WO2022038144A1

WO2022038144A1 - Novel nanopore-forming polypeptides

Info

Publication number: WO2022038144A1
Application number: PCT/EP2021/072846
Authority: WO
Inventors: Neža KORITNIK; Tomaž ŠVIGELJ; Gašper ŠOLINC; Marija SRNKO; Marjetka Podobnik; Gregor Anderluh
Original assignee: Kemijski inštitut
Priority date: 2020-08-18
Filing date: 2021-08-17
Publication date: 2022-02-24

Abstract

The present invention pertains to a novel polypeptide which is capable of forming a hexameric nanopore. The present invention further pertains to nanopores comprising said polypeptide as well as compositions, liposomes and systems comprising said nanopores. The present application also pertains to methods for producing the nanopores and systems of the invention.

Description

Novel nanopore-forming polypeptides

Field of the invention

The present invention generally relates to the field of pore-forming proteins, nanopores and the use thereof in various applications, such as analysis of biopolymers and other substances, typically by making electrical measurements during translocation through a nanopores. More specifically, the present invention pertains to a polypeptide which is capable of forming a nanopore, and more particularly a hexameric nanopore.

Background of the invention

Pore-forming proteins (PFPs) are produced by variety of organisms (from bacteria to humans) and take place in biological processes such as host attack and immune defense. Their common mode of action is binding to a membrane receptor (lipid, sugar or protein) followed by oligomerization and pore formation in the lipid membrane. PFPs are divided into a- and p- PFPs, whose pores in transmembrane region are built of either clusters of a-helices or a p- barrel, respectively [1],

Actinoporins are a family of a-PFPs and are mainly produced by sea anemones (Actiniaria). They are about 20 kDa hemolytic proteins with high isoelectric points and usually lack cysteine residues in their protein sequence [2], There are four representatives of actinoporins with known three-dimensional structure: fragaceatoxin C [3] (FraC; from Actinia fragacea), equinatoxin II [4, 5] (Eqtll; from Actinia equina), Stichiolysin I [6] and Sticholysin II [7] (Stnl and Stnll; from Stichodactyla helianthus). They possess very high structural similarity. The monomers consist of: N-terminal segment (around 30 amino acids) that contains a short p- strand, one turn of 3 -helix and a short a-helix. The N-terminal segment is followed by a p- sandwich (two sheets of five p-strands). There is also the second a-helix in the structure. The helices flank the two sides of the p-sandwich, and are positioned almost perpendicularly to each other.

All actinoporins bind sphingomyelin in lipid membranes, which is their specific receptor [8], After binding to the lipid, the monomers undergo a conformational change which encompasses only the N-terminal segment, while the other structural parts remain unchanged. The N- terminal segment rearranges into a single long N-terminal a-helix which final position in the membrane is approximately 180 ⁰ away to its original in the soluble monomer. This N-terminal a-helix is amphipathic and acts as an achor in the lipid membrane. Actinoporins form stable octameric pores, as deduced from the crystal structure of FraC [9], Nevertheless, oligomers of other stoichiometries were also detected [3], [10]-[15],

Actinoporin-like proteins are proteins similar to actinoporins (less than 30 % sequence identity). They are abundant in Hydrozoa, for example hydra's HALTs (hydra actinoporin-like toxins). They are also found in various snails, for example echotoxins (from Monoplex echo), coluporins (from Cumia reticulata), conoporins (from Conus), tereporins (from Mytilus coruscus, Cinguloterebra anilis, Terebra subulata). Other actinoporin-like protein are also found in moss (bryoporins), fish, fungi (lectins, aegerolysins) and plant pathogens (Nep-like proteins).

PFPs are applicable in various biotechnological and biomedical fields. They are used in nanopore technology for sequencing (DNA, RNA, proteins) and sensing of various metabolites [16]— [18]. They can also be used in detection of lipid composition of various biological membranes (for example, actinoporins for sphingomyelin detection [19]). They could also be used in cancer therapy (immunotoxins) [20] and transport systems for therapeutic agents.

Accordingly, it is an object of the present invention to provide a novel pore-forming protein (PFP) and nanopores composed thereof.

Summary of the invention

The above objective is solved by the present invention by providing in a first general aspect a polypeptide which is capable of forming a nanopore, particularly a hexameric nanopore. More specifically, the present invention provides a polypeptide having the amino acid sequence of SEQ ID NO: 1 and variants thereof.

The present invention provides in a further aspect a nanopore comprising a plurality of molecules of the polypeptide according to the present invention.

The present invention provides in a further aspect a composition comprising at least one nanopore according to the present invention.

The present invention provides in a further aspect a liposome comprising at least one nanopore according to the present invention within a lipid bilayer thereof.

The present invention provides in a further aspect a system comprising at least one nanopore according to the present invention comprised in a membrane. The present invention provides in a further aspect an isolated nucleic acid molecule comprising a nucleotide sequence encoding the polypeptide according to the present invention.

The present invention provides in a further aspect a DNA construct comprising an nucleotide sequence encoding the polypeptide according to the present invention.

The present invention provides in a further aspect a recombinant host cell, which comprises a DNA construct according to the present invention.

The present invention provide in a further aspect a method for producing a nanpore or system according to the present invention.

The present invention provide in a further aspect the use of a nanopore of the present invention, a liposome according to present invention or a system according to the present invention for sensing a biopolymer or metabolite.

The present invention provide in a further aspect the use of a nanopore of the present invention, a liposome according to present invention or a system according to the present invention for sequenceing a biopolymer.

The present invention can be summarized by the following items.

1. A polypeptide which is capable of forming a nanopore, particularly a hexameric nanopore.

2. The polypeptide according to item 1 selected from the group consisting of: a) a polypeptide having the amino acid sequence of SEQ ID NO: 1 ; b) a polypeptide having an amino acid sequence which has at least 75%, such as at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, sequence identity with the amino acid sequence of SEQ ID NO: 1 ; and c) a polypeptide having an amino acid sequence which corresponds to the amino acid sequence of SEQ ID NO: 1 , wherein 1 to 50, such as 1 to 40, 1 to 35, 1 to 30, 1 to 25, 1 to 20, 1 to 15, 1 to 10, 1 to 5, 1 to 3, amino acid residues are substituted; 1 to 20, such as 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3 or 1 to 2, amino acid residues are added to or deleted from the C-terminus; and/or 1 to 5, such as 1 to 4, 1 to 3 or 1 to 2, amino acid residues are added to or deleted from the N-terminus.

3. The polypeptide according to item 1 or 2, wherein the polypepetide has the amino acid sequence of SEQ ID NO: 1 .

4. The polypeptide according to item 1 or 2, wherein the polypepetide has an amino acid sequence which has at least 75% sequence identity with the amino acid sequence of SEQ ID NO: 1.

5. The polypeptide according to item 1 or 2, wherein the polypeptide has an amino acid sequence which corresponds to the amino acid sequence of SEQ ID NO: 1 , wherein 1 to 50, amino acid residues are substituted.

6. The polypeptide according to item 1 or 2, wherein the polypeptide has an amino acid sequence which corresponds to the amino acid sequence of SEQ ID NO: 1 , wherein 1 to 50, amino acid residues are substituted within the C-terminal half (amino acids 106 to 212) of the amino acid sequence of SEQ I D NO: 1.

7. The polypeptide according to item 5 or 6, wherein the amino acid substitution/s is/are a conservative substitution/s.

8. The polypeptide according to item 1 , 2, 5, 6 or 7, wherein the polypeptide has an amino acid sequence which corresponds to the amino acid sequence of SEQ ID NO: 1 , wherein 1 to 20 amino acid residues are added to the C-terminus.

9. The polypeptide according to item 1 , 2, 5, 6 or 7, wherein the polypeptide has an amino acid sequence which corresponds to the amino acid sequence of SEQ ID NO: 1 , wherein 1 to 20 amino acid residues are deleted from the C-terminus.

10. The polypeptide according to item 1 , 2, 5, 6, 7, 8 or 9, wherein the polypeptide has an amino acid sequence which corresponds to the amino acid sequence of SEQ ID NO: 1 , wherein 1 to 5 amino acid residues are added to the N-terminus.

11. A nanopore comprising a plurality of molecules of the polypeptide according to any one of items 1 to 10.

12. The nanopore according to item 11 , which is a hexameric nanopore.

13. The nanopore according to item 11 or 12, which is composed of 6 monomers of the polypeptide according to any one of items 1 to 10. 14. A composition comprising at least one nanopore according to any one of items 11 to 13.

15. The composition according to item 14, further comprising a membrane such a lipid bilayer or artificial membrane, wherein the nanopore is comprised in the membrane.

16. A liposome comprising at least one nanopore according to any one of items 11 to 13 within a lipid bilayer thereof.

17. The liposome according to item 16, which is composed of a glycerophospholipid.

18. The liposome according to item 17, wherein the glycerophospholipid is selected from the group consisting of 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1'-rac-glycerol), 1,2- dimyristoyl-sn-glycero-3-phospho-(1'-rac-glycerol), 1 ,2-dipentadecanoyl-sn-glycero-3- phospho-(T-rac-glycerol), 1 ,2-dipalmitoyl-sn-glycero-3-phospho-(1'-rac-glycerol), 1 ,2- distearoyl-sn-glycero-3-phospho-(1'-rac-glycerol), 1 ,2-dioleoyl-sn-glycero-3-phospho-(1'-rac- glycerol), 1,2-dielaidoyl-sn-glycero-3-phospho-(1'-rac-glycerol), 1 ,2-dilinoleoyl-sn-glycero-3- phospho-(T-rac-glycerol) and 1,2-dilinolenoyl-sn-glycero-3-phospho-(1'-rac-glycerol).

19. The liposome according to item 17, wherein the glycerophospholipid is 1-palmitoyl-2- oleoyl-sn-glycero-3-phospho-(1'-rac-glycerol).

20. The liposome according to any one of items 16 to 19, which is a multilamellar or unilamellar vesicle.

21. The liposome according to any one of items 16 to 20 or 15, which is a large unilamellar vesicle.

22. A system comprising at least one nanopore according to any one of items 11 to 13 comprised in a membrane such as a planar lipid bilayer or artificial membrane.

23. The system according to item 22, wherein the system is operative to detect a property of an analyte.

24. An isolated nucleic acid molecule comprising a nucleotide sequence encoding the polypeptide according to any one of items 1 to 10.

