Engineered microcompartment protein and related methods and systems of engineering bacterial systems for non-native protein expression and purification

1. An engineered microcompartment protein, comprising

an encapsulin protein having sequence SEQ ID NO: 1 or a sequence with at least 22% sequence identity or at least 40% sequence similarity to SEQ ID NO:1, and comprising a P-Domain (Peripheral Domain), an E-Loop (Elongated Loop) and an A-Domain (Axial Domain),

wherein

the P-Domain comprises

a first fragment of the P-domain having an N-terminus and a C-terminus and comprising residues configured to form, in a folded encapsulin protein, a secondary structure comprising in a direction N-terminus to C-terminus a 4 to 26 residues alpha helix α1, linked to an optional first non-structured region having 0 to 22 residues, linked to a 4 to 11 residues alpha helix α2, linked to a 3 to 9 residues beta strand β1, linked to a 3 to 13 residues second non-structured region,

a second fragment of the P-Domain having an N-terminus and a C-terminus and comprising residues configured to form, in a folded encapsulin protein, a secondary structure comprising a direction N-terminus to C-terminus a 9 to 15 residues beta strand β4, linked to a 6 to 15 residues alpha helix α3, linked to an optional first non-structured region having 0 to 10 residues, a 18 to 29 residues alpha helix α4, and a 9 to 21 residues second non-structured region, and

a third fragment of the P-domain having an N-terminus and a C-terminus and comprising residues configured to form, in a folded microcompartment protein, a secondary structure comprising in a direction N-terminus to C-terminus a 4 to 10 residues beta strand β9, linked to a 3 to 16 residues first non-structured region, linked to a 7 to 13 residues beta strand β10, linked to a 1 to 15 residues second non-structured region, linked to a 10 to 19 residues beta strand β11;

the E-Loop has an N-terminus and a C-terminus and comprises residues configured to form in a folded encapsulin protein, a secondary structure comprising in a direction N-terminus to C-terminus a 8 to 16 residues beta strand β2, linked to a 2 to 24 residues first non-structured region, linked to a 7 to 15 residues beta strand β3, linked to an optional second non-structured region having 0 to 6 residues; and

the A-Domain of the encapsulin protein has an N-terminus and a C-terminus and comprises residues configured to form in a folded encapsulin protein, a secondary structure comprising in a direction N-terminus to C-terminus an optional beta strand β5 having 0 to 8 residues, linked to a 1 to 15 residues first non-structured region, linked to a 16 to 23 residues alpha helix α5, linked to a 3 to 11 residues second non-structured region, linked to a 3 to 11 residues beta strand β6, linked to a 9 to 16 residues alpha helix α6, linked to a 1 to 24 third non-structured region, linked to an optional alpha helix α7 having 0 to 16 residues, linked to an optional fourth non-structured region having 0 to 8 residues, linked to a 1 to 10 residues beta strand β7, linked to a 1 to 12 residues fifth non-structured region, linked to a 3 to 10 residues beta strand β8, linked to a 2 to 12 residues sixth non-structured region;

and wherein the P-domain, A-domain and E-loop are arranged together in a configuration comprising, in a direction N-terminus to C-terminus, the first fragment of the P-domain linked to the E-loop linked to the second fragment of the P-domain linked to the A-domain linked to the third fragment of the P-domain;

the engineered microcompartment protein further comprising,

a target protein having an N-terminus, a C-terminus inserted at the N-terminus of the first fragment of the P-domain of the encapsulin protein alone or in combination with a tag and/or a linker;

at least one first protease cleavage site inserted between the C-terminus of the target protein and the N-terminus of the first fragment of the P-Domain of the encapsulin protein, alone or in combination with a tag and/or a linker; and

at least one second protease cleavage site inserted at an insertion site at the C terminus of the E-loop of the encapsulin protein, and/or within 1 to 17 amino acids adjacent to the C-terminal amino acid of the E-loop of the encapsulin protein and/or within 2-14 amino acids adjacent to the N-terminal amino acid of the A-domain of the encapsulin protein, alone or in combination with a tag and/or a linker to enable digestion of the encapsulin and release of the target protein.

2. The engineered microcompartment protein of claim 1 , wherein the target protein comprises at least one non-native antimicrobial peptide.

3. The engineered microcompartment protein of claim 2 , wherein the at least one non-native antimicrobial peptide is selected from Apidaecin la, HBCM-2, cecropins, magainins, melittin, protegrins, and nisins.

