Detection of low abundance or unstable proteins requires technology that is fast and highly sensitive. Nanopore biosensing is a rapidly evolving technology and one of the most attractive new techniques that enables protein identification at the single-molecule level. Facile sample preparation and portability of the MinION device make this technique ideal for in situ detection of proteins, increasing the chances of detecting unstable protein targets. This can be critical for biomarker or drug target discovery.
The main objective of this MSC action is to improve the properties of protein nanopores, which are important for their use in protein identification, through directed evolution and incorporation of noncanonical amino acids (ncAAs). For this purpose, two biological nanopores, an actinoporin and lysenin, were identified as suitable scaffolds. This research combines the researcher's knowledge of synthetic biology techniques, namely insertion of ncAA through genetic code expansion in E. coli, with the supervisor's expertise in natural cytolysins and nanopore biosensing. By integrating these individual areas of expertise, we were able to create stable cytolysin-derived nanopores with altered oligomerization parameters, modify the sensing region chemistry and diameter of these novel nanopores by incorporating five different ncAAs, obtain ncAA-containing nanopores with stable, low-noise open pore currents, and solve high-resolution structures of the engineered nanopores, thus achieving all of the scientific objectives outlined in the application.
In conclusion, the use of directed evolution approaches and the insertion of ncAAs can bring significant advances to nanopore biosensing. Nanopore-based detection of proteins is important for analysis of the human proteome and may be critical for identifying expression patterns and structure-function relationships that define healthy and pathological states.