25. A DNA construct comprising an nucleotide sequence encoding the polypeptide according to any one of items 1 to 10.

26. The DNA construct according to item 25, which is an expression cassette. 27. The DNA construct according to item 26, which is a vector, such as an expression vector.

28. A recombinant host cell which comprises a DNA construct according to any one of items 25 to 27.

29. The recombinant host cell according to item 28, which comprises an expression cassette according to item 26 or an expression vector according to item 27.

30. The recombinant host cell according to item 28 or 29, which is a bacterium or yeast.

31. The recombinant host cell according to any one of items 28 to 30, which is a bacterium.

32. A method for producing a nanpore according to any one of items 11 to 13, comprising the steps of:

(i) providing a plurality of molecules of the polypeptide according to any one of items 1 to 10;

(ii) contacting said a plurality of molecules of the polypeptide with at least one liposome, such as a multilamellar or large unilamellar vesicle, to assemble them into at least one nanopore;

(iii) optionally, recovering the at least one nanopore from the at least one liposome.

33. A method for producing a system according to any one of item 22 or 23; comprising the steps of:

(iii) recovering the at least one nanopore from the at least one liposome;

(iv) contacting the at least one nanopore with a membrane such as a planar lipid bilayer or artificial membrane.

34. The method according to item 32 or 33, wherein step (i) includes culturing a recombinant host cell according to any one of items 28 to 31 under suitable culture conditions allowing the production of the polypeptide by said host cell, and isolating said polypeptide from said host cell.

35. The method according to item 34, wherein the isolation includes a chromatographic procedure. 36. The method according to any one of items 32 to 35, wherein the liposome is composed of a glycerophospholipid.

37. The method according to item 36, wherein the glycerophospholipid is selected from the group consisting of wherein the glycerophospholipid is selected from the group consisting of 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1'-rac-glycerol), 1 ,2-dimyristoyl-sn-glycero-3- phospho-(T-rac-glycerol), 1 ,2-dipentadecanoyl-sn-glycero-3-phospho-(1 '-rac-glycerol), 1 ,2- dipalmitoyl-sn-glycero-3-phospho-(1 '-rac-glycerol), 1 ,2-distearoyl-sn-glycero-3-phospho-(1 '- rac-glycerol), 1 ,2-dioleoyl-sn-glycero-3-phospho-(1 '-rac-glycerol), 1 ,2-dielaidoyl-sn-glycero-3- phospho-(T-rac-glycerol), 1 ,2-dilinoleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) and 1 ,2- dilinolenoyl-sn-glycero-3-phospho-(1'-rac-glycerol).

38. The method according to item 36, wherein the glycerophospholipid is 1-palmitoyl-2- oleoyl-sn-glycero-3-phospho-(1'-rac-glycerol).

39. The method according to any one of items 32 to 38, wherein the liposome is a multilamellar or unilamellar vesicle.

40. The method according to any one of items 32 to 39, wherein the liposome is a large unilamellar vesicle.

41. The method according to any one of items 32 to 40, wherein step ii) includes mixing said a plurality of molecules of the polypeptide with said at least one liposome and incubating said mixture for a suitable time at a suitable temperature.

42. The method according to any one of items 32 to 40, wherein step ii) includes mixing said a plurality of molecules of the polypeptide with said at least one liposome and incubating said mixture for about 15 to about 60 minutes, such as for about 25 to about 35 minutes, such as for about 30 minutes, at a temperature from about 25°C to about 42°C, such as a temperature from 30°C to 42°C, such as at 37°C, under agitation (e.g. at about 250 rpm).

43. The method according to item 41 or 42, wherein said a plurality of molecules of the polypeptide is mixed with said at least one liposome at a molar ratio in the range of 1 :3 to 1 :2000, such as at a molar ratio of 1 :100, in a suitable buffer.

44. The method according to item 43, wherein the buffer is phosphate buffered saline.

45. The method according to any one of items 32 to 44, wherein step iii) includes incubating the at least one liposome containing said at least one nanopore in the presence of a suitable detergent for a suitable time at a suitable temperature. 46. The method according to any one of items 32 to 44, wherein step iii) includes incubating the at least one liposome containing said at least one nanopore in the presence of a polysorbate at a final concentration of about 0.5% v/v to about 5% v/v, such as about 2% v/v, for about 1 to about 20 minutes, such as for about 15 minutes, at a temperature from about 30°C to about 42°C, such as at about 37°C, under agitation (e.g. at about 250 rpm).

47. The method according to item 46, wherein the polysorabte is Polysorbat 80 (Polyoxyethylen(20)-sorbitan-monooleat; Tween® 80) or Polysorbat 20 (Polyoxyethylene (20) sorbitan monolaurate; Tween® 20).

48. The method according to item 46, wherein the polysorabte is Polysorbat 80 (Polyoxyethylen(20)-sorbitan-monooleat; Tween® 80).

49. The method according to any one of items 45 to 48, wherein step iii) further includes purifying the at least one nanopore after incubation.

50. The method according to item 49, wherein the purification includes centrifugation and/or a chromatographic procedure.

51. The method according to item 50, wherein the chromatographic procedure includes ion-exchange chromatography, such as cation-exchange chromatography.

52. The method according to item 50 or 51 , wherein the chromatographic procedure includes centrifuging the mixture containing the at least one liposome and the detergent after incubation, diluting the obtained supernatant with a suitable buffer solution containing a polyoxyethylene surfactant such as Polyoxyethylen (23) laurylether (Brij® 35) or Polyoxyethylen (20) cetylether (Brij® 58), such as a Tris buffer solution, and loading the suspension on an ion-exchange chromatography column.

53. The method according to item 52, wherein the buffer solution contains the polyoxyethylene surfactant such as Polyoxyethylen (23) laurylether or Polyoxyethylen (20) cetylether, at a final concentration of about 0.1 mM to about 0.5 mM, such as at about 0.25 mM.

54. The method according to item 52 or 53, wherein the buffer solution contains 50 mM Tris/HCI and 0,25 mM Polyoxyethylen (23) laurylether or Polyoxyethylen (20) cetylether (pH 7,4).

55. The method according to any one of items 51 to 54, further comprising eluting the at least one nanopore from the column by a salt gradient (e.g., at about 100 mM NaCI). 56. Use of a nanopore of any one of items 10 to 13, a liposome according to any one of items 16 to 21 or a system according to item 22 or 23 for sensing a biopolymer or metabolite.

57. The use according to item 53, wherein the biopolymer is a protein, a peptide, a nucleic acid, or combination thereof.

58. Use of a nanopore of any one of items 10 to 13, a liposome according to any one of items 16 to 21 or a system according to item 22 or 23 for sequencing a protein, a peptide or nucleic acid (such as DNA or RNA).

59. Use of a nanopore of any one of items 10 to 13, a liposome according to any one of items 16 to 21 or a system according to item 22 or 23 for targeted cellular permeabilization

60. Use of a nanopore of any one of items 10 to 13, a liposome according to any one of items 16 to 21 or a system according to item 22 or 23 for drug delivery.

Brief description of the drawings

Figure 1 : Amino acid sequence of the actinoporin-like polypeptide found in mediterranean mussel (Mytilus galloprovincialis).

Figure 2: Amino acid sequences of recombinant MiGa before (A) and after (B) the HisTag- removal. Black - MiGa protein sequence, red - the TEV protease recognition site, blue - HisTag, grey - translated remains of the plasmid vector (pET24a).

Figure 3: Purification of the protein from cell lysate. Protein bands in red squares represent MiGa. A) SDS-PAGE analysis of samples from the isolation steps. 1 - Cell culture before the induction, 2 - Cell culture after the induction, 3 - Supernatant of sonicated cell suspension, 4 - Pellet of sonicated cell suspension, 5 - Unbound chromatography fraction, 6 - Bound chromatography fraction with 68 mM imidazole, 7 - Bound chromatography fraction with 300 mM imidazole. B) Nickel-chelate affinity chromatography chromatogram. Peak numbers correspond the sample numbers in panel A.

Figure 4: HisTag removal by the TEV protease and purification of the reaction products. Black and red arrows mark protein bands representing MiGa with HisTag and without HisTag, respectively. A) SDS-PAGE analysis of samples before and after cleavage by the TEV protease. 1 - Sample of MiGa before the cleavage, 2 - The TEV protease, 3 - Sample after the cleavage, 4 - Unbound chromatography fraction (contains MiGa without HisTag), 5 - Bound chromatography fraction with 300 mM imidazole (contains MiGa with HisTag and the TEV protease). B) Nickel-chelate affinity chromatography chromatogram. Peak numbers correspond the sample numbers in Fig. A.

Figure 5: Gel filtration of MiGa sample. A) SDS-PAGE analysis of the chromatography fractions. B) Gel filtration chromatogram. MiGa was eluted at elution volume 75 ml. Peak numbers correspond the sample numbers in Fig. A. The degree of purity of the MiGa sample was very high.

Figure 6: Melting temperatures of MiGa at different pH-values and salt concentrations. The melting temperatures were obtained from melting curves of protein transition from native to denatured state by DSF.

Figure 7: CD spectra of MiGa in 5 mM phosphate buffer at different pH-values.

Figure 8: Hemolytic curves of MiGa. Measurements were performed at pH 7 and room temperature.

Figure 9: Binding of MiGa to various MLVs. A) SDS-PAGE analysis of samples after incubation of MiGa with MLVs. Presence of the protein in the supernatant (S) means lack of binding to the MLVs, while presence of the protein in the pellet (P) means binding to the MLVs. B) Diagram - distribution (%) of MiGa in supernatant and pellet at various MLVs. Percentages were calculated from optic densities of gel protein bands, which were obtained after analysing the gel picture by Imaged software. Salt concentration used in the experiments was 140 mM.

Figure 10: Calcein-release assay. Concentration is presented on logarithmic scale (x-axis). Salt concentration used in the experiments was 140 mM.

Figure 11 : Preparation and purification of MiGa pores. A) Native PAGE analysis of samples from preparation and purification steps. Red arrows mark oligomeric species that was purified, black arrows mark other oligomers that were not purified (probably due to precipitation). 1 - Monomeric MiGa, 2 - Sample after incubation with lipid vesicles and detergent, 3 - Unbound fraction of the IEC - Tween-80, 4 - Bound fraction of the IEC - MiGa pores, 5 - Tween-80. B) IEC chromatogram. Peak numbers correspond the sample numbers in Fig. A.

