Caspase-2 variants

Abstract

To provide a new modified caspase-2.SOLUTION: The problem is solved by a single-chain circularly permuted caspase-2 comprising the following structures from N - to C-terminus: I) the small subunit of caspase-2, or a functionally active variant thereof; and ii) the large subunit of caspase-2, or a functionally active variant thereof, wherein the cp-caspase-2 comprises one or more amino acid substitutions that increase the P1 ' - tolerance of said cp-caspase-2 compared to a cp-caspase-2 without amino acid substitutions.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates generally to the fields of molecular biology, biotechnology, or bioprocess engineering for the production and use of modified caspase-2, specifically circularly permuted caspase-2. The present invention further relates specifically to the production and isolation of recombinant protein constructs that use modified caspase-2 for the maturation of recombinant fusion proteins or polypeptides containing caspase recognition sites. [Background technology]

[0002] Despite all the recent advances in biotechnology, protein production remains challenging due to their diverse characteristics: protocols must usually be optimized for each protein, which is particularly problematic for large-scale production. The diverse characteristics of proteins make their purification challenging and prevent general protocols. For this reason, proteins are often fused to tags with specific binding properties. The first human proteins recombinantly expressed in Escherichia coli (E. coli), somatostatin [1] and insulin [2], were fusion proteins. Even today, protein tags are still widely used in recombinant protein production, not only to facilitate purification and detection but also to enhance expression and solubility. Popular tags that stabilize expression and increase solubility include GST (glutathione S-transferase), MBP (maltose-binding protein), SUMO (small ubiquitin-related modifier), or DsbA (protein disulfide isomerase I). Tags for affinity purification include His, HA (hemaglutinin antigen), Strep II, and FLAG tags, among others.

[0003] However, while tags are versatile and useful, their removal can be difficult. Many applications, particularly medical applications, require untagged proteins. Tags affect the structure and characteristics of proteins and thus alter the response to immunogens or may themselves initiate an immune response. [3] Especially in biopharmaceutical applications, proteases that efficiently cleave tags from products are essential. [4] A variety of proteases are available for tag removal. All of them cleave a defined recognition sequence inserted between the tag and the target protein. The protease itself usually carries the tag, which is subsequently removed in a second purification step. The most commonly used are endopeptidases such as factor Xa, thrombin, TEV (tobacco etch virus protease), and enterokinase. However, all of these proteases have one or several drawbacks: they cleave inefficiently or nonspecifically, do not accept all residues in the P1' position, leave overhanging residues at the N-terminus, or require specialized buffer conditions that are unfavorable for the target protein. Another major drawback in industrial applications is the high cost of these proteases [5]. Despite the fact that caspases have been studied more intensively than other protease classes and have very high specificity, they have rarely been explored for biotechnological purposes such as tag cleavage.

[0004] Caspase is an acronym for cysteinyl aspartate-specific proteases, a class of proteases defined by a conserved catalytic cysteine ​​and their strong preference for cleaving their substrates after aspartate residues [6]. The first caspases were described in the early 1990s, and since then, a total of 15 species have been discovered in mammals, 13 of which are found in humans [7]. They are well known for their roles in regulated cell death [8] and inflammatory responses [9]. More recently, they have been found to be involved in other processes, such as cell differentiation

[10] , cell cycle regulation

[11] , and even cell motility

[12] . MacKenzie and Clark investigated the role of dimerization in the ability of caspases to form fully functional proteases and noted that dimerization is required for active site formation because both caspase monomers contribute residues that allow for the formation of a fully functional active site (MacKenzie and Clark, Adv Exp Med Biol. (2012); 747:55-73).

[0005] Recently, reverse caspases have been developed in which the small subunit of caspase is located N-terminally of the large subunit of caspase (US Pat. No. 6,379,950). For example, Srinivasa et al. have described recombinant caspase 3 and 6 precursors that are constitutively active and whose small subunit precedes its large subunit (Srinivasa et al. Journal of Biological Chemistry, American Society for Biochemistry and Molecular Biology (1998), 273(17):10107-10111). Although circular permutation may offer potential benefits by rearranging the polypeptide chain of a protein, by connecting native protein termini through covalent linkers and introducing new termini through cleavage of existing peptide bonds, circular permutation may also disrupt local tertiary structure and protein dynamics, as well as introduce possible quaternary structural changes and problems (Yu and Lutz, Trends in Biotechnology (2011), 29(1):18-25).

[0006] WO2009 / 044988 describes a method for producing caspases using recombinant caspase expression vectors that can be overexpressed without cytotoxicity because the caspase autoactivation recognition site is replaced with a non-cysteine ​​protease recognition site to abolish the autoactivation activity during overexpression in E. coli. Three systems using caspases for tag removal have been published, which are difficult to compare because they use different fusion proteins, buffers, substrate-to-enzyme ratios, and incubation temperatures. Caspase-3 and engineered caspase-3 with a noncleavable propeptide (but with the wild-type order of subunits) have been used to cleave GST tags from fusion proteins. The engineered caspase was able to achieve complete tag cleavage in approximately 3 hours at 25°C (a caspase-to-substrate molar ratio of 1:80, a caspase-to-substrate mass ratio of 1:100)

[13] . In another system that also used caspase-3 to cleave the GST tag (a caspase-to-substrate mass ratio of 1:200), the process was more than 90% complete in 45 minutes, but the incubation time was 30°C

[14] .

[0007] A caspase-6-based system has also been published, which is more efficient and achieves complete substrate cleavage in approximately 30 minutes (caspase-to-substrate molar ratio of 1:500)

[15] . Although these caspase-based tag cleavage systems were published over a decade ago, they have not yet been adopted into the general repertoire for protein purification. The use of a caspase-3-based system has been published only once, for the expression of interleukins

[16] . The caspase-6 system has also only been used by two other groups, both at the Indian Institute of Immunology, for the purification of Mycobacterium tuberculosis

[17] and Helicobacter pylori proteins

[18] .

[0008] EP1597369 discloses, for example, a method for producing proteins using a fusion protein comprising a protein of interest and a protease recognition site, and cleaving the fusion protein at the recognition site using a protease such as a caspase. US7604980 also uses a fusion protein comprising the protein of interest and a caspase recognition site to produce the protein of interest, and discloses that caspase-6 is preferably used to cleave the fusion protein.

[0009] A key reason caspases have not become more popular in biotechnology may be the challenges they pose during recombinant production. Because native caspases are synthesized as inactive zymogens, there are two main possibilities for obtaining active enzymes. The subunits can be expressed separately and then mixed after purification, greatly complicating their production

[19] . Alternatively, procaspases can be expressed, which triggers autocatalytic activation. However, this process is often incomplete

[20] , and thus, enzyme activity can vary between batches

[21] . Furthermore, because caspases are active in E. coli, they may also cleave bacterial proteins

[22] , negatively impacting growth and yield. Furthermore, the substrate specificity of both caspase-3

[23] and caspase-6

[24] has been described as somewhat promiscuous. They are highly likely to cleave fusion proteins at undesired sites. Therefore, there is a pressing need for an industrially applicable platform technology that enables efficient and specific tag removal to improve the purification of recombinant protein products. Summary of the Invention

[0010] In recombinant protein production, treatment of fusion proteins with state-of-the-art enzymes to remove tags often results in non-authentic N- or C-termini due to the lack of specificity of these enzymes, which can also result in non-specific cleavage or proteolysis of the protein of interest. It is an object of the present invention to provide an improved system for the production of recombinant proteins using modified caspase-2, particularly circularly permuted caspase-2. This object is solved by the present invention. Specifically, provided herein are modified caspase-2s, specifically circularly permuted caspase-2s, that have significantly improved P1' tolerance. The caspase-2 variants provided herein are particularly useful in recombinant protein production, for generating proteins of interest that contain authentic N-termini by targeted cleavage of the N-terminal tag.

[0011] According to the present invention, the amino acid sequence of the amino acid sequence of the present invention is as follows: i. the small subunit of caspase-2, or a functionally active variant thereof, and ii. The large subunit of caspase-2 or a functionally active variant thereof A single-chain circularly permuted caspase-2 (cp caspase-2) comprising: Provided is a cp caspase-2 comprising one or more amino acid substitutions that increase the P1' tolerance of said cp caspase-2 compared to cp caspase-2 without the amino acid substitutions. Specifically, the cp caspase-2 provided herein is catalytically active, particularly when dimerized. Specifically, the cp caspase-2 described herein is catalytically active and can catalyze peptide bond cleavage when dimerized. Specifically, the cp caspase-2 described herein is a single-chain caspase-2 that does not require cleavage by an initiator caspase for activation. Specifically, the caspase-2 or cp caspase-2 provided herein is a functionally active variant of wild-type caspase-2 that contains improved P1' tolerance and is capable of cleaving substrates with high efficiency and specificity.

[0012] Specifically, as used herein, the following structure from N to C terminus: i. the small subunit of caspase-2 comprising SEQ ID NO:3, or a functionally active variant thereof comprising 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:112, SEQ ID NO:115, or SEQ ID NO:118, and optionally comprising up to 8, 9, or 10 amino acid substitutions, insertions, and / or deletions; and ii. The large subunit of caspase-2 comprising SEQ ID NO:4, or a functionally active variant thereof comprising SEQ ID NO:90, SEQ ID NO:93, SEQ ID NO:96, SEQ ID NO:99, SEQ ID NO:102, SEQ ID NO:105, SEQ ID NO:108, SEQ ID NO:111, SEQ ID NO:114, or SEQ ID NO:117, and optionally comprising up to 8, 9, or 10 amino acid substitutions, insertions, and / or deletions. and comprising one or more amino acid substitutions that increase the proteolytic activity of the single-chain caspase-2 compared to a caspase-2 comprising the same sequence as the single-chain caspase-2 but without the amino acid substitutions. The single-chain caspase-2 may also comprise one or more additional amino acid substitutions, insertions, or deletions.

[0013] In particular, said variant is of animal origin, in particular of mammalian, reptilian or fish origin, more in particular of human, marsupial, iguana or cartilaginous fish, chimaera or Tasmanian devil origin. According to certain embodiments, the cp caspase-2 provided herein comprises one or more amino acid substitutions at positions 171, 105, 172, 282, 225, 83, 185, 255, or 285 of SEQ ID NO:6, or at positions functionally equivalent to any of positions 171, 105, 172, 282, 225, 83, 185, 255, or 285 of SEQ ID NO:6, or any combination thereof. Specifically, cp caspase-2 comprising one or more of the amino acid substitutions, also referred to as "cp caspase-2 variants," comprises improved P1' tolerance compared to cp caspase-2 not comprising the substitutions. Specifically, the cp caspase-2 variants provided herein comprise improved P1' tolerance for at least one amino acid other than glycine.

[0014] According to further specific embodiments, the cp caspase-2 provided herein comprises a propeptide of the small caspase-2 subunit (SS propeptide) fused to the N-terminus of the small subunit. Specifically, the SS propeptide comprises one or more amino acid substitutions at the C-terminus of the SS propeptide. Specifically, the SS propeptide of the cp caspase-2 described herein is modified to prevent cleavage at its C-terminus. Specifically, the SS propeptide is substituted at position Asp of SEQ ID NO:2. 14 or Asp of SEQ ID NO: 11 347 It contains an amino acid substitution at a functionally equivalent position, specifically, Asp is substituted with Ala.

[0015] According to certain embodiments, the SS propeptide described herein comprises the amino acid sequence of SEQ ID NO:2, wherein X can be any amino acid other than D or E, and in particular, X is A or a variant thereof having 1, 2, 3, 4, or 5 point mutations or deletions. In particular, said variant is a functionally active variant. Preferably, the SS propeptide sequence comprises the amino acid sequence of SEQ ID NO:2, wherein X is not D or E.

[0016] According to further specific embodiments, the cp caspase-2 provided herein comprises one or more linker sequences, specifically, the linker sequence consists of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 or even more amino acid residues. Specifically, the linker can comprise more than 20 or 30 amino acids, or even more, as long as the caspase retains its functional activity as described herein. Specifically, the linker sequence comprises glycine, alanine, and / or serine residues. Specifically, the linker comprises at least one glycine and serine residue, more specifically, the linker is GS, GSG, GGSGG, GSGSGSGS, and / or GSAGSAAGSG.

[0017] Specifically, cp caspase-2 comprises a subunit linker sequence, which is the linker sequence between the cp caspase-2 small and large subunits described herein. According to further particular embodiments, the cp caspase-2 provided herein comprises one or more C- or N-terminal tags, in particular tags selected from the group consisting of affinity tags, solubility-enhancing tags, and monitoring tags. In particular, any tag known in the art can be fused to the cp caspase-2.

[0018] Specifically, the affinity tag is selected from the group consisting of a polyhistidine tag, a polyarginine tag, a peptide substrate for an antibody, a chitin-binding domain, RNAse S peptide, protein A, β-galactosidase, a FLAG tag, a Strep II tag, a streptavidin-binding peptide (SBP) tag, a calmodulin-binding peptide (CBP), glutathione S-transferase (GST), maltose-binding protein (MBP), an S-tag, an HA tag, a c-Myc tag, a SUMO tag, Escherichia coli thioredoxin, NusA, a chitin-binding domain CBD, a chloramphenicol acetyltransferase CAT, LysRS, ubiquitin, calmodulin, and lambda gpV; specifically, the tag is a His tag comprising one or more His, more specifically a hexahistidine tag.