4. The engineered microcompartment protein of claim 1 , wherein the target protein has a sequence up to 80 amino acids in length.

5. The engineered microcompartment protein of claim 1 , wherein the at least one second protease cleavage site is inserted within 1 to 17 amino acids adjacent to the C-terminal amino acid of the E-loop of the encapsulin protein and within the first non-structured region of the E-loop to provide a cage-forming engineered microcompartment protein.

6. The engineered microcompartment protein of claim 5 , wherein the at least one second protease cleavage site is inserted at positions 57 and/or 66 of SEQ ID NO: 1.

7. The engineered microcompartment protein of claim 6 , wherein the E-loop has sequence YAAHPLGEVEVLSDENEVVKWGLRKSLP (SEQ ID NO: 59), YTVVPEGRLKKIEDNPGNVCTGMYQVKP (SEQ ID NO: 60), YAAVNTGELRPIDDTPEDVDMKLRQVQP (SEQ ID NO: 61), YAAVNTGRRTALEDKAEGASIFQRQVLP (SEQ ID NO: 62), or FSALGTGHVSRVAADTPGVEALQRHVVR (SEQ ID NO: 63).

8. The engineered microcompartment protein of claim 1 , wherein the at least one second protease cleavage site is inserted within 1 to 17 amino acids adjacent to the C-terminal amino acid of the E-loop of the encapsulin protein and in the first non-structured region of the E-loop, and/or within 2-14 amino acids adjacent to the N-terminal amino acid of the A-domain of the encapsulin protein and in the β5 beta strand of the A-domain, and/or within 2-14 amino acids adjacent to the N-terminal amino acid of the A-domain of the encapsulin protein and in the alpha helix α5 of the A-Domain to provide a cage forming engineered microcompartment protein.

9. The engineered microcompartment protein of claim 8 , wherein the at least one second protease cleavage site is inserted at any one of positions 132 and 144 of SEQ ID NO: 1.

10. The engineered microcompartment protein of claim 8 , wherein the A-Domain has sequence LLSFEERKIECGSTPKDLLEAIVRALSIFSKDGIEGPYTLVINTDRWINFLKEEAGHYPLEKRV EECLRGGKIITTPRIEDALVVSER (SEQ ID NO: 64), LREGTSNPKLALPSSASDYPAAIAAALNQLRLAGVNGPYAVVLGAGVYTALSGGDDEGYP VFRHIESLIDGKIVWAPAIEGGFVLSTR (SEQ ID NO: 65) LLTEDGIVKFPISNWSEGENPFKDISIGLAKFIENG IVGRKALVVSPNLFVQLQRIQPGTGTT EYDRINKLLDGNIFSTPVLKDDKAVLVCSE (SEQ ID NO: 66), ILNAEGAQKLQISDWGQGENPYTDIVKAINMIREKGIVGRFVLCLSQSLYFDLQRIQQGTGM TEAQRISSMIGNLYNVPVIKGKKAALICAE (SEQ ID NO: 67), or LLTVKGSSKIKKSDWSQGENSFADITAGVAQLAKTGYLGRYALVVSPDLFLDLQRLQPNTG LLEIDRIKKLIGDNVYMTSVMGPGKAVLVCAE (SEQ ID NO: 68).

11. The engineered microcompartment protein of claim 1 , wherein the target protein is a protease sensitive target protein.

12. The engineered microcompartment protein of claim 1 , wherein the at least one second protease cleavage site is inserted within 1 to 17 amino acids adjacent to the C-terminal amino acid of the E-loop of the encapsulin protein and within the beta-strand β3 of the E-loop to provide a non-cage-forming engineered microcompartment protein.

13. The engineered microcompartment protein of claim 12 , wherein the encapsulin protein has SEQ ID NO: 47 and the at least one second protease cleavage site is inserted at position 71 of SEQ ID NO: 47.

14. The engineered microcompartment protein of claim 1 , wherein the target protein is a protease sensitive target protein and the engineered microcompartment protein further comprises a proline between the N-terminus of the first at least one protease cleavage site and the C terminus of the target protein.

15. The engineered microcompartment protein of claim 1 , wherein the at least one second protease cleavage site is inserted at any one of positions 57, 60, 71 and 139 of SEQ ID NO: 1.

16. The engineered microcompartment protein of claim 1 , wherein the at least one encapsulin protein has a sequence of SEQ ID NO: 47.

17. The engineered microcompartment protein of claim 1 , wherein the at least one encapsulin protein is from the PF04454 protein family.

