Spatial arrangement of proteins using scCro-tag: application for an in situ enzymatic microbead assay

In natural systems, various metabolic reactions are often spatially organized to increase enzyme activity and specificity. Thus, by spatially arranging enzyme molecules in synthetic systems to imitate these natural systems, it is possible to promote a high rate of enzymatic turnover. In this present study, a normal and mutant form of the scCro DNA-binding protein were shown to bind orthogonally to specific recognition sequences under appropriate conditions. Furthermore, these DNA-binding tags were used to establish an enzyme assay system based on the spatial arrangement of transglutaminase and its substrate at the molecular level. Together, the results of the present study suggest that the scCro-tag may be a powerful tool to facilitate the synthetic spatial arrangement of proteins on a DNA ligand. A cryptic plasmid pHM1519 contains repA gene encoding an essential replication initiator protein RepA. Altered RepA initiator by copA1 mutation induced elevation in copy number of pHM1519 ori-replicon plasmid.