Cellytic b 2x

The ROP18 gene was cloned and expressed as described previously
^{14 (link), 26} . In brief, an expression construct
starting from the sequence ERAQ (Glu83 based on the second ATG of GenBank
CAJ27113) and continuing through the C-terminus was cloned into pGEX-6p-1 vector
between BamH1 and SalI sites with the addition of a 6xHIS tag before the stop
codon. The plasmid was then transformed into E. coliBL21(DE3)V2RpAcYc-LIC+LamP-phosphotase cells ^{40 (link)}. A single colony was inoculated into 5
ml of TB with ampicillin (100 μg/ml) + chloramphenicol (34
μg/ml), and cultured overnight at 37°C. The culture was into 250
ml of fresh TB with ampicillin (100 μg /ml) + chloramphenicol
(34 μg /ml) and grown for 6 h at 37°C. This culture was then
supplemented with 1 mM IPTG and induced at 12°C overnight. The cell
pellet was lysed in CelLyticB 2x (Sigma-Aldrich) supplemented with benzonase
(SIGMA) and protease inhibitor cocktail (Sigma). The protein was purified with
glutathione Sepharose 4B (GE-Healthcare), and dialyzed in 250 mM NaCl, 10 mM
MgCl₂, 20 mM Tris-HCl pH 8.0. Glycerol was added to 20%
and the protein was stored at −80°C. Protein concentrations were
measured by SDS PAGE separation and staining with SYPRO-Ruby (Invitrogen) in
comparison to a BSA standard.