[0019] Specifically, the solubility-enhancing tag is selected from the group consisting of T7C, T7B, T7B1, T7B2, T7B3, T7B3, T7B4, T7B5, T7B6, T7B6, T7B7, T7B8, T7B9, T7B10, T7B11, T7B12, T7B13, T7A, T7A1, T7A2, T7A3, T7A4, T7A5, T7AC, T3, N1, N2, N3, N4, N5, N6, N7, calmodulin-binding peptide (CBP), poly-Arg, poly-Lys, G B1 domain, protein D, the Z domain of staphylococcal protein A, DsbA, DsbC, and thioredoxin. Preferably, the solubility-enhancing tag is selected from the group consisting of a T7A3 tag and a T7AC tag.

[0020] Specifically, the monitoring tag is selected from the group consisting of m-Cherry, GFP, and f-actin. According to certain embodiments, the cp caspase-2 described herein comprises multiple tag sequences, in particular affinity tags and solubility-enhancing tags. Specifically, the cp caspase-2 described herein comprises an affinity tag, a solubility-enhancing tag, and a monitoring tag. Specifically, the cp caspase-2 described herein comprises multiple tags of the same functionality, in particular multiple affinity tags, multiple solubility-enhancing tags, and / or multiple monitoring tags, and any combination thereof. Specifically, the cp caspase-2 described herein comprises a C-terminal and an N-terminal tag, each of which preferably comprises one or more tag sequences selected from affinity tags, solubility-enhancing tags, and monitoring tags. Specifically, the affinity tag is a hexahistidine tag and the solubility-enhancing tag is a T7AC tag.

[0021] According to further specific embodiments, the cp caspase-2 provided herein comprises a tag-linker sequence, which is a linker sequence between two tags or between a tag and the small subunit, large subunit, or SS propeptide of cp caspase-2. Specifically, the cp caspase-2 provided herein comprises one or more N-terminal tags and may comprise one or more tag-linker sequences between the tags or between the tag and the N-terminus of the small subunit or SS propeptide. Specifically, the cp caspase-2 provided herein comprises one or more C-terminal tags and may comprise one or more tag-linker sequences between the tags or between the tag and the C-terminus of the large subunit.

[0022] According to further embodiments, herein there is provided i. The small subunit of caspase-2 a) a first conserved region of the active center having at least 37.5% amino acid sequence identity with SEQ ID NO: 177 (first consensus: AAMRN TKR) or 100% sequence identity with XXXRNTXX (SEQ ID NO: 200), where X is any amino acid; and b) a second conserved region of the active center having at least 61.5% amino acid sequence identity with SEQ ID NO: 178 (second consensus: EGYAPGTEFHRCK) or 100% sequence identity with EGXXPGXXXHRCK (SEQ ID NO: 194), where X is any amino acid; Including, ii. The large subunit of caspase-2 is a) a third conserved region of the active center having at least 25.0% amino acid sequence identity with SEQ ID NO: 174 (third consensus: G-EKDLEFRSGGDVDH) or 100% sequence identity with X-XXXLXXRXGXXXDX (SEQ ID NO: 195), where X is any amino acid; b) a fourth conserved region of the active center having at least 53.3% amino acid sequence identity with SEQ ID NO: 175 (fourth consensus: LLSHGVEGGXYGVDG) or 100% sequence identity with XSSHGXXGXXYGXDG (SEQ ID NO: 196), where X is any amino acid; and c) a fifth conserved region of the active center having at least 50.0% amino acid sequence identity with SEQ ID NO: 176 (fifth consensus: QACRGDET) or 100% sequence identity with QACXGXXX (SEQ ID NO: 197), where X is any amino acid; The present invention provides a functionally active variant of cp caspase-2 or caspase-2, comprising:

[0023] According to certain embodiments, the cp caspase-2 provided herein comprises at least 1, 2, 3, 4, 5, and up to 10 or even more N- and / or C-terminal truncations, so long as the caspase retains its functional activity as described herein. According to further certain embodiments, the cp caspase-2 provided herein comprises at least 1, 2, 3, 4, 5, and up to 10 or even more N- and / or C-terminal extensions, so long as the caspase retains its functional activity as described herein. Specifically, the cp caspase-2 may comprise truncations and extensions.

[0024] Specifically, the small subunit of cp caspase-2 described herein comprises the amino acid sequence 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:112, SEQ ID NO:115, SEQ ID NO:118, or a functionally active variant thereof comprising at least 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, or 98% sequence identity. Specifically, the large subunit of cp caspase-2 described herein comprises the amino acid sequence of SEQ ID NO:4, SEQ ID NO:90, SEQ ID NO:93, SEQ ID NO:96, SEQ ID NO:99, SEQ ID NO:102, SEQ ID NO:105, SEQ ID NO:108, SEQ ID NO:111, SEQ ID NO:114, SEQ ID NO:117, or a functionally active variant thereof comprising at least 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, or 98% sequence identity.

[0025] According to certain embodiments, the cp caspase-2 variants provided herein comprise: Based on the position of SEQ ID NO:6 or a position functionally equivalent to the position of SEQ ID NO:6, iD, or Gly substituted with an amino acid selected from the group consisting of R, K, E, Q, N, A, S, T, P, H, and Y 171 , ii. Glu substituted with an amino acid selected from the group consisting of V, C, L, I, M, F, W, R, K, D, Q, and N 105 , iii. Glu substituted with an amino acid selected from the group consisting of V, C, L, I, M, F, W, R, K, D, Q, and N 172 , iv. Asp substituted with E or T, or an amino acid selected from the group consisting of R, K, Q, N, G, A, S, P, H, Y. 282 , vG, or Val substituted with an amino acid selected from the group consisting of A, S, T, P, H, Y, C, L, I, M, F, and W 225 , vi. Lys substituted with an amino acid selected from the group consisting of E, R, D, Q, and N 83 , vii. His substituted with an amino acid selected from the group consisting of A, G, S, T, P, and Y 185 , viii. Val substituted with an amino acid selected from the group consisting of M, C, L, I, F, and W 255 and / or ix. Asp 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 285 , The amino acid sequence of the present invention may contain one or more amino acid substitutions selected from the group consisting of:

[0026] Specifically, the selection of alternative amino acid exchanges at a given position that have a high potential for producing effects similar to the selected variants described is based on classifying all amino acids into distinct, non-overlapping groups according to their hydrophobicity characteristics, i.e., polar (R, K, E, D, Q, N), neutral (G, A, S, T, P, H, Y), and hydrophobic (C, V, L, I, M, F, W), as determined by Stapor et al. (Stapor K, et al. Machine Learning Paradigms - Advances in Data Analytics. Tsihrintzis GA, Sotiropoulos DN and Jain LC (eds.), Springer 2019 (ISSN 1868-4394), pp 101-128).

[0027] Specifically, the cp caspase-2 provided herein is i.His 185 and Asp 282 , specifically those containing the substitutions H185A and D282T; ii.Glu 105 and Asp 285 , specifically including the replacement of E105V and D285E; iii.Glu 105 , Gly 171, Val 225 , and Asp 282 , specifically those containing the substitutions E105V, G171D, V225G, and D282E; iv.Glu 105 , Gly 171 , Val 225 , Asp 282 , and Asp 285 , specifically those containing the substitutions E105V, G171D, V225G, D282E, and D285E; v.Lys 83 , Glu 105 , Glu 172 , Val 255 , and Asp 285 , specifically those containing the substitutions K83E, E105V, E172V, V255M, and D285Y; vi.Glu 105 and Gly 171 , specifically those containing E105V and G171D substitutions; vii.Glu 105 and Glu 172 , specifically including replacements for E105V and E172V; and viii.Gly 171 and Glu 172 , specifically those containing the G171D and E172V substitutions and comprising an amino acid substitution at a position in SEQ ID NO: 6 or a functionally equivalent position to a position in SEQ ID NO: 6 selected from The cp caspase-2 has increased P1' tolerance compared to cp caspase-2 without the respective amino acid substitution, and 14 or position Asp in SEQ ID NO: 11 347 It may also contain an SS propeptide containing an amino acid substitution with Ala at a position functionally equivalent to:

[0028] Specifically, the cp caspase-2 variant ms9 ProD, which contains the E105V, G171D, V225G, and D282E substitutions, exhibits excellent P1' tolerance. Specifically, the cp caspase-2 variant E105V G171D, which contains the E105V and G171D substitutions, exhibits increased P1' tolerance compared to the cp caspase-2 variant ms9 ProD. Specifically, the highest tolerance of the cp caspase-2 variant E105V G171D is for the amino acid residue proline. According to certain embodiments, the cp caspase-2 described herein comprises at least 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, or 98% sequence identity to SEQ ID NO:9 (Homo sapiens), SEQ ID NO:64 (Sarcophilus harrisii, Tasmanian devil), SEQ ID NO:66 (Anolis carolinensisilus), SEQ ID NO:68 (Elephant shark (Callorhinchus milii, chimaera), SEQ ID NO:76, or SEQ ID NO:77 (human), and comprises one or more amino acid substitutions at a position functionally equivalent to any of positions 171, 105, 172, 282, 225, 83, 185, 255, or 285 of SEQ ID NO:6, or any combination thereof.

[0029] Specifically, the cp caspase-2 variants described herein comprise SEQ ID NO:6 and one or more amino acid substitutions at positions 171, 105, 172, 282, 225, 83, 185, 255, or 285 of SEQ ID NO:6, or at positions functionally equivalent to positions 171, 105, 172, 282, 225, 83, 185, 255, or 285 of SEQ ID NO:6, or any combination thereof. Specifically, the cp caspase-2 variants described herein contain any one or more of the amino acid substitutions G171D, E105V, E172V, D282E, D282T, V225G, K83E, H185A, V255M, D285Y, and D285E, based on the numbering according to SEQ ID NO: 6. According to further particular embodiments, the cp caspase-2 variant described herein comprises SEQ ID NO: 6 or has at least 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97 or 98% sequence identity to SEQ ID NO: 6 and comprises the amino acid substitutions E105V, G171D, V225G, D282E and / or D285E relative to the numbering of SEQ ID NO: 6, wherein said cp caspase-2 has increased P1' tolerance.

[0030] According to further particular embodiments, the cp caspase-2 variant described herein comprises SEQ ID NO: 6 or has at least 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97 or 98% sequence identity to SEQ ID NO: 6 and comprises the amino acid substitutions K83E, E105V, E172V, V255M and / or D285Y relative to the numbering of SEQ ID NO: 6, wherein said cp caspase-2 has increased P1' tolerance. According to further particular embodiments, the cp caspase-2 variant described herein comprises SEQ ID NO: 6 or has at least 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97 or 98% sequence identity with SEQ ID NO: 6 and comprises the amino acid substitutions H185A and / or D282T relative to the numbering of SEQ ID NO: 6, wherein said cp caspase-2 has increased P1' tolerance, in particular towards branched P1' amino acid residues.

[0031] Specifically, the cp caspase-2 variants described herein are selected from the group consisting of SEQ ID NOs: 1, 13, 17, 18, 23, 24, 51, 52, 54, 70, 71, 72, 78, 79, 86, 87, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, and 192, or SEQ ID NOs: 1, 13, 17, 18, 23, 24, 51, 52, 54, 70, 71, 72, 78, 79, 86, 87, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, and 192. According to certain embodiments, the cp caspase-2 described herein comprises a C-terminal tag and amino acid substitutions at positions 285 and 292 of SEQ ID NO:6 or at positions functionally equivalent to positions 285 and 292 of SEQ ID NO:6, specifically substitutions to Glu and Ser, respectively (D285E and D292S).

[0032] Specifically, the caspase-2 or cp caspase-2 described herein is recruited by a proteolytic cleavage recognition site comprising five amino acids of the sequence P5 P4 P3 P2 P1, P1 can be any amino acid, preferably D or E; P2 can be any amino acid, preferably A; P3 can be any amino acid, preferably V; P4 can be any amino acid, preferably D; P5 can be any amino acid, preferably V. Specifically, the caspase-2 variants or cp caspase-2 variants described herein have increased specificity for the recognition site VDVAD (wherein P5 is V, P4 is D, P3 is V, P2 is A, and P1 is D) compared to wild-type (wt) caspase-2 comprising the amino acid sequence of SEQ ID NO: 11.

[0033] Specifically, the caspase-2 or cp caspase-2 described herein recognizes or can be further modified to recognize various recognition sites. According to specific examples, the caspase-2 variants or cp caspase-2 variants described herein recognize any one or more of the recognition sites LDESD, DVAD, DEVD, DEVE, ADVAD, VDTTD, DTTD, DVPD, VDVPD, VDQQD, or TDTSD. Preferably, the caspase-2 or cp caspase-2 described herein recognizes one recognition site and has high specificity therefor. According to further specific examples, the variants of caspase-2 or cpcaspase-2 described herein recognize the recognition sites DRKD, DAVD, VKVD, DTLD, EEPD, DETD, DATD, NKVD, DALD, DSVD, NAID, DKPD, IQLD, DNAD, DVVD, ENPD, DMAD, DLID, DGAD, DVKD, GYND, ELPD, DSTD, DRQD, HAVD, QERLD, LERD, MMPD, EEPD, VESID, EAMD, EDAD, EEED, AVLD, and / or EEGD. According to further embodiments, the variants of caspase-2 or cp caspase-2 described herein recognize the recognition sites TDTSD, LDEPD, and / or KDEVD.

