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Fig. 7 | Microbial Cell Factories

Fig. 7

From: Evaluation of factors influencing expression and extraction of recombinant bacteriophage endolysins in Escherichia coli

Fig. 7

Impact of the use of detergents and osmolytes during the extraction process. A, B E. coli C43(DE3) harboring pET-32a MatN/Cg was induced at 20 °C by adding 1 mM IPTG. Cells were harvested after 20 h of incubation with constant shaking and processed to obtain soluble (S) and insoluble (I) fractions. Cell pellets lysis was performed by a bead-beating approach in lysis buffer (50 mM Tris–HCl pH 8.0, 150 mM NaCl, 1% glycerol, and 1 mM PMSF) supplemented with various concentrations of N-lauroylsarcosine (N-LS), 25% glycerol, 0.75 M trehalose or 1 M glycine betaine. Equivalent protein amounts (10 µg) of each sample were loaded on 12% SDS-PAGE. Upon gel electrophoresis, gels were stained with Coomasie Brilliant Blue R-250 and unstained following standard protocols. C, D 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 (total MatN endolysin and ratio of soluble Cg endolysin vs. none additive group)

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