18. The engineered microcompartment protein of claim 1 , wherein the at least one first protease cleavage site and/or the at least one second protease cleavage site are selected from ENLYFQ\S(orG) (SEQ ID NO: 70) (TEV protease recognition), LEVLFQ/GP (SEQ ID NO: 13) (HRV 3C protease recognition), LVPR/GS (SEQ ID NO: 17) (thrombin recognition), DDDDK/ (SEQ ID NO: 14) (enterokinase recognition), and IEGR/ (SEQ ID NO: 15) (Factor Xa recognition).

19. The engineered microcompartment protein of claim 1 , wherein the target protein is fused to the N-terminus of the first segment of the P-domain of the encapsulin protein in combination with a linker and/or a tag.

20. The engineered microcompartment protein of claim 1 , wherein the at least one first protease cleavage site is fused to the target protein and the N-terminus of the first fragment of the P-Domain of the encapsulin protein together with a linker and/or a tag.

21. The engineered microcompartment protein of claim 1 , wherein the at least one second protease cleavage site is fused to the C-terminus of the E-loop and/or to the N-terminus of the A-domain of the encapsulin protein together with a linker and/or a tag.

22. The engineered microcompartment protein of claim 1 , wherein the tag is selected from His-Tag, Strep-Tag, FLAG-Tag, Avi-Tag, E-Tag, HA-Tag, Myc-Tag, and TC-Tag.

23. An engineered microcompartment comprising at least one engineered microcompartment protein of claim 1 .

24. A method to produce in a bacterial cell a protein non-native to the bacterial cell, the method comprising

introducing into the bacterial cell at least one first polynucleotide encoding at least one engineered microcompartment protein of claim 1 in which the target protein is the protein non-native to the bacterial cell;

wherein the at least one first polynucleotide is operatively linked to one or more first regulatory elements leading to the expression of the at least one engineered microcompartment protein in the bacterial cell; and

wherein the introducing is performed to obtain expression in the bacterial cell of the at least one engineered microcompartment protein to obtain the protein non-native to the bacterial cell within at least one engineered microcompartment formed by the at least one engineered microcompartment protein.

25. The method of claim 24 , wherein the at least one second protease cleavage site of the at least one engineered microcompartment protein is inserted within 1 to 17 amino acids adjacent to the C-terminal amino acid of the E-loop of the encapsulin protein and within the first non-structured region of the E-loop to provide a cage forming engineered microcompartment protein.

26. The method of claim 24 , wherein the at least one second protease cleavage site the at least one engineered microcompartment protein is inserted within 1 to 17 amino acids adjacent to the C-terminal amino acid of the E-loop of the encapsulin protein and within the beta-strand β3 of the E-loop to provide a non-cage forming engineered microcompartment protein.

27. The method of claim 24 , wherein the protein non-native to the bacterial cell is a toxic non-native protein capable of causing a cell damage.

28. The method of claim 24 , wherein the protein non-native to the bacterial cell is a non-native protein capable of being degraded within the bacterial cell.

29. A system to produce, in bacterial cell, a protein non-native to the bacterial cell, the system comprising

at least one first polynucleotide encoding at least one engineered microcompartment protein of claim 1 , wherein the target protein is the protein non-native to the bacterial cell, the at least one engineered microcompartment protein operatively linked to one or more first regulatory elements configured to enable the expression of the at least one engineered microcompartment protein in one or more bacterial cell, the at least one engineered microcompartment protein capable of assembling with one or more same and/or different engineered microcompartment proteins to form at least one microcompartment within the one or more bacterial cell,

the system further comprising at least one of:

the one or more bacterial cells capable of expressing the at least one first polynucleotide to provide an expressed engineered microcompartment protein;

at least one second polynucleotide encoding for at least one protease, the at least one second polynucleotide operably linked to one or more second regulatory elements leading to the expression of the at least one protease capable of targeting the protease cleavage sites of the engineered microcompartment protein to release the non-native protein from the engineered microcompartment protein in the bacterial cell to obtain the non-native protein; and

at least one protease capable of targeting the protease cleavage site of the engineered microcompartment protein to release the protein non-native to the bacterial cell from the engineered microcompartment protein in the bacterial cell.

30. A vector comprising at least one polynucleotide encoding for an engineered microcompartment protein of claim 1 , alone or in combination with regulatory elements in accordance with the disclosure.

31. A bacterial cell comprising at least one engineered microcompartment of claim 23 .