Fig. 5
(His)6 tag orientation on MatN soluble yields. A E. coli C43(DE3) harboring pET-32a N-term- (his)6 MatN or pET-22b C-term-(his)6 MatN were induced at 20 °C by addition of 1 mM IPTG. Cells were harvested after 20 h of incubation with constant shaking and processed to obtain soluble (S) and insoluble (I) fractions. Equivalent protein amounts (10 µg) of each sample were loaded on 12% SDS-PAGE. Upon gel electrophoresis, gels were stained with Coomassie Brilliant Blue R-250 and unstained following standard protocols. A sample of purified N-term-His MatN endolysin (57 kDa) was loaded on P lane. B Total amounts of recombinant endolysin (column height) and the ratio of soluble/insoluble protein (SP, gray dotted pattern; IP, diagonal lines pattern) were calculated after background normalization with ImageJ, open-source image processing software. Endolysin expression and solubility were analyzed in biological triplicates. Error bars represent SD of mean values. *p < 0.05 (comparison between soluble endolysin ratios)