Figure 12: Cryo-EM analysis of the MiGa pores. A) Cryo-EM micrograph. Scale bar size is 20 nm. B) 2D class averages of the pore particles. Scale bar size is 5 nm. C) Cryo-EM density map - a view from the side. D) Cryo-EM density map - a view from above. Detailed description of the invention

Unless specifically defined herein, all technical and scientific terms used have the same meaning as commonly understood by a skilled artisan in the fields of biochemistry, genetics and molecular biology.

All methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, with suitable methods and materials being described herein. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will prevail. Further, the materials, methods, and examples are illustrative only and are not intended to be limiting, unless otherwise specified.

The practice of the present invention will employ, unless otherwise indicated, conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, and recombinant DNA, which are within the skill of the art. Such techniques are explained fully in the literature. See, for example, Current Protocols in Molecular Biology (Frederick M. AUSUBEL, 2000, Wiley and son Inc, Library of Congress, USA); and Molecular Cloning: A Laboratory Manual, Third Edition, (Sambrook et al, 2001 , Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press).

Polypeptides of the invention

The present invention is based on the identification, isolation and characterization of an actinoporin-like polypeptide found in mediterranean mussel (Mytilus galloprovincialis). As will be described in more detail in the Example section below, the polypeptide has been shown to be unique in that it is capable of forming a stable hexameric nanopore opposed to most actinoporins or other actinoporin-like polypeptides which form stable octameric pores. Moreover, the nanopores formed by the polypeptide of the present invention are exclusively hexameric and can be isolated and purified. Because the hexameric pores are smaller (narrower) compared to their octameric and hepatmeric counterparts, they are particularly useful in applications such as fast high-resolution peptide mass determination and allow the identification of smaller analytes compared to octameric and heptameric pores.

The present invention thus provides in a first aspect a polypeptide which is capable of forming a hexameric nanopore. Particularly, the present invention provides a (isolated) polypeptide derived from Mytilus galloprovincialis (SEQ ID NO: 1) and variants thereof. Such variants may have an amino acid sequence which has at least about 75%, such as at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, sequence identity with the amino acid sequence of SEQ ID NO: 1. Such variants may also have an amino acid sequence corresponding to the amino acid sequence of SEQ ID NO: 1 , wherein 1 or more amino acid residues are substituted, deleted and/or inserted. Suitably, such variants will retain the functional properties of the parent polypeptide, e.g., the capability to form a hexameric nanopore.

Particularly, the present invention provides a polypeptide selected from the group consisting of: a) a polypeptide having the amino acid sequence of SEQ ID NO: 1 ; b) a polypeptide having an amino acid sequence which has at least 75%, such as at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, sequence identity with the amino acid sequence of SEQ ID NO: 1 ; and c) a polypeptide having an amino acid sequence which corresponds to the amino acid sequence of SEQ ID NO: 1 , wherein 1 to 50, such as 1 to 40, 1 to 35, 1 to 30, 1 to 25, 1 to 20, 1 to 15, 1 to 10, 1 to 5, 1 to 3, amino acid residues are substituted; 1 to 20, such as 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3 or 1 to 2, amino acid residues are added to or deleted from the C-terminus; and/or 1 to 5, such as 1 to 4, 1 to 3 or 1 to 2, amino acid residues are added to or deleted from the N-terminus.

According to some embodiments, the polypepetide has the amino acid sequence of SEQ ID NO: 1.

According to some embodiments, the polypepetide has an amino acid sequence which has at least 75% sequence identity with the amino acid sequence of SEQ ID NO: 1. According to some embodiments, the polypepetide has an amino acid sequence which has at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 1. According to some embodiments, the polypepetide has an amino acid sequence which has at least 85% sequence identity with the amino acid sequence of SEQ ID NO: 1. According to some embodiments, the polypepetide has an amino acid sequence which has at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1. According to some embodiments, the polypepetide has an amino acid sequence which has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1. Such polypeptide(s) suitably has the same functional property than the refence polypeptide of SEQ ID NO: 1 , i.e. the capability to form a nanopore, and particularly a hexameric nanopore.

According to some embodiments, the polypepetide has an amino acid sequence which corresponds to the amino acid sequence of SEQ ID NO: 1 , wherein 1 to 50, , such as 1 to 40, 1 to 35, 1 to 30, 1 to 25, 1 to 20, 1 to 15, 1 to 10, 1 to 5, 1 to 3, amino acid residues are substituted. In other words, 1 to 50 amino acid residues in the amino acid sequence of SEQ ID NO: 1 have been replaced by other amino acid residues. According to some embodiments, the polypepetide has an amino acid sequence which corresponds to the amino acid sequence of SEQ ID NO: 1 , wherein 1 to 40 amino acid residues are substituted. According to some embodiments, the polypepetide has an amino acid sequence which corresponds to the amino acid sequence of SEQ ID NO: 1 , wherein 1 to 20 amino acid residues are substituted. According to some embodiments, the polypepetide has an amino acid sequence which corresponds to the amino acid sequence of SEQ ID NO: 1 , wherein 1 to 10 amino acid residues are substituted. According to some embodiments, the polypepetide has an amino acid sequence which corresponds to the amino acid sequence of SEQ ID NO: 1 , wherein 1 to 5 amino acid residues are substituted. According to some embodiments, the polypepetide has an amino acid sequence which corresponds to the amino acid sequence of SEQ ID NO: 1 , wherein 1 to 3 amino acid residues are substituted. According to some embodiments, the polypepetide has an amino acid sequence which corresponds to the amino acid sequence of SEQ ID NO: 1 , wherein 1 or 2 amino acid residues are substituted. Such polypeptide(s) suitably has the same functional property than the refence polypeptide of SEQ ID NO: 1 , i.e. the capability to form a nanopore, and particularly a hexameric nanopore.

Any such amino acid substitution may occur at any position within the amino acid sequence of SEQ ID NO: 1 , but preferably is located within the C-terminal part of the polypeptide (amino acids 106 to 212). Moreover, while any such amino acid substitution may be conservative in nature, it is preferably a conservative substitution.

Alternatively, or in addition, the polypeptide may be modified to add or delete 1 to 20, such as 1 to 10; 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3 or 1 to 2, amino acid residues to/from the C-terminus. A non-limiting example of an amino acid addition is a 6-His-tag, alone or together with a recognition site for the TEV protease, which facilitates the purification of the polypeptide (see, e.g., SEQ ID NOs: 2 or 3). According to some embodiments, the polypepetide has an amino acid sequence which corresponds to the amino acid sequence of SEQ ID NO: 1 , wherein 1 to 20 amino acid residues are added to the C-terminus. According to some embodiments, the polypepetide has an amino acid sequence which corresponds to the amino acid sequence of SEQ ID NO: 1 , wherein 1 to 10, such as 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3 or 1 to 2, amino acid residues are added to the C-terminus. According to some embodiments, the polypepetide has an amino acid sequence which corresponds to the amino acid sequence of SEQ ID NO: 1 , wherein 1 to 20 amino acid residues are deleted from the C-terminus. According to some embodiments, the polypepetide has an amino acid sequence which corresponds to the amino acid sequence of SEQ ID NO: 1 , wherein 1 to 10, such as 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3 or 1 to 2, amino acid residues are deleted from the C-terminus. Such polypeptide(s) suitably has the same functional property than the refence polypeptide of SEQ ID NO: 1 , i.e. the capability to form a nanopore, and particularly a hexameric nanopore.

Alterntively, or in addition, the polypeptide may be modified to add or delete 1 to 5, such as 1 1 to 4, 1 to 3 or 1 to 2, amino acid residues to/from the N-terminus. According to some embodiments, the polypepetide has an amino acid sequence which corresponds to the amino acid sequence of SEQ ID NO: 1 , wherein 1 to 5, such as 1 to 4, 1 to 3 or 1 to 2, amino acid residues are added to the N-terminus. According to some embodiments, the polypepetide has an amino acid sequence which corresponds to the amino acid sequence of SEQ ID NO: 1 , wherein 1 to 5, such as 1 to 4, 1 to 3 or 1 to 2, amino acid residues are deleted from the N- terminus. Such polypeptide(s) suitably has the same functional property than the refence polypeptide of SEQ ID NO: 1 , i.e. the capability to form a nanopore, and particularly a hexameric nanopore.

A polypeptide’s capability to form a nanopore may be determined by any suitable technique. For example, it may be determined by a hemolysis assay similar to that described in the Example section below. Pore-forming is determined by way of hemolytic activity of the polypeptide in terms of attenuance on bovine erythrocytes, which had been washed in tripartite erythrocyte buffers (50 mM sodium acetate-Tris-g lycine, 140 mM NaCI) with various pH- values (5.5, 6, 7, 8 and 9). Bovine erythrocytes in tripartite erythrocyte buffers (100 pl) are added to the polypeptide dilutions to a final Q.D.630 of 0.5. The polypeptide is considered pore-forming if it causes the drop of Q.D.630 to 0.2 or lower after 20 minutes at the final polypeptide concentration of 2 mg/ml or lower.

A polypeptide’s capability to form a hexameric nanopore may be determined by any suitable technique for dermining subunit stoichiometry such as by cryo-EM analysis or SEC-MALS. For example, it may be determined by the cryo-EM analysis described in the Example section below.

Nanopores, compositions, liposomes and systems of the invention The present invention further provides a nanopore comprising a plurality of molecules (monomers) of the polypeptide according to the present invention. Particulary, the present invention provides a hexameric nanopore comprising a plurality of molecules of the polypeptide according to the present invention. More specifically, the present invention provides a nanopore which is composed of 6 molecules of the polypeptide of the present invention.

In a further aspect, the present invention provides a composition comprising at least one nanopore according to the present invention. The composition may further comprise a membrane, such as lipid bilayer or artificial membrane, which comprises the nanopore.

Lipid bilayers are models of cell membranes and serve as excellent platforms for a range of experimental studies. For example, lipid bilayers can be used for in vitro investigation of membrane proteins by single-channel recording. Alternatively, lipid bilayers can be used as biosensors to detect the presence of a range of substances. The lipid bilayer may be any lipid bilayer known in the art. Suitable lipid bilayers include, but are not limited to, a planar lipid bilayer, a supported bilayer or a liposome.

