Caspase-2 variants

1 . A single-chain circular permuted caspase-2 (cp caspase-2) comprising the following structure from N- to C-terminus:

i. a small subunit of a caspase-2; and

ii. a large subunit of a caspase-2,

wherein said cp caspase-2 comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 6, and

wherein said cp caspase-2 comprises one or more amino acid substitutions increasing P1′ tolerance of said cp caspase-2 compared to a cp caspase-2 without said amino acid substitutions.

2 . The cp caspase-2 of claim 1 comprising:

(A)

a) one or more amino acid substitutions at positions 171, 105, 172, 282, 225, 83, 185, 255, or 285 of SEQ ID NO: 6 or any combination thereof;

b) one or more amino acid substitutions, selected from

i. Gly171, substituted with D, or an amino acid selected from the group consisting of R, K, E, Q, N, A, S, T, P, H, and Y,

ii. Glu105, substituted with V, or an amino acid selected from the group consisting of C, L, I, M, F, W, R, K, D, Q, and N,

iii. Glu172, substituted with V, or an amino acid selected from the group consisting of C, L, I, M, F, W, R, K, D, Q, and N,

iv. Asp282, substituted with E, or T, or an amino acid selected from the group consisting of R, K, Q, N, G, A, S, P, H, and Y,

v. Val225, substituted with G, or an amino acid selected from the group consisting of A, S, T, P, H, Y, C, L, I, M, F, and W,

vi. Lys83, substituted with E, or an amino acid selected from the group consisting of R, D, Q, and N,

vii. His185, substituted with A, or an amino acid selected from the group consisting of G, S, T, P, and Y,

viii. Val255, substituted with M, or an amino acid selected from the group consisting of C, L, I, F, and W, and/or

ix. Asp285, substituted with E, or Y, or an amino acid selected from the group consisting of R, K, Q, N, G, A, S, T, P, and H,

with reference to the positions of SEQ ID NO: 6;

c) amino acid substitutions at positions of SEQ ID NO: 6 selected from the group consisting of

i. His185 and Asp282;

ii. Glu105 and Asp285;

iii. Val225 and Asp282;

iv. Val225, Asp282 and Asp285;

v. Lys83, Glu105,Glu172, Val255 and Asp285;

vi. Glu105 and Gly171;

vii. Glu105 and Glu172; and

viii. Gly171 and Glu172,

wherein said cp caspase-2 has increased P1′ tolerance compared to a cp caspase-2 without the respective amino acid substitution;

d) amino acid substitutions at positions of SEQ ID NO: 6 selected from the group consisting of

i. H185A and D282T substitutions;

ii. E105V and D285E substitutions;

iii. E105V, G171D, V225G and D282E substitutions;

iv. E105V, G171D, V225G, D282E and D285E substitutions;

v. K83E, E105V, E172V, V255M and D285Y substitutions;

vi. E105V and G171D substitutions;

vii. E105V and E172V substitutions; and

viii. G171 D and E172V substitutions,

wherein said cp caspase-2 has increased P1′ tolerance compared to a cp caspase-2 without the respective amino acid substitution; or

e) any one or more of amino acid substitutions at positions of SEQ ID NO: 6 selected from the group consisting of G171D, E105V, E172V, D282E, D282T, V225G, K83E, H185A, V255M, D285Y and D285E; or

(B) an amino acid sequence selected from the group consisting of SEQ ID NO: 1, 13, 17, 18, 23, 24, 51, 52, 54, 70, 71, 72, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191 and 192 or an amino acid sequence having at least 99% sequence identity with any one of SEQ ID NO: 1, 13, 17, 18, 23, 24, 51, 52, 54, 70, 71, 72, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191 and 192; and

wherein said amino acid substitution increases P1′ tolerance compared to a caspase-2 comprising the same sequence but not comprising said amino acid substitutions.

3 . The cp caspase-2 of claim 1 , further comprising:

(A) a C-terminal tag and an amino acid substitution at positions 285 and 292 of SEQ ID NO: 6;

(B) a C-terminal tag and an amino acid substitutions D285E and D292S of SEQ ID NO: 6; and/or

(C) (i) an N-terminal and/or C-terminal truncation; and/or (ii) an N-terminal and/or C-terminal extension.

4 . The cp caspase-2 of claim 3 , wherein the cp caspase 2 further comprises one or more linker sequences and at least one of the following conditions is met:

(A) the linker sequence:

i. consists of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 amino acid residues;

ii. comprises glycine and/or serine residues; and/or

iii. is GS, GGSGG (SEQ ID NO:278), GSAGSAAGSG (SEQ ID NO:279), (GS)n, GSG or G4S;

(B) the linker sequence is a subunit-linker sequence between the small subunit and the large subunit;

(C) the linker sequence is a tag-linker sequence, linking two tags or linking a tag and the small subunit, the large subunit or the small subunit propeptide of the cp caspase-2.

5 . The cp caspase-2 of claim 1 , wherein the cp caspase 2 further comprises one or more C-terminal or N-terminal tags and at least one of the following conditions is met:

(A) the one or more C-terminal or N-terminal tags are selected from the group consisting of affinity tags, solubility enhancement tags and monitoring tags, wherein:

i. the affinity tag is selected from the group consisting of poly-histidine tag, poly-arginine tag, peptide substrate for antibodies, chitin binding domain, RNAse S peptide, protein A, β-galactosidase, FLAG tag, Strep II tag, streptavidin-binding peptide (SBP) tag, calmodulin-binding peptide (CBP), glutathione S-transferase (GST), maltose-binding protein (MBP), S-tag, HA tag, c-Myc tag, SUMO tag, E. coli thioredoxin, NusA, chitin binding domain CBD, chloramphenicol acetyl transferase CAT, LysRS, ubiquitin, calmodulin, and lambda gpV; and/or

ii. the solubility enhancement tag is selected from the group consisting of T7C, T7B, T7B1, T7B2, T7B3, T7B4, T7B5, T7B6, T7B7, T7B8, T7B9, T7B10, T7B11, T7B12, T7B13, T7A, T7A1, T7A2, T7A3, T7A4, T7A5, T3, N1, N2, N3, N4, N5, N6, N7, T7AC, calmodulin-binding peptide (CBP), DsbA, DsbC, poly Arg, poly Lys, G B1 domain, protein D, Z domain of Staphylococcal protein A, and thioredoxin; and/or

iii. the monitoring tag is selected from the group consisting of m-Cherry, GFP and f-Actin;

(B) the cp caspase-2 further comprises more than one tag;

(C) the cp caspase-2 further comprises an affinity tag and a solubility enhancement tag;

(D) the cp caspase-2 further comprises an affinity tag and a solubility enhancement tag, wherein the affinity tag is a hexahistidine tag and the solubility enhancement tag is a T7AC or a T7A3 tag;

(E) the cp caspase 2 further comprises one or more N-terminal tags and optionally one or more tag-linker sequences between the tags or between a tag and the N-terminus of the small subunit or between a tag and a propeptide of a small caspase-2 subunit fused to the N-terminus of the small subunit;

(F) the cp caspase 2 further comprises one or more C-terminal tags and optionally one or more tag-linker sequences between the tags or between a tag and the C-terminus of the large subunit.

6 . A functionally active variant of the cp caspase-2 of claim 1 , wherein

(A) i. the small subunit of the cp caspase-2 comprises

a) a first conserved region of the active center with at least 37.5% amino acid sequence identity to SEQ ID No. 177 (1st consensus: AAMRNTKR) or 100% sequence identity to XXXRNTXX (SEQ ID No. 200), wherein X is any amino acid,

b) a second conserved region of the active center with at least 61.5% amino acid sequence identity to SEQ ID No. 178 (2nd consensus: EGYAPGTEFHRCK) or 100% sequence identity to EGXXPGXXXHRCK (SEQ ID No. 194), wherein X is any amino acid, and

ii. the large subunit of the cp caspase-2 comprises

a) a third conserved region of the active center with at least 25.0% amino acid sequence identity to SEQ ID No. 174 (3rd consensus: G-EKDLEFRSGGDVDH) or 100% sequence identity to X-XXXLXXRXGXXXDX (SEQ ID No. 195), wherein X is any amino acid,

b) a fourth conserved region of the active center with at least 53.3% amino acid sequence identity to SEQ ID No. 175 (4th consensus: LLSHGVEGGXYGVDG) or 100% sequence identity to XXSHGXXGXXYGXDG (SEQ ID No. 196), wherein X is any amino acid, and

c) a fifth conserved region of the active center with at least 50.0% amino acid sequence identity to SEQ ID No. 176 (5th consensus: QACRGDET) or 100% sequence identity to QACXGXXX (SEQ ID No. 197), wherein X is any amino acid; or

(B) the functionally active variant comprises at least 91, 92, 93, 94, 95, 96, 97 or 98% sequence identity to the cp caspase-2 as set forth in SEQ ID NO: 6; or

(C) the functionally active variant comprises at least 91, 92, 93, 94, 95, 96, 97 or 98% sequence identity to SEQ ID NO: 9, 6, 14, 15, 16, 80, 88, 25, 26, 27, 28, 29, 30, 35, 39, 41, 73, 74, 75, 76, 77, 81, 82 or 83.

7 . The cp caspase-2 of claim 1 , wherein:

i. the small subunit is selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 91, SEQ ID NO: 94, SEQ ID NO: 97, SEQ ID NO: 100, SEQ ID NO: 103, SEQ ID NO: 106, SEQ ID NO: 109, SEQ ID NO: 115 and SEQ ID NO: 118, or

ii. the large subunit is selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 90, SEQ ID NO: 93 and SEQ ID NO: 96.

8 . The cp caspase-2 of claim 1 , wherein said cp caspase-2 is recruited by a recognition site for proteolytic cleavage, comprising 5 amino acids of the sequence PS P4 P3 P2 P1, wherein

P1 can be any amino acid, optionally it is D or E,

P2 can be any amino acid, optionally it is A,

P3 can be any amino acid, optionally it is V,

P4 can be any amino acid, optionally it is D, and

P5 can be any amino acid, optionally it is V.

9 . An isolated nucleotide sequence encoding the cp caspase-2 of claim 1 .

10 . A vector comprising the nucleotide sequence of claim 9 or an expression cassette comprising the nucleotide sequence of claim 9 operably linked to regulatory elements.

11 . A host cell or a host cell line expressing the cp caspase-2 of claim 1 , wherein the host cells are selected from the group consisting of bacterial cells, yeast cells, insect cells, mammalian cells and plant cells.

12 . An expression system comprising (i) an expression vector, said vector comprising an isolated nucleotide sequence encoding the cp caspase-2 of claim 1 , or an expression cassette, said expression cassette comprising an isolated nucleotide sequence encoding the cp caspase-2 of claim 1 operably linked to regulatory elements, and (ii) a host cell expressing the cp caspase-2 of claim 1 , wherein the host cell is selected from the group consisting of bacterial cells, yeast cells, insect cells, mammalian cells and plant cells.

13 . A kit comprising the cp caspase-2 of claim 1 and an expression vector, said expression vector comprising a polynucleotide encoding a protein tag for enhanced expression of a protein of interest, wherein the protein tag comprises a solubility enhancement tag and the amino acid sequence VDVAD (SEQ ID NO:45), and wherein the sequence VDVAD is located at the C-terminus of the protein tag.