Localizing target enzymes to E.coli inner membrane
The membrane scaffold system has two indispensable elements. The phosphatidylglycerol::prolipoprotein diacylglyceryl transferase (Lgt), an E. coli transmembrane protein, is selected as the anchor module. Target enzymes can be fused with its N-terminus as a periplasmic enzyme or C-terminus as a cytoplasmic enzyme. Aside from Lgt, one short peptide named ssDsbA, the signal recognition particle (SRP)-dependent signaling sequence of DsbA, is necessary to orient the combined proteins onto the inner membrane of E. coli . To verify the function of the membrane anchor part, we fused β-lactamase and EGFP with the N-terminus of Lgt and with the C-terminus, respectively (Fig. 2A)[12]. The expression plasmid containing this construct was transformed to BL21 (DE3) and induced by L-arabinose (Fig. 2B). The BL21/pETara-Anchor strain exhibited a clear green fluorescence on the cell margin under the laser confocal microscope, confirming that the membrane anchor part was correctly localized. To further characterize the membrane anchor and define the optimal induction condition, we observed the growth phenotype of the BL21/pETara-Anchor strain at different ampicillin levels (Fig. S1) and quantitatively tested its growth status under different concentration combinations of L-arabinose and ampicillin (Fig. S2). The results revealed that 0.2% L-arabinose induction was the most satisfactory condition for membrane protein expression. In generally speaking, by fusing with the membrane anchor, the target proteins performed normal functions regardless of their periplasmic or cytoplasmic locations.