[0034] Specifically, the recognition site can be selected from the group consisting of DEXD (SEQ ID NO: 202) and DVXD (SEQ ID NO: 203), where X is any amino acid. Specifically, the recognition site comprises the sequence P5 P4 P3 P2 P1, where P5 is V, P4 is D, P3 is Q, P2 is Q, and P1 is D. Specifically, V at position P5 can be replaced with I, Y, L, T, N, or A, and / or D at position P4 can be replaced with S, and / or Q at position P3 can be replaced with V, E, or T, and / or Q at position P2 can be replaced with A, S, K, V, M, or L. Testing the recognition site library (P4-P1) for caspase 2 resulted in the following predominant amino acids: position P4: D, V; position P3: V, E, T; position P2: S, T; and position P1: D.

[0035] Specifically, the caspase-2 or cpcaspase-2 described herein can cleave at the cleavage site P1 / P1', where P1' can be any amino acid. Further provided herein, also referred to as "caspase-2 variants," are caspase-2s containing one or more amino acid substitutions at positions 212, 431, 213, 323, 266, 409, 226, 296, or 326 of SEQ ID NO: 11, or at positions functionally equivalent to any of positions 212, 431, 213, 323, 266, 409, 226, 296, or 326 of SEQ ID NO: 11, or combinations thereof, which amino acid substitutions increase P1' tolerance compared to a caspase-2 having the same sequence but not containing said substitutions. In other words, the caspase-2 to which the caspase-2 variant is compared has an identical sequence to the caspase-2 variant but does not contain any amino acid substitutions at positions 212, 431, 213, 323, 266, 409, 226, 296, or 326 of SEQ ID NO: 11, or at positions functionally equivalent to any of positions 212, 431, 213, 323, 266, 409, 226, 296, or 326 of SEQ ID NO: 11, which, according to the present invention, increase P1' tolerance. Specifically, the caspase-2 variants provided herein comprise improved P1' tolerance for at least one amino acid other than glycine, compared to caspase-2 that does not contain the respective amino acid substitution.

[0036] Specifically, the caspase-2 variants described herein comprise at least 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, or 98% sequence identity to SEQ ID NO:11, SEQ ID NO:89, SEQ ID NO:92, SEQ ID NO:95, SEQ ID NO:98, SEQ ID NO:101, SEQ ID NO:104, SEQ ID NO:107, SEQ ID NO:110, SEQ ID NO:113, or SEQ ID NO:116, and comprise one or more amino acid substitutions at positions 409, 431, 212, 213, 266, 296, 226, 323, or 326 of SEQ ID NO:11, or at a position functionally equivalent to any of positions 409, 431, 212, 213, 266, 296, 323, or 326 of SEQ ID NO:11, or a combination thereof. Specifically, the caspase-2 described herein includes at least a small caspase-2 subunit and a large caspase-2 subunit.

[0037] Specifically, the small subunit of caspase-2 described herein comprises the amino acid sequence 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:112, SEQ ID NO:115, SEQ ID NO:118, or a functionally active variant thereof comprising at least 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, or 98% sequence identity. Specifically, the large subunit of caspase-2 described herein comprises the amino acid sequence of SEQ ID NO:4, SEQ ID NO:90, SEQ ID NO:93, SEQ ID NO:96, SEQ ID NO:99, SEQ ID NO:102, SEQ ID NO:105, SEQ ID NO:108, SEQ ID NO:111, SEQ ID NO:114, SEQ ID NO:117, or a functionally active variant thereof comprising at least 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, or 98% sequence identity.

[0038] According to certain embodiments, the caspase-2 provided herein comprises one or more linker sequences, specifically, the linker sequence consists of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 or even more amino acid residues. Specifically, the linker can comprise more than 20 or 30 amino acids, or even more, as long as the caspase retains its functional activity as described herein. Specifically, the linker sequence comprises glycine, alanine, and / or serine residues. Specifically, the linker comprises at least one glycine and serine residue, more specifically, the linker is GS, GGSGG, and / or GSAGSAAGSG. Specifically, caspase-2 comprises a subunit linker sequence, which is a linker sequence between the small and large subunits of caspase-2 as described herein.

[0039] According to further specific embodiments, the caspase-2 provided herein comprises one or more C- or N-terminal tags, specifically tags selected from the group consisting of affinity tags, solubility-enhancing tags, and monitoring tags described herein. Specifically, any tag known in the art can be fused to caspase-2. According to certain embodiments, the caspase-2 provided herein comprises at least 1, 2, 3, 4, 5, and up to 10 or even more N- and / or C-terminal truncations, so long as the caspase retains its functional activity as described herein. According to further certain embodiments, the caspase-2 provided herein comprises at least 1, 2, 3, 4, 5, and up to 10 or even more N- and / or C-terminal extensions, so long as the caspase retains its functional activity as described herein. Specifically, the caspase-2 can comprise truncations and extensions.

[0040] According to certain embodiments, the caspase-2 variants provided herein comprise, relative to the position of SEQ ID NO: 11, or a position functionally equivalent to the position of SEQ ID NO: 11: iD, or Gly substituted with an amino acid selected from the group consisting of R, K, E, Q, N, A, S, T, P, H, and Y 212 , ii. Glu substituted with an amino acid selected from the group consisting of V, C, L, I, M, F, W, R, K, D, Q, and N 431 , iii. Glu substituted with an amino acid selected from the group consisting of V, C, L, I, M, F, W, R, K, D, Q, and N 213 , iv. Asp substituted with E or T, or an amino acid selected from the group consisting of R, K, Q, N, G, A, S, P, H, Y. 323 , vG, or Val substituted with an amino acid selected from the group consisting of A, S, T, P, H, Y, C, L, I, M, F, and W 266 , vi. Lys substituted with an amino acid selected from the group consisting of E, R, D, Q, and N 409 , vii. His substituted with an amino acid selected from the group consisting of A, G, S, T, P, and Y 226 , viii. Val substituted with an amino acid selected from the group consisting of M, C, L, I, F, and W 296 and / or ix. Asp 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 326 The amino acid sequence of the present invention may contain one or more amino acid substitutions selected from the group consisting of:

[0041] Specifically, the caspase-2 variants provided herein are i.His 226 and Asp 323 , specifically those containing the substitutions H226A and D323T; ii.Glu 431 , specifically including the replacement of E431V, iii.Glu 431 and Asp 326, specifically those containing the substitutions E431V and D326E; iv.Glu 431 , Gly 212 , Val 266 , and Asp 323 , specifically those containing the substitutions E431V, G212D, V266G, and D323E; v.Glu 431 , Gly 212 , Val 266 , Asp 323 , and Asp 326 , specifically those containing the substitutions E431V, G212D, V266G, D323E, and D326E; vi.Lys 409 , Glu 431 , Glu 213 , Val 296 , and Asp 326 , specifically those containing the substitutions K409E, E431V, E213V, V296M, and D326Y; vii.Glu 431 and Gly 212 , specifically those containing the substitutions E431V and G212D; viii.Glu 431 and Glu 213 , specifically those containing the substitutions E431V and E213V, and ix.Gly 212 and Glu 213 , specifically those containing the G212D and E213V substitutions The present invention comprises an amino acid substitution at a position in SEQ ID NO:11, or at a position functionally equivalent to a position in SEQ ID NO:11, selected from:

[0042] Further provided herein are methods for generating caspase variants, particularly caspase-2, and even more particularly circularly permuted caspase-2 that contain increased P1' tolerance. Specifically, the wild-type cp caspase-2 or cp caspase-2 variants described herein, or functionally active variants thereof, i. cloning a nucleotide sequence encoding caspase-2, particularly a circularly permuted caspase-2, into a vector, particularly said sequence under the control of a promoter; ii. transforming a host cell with the vector; iii. culturing the transformed host cells under conditions in which the caspase is expressed; iv. isolating the caspase from the host cell culture, optionally by disrupting the host cells; v. optionally purifying the caspase; The method includes:

[0043] Specifically, the nucleic acid sequence encoding caspase-2 described herein is operably linked to a promoter. Specifically, the promoter is an inducible or constitutive promoter. Specifically, the promoter is selected from the group consisting of T7, lac, tac, trc, lacUV5, trp, phoA, pL, XylS / Pm regulator / promoter system, Pm promoter, Pm promoter variant, araBAD, T3, T5, T4, T7A1, T7A2, T7A3, hybrid promoter, and strong constitutive HCD promoter. Specifically, the promoter is associated with one or more lac operators or corresponding other operators or regulators or additional regulatory elements, or the promoter is not associated with such regulatory elements. In a preferred embodiment, the promoter / regulator is a promoter / regulator selected from the group consisting of the T7 promoter / operator, the XylS / Pm regulator / promoter, a functionally active variant of the Pm promoter, the araBAD promoter / operator, the T5, T7A1, T7A2, T7A3 promoter / operator, the phoA promoter / regulator, and the trp promoter / operator system. Specifically, the promoter / regulator is of the T7 promoter / operator system.

[0044] Specifically, the host cell is a eukaryotic or prokaryotic microbial host cell. Specifically, the host cell is selected from the group consisting of a bacterial cell, a yeast cell, an insect cell, a mammalian cell, and a plant cell, and preferably the host cell is a bacterial or yeast cell selected from the group consisting of Escherichia coli, Pseudomonas species, Bacillus species, Streptomyces species, Saccharomyces species, Schizosaccharomyces species, Pichia species, Kluyveromyces species, and Hansenula species.

[0045] Even more specifically, the host cell is of the E. coli B or K strain, such as, but not limited to, BL21 or HMS174. Specifically, the host cell has incorporated into its genome a nucleotide sequence encoding T7 RNA polymerase and is capable of constitutive or inducible expression of caspase-2 as described herein. According to a specific example, the host cell is an E. coli BL21(DE3) or HMS174(DE3) cell or a cell derived from BL21(DE3) or HMS174(DE3) that contains a deletion of at least one essential lambda phage protein. The caspase-2 or cp caspase-2 described herein may contain a tag sequence within its sequence or fused to its N- or C-terminus.

[0046] The circularly permuted caspase-2 can optionally have an affinity tag fused to its N- or C-terminus, preferably a 6His tag. In a preferred embodiment, the 6His tag is at the N-terminus. Specifically, to increase the expression of soluble cpcaspase-2, cpcaspase-2 is fused to a solubility-enhancing tag at its C- or N-terminus. In a preferred embodiment, the solubility-enhancing tag is located at the N-terminus of cpcaspase-2. Preferably, the solubility tag is based on a highly charged peptide from a bacteriophage gene. Exemplary solubility tags and their sequences are listed in Table 1 of US Pat. No. 8,535,908. Specifically, the solubility tag is selected from the group consisting of TAG, T7C, T7B, T7B1, T7B2, T7B3, T7B3, T7B4, T7B5, T7B6, T7B6, T7B7, T7B8, T7B9, T7B10, T7B11, T7B12, T7B13, T7A, T7A1, T7A2, T7A3, T7A4, T7A5, T7AC, T3, N1, N2, N3, N4, N5, N6, N7, calmodulin binding peptide (CBP), DsbA, DsbC, poly-Arg, poly-Lys, G Bl domain, protein D, Z domain of staphylococcal protein A, and thioredoxin tag, and preferably comprises a T7AC or T7A3 tag. According to a specific example, the tag is a modified T7A3 tag, referred to herein as T7AC (SEQ ID NO: 43). Preferably, one or more T7A3 (SEQ ID NO: 37) and / or T7AC (SEQ ID NO: 43) tags or functional variants thereof, such as those having one to five amino acid substitutions, additions, deletions, etc., are used.

[0047] Specifically, caspases produced according to the methods described herein have one or more affinity tags and one or more solubility-enhancing tags fused to their N-terminus, with or without linker sequences between the tags or between the tags and the N-terminus of cpCaspase-2. Specifically, the caspases have a T7AC or T7A3 tag and a 6His tag fused to their N-terminus, where, from N to C-terminus, the 6His tag is the first tag and the T7AC or T7A3 tag is the second tag, or the T7AC or T7A3 tag is the first tag and the 6His tag is the second tag. Surprisingly, it has been found that the production of cp caspase-2 described herein can be significantly improved using a solubility-enhancing tag, such as T7A3 or T7AC, fused to the N-terminus of the enzyme, resulting in a significant increase in enzyme titer of about 2.5-fold or more.

[0048] According to a particular embodiment, the cp caspase-2 produced according to the methods described herein therefore has the following elements fused to its N-terminus in N to C-terminal order: a. an affinity tag, preferably a 6-His tag; b. optionally a linker; c. a solubility-enhancing tag, preferably T7AC or T7A3, and d. wild-type or variant cp caspase-2 as described herein Includes:

[0049] According to further particular embodiments, the cp caspase-2 produced according to the methods described herein has the following elements fused to its N-terminus in N to C-terminal order: a. a solubility-enhancing tag, preferably T7AC or T7A3; b. optionally a linker; c. an affinity tag, preferably a 6-His tag, and d. wild-type or variant cp caspase-2 as described herein Includes:

[0050] Specifically, the expression cassette for expression of cp-caspase-2 comprises a nucleotide sequence encoding cp-caspase-2 under the control of a promoter. The expression cassette may further comprise a nucleotide sequence encoding an affinity tag, in preferred embodiments a 6His tag and / or a T7AC or T7A3 tag, as well as a nucleotide sequence encoding a linker sequence between the tags and / or between the tag and cp-caspase-2. In another embodiment, the expression cassette is flanked by two sequences homologous to sequences in the genome of a host cell, preferably a microbial cell, more preferably a bacterial cell, more preferably E. coli, for integration of the expression cassette into the genome of the host cell by homologous recombination.

