Plant Hsp90 Proteins Interact with B-Cells and Stimulate Their Proliferation
Figure 2
Analysis of the expression of plant Hsp90.
(A) Expression and purification of recombinant A. thaliana Hsp81.2. (B) Expression and purification of recombinant N. benthamiana Hsp90.3. The expression and purification of the recombinant proteins were checked by Coomassie blue-stained polyacrylamide gel and western blot using an anti-rAtHsp81.2. Lane M: protein mass markers; lane 1: lysate of E. coli Rosetta (DE3) without induction; lane 2: lysate of E. coli Rosetta (DE3) after IPTG induction; lane 3: elute after Ni+ affinity purification; Lane 4: western blot from elute after Ni+ affinity purification using a polyclonal antibody anti-rAtHsp81.2. Arrows indicate the bands that were sent to analyze by MALDI-TOF-TOF. (C) Western blot analysis, where the anti-AtHsp81.2 was able to recognize the recombinant LiHsp83 protein and the Hsp90 proteins from plant extract. Nb: protein extracts from N. benthamiana leaves; At: protein extracts from A. thaliana leaves; rLiHsp83: recombinant LiHsp83 expressed and purified from E. coli. M: protein mass markers. Arrows indicate the bands detected by anti-AtHsp81.2. (D) The ATPase activity of rAtHsp81.2 was determined using varying concentrations of ATP (0–3000 µM). The amount of phosphate released was determined colorimetrically. The kinetic parameters KM and Vmax were determined using a Lineweaver–Burke plot. The inset shows the Michaelis–Menten plot generated with the data. All points are the means triplicate samples of three independent experiments.