An artificial membrane is a membrane which is electrically resistant similar to lipid bilayers, and which can be used to produce a planar membrane, such as polymeric membranes from cellulose acetate, Nitrocellulose, cellulose esters (CA, CN, and CE), polysulfone (PS), polyethersulfone (PES), polyacrylonitrile (PAN), polyamide, polyimide, polyethylene and polypropylene (PE and PP), polytetrafluoroethylene (PTFE), polyvinylidene fluoride (PVDF) or polyvinylchloride (PVC).

The present invention further provides a liposome comprising at least one nanopore according to the present invention within a lipid bilayer thereof.

A liposome is a spherical vesicle having at least one lipid bilayer. The liposome may be composed of a phospholipid, such as glycerophospholipid, cardiolipin or glycero-3-phospho- L-serine, especially glycerophospholipid. Non-limiting examples of glycerophospholipid include 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1'-rac-glycerol), 1 ,2-dimyristoyl-sn- glycero-3-phospho-(1'-rac-glycerol), 1 ,2-dipentadecanoyl-sn-glycero-3-phospho-(1'-rac- glycerol), 1 ,2-dipalmitoyl-sn-glycero-3-phospho-(1'-rac-glycerol), 1 ,2-distearoyl-sn-glycero-3- phospho-(T-rac-glycerol), 1 ,2-dioleoyl-sn-glycero-3-phospho-(1'-rac-glycerol), 1 ,2-dielaidoyl- sn-glycero-3-phospho-(1'-rac-glycerol), 1 ,2-dilinoleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) and 1 ,2-dilinolenoyl-sn-glycero-3-phospho-(1'-rac-glycerol). Suitable, the phospholipid is a negatively charged lipid. According to some embodiments, the liposome is composed of a phospholipide which is not a sphingophospholipide.

According to some emodiments, the liposome is composed of a glycerophospholipid.

According to some emodiments, the liposome is composed of a glycerophospholipid is selected from the group consisting of 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1'-rac- glycerol), 1 ,2-dimyristoyl-sn-glycero-3-phospho-(1 '-rac-glycerol), 1 ,2-dipentadecanoyl-sn- glycero-3-phospho-(1'-rac-glycerol), 1 ,2-dipalmitoyl-sn-glycero-3-phospho-(1'-rac-glycerol), 1 ,2-distearoyl-sn-glycero-3-phospho-(1'-rac-glycerol), 1 ,2-dioleoyl-sn-glycero-3-phospho-(1'- rac-glycerol), 1 ,2-dielaidoyl-sn-glycero-3-phospho-(1 '-rac-glycerol), 1 ,2-dilinoleoyl-sn-glycero- 3-phospho-(1 '-rac-glycerol) and 1 ,2-dilinolenoyl-sn-glycero-3-phospho-(1 '-rac-glycerol).

According to some embodiments, the lipsome is composed of 1-palmitoyl-2-oleoyl-sn-glycero- 3-phospho-(1 '-rac-glycerol).

The liposome may be a multilamellar vesicle (MLV, with several lamellar phase lipid bilayers) or unilamellar liposome vesicle (with one lipid bilayer), such as a large unilamellar vesicle (LUV). According to some embodiments, the lipsome is a large unilamellar vesicle (LUV).

The present invention further provides a system comprising at least one nanopore according to the present invention comprised in membrane, such as a planar lipid bilayer or artificial membrane.

The planar bilayer lipid membrane (BLM) is a simple model of a lipid system. It is usually formed across a small aperture in a hydrophobic partition that separates two compartments filled with aqueous solutions. The advantage of the BLM is that bothsides of the membrane can be easily altered and probed by electrodes.

The system may be operative to detect a property of an analyte. According to some embodiments, the system is operative to detect a property of an analyte comprising subjecting the nanopore to an electric field such that the analyte interacts with the nanopore. The applied potential (which creates the electric field) is necessary to have a current. The current is the output signal. For example, the system is operative to detect a property of the analyte comprises subjecting the nanopore to an electric field such that the analyte electrophoretically and/or electro-osmotically translocates through and/or is trapped in the nanopore. The property may be an electrical, chemical, or physical property of the analyte.

DNA constructs and host cells of the invention The present invention further provides a (isolated) nucleic acid molecule comprising a nucleotide sequence encoding the polypeptide according to the present invention.

More specifically, the present invention further provides a DNA construct comprising an nucleotide sequence encoding the polypeptide according to the present invention.

The DNA construct may comprise at least one genetic element for facilitating expression of the polypeptide encoding nucleotide sequence, such as at least one promoter. Suitably, the at least one promoter is operably linked to the nucleotide sequence encoding the polypeptide.

Promoters useful in accordance with the invention are any known promoters that are functional in a given host cell to cause the production of an mRNA molecule. Many such promoters are known to the skilled person. Such promoters include promoters normally associated with other genes, and/or promoters isolated from any bacteria. The use of promoters for protein expression is generally known to those of skilled in the art of molecular biology, for example, see Sambrook et al. (Sambrook, Russell 2001). The promoter employed may be inducible, such as a temperature inducible promoter (e.g., a pL or pR phage lambda promoters, each of which can be controlled by the temperature-sensitive lambda repressor c1857). The term “inducible” used in the context of a promoter means that the promoter only directs transcription of an operably linked nucleotide sequence if a stimulus is present, such as a change in temperature or the presence of a chemical substance (“chemical inducer”). As used herein, “chemical induction” according to the present invention refers to the physical application of an exogenous or endogenous substance (incl. macromolecules, e.g., proteins or nucleic acids) to a host cell. This has the effect of causing the target promoter present in the host cell to increase the rate of transcription. Alternatively, the promoter employed may be constitutive. The term “constitutive” used in the context of a promoter means that the promoter is capable of directing transcription of an operably linked nucleotide sequence in the absence of stimulus (such as heat shock, chemicals etc.). Examples of promoters that have been commonly used to express heterologous polypeptides, include, without limitation, PermE* promoter, Pm promoter, lac promoter, trp promoter, tac promoter, ApL promoter, T7 promoter, phoA promoter, araC promoter, xapA promoter, cad promoter and recA promoter.

Non-limiting examples of promoters functional in bacteria, such as Bacillus subtilis, Lactococcus lactis or Escherichia coli, include both constitutive and inducible promoters such as T7 promoter, the beta-lactamase and lactose promoter systems; alkaline phosphatase (phoA) promoter, a tryptophan (trp) promoter system, tetracycline promoter, lambda-phage promoter, ribosomal protein promoters; and hybrid promoters such as the tac promoter. Other bacterial and synthetic promoters are also suitable. Non-limiting examples of promoters functional in yeast, such as Saccharomyces cerevisiae, include xylose promoter, GAL1 and GAL10 promoters, TEF1 promoter, and pgk1 promoter.

Besides a promoter, the DNA construct may further comprise at least one genetic element selected from a 5’ untranslated region (5’UTR) and 3’ untranslated region (3’ UTR). Many such 5’ UTRs and 3’ UTRs derived from prokaryotes are well known to the skilled person. Such genetic elements include 5’ UTRs and 3’ UTRs normally associated with other genes, and/or 5’ UTRs and 3’ UTRs isolated from any prokaryotes, notably bacteria. Usually, the 5’ UTR contains a ribosome binding site (RBS), also known as the Shine Dalgarno sequence which is usually 3-10 base pairs upstream from the initiation codon. The ribosome binding site may be an RBS naturally associated with a prokaryotic gene or may be synthetic.

Further genetic elements may include, but are not limited to, an enhancer, a response element, a terminator sequence, a polyadenylation sequence, and the like.

The DNA construct may be a vector, such as an expression vector, or part of a vector, such as an expression cassette. Normally, such a vector remains extrachromosomal within the host cell which means that it is found outside of the nucleus or nucleoid region of the cell. However, it is also contemplated by the present invention that the DNA construct is stably integrated into at least one chromosome of a host cell. Means for stable integration into a chromosome of a host cell, e.g., by homologous recombination, are well known to the skilled person. For example, the DNA construct may contain one or more integration elements facilitating the integration into the chromosome of a host cell.

According to some embodiments, the DNA construct is a vector, such as an expression vector. According to particular embodiments, the vector is a plasmid, such as a cosmid. The vector may be an integrative vector, such as an integrative plasmid.

According to some embodiments, the DNA construct is an expression cassette.

The present invention further provides a recombinant host cell comprising (e.g. modified to express) the polypeptide according to the present invention or a nucleic acid molecule comprising a nucleotide sequence encoding the polypeptide according to the present invention.

According to some embodiments, the recombinant host cell comprises a DNA construct of the present invention. Generally, the DNA construct will be heterologous to said host cell. Recombinant host cells in accordance with the present invention can be produced from any suitable host organism, including single-celled or multicellular microorganisms such as bacteria, yeast, fungi, algae and plant.

According to some embodiments, the recombinant host cells in accordance with the invention is selected from the group consisting of bacteria and yeast.

Bacterial host cells are selected from Gram-positive and Gram-negative bacteria. Non-limiting examples for Gram-negative bacterial host cells include species from the genera Escherichia, Erwinia, Klebsiella and Citrobacter. Non-limiting examples of Gram-positive bacterial host cells include species from the genera Bacillus, Lactococcus, Lactobacillus, Clostridium, Corynebacterium, Streptomyces, Streptococcus, and Cellulomonas.

According to some embodiments, the recombinant host cell is a bacterium, which may be a bacterium of the genus Bacillus, Lactococcus, Lactobacillus, Clostridium, Corynebacterium, Geobacillus, Thermoanaerobacterium, Streptococcus, Pseudomonas, Streptomyces, Escherichia, Shigella, Acinetobacter, Citrobacter, Salmonella, Klebsiella, Enterobacter, Erwinia, Kluyvera, Serratia, Cedecea, Morganella, Hafnia, Edwardsiella, Providencia, Proteus, or Yersinia.

For example, the recombinant host cell of the present invention may be a bacterium selected from the group consisting of: Escherichia coli, Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus licheniformis, Bacillus mojavensis, Lactococcus lactis, Corynebacterium glutamicum, Streptomyces lividans, Streptomyces coelicolor, Streptomyces griseus, Pseudomonas putida, Geobacillus thermoglucosidasius, Geobacillus stearothermophilus, and Thermoanaerobacterium thermosaccharolyticum.

