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.