This approach would also enable the analysis of GST-fusion protein expression levels by Western Blotting, using NU7441 chemical structure anti-GST antibodies (see
below). To achieve this, a DNA cassette that included the Ptac promoter, consensus ribosomal binding site, gst gene, multiple cloning site (MCS) and downstream terminator (Term) sequence (Ptac–gst–MCS–Term); was inserted into pZ7C to produce pZ7-GST (Figure 2). The (heterologous) genes of interest may be cloned into the pZ7-GST expression vector via a variety of commonly-used restriction sites present in the MCS. In this plasmid, the Ptac–gst–MCS–Term cassette PF-6463922 cell line is inserted in the opposite orientation to the Plac promoter that originates from the pUC18 backbone. This ensured that transcription of the GST–heterologous gene fusions would be under the primary control of the Ptac promoter. As the lacI gene, which encodes the LacI repressor protein was not included on the pZ7-GST plasmid; selleck chemical gene expression would not be expected to be repressed under normal growth conditions. Analysis of plasmid-based Glutathione S-Transferase (GST) expression in E. coli, Z. mobilis ATCC 29191 and CU1
Rif2 strains To determine the effectiveness of this gene-expression strategy, we first analyzed GST protein expression levels from the pZ7-GST plasmid established within E. coli BL21 (DE3) and Z. mobilis ATCC 29191 and CU1 Rif2 cells. The cell lysate proteins captured by glutathione-affinity chromatography were analyzed by SDS-PAGE (see Additional file 6, Panels A-D). It was found that the fractions eluted from the affinity-columns loaded with the E. coli BL21 (DE3)/pZ7-GST (Panel A), Z. mobilis ATCC 29191/pZ7-GST (Panel B) and CU1 Rif2/pZ7-GST (Panel C) cell lysates, all contained a band at ca. 26 kDa. Analysis via mass spectrometry confirmed that this band corresponded to recombinant (plasmid-derived) GST.
The weak band at ca. 29 kDa which was apparent in the lysate prepared from wild type Z. mobilis ATCC 29191 (Additional file 6, Panel D), was Liothyronine Sodium identified as endogenous glutathione S-transferase (ZM-GST) from Z. mobilis ATCC 29191 (glutathione S-transferase domain protein, ZZ6_0208; 223 aa). This protein was not observable in the fractions eluted from Z. mobilis ATCC 29191/pZ7-GST, presumably due to its relatively low abundance compared to the recombinant GST. The fractions eluted from the affinity-columns loaded with Z. mobilis ATCC 29191, ATCC 29191/pZ7-GST and CU1 Rif2/pZ7-GST cell lysates all contained a common protein band with a molecular mass of ca. 12 kDa (Additional file 6; Panels B, C and D), which did not appear in the purified E. coli fractions (Additional file 6, Panel A). This was subsequently identified as the 13.5 kDa glyoxalase/bleomycin resistance protein/dioxygenase (Glo, ZZ6_1397; 128 aa).