[0051] Specifically, cells are transformed with a vector containing an expression cassette, specifically cp Caspase-2, with or without a tag as described herein, is expressed from one or more plasmids or from one or two copies of a nucleic acid sequence integrated into the genome of the host cell. Specifically, cp caspase-2, with or without the tags described herein, can be produced in a bioreactor (fermentor) by culturing host cells and inducing expression by adding an inducer, such as IPTG when using a T7 promoter / operator system. Specifically, the culturing in step (iii) of the method for producing cp caspase-2 described herein comprises a fed-batch stage for expression of cp caspase-2, including specific growth rate and induction of expression, preferably using IPTG.

[0052] Specifically, the culturing in step (iii) of the method for producing cp caspase-2 described herein comprises a fed-batch stage for the expression of cp caspase-2, the fed-batch stage being for about 0.01 to 0.1 hours. -1Specifically, this involves inducing expression of cpCaspase-2 by the addition of IPTG at a specific growth rate of μg / g and a concentration of approximately 0.01-1.5 μmol / g actual CDM (cell dry mass). Specifically, an IPTG concentration of μmol / g actual CDM refers to the concentration of IPTG in the fermentor at a particular time point during the feeding phase relative to g of CDM at that particular time point. According to a particular embodiment, the growth rate μ is about 0.01 to 0.07 h -1 and preferably about 0.01 to 0.03 hours -1 or 0.01 to 0.05 o'clock -1 or 0.02 to 0.05 o'clock -1 or 0.03 to 0.05 o'clock -1 or 0.03 to 0.07 o'clock -1 or 0.05 to 0.07 o'clock -1 and preferably about 0.03, 0.05 or 0.07 hours -1 It is one of the following.

[0053] According to further specific embodiments, the IPTG concentration is about 0.1-1.5 μmol / g or 0.1-1.3 μmol / g or 0.2-1.3 μmol / g or 0.3-1.3 μmol / g or 0.5-1.3 μmol / g actual CDM, preferably about 0.5-0.9 μmol / g actual CDM or about 0.9-1.3 μmol / g actual CDM, preferably about 0.5, 0.9 or about 1.3 μmol / g CDM.

[0054] According to a further particular embodiment, the culturing of step (ii) further comprises a first fed-batch stage for the generation of biomass before the fed-batch stage for the expression of cp caspase-2, said first fed-batch stage lasting for about 0.05 to 0.5 hours. -1 or 0.05 to 0.4 o'clock -1 or 0.07 to 0.3 o'clock -1 Specifically, the growth rate μ is about 0.1 to 0.3 hr. -1 Or 0.1 to 0.2 hours -1, or 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, or 0.20 o'clock -1 , preferably about 0.13 to 0.21 hours -1 , and even more preferably about 0.16 to 0.18 hours -1 and most preferably about 0.17 hours -1 Specifically, the first fed-batch step is followed by a second fed-batch step for expression of the recombinant protein, which is preferably initiated by the addition of an expression inducer such as IPTG and typically involves a slower growth rate.

[0055] Specifically, as used herein, the following structure from N to C terminus: i. a tag sequence comprising a caspase recognition site that is specifically recognized by cp caspase-2 or caspase 2 as described herein; ii. cleavage site P1 / P1', and iii. Protein or Polypeptide of Interest (POI) The present invention provides a fusion protein comprising: Specifically, P1' is the N-terminal amino acid of the protein of interest (POI). Specifically, the tag sequence of the fusion protein described herein further comprises one or more tags selected from the group consisting of affinity tags, solubility-enhancing tags, and monitoring tags. Specifically, any tag having any function known in the art can be fused to the POI. Specifically, the fusion protein further comprises one or more linker sequences. Specifically, the fusion protein comprises a caspase recognition site and cleavage site P1 / P1', which comprises five amino acids of the sequence P5 P4 P3 P2 P1, where P1' is the N-terminal amino acid of the POI.

[0056] According to certain embodiments, the fusion proteins provided herein comprise within their sequence a cp caspase-2 or caspase 2 as described herein. Specifically, the fusion proteins comprise a cp caspase-2 or caspase 2 as described herein fused to the N- or C-terminus of the fusion protein. Specifically, such fusion proteins are used to generate POIs containing authentic N-termini by cleaving the fusion protein at the N-terminus of the POI using cp caspase-2 or caspase-2 as described herein. In one embodiment, the POI comprises an N-terminal tag comprising at least a caspase recognition site, with P1, the C-terminal amino acid of the recognition site, being the last (C-terminal) amino acid residue of the tag, and the N-terminal amino acid of the POI being the P1' residue of the caspase cleavage site. In this embodiment, the tag sequence comprising the recognition site is released from the POI through proteolytic cleavage by a caspase, generating a POI comprising an authentic N-terminus.

[0057] Further provided herein are methods for producing proteins or polypeptides of interest (POIs) using the cp caspase-2 or caspase-2 described herein. Specifically, the cp caspase-2 described herein is used to produce POIs that contain authentic N-termini. Specifically, provided herein are methods for generating POIs that contain authentic N-termini using the fusion proteins described herein and the caspase-2 or cpcaspase-2 described herein.

[0058] Specifically, provided herein are methods for generating a POI comprising an authentic N-terminus using a fusion protein described herein, wherein the fusion protein comprises a caspase-2 or cpcaspase-2 described herein at its N- or C-terminus. Specifically, the fusion proteins used in the methods described herein have the following structure from N to C terminus: i. one or more N-terminal tags; ii. optionally one or more tag-linker sequences, and iii. a caspase recognition site comprising five amino acids of the sequence P5 P4 P3 P2 P1; iv. cleavage site P1 / P1', and v.POI wherein the recognition site is specifically recognized by caspase-2 or cp caspase-2 as described herein. Specifically, P1' is the N-terminal amino acid of the POI.

[0059] Specifically, provided herein are methods for producing POI in vivo. Specifically, the in vivo method for generating a POI containing an authentic N-terminus includes: expressing in the same host cell a fusion protein comprising, from N- to C-terminus, optionally one or more tags, optionally one or more tag-linker sequences, and a caspase recognition site fused at the N-terminus to a POI, and a caspase-2 or cp-caspase-2 as described herein that specifically recognizes the recognition site of the fusion protein; ii. optionally, the fusion protein and caspase-2 or cp caspase-2 are under the control of the same promoter; iii. culturing the host cells, wherein the caspase-2 or cp caspase-2 cleaves the fusion protein in the culture; iv. isolating the POI from the cells and optionally purifying the POI; Includes:

[0060] Specifically, the host cell is selected from the group consisting of a bacterial cell, a yeast cell, an insect cell, a mammalian cell, and a plant cell, and preferably the host cell is a bacterial or yeast cell selected from the group consisting of Escherichia coli, Pseudomonas spp., Bacillus spp., Streptomyces spp., Saccharomyces spp., Schizosaccharomyces spp., Pichia spp., Kluyveromyces spp., and Hansenula spp. According to certain embodiments, the fusion protein and the caspase described herein are under the transcriptional control of different promoters, and expression of the caspase is induced after expression of the fusion protein. According to different embodiments, the fusion protein and the caspase are under the transcriptional control of the same promoter, in particular, they are expressed simultaneously. Specifically, the caspases contain N- or C-terminal tags that can be used to separate the caspase from the POI.

[0061] According to further particular embodiments, the caspase described herein is part of a fusion protein expressed in a host cell and cleaves the fusion protein to release the POI containing the authentic N-terminus into the host cell. Specifically, the fusion protein, POI, and / or caspase are isolated using a column, specifically a chromatography column, more specifically an immobilized metal affinity chromatography column (IMAC). Specifically, provided herein are methods for producing POI in vitro.

[0062] Specifically, an in vitro method for producing a protein of interest (POI) containing an authentic N-terminus includes: providing a fusion protein comprising, from N- to C-terminus, one or more tags, optionally one or more tag-linker sequences, and a caspase recognition site fused at the N-terminus to a POI, wherein said caspase recognition site is specifically recognized by caspase-2 or cpcaspase-2 as described herein; ii. contacting the fusion protein with the caspase-2 or cp caspase-2 for a period of time sufficient for the caspase-2 or cp caspase-2 to cleave the fusion protein; iii. optionally purifying the POI; Includes:

[0063] Specifically, the method for generating a POI described herein comprises: From N to C-terminus, the structure is: an N-terminal affinity tag, b. optionally a linker sequence; c. caspase recognition site, d. cleavage site P1 / P1', and e.POI expressing in a host cell a fusion protein comprising: P1' being the N-terminal amino acid of the POI; and said recognition site being specifically recognized by caspase-2 or cpcaspase-2 (caspase) as described herein; ii. isolating the fusion protein; iii. purifying the fusion protein using an N-terminal affinity tag; iv. providing a caspase as described herein that specifically recognizes the recognition site of the fusion protein; v. contacting the fusion protein with the caspase for a period of time sufficient for the caspase to cleave the fusion protein; vi. optionally removing the cleaved affinity tag and optionally the uncleaved fusion protein using the affinity tag and a caspase; vii. Optionally, further purifying the POI; Includes:

[0064] Specifically, the caspase used in such a method comprises an affinity tag at its N- or C-terminus that is identical to or similar to the affinity tag of the fusion protein, and the caspase, the cleaved affinity tag, and any uncleaved fusion protein are removed using the affinity tag in step vi. Specifically, the fusion protein is purified using the tag, for example, by affinity chromatography, more specifically, by immobilized metal affinity chromatography, where the captured fusion protein is released and the N-terminal tag is removed in solution or in an immobilized enzyme reactor, and the caspase is immobilized in a column or on a carrier. According to a further particular embodiment, the fusion protein and the caspase are bound on a column.

[0065] Specifically, the method for generating POIs containing authentic N-termini using columns involves: i. expressing in a host cell a fusion protein comprising one or more N-terminal affinity tags, optionally one or more tag-linker sequences, a caspase recognition site and a cleavage site P1 / P1′, where P1′ is the N-terminal amino acid of the POI, and the POI; ii. isolating the fusion protein from the host cell and capturing / binding the fusion protein onto a solid support using an affinity tag; iii. providing a caspase-2 or cpcaspase-2 (caspase) as described herein that specifically recognizes the recognition site of the fusion protein; iv. contacting the caspase-bound fusion protein for a period of time sufficient for the caspase to cleave the fusion protein and release the POI from the solid support, while the tag and, optionally, uncleaved fusion protein remain bound; v. isolating and optionally further purifying the POI; Includes:

[0066] In particular, the caspase comprises a tag sequence, in particular an affinity tag, to allow separation of the caspase from the POI after cleavage. In certain embodiments, the caspase-2 or cp caspase-2 described herein comprises an affinity tag and is immobilized on a solid support or column, and the fusion protein is contacted with the immobilized caspase. Specifically, the fusion protein is passed through the column, thereby contacting the fusion protein with the immobilized caspase in the column. Specifically, the cleaved tag is separated from the POI using the affinity tag in the tag sequence.

[0067] In further particular embodiments, the caspase and the fusion protein contain the same N-terminal affinity tag, allowing the fusion protein and the caspase to be immobilized on a solid support. Upon cleavage of the POI by the caspase, the POI is released from the column, while the tag sequence, as well as the caspase and any uncleaved fusion protein, are retained in the column. In particular, the solid support is a column, in particular a chromatography column, more particularly an immobilized metal affinity chromatography column (IMAC) or an activated NHS column that allows the immobilization of polypeptides through amine coupling.

[0068] Specifically, a flow reactor is used that contains immobilized caspase-2, cp caspase-2, or a fusion protein described herein. Specifically, the flow reactor is a plug flow reactor. Further provided herein is an isolated nucleotide sequence encoding a caspase-2 or cpcaspase-2 described herein. Further provided herein is a vector comprising the isolated nucleotide sequence described herein, specifically, the vector is a bacterial expression vector. More specifically, the vector is a plasmid. In another embodiment, the vector is a linear vector and is flanked by homologous regions for homologous integration of the nucleotide sequence encoding caspase-2 or cp-caspase-2 described herein into the chromosome of a host cell. Further provided herein is an expression cassette comprising a nucleotide sequence operably linked to a regulatory element, such as a promoter, an operator, a terminator, etc. Specifically, the regulatory element is one or more promoters or expression-enhancing elements.

[0069] Further provided herein is a host cell or host cell line expressing caspase-2 or cp caspase-2 as described herein, wherein the host cell is selected from the group consisting of a bacterial cell, a yeast cell, an insect cell, a mammalian cell, and a plant cell, preferably the host cell is a bacterial or yeast cell selected from the group consisting of Escherichia coli, Bacillus spp., Streptomyces spp., Saccharomyces spp., Schizosaccharomyces spp., Kluyveromyces spp., and Pichia spp. According to certain embodiments, the expression cassettes and host cells or host cell systems described herein are comprised in an expression system. Further provided herein is an expression system comprising an expression cassette and a host cell or host cell system described herein.

