Investigating the Chaperone Properties of a Novel Heat Shock Protein, Hsp70.c, from Trypanosoma brucei
Figure 2
Expression and purification of recombinant TbHsp70.c, TcHsp70B, and Tbj2. (a) SDS-PAGE of a TbHsp70.c purification. Upper panel: Lanes 1 - E. coli BB1994 [pQE80TbHsp70.c] whole lysate 3 hr post IPTG induction, 2 - fraction unbound to Ni-NTA column, 3 - wash using 20 mM imidazole, 4 - first TbHsp70.c elution (500 mM imidazole). Lower panel: western analysis of TbHsp70.c at 73 kDa using anti-TbHsp70.c peptide antibody. (b) SDS-PAGE analysis of a TcHsp70B purification. Upper panel: Lanes 1 - E. coli BL21(DE3) [pET14bTcHsp70B] whole cell lysate 5 hours post IPTG induction, 2 - fraction unbound to Ni-NTA column, 3 - wash using 20 mM imidazole, 20% glycerol and 10 mM ATP, 4 - TcHsp70B first elution (500 mM imidazole). Second panel: western analysis of copurified DnaK at 70 kDa using anti-DnaK antibody. Third panel: TcHsp70B detected at 70 kDa by western analysis using anti-His antibody. (C) SDS-PAGE analysis of a Tbj2 purification. Upper panel: Lanes 1 - E. coli BL21(DE3) [pET28aTbj2] whole cell lysate 5 hours post IPTG induction, 2 - fraction unbound to Ni-NTA column, 3 - wash containing 20 mM imidazole, 20% glycerol and 10 mM ATP, 4 - Tbj2 first elution (500 mM imidazole). Second panel: western analysis of co-purified DnaK at 70 kDa using anti-DnaK antibody. Third panel: Tbj2 detected at 44 kDa by western analysis using anti-His antibody. Numbers to the left of the panels indicate the protein marker ladder, the Peqlab peqGOLD Protein Marker II in (a) and the Fermentas Pierce Unstained Protein MW Marker in (b) and (c).
