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.