[0070] Further described herein is the use of caspase-2 or cp caspase-2 described herein for in vivo cleavage of a substrate in a non-human organism, particularly a prokaryotic organism, particularly Escherichia coli. In this specification, i. caspase-2 or cp caspase-2 as described herein; ii. an expression vector optionally comprising a nucleotide sequence encoding an affinity tag, preferably a 6His tag, a linker sequence, and / or a recognition site, preferably VDVAD; Further provided is a kit comprising:

[0071] The kit may further comprise chromatography material for affinity chromatography, preferably IMAC (immobilized metal affinity chromatography) material, preferably Ni-NTA (Ni-nitrilotriacetic acid) chromatography material, preferably pre-packed in a chromatography column. The kit may further include a plasmid comprising, from 5' to 3', a nucleotide sequence encoding an affinity tag, preferably a 6His tag, optionally a linker sequence, and a recognition site, preferably a nucleotide sequence encoding VDVAD. Via the multiple cloning site, the DNA sequence encoding the POI can be inserted into the plasmid in direct fusion with the nucleotide sequence encoding the recognition site. Further described herein are pharmaceutical compositions comprising the caspase-2 or cp caspase-2 provided herein, and optionally one or more excipients.

[0072] Further described herein is the use of caspase-2 or cp caspase-2 provided herein to prepare a pharmaceutical composition. In particular, the caspase-2 or cp caspase-2 described herein is provided for use in the treatment of diseases, such as cancer, osteoporosis, Alzheimer's disease, Parkinson's disease, inflammatory diseases, or autoimmune diseases, particularly through proteolytic attack of proteins associated with the respective diseases.

[0073] Specifically, the caspase-2 or cp caspase-2 described herein is provided for the manufacture of a medicament for the treatment of cancer, Alzheimer's disease, Parkinson's disease, or inflammatory disease.The present invention further provides a protein tag for enhancing the expression of POI, the protein tag comprising a solubility-enhancing tag and the amino acid sequence VDVAD (SEQ ID NO: 45).Specifically, the sequence VDVAD is located at the C-terminus of the protein tag described herein, and is specifically directly linked to the N-terminus of POI. Surprisingly, we have found that including the amino acid sequence VDVAD in a protein tag can significantly increase the expression of a target protein fused to the protein tag. Importantly, this increase in expression persists despite the addition of a histidine tag sequence to the protein tag. The use of a histidine affinity tag, such as 6-His, typically significantly reduces the expression rate of a target protein. We have surprisingly found that including the sequence VDVAD in a protein tag can reverse this effect and provide increased expression titers.

[0074] According to certain embodiments, the solubility-enhancing tag is selected from the group consisting of T7C, T7B, T7B1, T7B2, T7B3, T7B3, T7B4, T7B5, T7B6, 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, the Z domain of staphylococcal protein A, and thioredoxin tags. Specifically, the solubility-enhancing tag is T7AC or T7A3.

[0075] According to further particular embodiments, the protein tags described herein further comprise a histidine tag sequence, preferably comprising 1 to 20 histidine residues, and even more preferably a 1-His, 2-His, 3-His, 4-His, 5-His, 6-His, 7-His, 8-His, 9-His, 10-His, 11-His, 12-His, 13-His, 14-His, 15-His, 16-His, 17-His, 18-His, 19-His, or 20-His tag sequence. Specifically, the solubility-enhancing tag is located at the N-terminus of the protein tag described herein. Specifically, the histidine tag sequence is located at the N-terminus of the protein tag described herein.

[0076] According to certain embodiments, the protein tags described herein further comprise one or more linker sequences comprising one or more amino acid residues. Specifically, the linker sequence comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 or more amino acid residues. Specifically, the linker can comprise more than 20 or 30 amino acids, or even more, as long as the caspase retains its functional activity as described herein. Specifically, one or more amino acid residues of the linker sequence are any of naturally occurring amino acids or derivatives thereof, preferably selected from the group consisting of G, S, T, N, and A. Specifically, the linker sequence comprises glycine, alanine, and / or serine residues. Specifically, the linker comprises at least one glycine and serine residue, more specifically, the linker is GS, GSG, GGSGG, GSGSGSG, and / or GSAGSAAGSG. Specifically, the one or more linker sequences are located between the VDVAD sequence and the solubility-enhancing tag or histidine tag sequence.

[0077] According to certain embodiments, the protein tags described herein further comprise a signal peptide at their N-terminus.Signal peptides are known to those skilled in the art and include, for example, those described by Choi and Lee, Appl Microbiol Biotechnol (2004); 64:625-635 or Karyolaimos et al. Frontiers in Microbiology (2019); 10:1-11. Specifically, the signal peptide is selected from the group consisting of ompA (outer membrane protein A), DsbA (thiol:disulfide exchange protein), MalE (maltose binding protein), PelB (pectate lyase B) from Erwinia carotovora, PhoA (alkaline phosphatase), OmpC (outer membrane protein C), OmpF (outer membrane protein F), OmpT (protease VII), endoxylanase from Bacillus species, LamB (lambda receptor protein), Lpp (murein lipoprotein), LTB (heat-labile enterotoxin subunit B), PhoE (outer membrane pore protein E), and StII (heat-stable enterotoxin 2).

[0078] The use of a signal sequence (also referred to herein as a signal peptide, leader sequence, or leader peptide), such as the ompA signal peptide, which directs proteins through the inner membrane into the periplasm of bacteria, e.g., E. coli, and is fused to the N-terminus of the protein tag described herein, enables successful production of the POI and fusion proteins described herein in the periplasm of E. coli. This is surprising, since expression enhancers are typically located at the N-terminus of all fusion proteins described herein (respectively, expression constructs, genes encoding the respective fusion proteins). As demonstrated in Example 10.2 and Figure 31, very high titers of over 5 g / L of recombinant protein expressed in the periplasm of E. coli can be achieved using a signal peptide located at the N-terminus of the protein tag.

[0079] Specifically, the protein tags described herein have the following structure from N to C terminus: a. T7AC-6-His-VDVAD; b. T7A3-6-His-VDVAD; c. T7AC-6-His-GSG-VDVAD; d. T7A3-6-His-GSG-VDVAD; e. T7AC-6-His-GSGSGSG-VDVAD; f. T7A3-6-His-GSGSGSG-VDVAD; g. 6-His-T7AC-VDVAD; h. 6-His-T7A3-VDVAD; i. 6-His-T7AC-GSG-VDVAD; j. 6-His-T7A3-GSG-VDVAD; k. 6-His-T7AC-GSG-VDVAD; l. 6-His-T7A3-GSG-VDVAD Contains one of the following:

[0080] Specifically, the protein tags described herein have the following structure from N to C terminus: a. ompA signal peptide-T7AC-6-His-VDVAD; b. ompA signal peptide-T7A3-6-His-VDVAD; c. ompA signal peptide-T7AC-6-His-GSG-VDVAD; d. ompA signal peptide-T7A3-6-His-GSG-VDVAD; e. ompA signal peptide-T7AC-6-His-GSGSGSG-VDVAD; f. ompA signal peptide-T7A3-6-His-GSGSGSG-VDVAD; g. ompA signal peptide-6-His-T7AC-VDVAD; h. ompA signal peptide-6-His-T7A3-VDVAD; i. ompA signal peptide-6-His-T7AC-GSG-VDVAD; j. ompA signal peptide-6-His-T7A3-GSG-VDVAD; k. ompA signal peptide-6-His-T7AC-GSGSGSG-VDVAD; l. ompA signal peptide-6-His-T7A3-GSGSGSG-VDVAD Contains one of the following:

[0081] The present specification also provides a fusion protein comprising the protein tag and POI described herein.Specifically, the N-terminus of POI is fused to the C-terminus of said protein tag.More specifically, the N-terminus of POI is directly fused to the C-terminus of the protein tag, which is the sequence VDVAD, that is, the N-terminal amino acid of POI is directly linked to the C-terminal D of the VDVAD sequence of the protein tag. There are no limitations regarding the POI: the POI can be any polypeptide, including, for example, a caspase as described herein.

[0082] Further provided herein is a method for generating a POI, comprising: i. providing a fusion protein as described herein comprising a protein tag as described herein comprising a POI; ii. contacting the fusion protein with circularly permuted caspase-2 (cp caspase-2) for a period of time sufficient for the cp caspase-2 to cleave the fusion protein, thereby liberating the POI; iii. optionally purifying the POI; Also provided is a method comprising:

[0083] According to certain embodiments, the method for generating a POI described herein comprises: i. cloning a nucleotide sequence encoding a fusion protein described herein, comprising a protein tag described herein, under the control of a promoter into an expression vector; ii. transforming a host cell with the vector; iii. culturing the transformed host cells under conditions in which the fusion protein is expressed; iv. optionally isolating said fusion protein from the host cell culture, optionally by disrupting the host cells; v. purifying the fusion protein using IMAC chromatography; vi. contacting the fusion protein with circularly permuted caspase-2 (cp caspase-2) for a period of time sufficient for the cp caspase-2 to cleave the fusion protein, thereby liberating the POI; vii. Optionally, further purifying the POI; viii. Optionally modifying the POI; ix. optionally formulating the POI; Includes:

[0084] Specifically, the promoter is selected from the group consisting of the T7 promoter / operator, the XylS / Pm regulator / promoter or a variant of the Pm promoter, the araBAD promoter / operator, the T5, T7A1, T7A2, T7A3 promoter / operator, the phoA promoter / regulator, and the trp promoter / operator system. [Brief explanation of the drawings]

