(A) Western blot analysis of expression levels yielded by 3 different His-tagged MntH2 constructs. The indicated constructs in E. coli strain BL21 Star™ (DE3) cells harbouring pRARE2 were autoinduced for 24 h as detailed in the text and then cell lysates analysed by SDS-PAGE/western blotting. Each lane contained 20 μg total protein. The amounts of His-tagged TEV protease standards blotted in parallel are indicated. The blot was stained for the presence of oligohistidine tags with HRP-labelled monoclonal anti-6 × polyhistidine antibody. The mobilities of marker proteins of known molecular mass are shown on the left. (B) Western blot analysis of samples from expression trials performed using pL21-AXZIP. Expression was performed in E. coli strains BL21Star either with IPTG induction (IPTG) or autinduction for amount of times indicated under the figure. The blot was stained with HRP-labelled monoclonal anti-6 × polyhistidine antibody. (C) Purification of MntH2 a
(A) Western blot analysis of expression levels yielded by 3 different His-tagged MntH2 constructs. The indicated constructs in E. coli strain BL21 Star™ (DE3) cells harbouring pRARE2 were autoinduced for 24 h as detailed in the text and then cell lysates analysed by SDS-PAGE/western blotting. Each lane contained 20 μg total protein. The amounts of His-tagged TEV protease standards blotted in parallel are indicated. The blot was stained for the presence of oligohistidine tags with HRP-labelled monoclonal anti-6 × polyhistidine antibody. The mobilities of marker proteins of known molecular mass are shown on the left. (B) Western blot analysis of samples from expression trials performed using pL21-AXZIP. Expression was performed in E. coli strains BL21Star either with IPTG induction (IPTG) or autinduction for amount of times indicated under the figure. The blot was stained with HRP-labelled monoclonal anti-6 × polyhistidine antibody. (C) Purification of MntH2 and AXZIP. Purified proteins were loaded on a gel and either stained with Coomassie Blue (Co) or transferred on a nitrocellulose membrane and stained with anti-His antibody (IB).