According to some embodiments, the recombinant host cell is a bacterium of the genus Escherichia. A non-limiting example of a bacterium of the genus Escherichia is Escherichia coli. According to some embodiments, the recombinant host cell is Escherichia coli.

Yeast host cells may be derived from e.g., Saccharomyces, Pichia, Schizosacharomyces, Zygosaccharomyces, Hansenula, Pachyosolen, Kluyveromyces, Debaryomyces, Yarrowia, Candida, Cryptococcus, Komagataella, Lipomyces, Rhodospiridium, Rhodotorula, or Trichosporon.

According to certain embodiments, the recombinant host cell is a yeast, which may be a yeast of the genus Saccharomyces, Pichia, Schizosacharomyces, Zygosaccharomyces, Hansenula, Pachyosolen, Kluyveromyces, Debaryomyces, Yarrowia, Candida, Cryptococcus, Komagataella, Lipomyces, Rhodospiridium, Rhodotorula, or Trichosporon. According to particular embodiments, the recombinant host cell is a yeast of the genus Saccharomyces. A non-limiting example of a yeast of the genus Saccharomyces is Saccharomyces cerevisiae. According to some embodiment, the recombinant host cell is Saccharomyces cerevisiae.

The recombinant host cell of the present invention may be cultivated in an conventional medium suitable for culturing the host cell in question, which may be composed according to the principles of the prior art. The culture medium will usually contain all nutrients necessary for the growth and survival of the respective host cell, such as carbon and nitrogen sources and other inorganic salts. Suitable media, e.g. minimal or complex media, are available from commercial suppliers, or may be prepared according to published receipts, e.g. the American Type Culture Collection (ATCC) Catalogue of strains. Non-limiting standard medium well known to the skilled person include Luria Bertani (LB) broth, Sabouraud Dextrose (SD) broth, MS broth, Yeast Peptone Dextrose, BMMY, GMMY, or Yeast Malt Extract (YM) broth, which are all commercially available. A non-limiting example of suitable media for culturing bacterial cells, such as B. subtilis, L. lactis or E. coli cells, including minimal media and rich media such as Luria Broth (LB), M9 media, M17 media, SA media, MOPS media, Terrific Broth, YT and others. Suitable media for culturing eukaryotic cells, such as yeast cells, are RPM1 1640, MEM, DMEM, all of which may be supplemented with serum and/or growth factors as required by the particular host cell being cultured. The medium for culturing eukaryotic cells may also be any kind of minimal media such as Yeast minimal media.

Suitable conditions for culturing the respective host cell are well known to the skilled person. Typically, the recombinant host cell is cultured at a temperature ranging from about 23°C to about 60°C, such as from about 25°C to about 42°C, such as at about 30°C to about 40°C, such as about 37°C. The pH of the medium may range from about pH 5.0 to about pH 10.0, but in most cases will be from about pH 6 to about pH 8, e.g. from about pH 7.0 to about pH 7.5.

Methods of the invention

The present invention further provides methods for producing a nanopore or system according to the invention as well as uses thereof.

Particularly, the present invention provides a method for producing a nanpore according to the present invention, comprising the steps of:

(i) providing a plurality of molecules of the polypeptide according to the present invention; (ii) contacting said a plurality of molecules of the polypeptide with at least one liposome, such as a multilamellar or large unilamellar vesicle, to assemble them into at least one nanopore;

Moreover, the present invention provides a method for producing a system according to the present invention; comprising the steps of:

(i) providing a plurality of molecules of the polypeptide according to the present invention;

(iii) recovering the at least one nanopore from the at least one liposome;

(iv) contacting the at least one nanopore with a planar lipid bilayer.

The polypeptide of the present invention may be chemically synthesized or produced by a suitable host cell using recombinant technology. Thus, according to some embodiments, step (i) includes culturing a recombinant host cell according to the present invention under suitable culture conditions allowing the production of the polypeptide by said host cell, and isolating said polypeptide from said host cell. Examples of suitable conditions have been described above. The isolation may include the purification of the polypeptide. The polypeptide may be isolated and purified by conventional methods for the isolation and purification of polypeptides. Well-known purification procedures include centrifugation or filtration, precipitation, and chromatographic procedures such as e.g. ion exchange chromatography, gel filtration chromatography, etc. According to some embodiments, the isolation includes a chromatographic procedure.

The liposome used in step (ii) may be a liposome as described above. Particularly, the lipomsome may be composed of a glycerophospholipid, such as one selected from the group consisting of 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1'-rac-glycerol), 1 ,2-dimyristoyl-sn- glycero-3-phospho-(1'-rac-glycerol), 1 ,2-dipentadecanoyl-sn-glycero-3-phospho-(1'-rac- glycerol), 1 ,2-dipalmitoyl-sn-glycero-3-phospho-(1'-rac-glycerol), 1 ,2-distearoyl-sn-glycero-3- phospho-(T-rac-glycerol), 1 ,2-dioleoyl-sn-glycero-3-phospho-(1'-rac-glycerol), 1 ,2-dielaidoyl- sn-glycero-3-phospho-(1'-rac-glycerol), 1 ,2-dilinoleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) and 1 ,2-dilinolenoyl-sn-glycero-3-phospho-(1'-rac-glycerol). According to some embodiments, the glycerophospholipid is 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1'-rac-glycerol). According to some embodiments, step ii) includes mixing said a plurality of molecules of the polypeptide with said at least one liposome and incubating said mixture for a suitable time at a suitable temperature.

A suitable time is any time which allows the plurality of molecules of the polypeptide to assemble into at least one nanopore within the liplid bilayer of the liposome. For example, the mixture may be incubated for about 15 to about 60 minutes, such as for about 20 to about 40 minutes. According to some embodiments, the mixture is incubated for about 25 to about 35 minutes. According to some embodiments, the mixture is incubated for about 30 minutes.

A suitable temperature is any temperature which allows the plurality of molecules of the polypeptide to assemble into at least one nanopore within the liplid bilayer of the liposome. For example, the mixture may be incubated at a temperature from about 25°C to about 42°C, such as a temperature from about 30°C to about 42°C. According to some embodiments, the mixture is incubated at a temperature from about 35°C to about 40°C. According to some embodiments, the mixture is incubated at about 37°C.

The incubation may further include agitating the mixture. Thus, according to some embodiments, the mixture is incubated under agitation, e.g., using a shaker or the like. For example, the mixture may be incubated under agitation at about 50 to about 500 rpm, such as at about 250 rpm.

According to some embodiments, step ii) includes mixing said a plurality of molecules of the polypeptide with said at least one liposome and incubating said mixture for about 25 to 35 minutes at about about 35°C to about 40°C at about 250 rpm.

The plurality of molecules of the polypeptide may be mixed with said at least one liposome at any suitbale molar ratio, typically in the range of 1 :3 to 1 :2000, such as at a molar ratio in the range of 1 :10 to 1 :1000, in a suitable buffer, such as a balanced salt solution. Accoridng to some embodiments, the plurality of molecules of the polypeptide are mixed with said at least one liposome at a molar ratio in the range of 1 :50 to 1 :500, such as at a molar of 1 :100. Typically, the buffer has a pH in the range from about 6 to about 8.5, such as from about 7 to about 7.5. Non-limiting examples of a suitable buffer are phosphate buffered saline (PBS), TRIS buffered saline (TBS), and MES. According to some embodiments, the buffer is phosphate buffered saline.

According to some embodiments, step iii) includes incubating the at least one liposome containing said at least one nanopore in the presence of a suitable detergent for a suitable time at a suitable temperature. A suitable detergent is any non-ionic surfactant or mixture of non-inonic surfactants which allows the solubilisation of lipids forming the liposome, while retaining the integrity of the nanopores. Non-limiting examples of a suitable detergent are polysorbates such as Polysorbat 80 (Polyoxyethylen(20)-sorbitan-monooleat; Tween® 80) or Polysorbat 20 (Polyoxyethylene (20) sorbitan monolaurate; Tween® 20). According to some embodiments, the detergent is a polysorbate. According to some embodiments, the detergent is Polysorbat 80 or Polysorbat 20. Particularly, Polysorbat 80 has been shown to be very effective. According to some embodiments, the detergent is Polysorbat 80.

The detergent may be employed at any suitable concentration. Typically, the concentration will be in the range of about 0.5% v/v to about 5% v/v, such as in the range of 1 % v/v to 3% v/v. High concentrations may also be used. However, this may require an additional purification step to remove the detergent after the incubation.

A suitable time is any time which allows the lipids forming the liposome to solubized so to release the nanopore(s). For example, the mixture of liposome and detergent may be incubated for about 1 to about 20 minutes, such as for about 10 to about 20 minutes. According to some embodiments, the mixture is incubated for about 10 to about 15 minutes. According to some embodiments, the mixture is incubated for about 15 minutes.

A suitable temperature is any temperature which allows the lipids forming the liposome to solubized so to release the nanopore(s). For example, the mixture of liposome and detergent may be incubated at a temperature from about 25°C to about 42°C, such as a temperature from about 30°C to about 42°C. According to some embodiments, the mixture is incubated at a temperature from about 35°C to about 40°C. According to some embodiments, the mixture is incubated at about 37°C.

The incubation may further include agitating the mixture. Thus, according to some embodiments, the mixture of liposome and detergent is incubated under agitation, e.g., using a shaker or the like. For example, the mixture of liposome and detergent may be incubated under agitation at about 50 rpm to about 500 rpm, such as at at about 250 rpm.

According to some embodiments, step iii) includes incubating the at least one liposome containing said at least one nanopore in the presence of a polysorbate at a final concentration of 1 to 3% v/v for 10 to 20 minutes at a temperature from 35 to 40°C, such as at 37°C at about 250 rpm.

Follwing the incubation, the at least one nanopore may be purified. The purification may be any well-know purification procedure include centrifugation and chromatographic procedures such as e.g. ion exchange chromatography, gel filtration chromatography, etc. According to some embodiments, step iii) further includes centrifuging the mixture containing the at least one liposome and the detergent after incubation. According to some embodiments, step iii) further includes purifying the at least one nanopore by a chromatographic procedure. Accoring to some embodiments, the chromatographic procedure includes ion-exchange chromatography, such as cation-exchange chromatography.

