terminal 6× His-tag was expressed
in Escherichia coli BL21 Codon Plus RP competent
cells. Cells were grown at 37 °C in LB media until OD at 600
nm reached 0.7. Cells were pelleted and transferred to M9 media containing
1 g of ¹⁵N-ammonium chloride and 2 g of ¹³C
uniformly label glucose (Cambridge Isotope Laboratories). After transfer,
cells were incubated at 37 °C for 30 min before adding IPTG to
a final concentration of 0.4 mM to induce protein expression for 5
h. Cells were then pelleted and resuspended in a lysis buffer (20
mM Tris-HCl pH 8.0, 500 mM NaCl) for cell lysis using a French press. n-Dodecylphosphocholine (DPC; Anatrace) was added to the
lysate to a final concentration of 2% (wt/vol) and then incubated
overnight at 4 °C with agitation. Cell lysate was centrifuged
at 250 000g for 30 min. Protein purification
was performed using Ni-NTA resin (Qiagen) equilibrated with the lysis
buffer containing 20 mM imidazole. The column was washed using the
lysis buffer containing 0.5% (wt/vol) DPC and 60 mM imidazole. Protein
was eluted with the same buffer but containing 400 mM imidazole.