박테리아 공동발현 및 즉각적 이황화 결합에 의한 단백질 자가조립체의 안정성 증가

Abstract

Research on the utilization and application of biomolecular tools is being actively conducted. Currently, there is a demand for excellent molecular tools that require greater biocompatibility and stability than existing molecular tools. The self-assembled coiled coil, which can be one of the candidates, is one of the motifs that are very abundant in nature and has the characteristic that complementary coils are specifically assembled with each other. However, self-assembled coiled coils can sometimes become unstable in harsh biological environments, and rapid dissociation by dilution becomes a fatal problem. Formation of disulfide bonds by cysteine is often used as a strategy to increase structural stability by artificially inserting into proteins. However, due to the non-specific binding of thiol groups, a complicated post-processing process is required compared to conventional protein purification. Here we report a cysteine-coupled self-coupling coiled coil that introduces cysteine to form a disulfide bond to overcome the rapid dissociation of self-assembled coiled coil and is co-expressed in bacteria to overcome the defect of non-specific coupling between cysteines. This scaffold, co-expressed in E.coli SHuffle, formed heterodimers by self-assembly and then coupled with disulfide bonds, showed improved bonding stability in vitro compared to wild-type coiled coils. These results suggest that engineered self-assembled coiled coil-based scaffolds can be promising tools in various biotechnology fields in the future.