[0085] [Figure 1] SEQ ID NOs for the amino acid and nucleotide sequences referred to herein. Bold and / or underlined letters in amino acid sequences indicate amino acid substitutions. [Figure 2]Schematic diagram of wild-type and circularly permuted caspase-2 structures: (A) human wild-type pro-caspase-2 (unprocessed) (SEQ ID NO: 11), (B) canonical cp-caspase-2 (SEQ ID NO: 6) based on human wild-type caspase-2 and containing a modified SS propeptide, a His tag, and a GS linker between the SS and LS, (C) canonical cp-caspase-2 (SEQ ID NO: 9) containing a modified SS propeptide and a GS linker between the SS and LS, and (D) canonical cp-caspase-2 (SEQ ID NO: 76). [Figure 3] Schematic representation of the mature enzymes of (A) human wild-type caspase-2, processed, (B) canonical cp-caspase-2 (SEQ ID NO: 6) based on human wild-type caspase-2 and containing a modified SS propeptide and a His tag and a GS linker between the SS and LS, (C) canonical cp-caspase-2 (SEQ ID NO: 9) containing a modified SS propeptide and a GS linker between the SS and LS, and (D) canonical cp-caspase-2 (SEQ ID NO: 76) containing a His tag and a GS linker between the SS and LS. [Figure 4] Standard cleavage assay using cp-caspase-2 (SEQ ID NO: 6) and VDVAD-E2 (SEQ ID NO: 33) with a P1' glycine. Lane 1: molecular weight marker; Lane 2: substrate cleavage after 1 minute of reaction time; Lane 3: substrate cleavage after 2.5 minutes of reaction time; Lane 4: substrate cleavage after 5 minutes of reaction time. E2: untagged E2. B: Standard cleavage assay using cp-caspase-2 (SEQ ID NO: 6) and VDVAD-SOD (SEQ ID NO: 193). Lane 1: molecular weight marker; Lanes 2-8: substrate cleavage after 0, 2, 3, 4, 5, and 6 hours of reaction time, respectively. Lanes 9-10: caspase-free substrate VDVAD-SOD incubated for 0 and 6 hours, respectively. 6His-SOD: SOD with an N-terminal 6His tag and the recognition site VDVAD fused directly to the N-terminus of SOD; SOD: untagged SOD. [Figure 5] cp Diagrammatic representation of the C-terminal sequence of caspase-2. [Figure 6]Alignment of the native sequences of homologous caspase-2 from different species (01 human (SEQ ID NO: 11), 02 mouse (SEQ ID NO: 89), 03 sheep (SEQ ID NO: 92), 03 Tasmanian devil (SEQ ID NO: 95), 05 chicken (SEQ ID NO: 98), 06 anole (SEQ ID NO: 101), 07 alligator (SEQ ID NO: 104), 08 xenopus (SEQ ID NO: 107), 09 danio (SEQ ID NO: 110), 10 chimaera (SEQ ID NO: 113), 11 ascidian (SEQ ID NO: 116)). The unprocessed protein consists of a large subunit (LS) containing a CARD domain, two catalytic centers, a small subunit propeptide (SS Propept), and a small subunit (SS). Active sites 1-5 interact with the substrate. [Figure 7] Alignment of the active sites of native caspase-2 sequences from different species. The active sites interact with the substrate and are relatively conserved. See Tables 3 and 4 for definitions of subunits and active sites. The number before the first active site indicates the starting position of the first active site. Bold letters in the amino acid sequences indicate amino acids that are identical in all species in each active site. [Figure 8] Michaelis-Menten kinetic parameters kcat / KM for cp caspase-2 and its variants: cpCasp2D (SEQ ID NO: 6), T7AC_cpCasp2D (SEQ ID NO: 41), S9 (SEQ ID NO: 51), G171D (SEQ ID NO: 190), T7AC_mS9ProE (SEQ ID NO: 71), T7AC_mS9ProD (SEQ ID NO: 72). [Figure 9] Cleavage of DEVD-E2 (SEQ ID NO: 57) by cp caspase-2 (SEQ ID NO: 6) and wild-type caspase-2: Decreased activity when DEVD is used instead of VDVAD as the recognition site for cp caspase-2 and wild-type caspase (DEVD-E2 instead of VDVAD-E2 as the substrate). [Figure 10]Laboratory-scale fermentation of E. coli BL21(DE3)(pET30a_6H-cpCasp2D) (A, two graphs on the left) and BL21(DE3)(pET30a_T7AC-6H-cpCasp2D) (B, two graphs on the right): Expression of soluble and insoluble 6H-cpCaspase-2D (cpCasp2) (A) and T7AC-6H-cpCaspase-2D (T7AC-6H-cpCasp2) (B) over time as specific yield [mg / g] and volumetric yield [g / L]: with (T7AC_6H-cpCasp2, B) and without (cpCasp2, A) the solubility tag T7AC. [Figure 11] Laboratory-scale fermentation of E. coli BL21(DE3)(pET30a_6H-cpCasp2D) and BL21(DE3)(pET30a_T7AC-6H-cpCasp2D): biomass process. [Figure 12] Biomass process of lab-scale fermentation of three cp-caspase-2 strains (cp-caspase-2, mS9 Pro D285E, and mS9 Pro D285) with and without the T7AC solubility tag in E. coli BL21(DE3) with the pET30a vector. Total CDM is shown as the average of all six fermentations with standard deviation compared to expected growth (calc.CDM). [Figure 13] Standardized soluble production of three different cp-caspase-2s (cp-caspase-2 (cpCasp2D), mS9 Pro D285E (mS9ProE), and mS9 Pro (mS9ProD)) with and without the T7AC solubility tag in E. coli BL21(DE3) harboring the pET30a vector. [Figure 14] Growth kinetics of E. coli BL21(DE3)(pET30a_T7AC_6H-cpCasp2D) in carbon-limited two-stage fed-batch culture (μ = 0.17 followed by 0.03 h-1 during induction) at three different IPTG induction intensities; process of CDM production; CDM in [g / L]. [Figure 15]E. coli BL21(DE3)(pET30a_T7AC_6H-cpCasp2D) in a carbon-limited, two-stage fed-batch culture (μ = 0.17 followed by 0.03 h-1 during induction) at three different IPTG induction intensities. Volumetric soluble cpCaspase-2 titers (sol.POI [g / L]) obtained by culturing at the lowest growth rate (μ = 0.03 h-1) and inducing with different IPTG levels. cpCaspase-2 was quantified by SDS-PAGE. Mean values ​​and standard deviations for individual determinations are shown (n = 3). [Figure 16] Exemplary Michaelis-Menten kinetics measured by FRET assay. [Figure 17] Cleavage kinetics for 2.9 g / L hFGF-2 fusion proteins incubated with 0.055 g / L T7AC_cpCasp2D (SEQ ID NO: 41), T7AC_mS9ProE (SEQ ID NO: 71) and T7AC_mS9ProD (SEQ ID NO: 72). [Figure 18] Cleavage kinetics for hFGF-2 fusion proteins incubated at varying concentrations with cp caspase-2 (cpCasp2, SEQ ID NO: 6). [Figure 19] Cleavage kinetics for 2.4 g / L of TNF-alpha fusion protein incubated with 0.046 g / L of cp caspase-2 (T7AC-cpCasp2D, SEQ ID NO: 41) or variant mS9 Pro D285E (T7AC_mS9ProE, SEQ ID NO: 71). [Figure 20] Cleavage kinetics for 9.1 g / L of GFP fusion protein incubated with 0.11 g / L of cp caspase-2 variant mS9 Pro D285E. (T7AC_mS9ProE, SEQ ID NO: 71). [Figure 21] Percentage of cleavage as described in Example 9.3.6 at varying residence times performed with hFGF-2 fusion protein as substrate with a concentration of 50 μM. [Figure 22]Direct comparison between T7AC-6H-cpCasp2D and T7AC-6H-mS9 ProD production during carbon-limited two-stage fed-batch cultivation (μ = 0.17 followed by 0.03 h-1 during induction) using a constant 0.9 μmol IPTG / g CDM:biomass process. [Figure 23] Direct comparison between T7AC-6H-cpCasp2D and T7AC-6H-mS9 ProD production during carbon-limited two-stage fed-batch cultivation (μ = 0.17 followed by 0.03 h during induction) with a constant 0.9 μmol IPTG / g CDM: Expression of soluble (sol) and insoluble (IB) cpCaspase-2 over time as specific yield [mg / g] (top) and volumetric yield [g / L] (bottom). [Figure 24] Direct comparison between T7AC-6H-cpCasp2D and T7AC-6H-mS9 ProD production during carbon-limited two-stage fed-batch cultivation (μ = 0.17 followed by 0.05 h-1 during induction) using a constant 0.9 μmol IPTG / g CDM:biomass process. [Figure 25] Direct comparison between T7AC-6H-cpCasp2D and T7AC-6H-mS9 ProD production during carbon-limited two-stage fed-batch cultivation (μ = 0.17 followed by 0.03 h during induction) with a constant 0.9 μmol IPTG / g CDM: Expression of soluble and insoluble cpCaspase-2 over time as specific yield [mg / g] (left) and volumetric titer [g / L] (right). [Figure 26] Laboratory-scale fermentation of E. coli BL21(DE3)(pET30a_casp2-6H): Expression of soluble and insoluble wild-type caspase-2 over time (23 and 29 hours after induction) assessed via Western blot using an anti-caspase-2 antibody. Lane 6: Positive control 6H-cpCasp2D (29 hours after induction, diluted 1:4). [Figure 27] Biomass process of laboratory-scale fermentation of 6H-cpCasp2D (6H-cpcaspase-2D) and wtcaspase-2-6H in E. coli BL21(DE3) with pET30a vector. [Figure 28] Biomass processing in benchtop fermentation of four different cp caspase homologues. [Figure 29] Benchtop fermentation of two different cp-caspase homologs: Expression of soluble and insoluble (IB) cp-caspase-2 homologs over time. Left: wild-type-like homolog, T7AC-6H-cpCasp2_sar; Right: P1'-permissive cp-caspase-2 variant, T7AC-6H-cpCasp2_sat_mut. [Figure 30] Comparison of different fermentation conditions and expression tags for the production of cpCaspase-2D (see Table 40): Titer (volumetric yield) of soluble POI cpCaspase-2D with either the 6H or T7AC-6H tag in [g / L]. 6H_cpCasp2D: 6H-cpCaspase-2D fermented as described in Example 9, Section 9.1.2.2; T7AC_6H_cpCasp2D: T7AC-6H-cpCaspase-2D fermented as described in Example 9, Section 9.1.2.2; DoE: T7AC-6H-cpCaspase-2D fermented as described in Example 9, Section 9.1.2.3; Optimization run: T7AC-6H-cpCaspase-2D fermented as described in Example 9, Section 9.1.2.9. [Figure 31] Pathway of fermentation of the fusion protein T7AC-6H-GSG-VDVAD-rhGH performed as described in Example 10, section 10.2 (the fusion protein is expressed with an N-terminal signal peptide (leader peptide), the ompA leader peptide, to direct the fusion protein into the periplasm of the host cell): Left: Formation of biomass (as CDM (cell dry mass)) in [g / L] compared to calculated CDM, Right: Volumetric titer of soluble fusion protein in [g / L]. [Figure 32] Pathway of the fermentation of the fusion protein T7AC-6H-GSG-VDVAD-PTH carried out as described in Example 10, section 10.2 and Table 53; left: formation of biomass (as CDM (cell dry mass)) in [g / L] compared to calculated CDM, right: volumetric titer of soluble fusion protein in [g / L]. [Figure 33]Pathway of the fermentation of the fusion protein T7AC-6H-GSG-VDVAD-TNFα carried out as described in Example 19, section 19.2 and Table 53; left: formation of biomass (as CDM (cell dry mass)) in [g / L] compared to calculated CDM, right: volumetric titer of soluble fusion protein in [g / L]. [Figure 34] Pathway of the fermentation of the fusion protein 6H-GSG-VDVAD-TNFα carried out as described in Example 19, section 19.2 and Table 53; left: formation of biomass (as CDM (cell dry mass)) in [g / L] compared to calculated CDM, right: volumetric titers of soluble and insoluble (IB) fusion protein in [g / L]. [Figure 35] Pathway of the fermentation of the fusion protein 6H-GSG-VDVAD-BIWA4 (scFv) performed as described in Example 19, section 19.2 and Table 53; left: formation of biomass (as CDM (cell dry mass)) in [g / L] compared to calculated CDM, right: volumetric titer of insoluble (IB) fusion protein in [g / L]. [Figure 36] Pathway of the fermentation of the fusion protein 6H-GSG-VDVAD-GFPmut3.1 (=6H-GSG-VDVAD-GFP) performed as described in Example 19, section 19.2 and Table 53; left: formation of biomass (as CDM (cell dry mass)) in [g / L] compared to calculated CDM, right: volumetric titers of soluble and insoluble (IB) fusion protein in [g / L]. [Figure 37] Pathways of fermentation of protein hFGF-2 and fusion proteins 6H-hFGF-2, 6H-GSG-VDVAD-hFGF-2, T7AC-6H-GSG-VDVAD-hFGF-2 and T7A3-6H-GSG-VDVAD-hFGF-2 performed as described in Example 19, Section 19.2 and Table 53; Biomass (as CDM (cell dry mass)) in [g / L] compared to calculated CDM. [Figure 38]Fermentation pathways of protein hFGF-2 and fusion proteins 6H-hFGF-2, 6H-GSG-VDVAD-hFGF-2, T7AC-6H-GSG-VDVAD-hFGF-2 and T7A3-6H-GSG-VDVAD-hFGF-2 performed as described in Example 19, Section 19.2 and Table 53; volumetric titers in [g / L] of soluble protein resp. fusion proteins. [Figure 39] Pathway of the fermentation of the fusion protein T7AC-6H-GSG-VDVAD-GCSF carried out as described in Example 19, section 19.2 and Table 53; left: formation of biomass (as CDM (cell dry mass)) in [g / L] compared to calculated CDM, right: volumetric titer of soluble fusion protein in [g / L]. [Figure 40] Comparison of Michaelis-Menten kinetics depending on the recognition site of the cleavage tag with T7AC-6H-mS9ProD. The gray trace and data points correspond to the cleavage kinetics of T7AC-6H-GSG-VDVAD-hFGF2. The black trace and data points correspond to the cleavage kinetics of T7AC-6H-GSG-VDSAD-hFGF2. Circles represent measured data, solid lines represent model fits, and dotted lines represent 95% confidence intervals of the model fits. [Figure 41] IMAC capture of 6H_GSG_VDVAD-TNFα. 3 L of cell lysis supernatant was loaded. [Figure 42] SDS-PAGE of 6H_GSG_VDVAD-TNFα IMAC capture. 1: Marker; 2: Cell lysis supernatant (1 to 5); 3: Flow-through (1 to 5); 4: Wash; 5–17: Elution fractions. [Figure 43] IMAC capture of T7AC_6H_GSG_VDVAD-TNFα. 3 L of cell lysis supernatant was loaded. [Figure 44]SDS-PAGE of T7AC_6H_GSG_VDVAD-TNFα IMAC capture. 1: Marker; 2: Cell lysis supernatant (1:5); 3: Flow-through (1:5); 4: Wash; 5-6: Elution fractions; 7: Elution fraction (1:2); 8-17: Elution fractions. The main peak in lanes 5-17 represents the fusion protein T7AC_6H_GSG_VDVAD-TNFα. [Figure 45] Comparison of Michaelis-Menten kinetics depending on the cleavage tag with 6H-cpCasp2D. The gray trace and data points correspond to the cleavage kinetics of T7AC-6H-GSG-VDVAD-hFGF2. The black trace and data points correspond to the cleavage kinetics of 6H-GSG-VDVAD-hFGF2. Circles represent measured data, solid lines represent model fits, and dotted lines represent the 95% confidence intervals of the model fits. [Figure 46] Comparison of Michaelis-Menten kinetics as a function of cleavage tag with T7AC-6H-cpCasp2D. Gray trace and data points correspond to the cleavage kinetics of T7AC-6H-GSG-VDVAD-hFGF2. Black trace and data points correspond to the cleavage kinetics of 6H-GSG-VDVAD-hFGF2. Circles represent measured data, solid lines represent model fits, and dotted lines represent 95% confidence intervals of the model fits. [Figure 47] Comparison of Michaelis-Menten kinetics depending on the cleavage tag with T7AC-6H-mS9ProD. The gray trace and data points correspond to the cleavage kinetics of T7AC-6H-GSG-VDVAD-hFGF2. The black trace and data points correspond to the cleavage kinetics of 6H-GSG-VDVAD-hFGF2. Circles represent measured data, solid lines represent model fits, and dotted lines represent 95% confidence intervals of the model fits. [Figure 48]Comparison of Michaelis-Menten kinetics depending on the cleavage tag with T7AC-6H-mS9ProE. The gray trace and data points correspond to the cleavage kinetics of T7AC-6H-GSG-VDVAD-hFGF2. The black trace and data points correspond to the cleavage kinetics of 6H-GSG-VDVAD-hFGF2. Circles represent measured data, solid lines represent model fits, and dotted lines represent 95% confidence intervals of the model fits. [Figure 49] Comparison of Michaelis-Menten kinetics depending on the cleavage tag with T7AC-6H-mS9ProD. The light gray trace and triangle data points correspond to the cleavage kinetics of T7AC-6H-GSGSGSG-VDVAD-hFGF2. The dark gray trace and square data points correspond to the cleavage kinetics of T7AC-6H-GSG-VDVAD-hFGF2. The black trace and circle data points correspond to the cleavage kinetics of T7AC-6H-VDVAD-hFGF2. Measured data are shown as circles, squares, or triangles; the solid line represents the model fit, and the dotted lines represent the 95% confidence interval of the model fit. [Figure 50]Cleavage reactions of T7AC_6H_GSG_VDVAD-hFGF2 and T7AC_6H_GSG_VDVAD-TNFα with T7AC_6H-cpCasp2D, T7AC_6H-mS9ProE, and T7AC_6H-mS9ProD. Lane 1: Marker; Lane 2: T7AC_6H_GSG_VDVAD-hFGF2; Lane 3: T7AC_6H_GSG_VDVAD-hFGF2 + T7AC_6H-cpCasp2D 100:1 (M / M) 1h; Lane 4: T7AC_6H_GSG_VDVAD-hFGF2 + T7AC_6H-mS9ProE 100:1 (M / M) 1h; Lane 5: T7AC_6H_GSG_VDVAD-hFGF2 + T7AC_6H-mS9ProD 100:1 (M / M) 1h; Lane 6: T7AC_6H_GSG_VDVAD-TNFα; Lane 7: T7AC_6H_GSG_VDVAD-TNFα + T7AC_6H-cpCasp2D Lane 8: T7AC_6H_GSG_VDVAD-TNFα + T7AC_6H-mS9ProE 100 to 1 (M / M) 1 h; Lane 9: T7AC_6H_GSG_VDVAD-TNFα + T7AC_6H-mS9ProD 100 to 1 (M / M) 1 h; Lane 10: T7AC_6H-cpCasp2D, T7AC_6H-mS9ProD, T7AC_6H-mS9ProE. The main peak in lane 2 represents the uncleaved fusion protein, T7AC_6H_GSG_VDVAD-hFGF2; the peaks in lanes 3-5, which have the same migration as the main peak in lane 2, represent the uncleaved fusion protein, T7AC_6H_GSG_VDVAD-hFGF2; the lower peaks in lanes 3-5, which have a migration of 14-17 kDa, represent the released protein of interest, hFGF-2. The main peak in lane 6 represents the uncleaved fusion protein, T7AC_6H_GSG_VDVAD-TNFα; the peaks in lanes 7-9, which have the same migration as the main peak in lane 6, represent the uncleaved fusion protein, T7AC_6H_GSG_VDVAD-TNFα; the lower peaks in lanes 7-9, which have a migration of 14-17 kDa, represent the released protein of interest, TNFα. [Figure 51]Cleavage reactions of T7AC_6H_GSG_VDVAD-rhGH and T7AC_6H_GSG_VDVAD-GCSF with T7AC_6H-cpCasp2D, T7AC_6H-mS9ProE, and T7AC_6H-mS9ProD. Lane 1: Marker; Lane 2: T7AC_6H_GSG_VDVAD-rHGH; Lane 3: T7AC_6H_GSG_VDVAD-rHGH + T7AC_6H-cpCasp2D 100:1 (M / M) 2h; Lane 4: T7AC_6H_GSG_VDVAD-rHGH + T7AC_6H-mS9ProE 100:1 (M / M) 2h; Lane 5: T7AC_6H_GSG_VDVAD-rHGH + T7AC_6H-mS9ProD 100:1 (M / M) 2h; Lane 6: T7AC_6H_GSG_VDVAD-GCSF; Lane 7: T7AC_6H_GSG_VDVAD-GCSF + T7AC_6H-cpCasp2D Lane 8: T7AC_6H_GSG_VDVAD-GCSF+T7AC_6H-mS9ProE 100 to 1 (M / M) 2 h; Lane 9: T7AC_6H_GSG_VDVAD-GCSF+T7AC_6H-mS9ProD 100 to 1 (M / M) 2 h; Lane 10: T7AC_6H-cpCasp2D, T7AC_6H-mS9ProD, T7AC_6H-mS9ProE. The main peak in lane 2 represents the uncleaved fusion protein, T7AC_6H_GSG_VDVAD-rhGH; the peaks in lanes 3-5, which have the same migration as the main peak in lane 2, represent the uncleaved fusion protein, T7AC_6H_GSG_VDVAD-rhGH; the lower peaks in lanes 3-5, which have a migration of approximately 17 kDa, represent the released protein of interest, rhGH. The main peak in lane 6 represents the uncleaved fusion protein, T7AC_6H_GSG_VDVAD-GCSF; the peaks in lanes 7-9, which have the same migration as the main peak in lane 6, represent the uncleaved fusion protein, T7AC_6H_GSG_VDVAD-GCSF; the lower peaks in lanes 7-9, which have a migration of 14-17 kDa, represent the released protein of interest, GCSF. [Figure 52]T7AC_6H_GSG_VDVAD-GCSF and T7AC_6H_GSG_VDVAD-PTH cleavage reactions with T7AC_6H-cpCasp2D, T7AC_6H-mS9ProE, and T7AC_6H-mS9ProD. Lane 1: Marker; Lane 2: T7AC_6H_GSG_VDVAD-GCSF; Lane 3: T7AC_6H_GSG_VDVAD-GCSF + T7AC_6H-cpCasp2D 50:1 (M / M) 2h; Lane 4: T7AC_6H_GSG_VDVAD-GCSF + T7AC_6H-mS9ProE 50:1 (M / M) 2h; Lane 5: T7AC_6H_GSG_VDVAD-GCSF + T7AC_6H-mS9ProD 50:1 (M / M) 2h; Lane 6: T7AC_6H_GSG_VDVAD-PTH; Lane 7: T7AC_6H_GSG_VDVAD-PTH + T7AC_6H-cpCasp2D 50 to 1 (M / M) 2h; Lane 8: T7AC_6H_GSG_VDVAD-PTH+T7AC_6H-mS9ProE 50 to 1 (M / M) 2h; Lane 9: T7AC_6H_GSG_VDVAD-PTH+T7AC_6H-mS9ProD 50 to 1 (M / M) 2h; lane 10: T7AC_6H-cpCasp2D, T7AC_6H-mS9ProD, T7AC_6H-mS9ProE. The main peak in lane 2 represents the uncleaved fusion protein, T7AC_6H_GSG_VDVAD-GCSF; the peaks in lanes 3-5, which have the same migration as the main peak in lane 2, represent the uncleaved fusion protein, T7AC_6H_GSG_VDVAD-GCSF; the lower peaks in lanes 3-5, which have a migration of approximately 14-17 kDa, represent the released protein of interest, GCSF; the main peak in lane 6 represents the uncleaved fusion protein, T7AC_6H_GSG_VDVAD-PTH; the peaks in lanes 7-9, which have the same migration as the main peak in lane 6, represent the uncleaved fusion protein, T7AC_6H_GSG_VDVAD-PTH; the lower peaks in lanes 7-9, which have a migration of 6-14 kDa, represent the released protein of interest, PTH. [Figure 53]IMAC capture of 6H_GSG_VDVAD_hFGF-2. Elution is observed between 80 and 100 mL. Although a split peak was observed, SDS-PAGE analysis revealed that both halves of the peak contained the majority of the fusion protein, 6H-GSG-VDVAD-hFGF-2. [Figure 54] Subtractive IMAC polish of 6H_GSG_VDVAD_hFGF-2. The product elutes during loading (approximately 0–15 mL). [Figure 55] SDS-PAGE of the hFGF-2 platform process. M: Marker; SN: Clarified lysis supernatant; CF: Capture IMAC flow-through; CWA: Capture IMAC wash; CEL: Capture IMAC eluate; BX: UF / DF buffer exchange; ETR: Enzymatic tag removal; SFT: Subtractive IMAC flow-through; SWA: Subtractive IMAC wash; SEL: Subtractive IMAC eluate. The main peaks of CEL and BX represent the uncleaved fusion protein 6H-GSG-VDVAD-hFGF2, and the main peaks of ETR and SFT represent hFGF2 with the native N-terminus from which the tag has been cleaved. [Figure 56] Intact mass spectra of hFGF-2 after tag removal and flow-through IMAC purification. (A) shows the full deconvoluted MS spectrum, and (B) shows the expanded spectrum. [Figure 57] Sequence logos of 79 selected recognition sites. Letter size represents the probability of the presence of amino acids at positions P1–P5 of the caspase recognition site. [Figure 58] Pathway of the fermentation of the fusion protein T7AC-6H-GSG-VDVAD-BIWA4 (scFv) performed as described in Example 19, section 19.2 and Table 53; left: formation of biomass (as CDM) in [g / L] compared to calculated CDM (cell dry mass), right: volumetric titer of insoluble (IB) fusion protein in [g / L]. [Figure 59]Cleavage of 1 mg / ml VDVAD-β-galactosidase (SEQ ID NO: 34) incubated with 0.1 mg / ml cp-caspase-2 (SEQ ID NO: 6) for 24 hours. "+" indicates incubation with cp-caspase, and "-" indicates incubation without cp-caspase. No nonspecific cleavage is observed in lane "+" as no additional band is seen in lane "-" compared to lane "-". Cleavage of the β-galactosidase fusion protein is not observed in this SDS-Page as the migration difference between the cleaved and uncleaved fusion protein is not detectable by this SDS-Page method. [Figure 60] Cleavage of 1 mg / ml VDTTD-E2 (SEQ ID NO: 19) fusion protein and 1 mg / ml VDVAD-E2 (SEQ ID NO: 33) fusion protein incubated with 0.003 mg / ml cp caspase-2 (SEQ ID NO: 6) for 30 minutes. DETAILED DESCRIPTION OF THE INVENTION