According to some embodiments, step iii) includes centrifuging the mixture containing the at least one liposome and the detergent after incubation, diluting the obtained supernatant with a suitable buffer solution containing a polyoxyethylene surfactant such as Polyoxyethylen (23) laurylether (Brij® 35) or Polyoxyethylen (20) cetylether (Brij® 58), such as a Tris buffer solution, and loading the suspension on an ion-exchange chromatography column, such as an cation-exchange chromatography column. According to some embodiments, the buffer solution contains the polyoxyethylene surfactant such as Polyoxyethylen (23) laurylether or Polyoxyethylen (20) cetylether at a final concentration of about 0,1 mM to about 0,5 mM, such as at about 0,25 mM. According to some embodiments, the buffer solution contains 50 mM Tris/HCI and 0,25 mM Polyoxyethylen (23) laurylether or Polyoxyethylen (20) cetylether (pH 7,4).

As a final step in the chromatographic procedure, the at least one nanopore is eluted from the chromatography column, e.g., by using a salt gradient (e.g., at about 100 mM NaCI).

Once recovered, the at least one nanopore may be directly processed. Alterntively, it may be stored under suitbale cooling conditions, such as at about 4° to about 6°C, until further use.

Uses of the invention

The nanopore, liposome or system of the present invention may be used in a variety of downstream applications, in particular as biosensors. The nanopore, liposome or system of the present invention may particularly be used in pore-based mapping or partial sequencing of unfolded proteins, label-free sensing of bioanalytes, such as folded proteins, small peptides and oligo saccharides, targeted cellular permeabilization, or drug delivery.

The present invention thus further provides the use of a nanopore, liposome or system of the present invention for sensing a biopolymer, such as a protein, a peptide or a nucleic acid (such as DNA or RNA), or metabolite.

The present invention further provides the use of a nanopore, liposome or system of the present invention for sequencing a protein, a peptide or nucleic acid (such as DNA or RNA). The present invention further provides the use of a nanopore, liposome or system of the present invention for targeted cellular permeabilization.

The present invention further provides the use of a nanopore, liposome or system of the present invention for drug delivery.

Certain definitions

“Polypeptide," or "protein" are used interchangeably herein to denote a polymer of at least two amino acids covalently linked by an amide bond, regardless of length or post- transiational modification (e.g., glycosylation, phosphorylation, lipidation, myristilation, ubiquitination, etc.). Included within this definition are D- and L-amino acids, and mixtures of D- and L-amino acids.

"Nucleic acid" or "polynucleotide" are used interchangeably herein to denote a polymer of at least two nucleic acid monomer units or bases (e.g., adenine, cytosine, guanine, thymine) covalently linked by a phosphodiester bond, regardless of length or base modification.

"Recombinant" or "non-naturally occurring" when used with reference to, e.g., a host cell, nucleic acid, or polypeptide, refers to a material, or a material corresponding to the natural or native form of the material, that has been modified in a manner that would not otherwise exist in nature, or is identical thereto but produced or derived from synthetic materials and/or by manipulation using recombinant techniques. Non-limiting examples include, among others, recombinant host cells expressing genes that are not found within the native (nonrecombinant) form of the cell or express native genes that are otherwise expressed at a different level.

“Substitution” or “substituted” refers to modification of the polypeptide by replacing one amino acid residue with another, for instance the replacement of an Arginine residue with a Glutamine residue in a polypeptide sequence is an amino acid substitution. A substitution may be conservative or non-conservative.

"Conservative substitution" refers to a substitution of an amino acid residue with a different residue having a similar side chain, and thus typically involves substitution of the amino acid in the polypeptide with amino acids within the same or similar class of amino acids. By way of example and not limitation, an amino acid with an aliphatic side chain may be substituted with another aliphatic amino acid, e.g., alanine, valine, leucine, and isoleucine; an amino acid with hydroxyl side chain is substituted with another amino acid with a hydroxyl side chain, e.g., serine and threonine; an amino acid having an aromatic side chain is substituted with another amino acid having an aromatic side chain, e.g., phenylalanine, tyrosine, tryptophan, and histidine; an amino acid with a basic side chain is substituted with another amino acid with a basic side chain, e.g., lysine and arginine; an amino acid with an acidic side chain is substituted with another amino acid with an acidic side chain, e.g., aspartic acid or glutamic acid; and a hydrophobic or hydrophilic amino acid is replaced with another hydrophobic or hydrophilic amino acid, respectively.

"Non-conservative substitution" refers to substitution of an amino acid in a polypeptide with an amino acid with significantly differing side chain properties. Non-conservative substitutions may use amino acids between, rather than within, the defined groups and affects (a) the structure of the peptide backbone in the area of the substitution (e.g., proline for glycine) (b) the charge or hydrophobicity, or (c) the bulk of the side chain. By way of example and not limitation, an exemplary non-conservative substitution can be an acidic amino acid substituted with a basic or aliphatic amino acid; an aromatic amino acid substituted with a small amino acid; and a hydrophilic amino acid substituted with a hydrophobic amino acid.

"Deletion" or “deleted” refers to modification of the polypeptide by removal of one or more amino acids in the reference polypeptide. Deletions can comprise removal of 1 or more amino acids, 2 or more amino acids, 5 or more amino acids, 10 or more amino acids, 15 or more amino acids, or 20 or more amino acids, up to 10% of the total number of amino acids, or up to 20% of the total number of amino acids making up the polypeptide while retaining its functionality. Deletions can be directed to the internal portions of the polypeptide, or to the carboxy and/or amino terminus of the polypeptide. In various embodiments, the deletion can comprise a continuous segment or can be discontinuous.

"Insertion" or “inserted” refers to modification of the polypeptide by addition of one or more amino acids to the reference polypeptide. Insertions can comprise addition of 1 or more amino acids, 2 or more amino acids, 5 or more amino acids, 10 or more amino acids, 15 or more amino acids, or 20 or more amino acids. Insertions can be in the internal portions of the polypeptide, or to the carboxy or amino terminus. The insertion can be a contiguous segment of amino acids or separated by one or more of the amino acids in the reference polypeptide.

“Isolated”, as used herein, means that a a biological component, such as a polypeptide or polynucleotide, has been separated from its original environment (e.g., the environment in which it naturally occurs). Particularly, a polynucleotide or polypeptide which has been separated from some or all of the coexisting materials in the natural system is considered isolated.

"Host cell" as used herein refers to a living cell or microorganism that is capable of reproducing its genetic material and along with it recombinant genetic material that has been introduced into it - e.g., via heterologous transformation. “Heterologous”, as used herein, means that a polynucleotide or polypeptide is normally not found in or made (i.e. expressed) by the host cell, but derived from a different organism or made synthetically. Moreover, a host cell transformed with an expression vector as described herein which is not normally present in the host cell would be considered heterologous for the purpose of the present invention.

"Expression" includes any step involved in the production of a polypeptide including, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

As used herein, "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid molecule to which it has been linked. One type of vector is a "plasmid", which refers to a circular double stranded nucleic acid loop into which additional nucleic acid segments can be ligated. Certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as "expression vectors". Certain other vectors are capable of facilitating the insertion of a exogenous nucleic acid molecule into a genome of a host cell. Such vectors are referred to herein as "transformation vectors". In general, vectors of utility in recombinant nucleic acid techniques are often in the form of plasmids. In the present specification, "plasmid" and "vector" can be used interchangeably as the plasmid is the most commonly used form of a vector. Large numbers of suitable vectors are known to those of skill in the art and commercially available.

As used herein, "promoter" refers to a sequence of DNA, usually upstream (5') of the coding region of a structural gene, which controls the expression of the coding region by providing recognition and binding sites for RNA polymerase and other factors which may be required for initiation of transcription. The selection of the promoter will depend upon the nucleic acid sequence of interest. A "promoter functional in a host cell" refers to a "promoter" which is capable of supporting the initiation of transcription in said cell, causing the production of an mRNA molecule.

As used herein, "operably linked" refers to a juxtaposition wherein the components described are in a relationship permitting them to function in their intended manner. A control sequence "operably linked" to a coding sequence is ligated in such a way that expression of the coding sequence is achieved under conditions compatible with the control sequence. A promoter sequence is "operably-linked" to a gene when it is in sufficient proximity to the transcription start site of a gene to regulate transcription of the gene.

The term “expression cassette” refer to a segment of DNA that can be inserted into a target nucleic acid molecule, such as a vector or genomic DNA, at specific restriction sites or by homologous recombination. The segment of DNA comprises a nucleotide sequence that 1 encodes a polypeptide of interest, and the cassette and restriction sites are designed to ensure insertion of the cassette in the proper reading frame for transcription and translation. An expression cassette of the invention may also comprise one or more elements that allow for expression of a nucleotide sequence encoding a polypeptide of interest in a host cell. These elements may include, but are not limited to: a promoter, an enhancer, a response element, a terminator sequence, a polyadenylation sequence, and the like.

"Percentage of sequence identity," "% sequence identity" and "percent identity" are used herein to refer to comparisons between an amino acid sequence and a reference amino acid sequence. The “% sequence identify”, as used herein, is calculated from the two amino acid sequences as follows: The sequences are aligned using Version 9 of the Genetic Computing Group's GAP (global alignment program), using the default BLOSUM62 matrix (with a gap open penalty of -12 (for the first null of a gap) and a gap extension penalty of -4 (for each additional null in the gap). After alignment, percentage identity is calculated by expressing the number of matches as a percentage of the number of amino acids in the reference amino acid sequence.

As used herein, the indefinite articles "a" and "an" mean "at least one" or "one or more" unless the context clearly dictates otherwise.

As used herein, the terms "comprising", "including", "having" and grammatical variants thereof are to be taken as specifying the stated features, steps or components but do not preclude the addition of one or more additional features, steps, components or groups thereof.

As used herein, the term "about" means plus or minus 10% of the numerical value of the number with which it is being used.

Where a numerical limit or range is stated herein, the endpoints are included. Also, all values and sub ranges within a numerical limit or range are specifically included as if explicitly written out.

Having generally described this invention, a further understanding can be obtained by reference to certain specific examples, which are provided herein for purposes of illustration only, and are not intended to be limiting unless otherwise specified. Examples

Example 1

Summary

The present invention is based on the identification, isolation and characterization of an actinoporin-like polypeptide found in mediterranean mussel (Mytilus galloprovincialis). It has been named »MiGa«. The amino acid sequence of the polypeptide is shown in Figure 1.

