nanoEx · Towards nanopore proteomics: enhancing cytolysin performance through genetically encoded noncanonical amino acids
Horizon 2020 — Marie Skłodowska-Curie Actions
- Duration
- 2020-04-01 → 2022-03-31
- EU contribution
- €162,040
- Participants
- 1
- Scheme
- MSCA-IF-EF-RI
Lines connect the coordinator with its partners.
Results in brief
Towards nanopore proteomics: enhancing cytolysin performance through genetically encoded noncanonical amino acids
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.
Data: CORDIS, © European Union
Project objective
Accurate detection of low-abundance proteins in biological samples obtained from patients or invasive species relies on appropriate handling and fixation techniques. Due to their inherent instability and propensity to denature upon freeze-thaw treatments, important protein markers tend to escape detection. Analogous challenges in RNA sequencing have been mitigated by the use of nanopore technology. European academic research and industry actions led to the creation of the widely used MinION devices that employ nanopore technology to enable DNA and RNA sequencing to be executed on site. Through this application I seek to improve nanopores as biosensors for the detection of proteins that will facilitate immediate protein analysis from clinical or ecological samples. I will use my expertise in genetic code expansion (GCE) techniques to engineer two well-characterized cytolysins with distinct architectures: an α-helical actinoporin and β-barrel containing lysenin. To improve these pores for protein identification and sequencing, the proposed research will advance through four stages: Incorporation of noncanonical amino acid (ncAA) to covalently stabilize smaller pores (1) and modulate the pore’s sensing region (2), directed evolution of residues lining the channel walls (3) and structural characterization of the identified variants (4). This is a multidisciplinary project that combines GCE techniques with advanced biophysical and structural characterization of the evolved nanopores. The host’s expertise in nanopore analysis together with my experience with GCE and directed evolution methods provide an important two-way transfer of knowledge. Action includes a training-through-research essential to advance my future academic career and enhance my employability in the biomedical industry. I anticipate the results of nanoEx project will have a major impact on the progress of nanopore biosensing, increasing the competitiveness of the European industry in protein sequencing.
Original text from CORDIS.
Participants
- KEMIJSKI INSTITUT · LjubljanaCoordinatorSlovenia
Links
- View on CORDIS
- DOI: 10.3030/896849
- https://www.ki.si/en/departments/d11-department-of-molecular-biology-and-nanobiotechnology/
Data: CORDIS, © European Union