[0086] Unless otherwise specified or defined, all terms used herein have their ordinary meaning in the art, which will be apparent to those skilled in the art. For example, refer to standard manuals such as Sambrook et al., "Molecular Cloning: A Laboratory Manual" (2nd Ed.), Vols. 1-3, Cold Spring Harbor Laboratory Press (1989), Lewin, "Genes IV", Oxford University Press, New York, (1990), and Janeway et al., "Immunobiology" (5th Ed., or more recent editions, Garland Science, New York, 2001).

[0087] The claimed subject matter specifically refers to artificial products, or methods of using or producing such artificial products, which may be variants of native (wild-type) products. While a degree of sequence identity to native structures may exist, it will be appreciated that the materials, methods, and uses of the invention, specifically referring to isolated nucleic acid sequences, amino acid sequences, fusion constructs, expression constructs, transformed host cells, and engineered proteins, including enzymes, are "man-made" or synthetic and, therefore, are not considered the result of the "laws of nature."

[0088] As used herein, the terms "comprise," "containing," "having," and "include" can be used interchangeably and should be understood as an open definition allowing for additional members or parts or elements. "Consisting of" is considered the closest definition without additional elements of the defining feature it constitutes. Thus, "comprise" is broader and encompasses the definition of "consisting of." As used herein, the term "about" refers to the same value or a value that differs by + / - 5% from a given value. As used herein, amino acid refers to the 20 naturally occurring amino acids encoded by 61 triplet codons. These 20 amino acids can be divided into neutral, positive, and negatively charged amino acids.

[0089] The "neutral" amino acids are shown below with their respective three-letter and one-letter codes and polarities: Alanine: (Ala, A) nonpolar, neutral, Asparagine: (Asn, N) polar, neutral, Cysteine: (Cys, C) nonpolar, neutral, Glutamine: (Gln, Q) polar, neutral, Glycine: (Gly, G) nonpolar, neutral, Isoleucine: (Ile, I) nonpolar, neutral, Leucine (Leu, L) nonpolar, neutral, Methionine: (Met, M) nonpolar, neutral, Phenylalanine: (Phe, F) nonpolar, neutral, Proline: (Pro, P) nonpolar, neutral, Serine: (Ser, S) polar, neutral, Threonine: (Thr, T) polar, neutral, Tryptophan: (Trp, W) nonpolar, neutral, Tyrosine: (Tyr, Y) polar, neutral, Valine: (Val, V) nonpolar, neutral, and Histidine: (His, H) Polar, positive (10%) neutral (90%).

[0090] "Positively" charged amino acids are Arginine: (Arg, R) polar, positive, and Lysine: (Lys, K) polar, positive is. "Negatively" charged amino acids are Aspartic acid: (Asp, D) polar, negative, and Glutamic acid: (Glu, E) polar, negative is.