It has a molecular mass of 22.7 kDa, and exhibits hemolytic activity towards the bovine erythrocytes. It possesses high isoelectric point and contains two cysteine residues in its protein sequence. The protein has sequence similarity to actinoporins (around 20 % identity) and actinoporin-like proteins (up to 35 % sequence identity). The polypeptide is unique (distinct from actinoporins) by the following features:

• receptor specificity - the protein binds well to POPG (1-palmitoyl-2-oleoyl-sn-glycero- 3-phospho-(1 '-rac-glycerol)), a negatively charged lipid, while its affinity to sphingomyelin is poor

• subunit stoichiometry - cryo-EM analyses showed hexameric subunit stoichiometry, and not octameric as expected.

Materials

KOD Hot Start DNA-Polymerase from Novagen was used for PCR. The restriction enzymes Ndel and Hindlll were from New England BioLabs. T4 DNA ligase was from Thermo Fisher Scientific. The TEV protease and fragaceatoxin C (FraC) were recombinant proteins prepared by the standard protocols of the National Institute of Chemistry (Slovenia). All lipids were from Avanti Polar Lipids.

Cloning, expression and purification of monomeric MiGa cDNA sequence of MiGa was amplified by PCR using sense (5'- TCACATATGCTGATTGACTGGGCTGC-3'; SEQ ID NO: 4) and antisense (5'- TATAAGCTTACCCACGATCGGGACTTTTTC-3' SEQ ID NO: 5) oligonucleotides containing Nde\ and /7/ndlll restriction sites, respectively. Amplified cDNA was inserted into a modified pET24a vector that contained an encoded recognition site for the TEV protease, which allowed the expression of the protein with a TEV-cleavage site followed by a His-Tag at its C-terminus (Figure 2). The DNA construct was verified by nucleotide sequencing. PCR, restriction and ligation reactions, cloning and agarose gel electrophoresis were done following the standard protocols. The vector containing MIGA sequence was used to transform an Escherichia coli strain BL21(DE3).

A colony grown overnight at 37 °C on Luria-Bertani plates with kanamycin (30 pg/ml) was inoculated into 100 ml of Luria-Bertani medium with kanamycin (30 pg/ml) and incubated overnight at 37 °C. A 60 ml sample of overnight culture was inoculated into 3 I of Terrific broth medium with kanamycin (30 pg/ml) and incubated at 37 °C until the O.D.eoo of 0.7. Expression of recombinant protein was induced by 0.2 mM isopropyl p-D-1-thiogalactopyranoside. The protein was expressed overnight at 18 °C. Cells were centrifuged at 6000 rpm for 6 min and resuspended in approximately 100 ml of cell lysis buffer (50 mM Tris/HCI, 300 mM NaCI, 5 % (v/v) glycerol, pH 7).

Cell suspension was sonicated on ice two times for 7 min using Cole-Parmer Ultrasonic Processor and centrifuged at 50000 ref and 4 °C for 20 min. Supernatant was put through a filter with pore size 0.22 pm and loaded on a nickel-chelate affinity column Tricorn 16/20 (GE Healthcare Life Sciences) filled with 30 ml of Ni-NTA Superflow resin (Qiagen). The column was connected to AKTA Purifier chromatography system (GE Healthcare Life Sciences) and equilibrated with phosphate-buffered saline (1.8 mM KH2PO4, 10.1 mM Na2HPO4, 140 mM NaCI, 2.7 mM KCI) containing additional 150 mM NaCI, 5 % (v/v) glycerol and 10 mM imidazole (pH 7.0), which was used to wash the unbound proteins. MiGa was eluted from the column by 300 mM imidazole in the same phosphate/saline buffer (Figure 3) and then dialysed overnight at 4 °C in phosphate-buffered saline with additional 150 mM NaCI and 5 % (v/v) glycerol (pH 7.0).

The TEV protease (final concentration was 25-times the estimated molar concentration of MiGa) and dithiothreitol (final concentration was 1 mM) were added to the protein. The reaction mixture was incubated overnight at 4 °C and then separated by a nickel-chelate chromatography as described above, after which MiGa lacking the His-Tag was collected in the unbound fraction and the TEV protease in the bound fraction (Figure 4). The unbound fraction was purified by gel filtration using HiLoad 16/60 Superdex 75 prep grade column (GE Healthcare Life Sciences) that was connected to AKTA Purifier chromatography system (GE Healthcare Life Sciences). MiGa with high degree of purity was collected in buffer consisting of 50 mM sodium acetate, 300 mM NaCI and 5 % (v/v) glycerol (pH 5). The results are shown in Figure 5. Differential scanning fluorimetry (DSF)

We performed DSF to determine thermal stability of MiGa at different pH-values and salt concentrations. Fluorescence measurements were done using LightCycler® 480 System (Roche) and temperature range 20-95 °C (gradient 1.8 °C/min). Test solutions (30 pl) were prepared in 96-Well PCR Plates (Thermo Scientific) and contained MiGa (final concentration was 0.2 mg/ml) and Spyro™ Orange fluorescent dye (Invitrogen) in 50 mM tripartite sodium acetate-Tris-glycine buffer. Fluorescent dye (5000x stock concentration) was diluted in ultrapure water and 10x dye solution was used as a final concentration in the wells. Buffers in wells contained different salt concentrations (0, 100, 200, 300, 500, 750, 1000 and 1500 mM NaCI) and different pH-values (from 4 to 9.5, using 0.5 unit intervals). Data was processed using the Boltzmann function of the Origin 8.1 software (OriginLab Corporation) to obtain the melting temperatures (TM), i.e. the transition midpoints between native and denatured protein. All measurements were performed twice. The results are shown in Figure 6.

MiGa is thermally most stable at low pH (pH-optimum is 5) and high salt concentration, while high pH and low salt concentration decrease its thermal stability. We measured the highest melting temperature at pH 5 and 1 .5 M NaCI (57.6 °C) and the lowest at pH 9 and 0 mM NaCI (33.1 °C).

Circular dichroism (CD)

CD-spectra were measured using Chirascan™ CD-spectrometer (Applied Photophysics). Spectra were scanned in far-UV region from 195 to 250 nm at 25 °C. Bandwidth was set to 0.5 nm and time-per-point to 1s. Samples contained MiGa in 5 mM phosphate buffers with various pH-values (5, 6, 7, 8, and 9). The protein concentration in the samples was 0.2 mg/ml. 1-mm-pathlength cuvette was used for the measurements. All spectra are were scanned 10 times. The results are shown in Figure 7.

The shape of MiGa's circular dichroism spectra (estimation) reveal the presence of a- and - structural elements. Different pH-values (5-9) do not affect the protein's structural properties.

Hemolysis assay

Hemolytic activity of MiGa was measured in terms of attenuance on bovine erythrocytes, which had been washed in tripartite erythrocyte buffers (50 mM sodium acetate-Tris-glycine, 140 mM NaCI) with various pH-values (5.5, 6, 7, 8 and 9). 2-fold serial dilutions of the protein in tripartite erythrocyte buffers (100 pl) were prepared in 96-well microplates (Costar). Bovine erythrocytes in tripartite erythrocyte buffers (100 pl) were added to the protein dilutions to a final O.D.630 of 0.5. Final concentration of the protein in the first well was 0.3 mg/ml. Attenuance at A63o was measured for 20 min (using 20 s intervals) at 25 °C with Synergy™ MX microplate reader (BioTek). Experimental curves were processed by a linear regression with 3 data points using Gen5 software (BioTek) to determine the maximum velocity of hemolysis (V_max). All measurements were performed three times. The results are shown in Figure 8.

MiGa exibits hemolytic activity towards bovine erythrocytes.

Sedimentation assay

Multilamellar vesicles (MLVs) were prepared in phosphate-buffered saline (1.8 mM KH2PO4, 10.1 mM Na2HPO4, 140 mM NaCI, 2.7 mM KCI, pH 7.4) and were composed of the following lipids: (1) 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (POPG), (2) 1-palmitoyl- 2-oleoyl-sn-glycero-3-phosphocholine (POPC) and cholesterol (Choi) at molar ratio 1 : 1 , (3) POPC and sphingomyelin (SM) at molar ratio 1 : 1 and (4) POPC. Lipid mixtures were dissolved in chloroform, which was then evaporated using Rotavapor R-300 (Buchi Labortechnik AG) to generate a lipid film. The lipid film was dissolved in phosphate-buffered saline and flash frozen in liquid nitrogen and then thawed five times. MiGa (2 pg) was incubated with MLVs in phosphate-buffered saline at molar ratio 1 : 2000 for one hour at 25 °C and 250 rpm. Final protein concentration was 4.2 pM. Reaction mixtures were centrifuged at 16100 ref for 10 min to generate supernatant and pellet, both of which were then analyzed by gel electrophoresis in the presence of sodium dodecyl sulphate. Protein bands were analysed with Imaged in order to quantify the distribution of the protein in supernatant and pellet. All experiments were performed three times. The results are shown in Figure 9.

The assay shows that MiGa binds to POPG, and not to sphingomyelin, the known actinoporin lipid receptor.

Calcein release assay

Large unilamellar vesicles (LUVs) in phosphate-buffered saline with 50 mM calcein/NaOH were prepared by extrusion of MLVs using LipoFast™ lipid extruder (Avestin) and polycarbonate membranes with 100 nm pores (ipPORE™ Track Etched Membrane, Ion Track Technology 4 Innovative Products). MLVs, which had been prepared as described above, were pushed through the membranes 20 times. LUVs were composed of the following lipids: (1) 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (POPG), (2) 1-palmitoyl-2- oleoyl-sn-glycero-3-phosphocholine (POPC) and POPG at molar ratio 1 : 1 , (3) POPC and sphingomyelin (SM) at molar ratio 1 : 1 and (4) POPC. A 10-ml-Sephadex G-50 Superfine column (GE Healthcare) was used to remove the external calcein pool from the LUV suspension. Lipid concentration was determined by colorimetric assay LabAssay™ Phospholipid (Fujifilm Wako Diagnostics). LLIVs were also analysed by fluorescence measurements before and after the addition of detergent Triton-X100. All fluorescence measurements were done with Synergy™ MX microplate reader (BioTek) using excitation and emission wavelengths 485 nm and 520 nm, respectively. Calcein leakage from the LLIVs in the presence of MiGa was measured in black 96-well microtiter plates (Costar) for 30 min (using 31 s intervals) at 25 °C. Prior to the measurements, 100 pl of LUV suspensions were added to 2-fold serial dilutions of the protein in phosphate-buffered saline (100 pl) in the wells. Final concentration of the protein in the first well was 16 pM and final concentration of LUVs in all wells was 50 pM. After the measurements, 2 pl of 200 mM detergent Triton X-100 was added to the wells and fluorescence was measured for additional 3 min. The data was presented as a percentage of calcein release (equation 1) against protein concentration. All measurements were performed three times. The results are shown in Figure 10.

