For Industry

Nanopore technology is important in biotechnology for applications such as nanopore sensing and nanopore sequencing. Nanopore-based devices contain a nanopores, embedded into an artificial membrane, that are used for sensing analytes that pass through the nanopore by measuring changes in ionic pore current. Biological nanopores can be prepared using various membrane proteins, including pore-forming proteins. The latter are characterized by their expression as soluble monomers that oligomerize after their contact with target lipid membrane and form transmembrane pores. Pore-forming proteins can also be edited using protein engineering methods in order to gain a larger set of biotechnologically applicable nanopores with different properties. This is important for producing nanopores with new and better functions in terms of sensing (specificity, stability, affinity, etc.). Our research group prepared a novel recombinant pore-forming protein that forms hexameric pores in negatively charged lipid membranes.

Technology

We prepared novel recombinant actinoporin-like proteins from mediterranean mussel (Mytilus galloprovincialis) that we named »MiGa«. Actinoporins are pore-forming proteins from sea anemones that act on sphingomyelin-rich biological membranes. Stoichiometry of their pores is mostly octa- or nonameric. While studying potential lipid receptors of the novel protein MiGa we showed its low activity towards sphingomyelin-containing membranes and high activity towards negatively charged POPG (1-palmitoyl-2-oleoyl-sn-glycero-3(phospho-rac-(1-glycerol))) membranes. In the latter case, we showed that protein-lipid interactions were electrostatic. MiGa pores were then isolated from large unilamellar POPG vesicles with detergent addition. Hexameric pore stoichiometry was determined using cryo-electron microscopy.

Main advantages

• We prepares a novel biological actinoporin-like protein nanopores and showed pores characteristics that have not yet been shown for actinoporins.
• Novel pore properties, such as its preparation through monomer oligomerization in negatively charged lipid membranes and its hexameric stoichiometry, open new possibilities for design of novel biotechnologically applicable nanopores.

Fields of use: Biotechnology, Protein sequncing

Technology Readiness Level: TRL3

Intellectual property: Patent pending

Partner sought: R&D collaboration to further develop the technology, licensing or sell of IP rights

Next steps needed: Partner search for further development

Contact:

knowledge.transfer(at)ki.si