Clustering target enzymes on the membrane through mammalian
protein–protein interaction domains and ligands
Given that the cell membrane has a more compact space than the volume of
the cytoplasm, we chose the inner cell membrane of E. coli as the
scaffold. However, whether the proteins anchored onto the membrane can
be clustered to improve the metabolic flux, as in the case of other
artificial scaffolds, remains uncertain. To confirm that our membrane
scaffold equipped with metazoan interacting proteins could be clustered,
we conducted fluorescence complementation assay (Figs. 3A and 3B) assay[3, 13]. First,
protein–protein
interaction domains and ligands from metazoan cells (mouse SH3 and PDZ
domains and rat GBD domain) were utilized on the basis of the
combination of the protein domains and their corresponding cognate
ligands to rationally assemble and arrange enzymes onto the inner
membrane of E. coli . These three groups of interacting proteins
were fused with the N- or C-terminus of Lgt to create the desired
protein complexes on the membrane (Fig. 3B).
In the fluorescence complementation assay, the fluorescent protein EGFP
was split into two halves (1EGFP and 2EGFP), and the proteins that were
postulated to interact were fused with the unfolded complementary
fragments of EGFP and expressed in E. coli . The interaction
between protein domain and the ligand brought the fluorescent fragments
within proximity, allowing the reporter protein to restore its native 3D
structure and emit a fluorescent signal. Therefore, fluorescence could
be observed if an interaction occurred between 1EGFP and 2EGFP.
Otherwise, no fluorescence could be detected. The split EGFP parts were
fused with Lgt without interacting proteins as the negative control or
fused with Lgt interaction groups to test the protein clustering (Fig.
3B). In Fig. 3C, the detected green fluorescence signal implied that all
of the three groups of Lgt fusion proteins successfully developed a
functional EGFP on the membrane, demonstrating that the membrane
proteins with interacting proteins could dimerize with one another.
Thus, we could easily use the inner membrane as a scaffold to cluster
target proteins by recruiting Lgt, which is the native membrane protein
of E. coli , interacting proteins (SH3, PDZ, and GBD), and the
desired enzymes.