In the presence of MiGa, we detected calcein release from the vesicles with the following lipid composition: (1) POPG, (2) POPC and POPG at molar ration 1 :1 and (3) POPC and SM at molar ration 1 :1. The highest calcein release was observed from the POPG-vesicles (1). The reason for the activity towards the SM-containing vesicles was probably very high molar ratio proteinlipid (1 :25 and higher).

Equation 1

F - F_B

Calcein release [%] = — - — x 100

FT ~ F_B

F... Fluorescence

FB... Baseline fluorescence

FT... Total fluorescence - fluorescence after detergent addition

Preparation of MiGa pores

Monomeric MiGa (300 pg) was mixed with 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1'-rac- glycerol) (POPG) large unilamellar vesicles (LUVs) at molar ratio 1 : 100 in phosphate-buffered saline (1.8 mM KH₂PO₄, 10.1 mM Na₂HPO₄, 140 mM NaCI, 2.7 mM KOI, pH 7.4). LUVs were prepared in the phosphate-buffered saline as described above. The mixture was incubated for 30 min at 37 °C and 250 rpm. Lipids were solubilized by addition of detergent Tween-80 (in a final percentage of 2 % (v/v)) and incubation at 37 °C and 250 rpm for 15 min. The mixture was centrifuged for 3 min at 16100 ref, diluted 40 times in buffer consisting of 50 mM Tris/HCI and 0.25 mM Brij-35/58 (pH 7.4) and loaded on cation-exchange chromatography column Resource S (GE Healthcare Life Sciences). MiGa pores were eluted from the column by salt gradient at approximately 100 mM NaCI. The results are shown in Figure 11.

As seen from the Native PAGE gel (Figure 12 A), the pores (homogenic species) were collected in the ion-exchange cromatography bound fraction (Figure 12 B). Their protein band appers higher on the gel compared to the protein band of the monomer. Detergent Tween-80 eluted from the column in the unbound fraction.

Cryo-transmission electron microscopy

3 pl of MiGa pore solution at the concentration of 0.5 mg/ml was applied to a glow-discharged Quantifoil R1.2/1.3 300-mesh copper holey carbon grid (Quantifoil), blotted under 100 % humidity at 4 °C, for 6-7 s and plunged into liquid ethane using a Mark IV Vitrobot (Thermo Fisher Scientific-TFS). Micrographs were collected on a Glacios (TFS) with a Falcon 3EC direct electron detector (TFS) operated at 200 kV using the EPU software (TFS). Images were recorded in counting mode with the pixel size of 0.98 A. Micrographs were dose-fractioned into 38 frames with a dose rate of 0.79 e-/A/frame.

All steps of data processing were performed in cryoSPARC 2.4 with build-in algorithms. After dose-weighting, motion-correction, and CTF estimation 200 particles were handpicked to create a 2D reference. Reference-based auto picking produced 420 000 particles. 2D classification and 2D class inspection resulted in 180 000 good particles.

The results are shown in Figure 13.

Isolated MiGa pores are clearly visible under cryo-EM (Figure 13A). Top views of 2D classes after unbiased 2D classification (Figure 13B) all have 6 subunits, which is better visible in 3D reconstructed density map shown in Figure 13C and 13D (the latter has numbered subunits).

List of certain references cited in the

[1] M. D. Peraro in F. G. Van Der Goot, „Pore-forming toxins: ancient, but never really out of fashion", Nat. Rev. Microbiol., vol. 14, no. 2, pages 77-92, 2015.

[2] S. Ramirez-Carreto, B. Miranda-Zaragoza, in C. Rodriguez-Almazan, „Actinoporins: From the structure and function to the generation of biotechnological ant therapeutic tools", Biomolecules, vol. 10, no. 4, page E539, 2020.

[3] A. E. Mechaly idr. , ..Structural insights into the oligomerization and architecture of eukaryotic membrane pore-forming toxins", Structure, vol. 19, no. 2, pages 181-191 , 2011.

[4] A. Athanasiadis, G. Anderluh, P. Macek, in D. Turk, ..Crystal structure of the soluble form of equinatoxin II, a pore-forming toxin from the sea anemone Actinia equina", Structure, vol. 9, no. 4, pages 341-346, 2001.

[5] M. G. Hinds, W. Zhang, G. Anderluh, P. E. Hansen, in R. S. Norton, ..Solution structure of the eukaryotic pore-forming cytolysin equinatoxin II: Implications for pore formation", J. Mol. Biol., vol. 315, no. 5, pages. 1219-1229, 2002.

[6] I. Castrillo, J. Alegre-Cebollada, A. Martinez Del Pozo, J. G. Gavilanes, J. Santoro, in M. Bruix, „1H, 13C, and 15N NMR assignments of the actinoporin Sticholysin i“, Biomol. NMR Assign., vol. 3, no. 1, pages 5-7, 2009.

[7] J. M. Mancheno, J. Martin-Benito, M. Martinez-Ripoll, J. G. Gavilanes, in J. A. Hermoso, ..Crystal and electron microscopy structures of sticholysin II actinoporin reveal insights into the mechanism of membrane pore formation", Structure, vol. 11, no. 11, pages 1319-1328, 2003.

[8] B. Bakrac idr., ..Molecular determinants of sphingomyelin specificity of a eukaryotic pore-forming toxin", J. Biol. Chem., vol. 283, no. 27, pages 18665-18677, 2008.

[9] K. Tanaka, J. M. M. Caaveiro, K. Morante, J. M. Gonzalez-Manas, in K. Tsumoto, ..Structural basis for self-assembly of a cytolytic pore lined by protein and lipid", Nat. Commun., vol. 6, page 6337, 2015.

[10] Y. P. Hervis idr., ..Architecture of the pore forming toxin sticholysin I in membranes", J. Struct. Biol., vol. 208, no. 1, pages 30-42, 2019.

[11] Mechaly AE, Bellomio A, Gil-Carton D, et al. Structural insights into the oligomerization and architecture of eukaryotic membrane pore-forming toxins. Structure, vol. 19, no. 2, pages 181-191, 2011

[12] Huang G, Voet A, Maglia G. FraC nanopores with adjustable diameter identify the mass of opposite-charge peptides with 44 dalton resolution. Nat Commun., vol. 10, no. 1, page 835

[13] Hervis YP, Valle A, Dunkel S, et al. Architecture of the pore forming toxin sticholysin I in membranes. J Struct Biol., vol. 208, no. 1 , pages 30-42, 2019 [14] Mancheno JM, Martin-Benito J, Martinez-Ripoll M, Gavilanes JG, Hermoso JA. Crystal and electron microscopy structures of sticholysin II actinoporin reveal insights into the mechanism of membrane pore formation. Structure, vol. 11, no. 11, pages 1319-1328, 2003

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Claims

1. A polypeptide which is capable of forming a nanopore selected from the group consisting of: a) a polypeptide having the amino acid sequence of SEQ ID NO: 1 ; b) a polypeptide having an amino acid sequence which has at least 75% sequence identity with the amino acid sequence of SEQ ID NO: 1 , wherein the polypeptide is capable of forming a nanopore; and c) a polypeptide having an amino acid sequence which corresponds to the amino acid sequence of SEQ ID NO: 1 , wherein 1 to 50 amino acid residues are substituted; 1 to 20 amino acid residues are added to or deleted from the C-terminus; and/or 1 to 5 amino acid residues are added to or deleted from the N-terminus; and wherein the polypeptide is capable of forming a nanopore.

2. The polypeptide according to claim 1 , wherein the polypepetide has the amino acid sequence of SEQ I D NO: 1.

3. The polypeptide according to claim 1 , wherein the polypepetide has an amino acid sequence which has at least 75% sequence identity with the amino acid sequence of SEQ ID NO: 1.

4. The polypeptide according to claim 1 , wherein the polypeptide has an amino acid sequence which corresponds to the amino acid sequence of SEQ ID NO: 1 , wherein 1 to 50, amino acid residues are substituted.

5. The polypeptide according to claim 1 , 3 or 4, wherein the polypeptide has an amino acid sequence which corresponds to the amino acid sequence of SEQ ID NO: 1 , wherein 1 to 20 amino acid residues are added to the C-terminus or wherein the polypeptide has an amino acid sequence which corresponds to the amino acid sequence of SEQ ID NO: 1 , wherein 1 to 20 amino acid residues are deleted from the C-terminus.

6. The polypeptide according to claim 1 , 3, 4 or 5, wherein the polypeptide has an amino acid sequence which corresponds to the amino acid sequence of SEQ ID NO: 1 , wherein 1 to 5 amino acid residues are added to the N-terminus.

7. A nanopore comprising a plurality of molecules of the polypeptide according to any one of claims 1 to 6.

8. A composition comprising at least one nanopore according claim 7.

37

9. A liposome comprising at least one nanopore according to claim 7 within a lipid bilayer thereof.

10. A system comprising at least one nanopore according to claim 7 comprised in a membrane such as a planar lipid bilayer or artificial membrane.

11. A DNA construct comprising an nucleotide sequence encoding the polypeptide according to any one of claims 1 to 6.

12. A recombinant host cell which comprises a DNA construct according to claim 11 .

13. A method for producing a nanpore according to claim 7, comprising the steps of:

(i) providing a plurality of molecules of the polypeptide according to any one of claims 1 to 6;

14. A method for producing a system according to claim 10; comprising the steps of:

(iii) recovering the at least one nanopore from the at least one liposome;

(iv) contacting the at least one nanopore with a planar lipid bilayer.

15. Use of a nanopore according to claim 7, a liposome according to claim 9 or a system according to claim 10 for sensing a biopolymer or metabolite, for sequencing a protein, a peptide or nucleic acid, for targeted cellular permeabilization or for drug delivery.

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