[0091] Caspases are key enzymes in the initiation and execution of apoptosis and inflammation, and therefore their activity must be tightly regulated. Although caspase sequences differ (e.g., human caspase-1 and -2 share only 27% amino acid identity and 52% similarity), their active sites and tertiary structures are highly conserved. All caspases are synthesized as relatively inactive single-chain zymogens (procaspases), containing a prodomain (2–25 kDa) and large and small subunits of 17–21 kDa and 10–13 kDa, respectively. Executioner caspases (caspase-3, -6, and -7) and caspase-14 have short prodomains, whereas all other caspases have long prodomains. To become fully active, wild-type caspases must first dimerize through hydrophobic interactions; then, their intersubunit linker is cleaved and the prodomain is removed by proteolytic cleavage after an aspartic acid residue. The main difference in activation between executioner and initiator caspases is that the latter are already active after dimerization, and autocatalytic separation of their subunits is only necessary for stabilization. Active wild-type caspases are homodimers of heterodimers. Each heterodimer consists of a large and a small subunit derived from a single protein chain. The enzyme is formed by a central 12-stranded β-sheet, to which each of the four subunits contributes. Four loops protrude from this core, which contain the active site and form a binding pocket. In all caspases, the catalytic center is located in the large subunit. The substrate recognition site is formed by amino acids from both subunits, but the small subunit contributes key residues, which are responsible for the different substrate specificities among caspases. Cleavage of the intersubunit linker triggers rearrangement of the active site loops, thereby forming a binding pocket and making the active cysteine ​​solvent accessible.

[0092] As used herein, the term "recognition site" or "caspase recognition site" refers to an amino acid sequence of at least three, preferably at least four or five, amino acid residues of a substrate that is specifically recognized by caspase-2 or cpcaspase-2 described herein. Specifically, at least three substrate amino acids that are targeted and bound by the caspases provided herein to form a recognition site are referred to as P3-P1 or P3 P2 P1, a recognition site containing four substrate amino acids is referred to as P4-P1 or P4 P3 P2 P1, a recognition site containing five substrate amino acids is referred to as P5-P1 or P5 P4 P3 P2 P1, a recognition site containing six substrate amino acids is referred to as P6-P1 or P6 P5 P4 P3 P2 P1, a recognition site containing seven substrate amino acids is referred to as P7-P1 or P7 P6 P5 P4 P3 P2 P1, etc. As described herein, the caspases provided herein interact with their substrates in a target-specific manner by specifically recognizing and binding to a recognition site comprising at least 3, 4, 5, 6, 7, 8, 9, 10, or more amino acid residues contained within the substrate's sequence. The amino acid residues of the recognition site occupy specific pockets on the caspase, numbered with a corresponding S designation (S1, S2, S3, S4, S5, etc., S1', S2', etc.), each of which can be constructed from several amino acid residues. The purpose of this interaction mode, which almost always binds the cleavage region in an extended peptide conformation, is to precisely align the substrate to match the catalytic mechanism.

[0093] Specifically, the caspase-2 or cp caspase-2 provided herein is not limited to the wild-type caspase-2 recognition site, VDVAD (SEQ ID NO: 45). Further provided herein are caspase-2 variants that target recognition sites other than VDVAD with high specificity and efficiency. Specifically, provided herein are caspase-2 variants that target any one or more of the recognition sites described herein. Preferably, the caspases described herein have high specificity for a single recognition site, although embodiments are contemplated in which a caspase recognizes multiple recognition sites, e.g., for cleavage at multiple sites in a single protein, or for simultaneous cleavage of different proteins containing different recognition sites.

[0094] The selection method described herein can be used to select caspase-2 variants that specifically recognize any one or more recognition sites. Specifically, any of the caspase variants described herein that contain any one or more of the amino acid substitutions that increase P1' tolerance as described herein can be subjected to the selection method described herein. In this way, for example, caspase-2 variants that contain increased P1' tolerance and target specificity for a specific recognition site can be selected. According to further particular embodiments, the recognition site specificity of the caspase variants described herein can be influenced by introducing amino acid substitutions, additions, or deletions known to increase or decrease specificity for a particular recognition site. Specifically, the caspase-2 or cp caspase-2 described herein recognizes a recognition site comprising the sequence XDXXD (SEQ ID NO: 201), where X can be any amino acid. Specifically, the recognition site can be selected from the group consisting of DEXD (SEQ ID NO: 202) and DVXD (SEQ ID NO: 203), where X is any amino acid.

[0095] According to specific examples, the caspase-2 or cp caspase-2 described herein recognizes any one or more of the recognition sites LDESD (SEQ ID NO: 204), DVAD (SEQ ID NO: 205), DEVD (SEQ ID NO: 206), DEVE (SEQ ID NO: 207), ADVAD (SEQ ID NO: 208), VDTTD (SEQ ID NO: 209), DTTD (SEQ ID NO: 210), DVPD (SEQ ID NO: 211), VDVPD (SEQ ID NO: 212), VDQQD (SEQ ID NO: 213), or TDTSD (SEQ ID NO: 214).

[0096] According to further specific examples, the caspase-2 or cpcaspase-2 described herein has the recognition sites DRKD (SEQ ID NO: 215), DAVD (SEQ ID NO: 216), VKVD (SEQ ID NO: 217), DTLD (SEQ ID NO: 218), EEPD (SEQ ID NO: 219), DETD (SEQ ID NO: 220), DATD (SEQ ID NO: 221), NKVD (SEQ ID NO: 222), DALD (SEQ ID NO: 223), DSVD (SEQ ID NO: 224), NAID (SEQ ID NO: 225), DKPD (SEQ ID NO: 226), IQLD (SEQ ID NO: 227), DNAD (SEQ ID NO: 228), DVVD (SEQ ID NO: 229), ENPD (SEQ ID NO: 230), DMAD (SEQ ID NO: 231), DMAD (SEQ ID NO: 232), DMAD (SEQ ID NO: 233), DMAD (SEQ ID NO: 234), DMAD (SEQ ID NO: 235), DMAD (SEQ ID NO: 236), DMAD (SEQ ID NO: 237), DMAD (SEQ ID NO: 238), DMAD (SEQ ID NO: 239), DMAD (SEQ ID NO: 240), DMAD (SEQ ID NO: 241), DMAD (SEQ ID NO: 242), DMAD (SEQ ID NO: 243), DMAD (SEQ ID NO: 244), DMAD (SEQ ID NO: 245), DMAD (SEQ ID NO: 246), DMAD (SEQ ID NO: 247), DMAD (SEQ ID NO: 248), DMAD (SEQ ID NO SEQ ID NO: 231), DLID (SEQ ID NO: 232), DGAD (SEQ ID NO: 233), DVKD (SEQ ID NO: 234), GYND (SEQ ID NO: 235), ELPD (SEQ ID NO: 236), DSTD (SEQ ID NO: 237), DRQD (SEQ ID NO: 238), HAVD (SEQ ID NO: 239), QERLD (SEQ ID NO: 240), LERD (SEQ ID NO: 241), MMPD (SEQ ID NO: 242), EEPD (SEQ ID NO: 243), VESID (SEQ ID NO: 244), EAMD (SEQ ID NO: 245), EDAD (SEQ ID NO: 246), EEED (SEQ ID NO: 247), AVLD (SEQ ID NO: 248), and / or EEGD (SEQ ID NO: 249).

[0097] According to further embodiments, the caspase-2 or cp caspase-2 described herein recognizes the recognition sites TDTSD, LDEPD (SEQ ID NO: 250), and / or KDEVD (SEQ ID NO: 251). As used herein, the term "cleavage site" refers to amino residues P1 / P1', where cleavage occurs at residue P1 at the amino terminal scissile bond and residue P1' on the carboxy terminal side.

[0098] Proteolytic cleavage of the substrate occurs after the P1 residue. Specifically, the amino acids after the P1 residue are referred to as the P1'-P4' residues, also known as the prime side. The prime side of the substrate, specifically the P1' residue, is important for substrate processing. Under certain conditions, the P1'-P4' residues can affect binding through steric hindrance. P1' residues close to the active site, especially branched (e.g., leucine or valine) and polar amino acids (e.g., threonine or aspartic acid) at this position, can compete spatially with the catalytic cysteine ​​and negatively affect cleavage.

[0099] Wild-type caspases have a strong preference for aspartic acid at the P1 position. The P2 and P3 positions are less selective and can accommodate a variety of residues, but many caspases have the highest activity when a glutamic acid residue is present at the P3 position. The P4 position is crucial for distinguishing between caspase classes: inflammatory caspases and caspase-14 prefer hydrophobic residues, initiator caspases and caspase-6 prefer aliphatic residues, and executioner caspases and wild-type caspase-2 prefer aspartic acid. While the prime position of the substrate has not been investigated as thoroughly, studies have shown that the P1' site influences cleavage, with certain residues reducing activity by up to 1000-fold. All wild-type caspases prefer substrates with small residues (glycine, serine, alanine), but large hydrophobic amino acids (phenylalanine, tyrosine) are also tolerated surprisingly well. It is highly likely that the P1' site is not required for efficient substrate binding, although certain residues can interfere with it. Prime sites further away from the cleavage site (P2'-P4') have less of an effect. However, because many proteins are processed at non-canonical sites, whether a substrate is cleaved by a caspase does not depend solely on the presence or absence of a recognition site. The secondary and tertiary structure of the substrate are very important in recognition. In vivo proteins are preferably cleaved at solvent-accessible loops, although a significant amount is also cleaved within α-helices.

[0100] Specifically, wild-type caspase-2 has a strong preference for a glycine residue at the P1' site. According to certain embodiments, cp caspase-2 and / or caspase-2 variants can be selected for increased P1' tolerance as described herein. Specifically, functionally active variants of cp caspase-2 having at least 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity with SEQ ID NO:6, preferably at least 85, 90, or 95% sequence identity with SEQ ID NO:6, include improved tolerance for P1' residues other than glycine. According to further specific embodiments, caspase-2 variants described herein can be selected for specific cleavage of recognition sites other than VDVAD.

[0101] Thus, there are no limitations regarding the substrate. Substrates for the caspases provided herein can be any protein or polypeptide, a naturally occurring protein or polypeptide that naturally contains a recognition site that is specifically targeted by the caspases described herein, or a heterologous protein or polypeptide that has been engineered to contain a recognition site within its sequence or at or near its N- or C-terminus. According to certain embodiments, the substrate comprises a protein of interest as described herein.

[0102] Caspase-2 was first described as an apoptotic protein in 1994 due to its similarity to the cell death protein CED-3 in Caenorhabditis elegans and human caspase-1. Procaspase-2 consists of a CARD followed by large and small subunits (see Figure 2A). Its structure is most similar to caspase-9; however, unlike other initiator caspases, caspase-2 does not activate executioner caspases. Instead, it initiates apoptosis by liberating cytochrome c from mitochondria, thereby initiating the intrinsic pathway of caspase-9 activation. Like all caspases, active caspase-2 is a dimer of heterodimers. The large (p19) and small (p12) subunits form the caspase heterodimer, which together comprise the complete enzyme. Wild-type caspase-2 contains two active sites, one in each heterodimer. The two wt heterodimers are linked by two cysteines in the small subunit (Cys in SEQ ID NO: 11). 436 ) are linked by disulfide bridges formed by the large and small subunits. No other caspases have such an intermolecular covalent bond that allows them to exist as stable dimers in solution. Interestingly, disulfide bridges can only form after separation of the large and small subunits via cleavage.

[0103] The substrate binding site of wild-type caspase-2 is primarily formed by three protein loops. The first loop (residues 212-221 of SEQ ID NO:11, large subunit) interacts with the substrate prime site (P1'-P4'), while the second loop (residues 373-382 of SEQ ID NO:11, small subunit) binds the entire substrate (P5-P4'). The third loop (residues 419-431 of SEQ ID NO:11, small subunit) interacts with the recognition site (P5-P1). Wild-type caspase-2 has a near-absolute requirement for aspartic acid residues at both the P1 and P4 positions of the recognition site, while many residues are tolerated at the P2 and P3 positions. The S1 pocket is positively charged, and the substrate residues are stabilized by two arginine residues. The S4 pocket is deep and narrow, and therefore highly specific. Similarly, the P3 and P5 residues bind with this unique pocket, while the P2 residue alone does not bind individually. Wild-type caspase-2 is unique in that it recognizes a pentapeptide rather than a tetrapeptide like all other caspases. VDVAD is considered the preferred cleavage site for wild-type caspase-2.

[0104] As used herein, the term "wild type" generally refers to the phenotype, genotype, or gene that predominates in a natural population of an organism or strain of an organism, as opposed to a naturally occurring or recombinant mutant variant. In other words, "wild type" refers to the form or forms of a gene that typically occur naturally in a given species. As used herein, the term "wild type" with respect to caspase-2 and cpcaspase-2 refers to the amino acid or nucleotide sequence of caspase-2, or a domain thereof, such as the small and large subunits, that typically occur naturally in different species. In the context of the present invention, the term "caspase" is understood to refer generally to "caspase-2" and functionally active variants thereof.

[0105] As used herein, the term "cp-caspase-2" refers to a circularly permuted caspase-2 described herein, which is a single-chain caspase-2 containing a small subunit N-terminal to the large subunit of caspase-2, as further described herein. "cp-caspase-2" includes the small and large subunits of caspase-2 from different species, as well as functionally active variants thereof. Specifically, wild-type caspase-2 from different species contains several, specifically four, domains. The terms "wild-type caspase-2" or "wt caspase-2" and "wild-type cp caspase-2" or "wt cp caspase-2" encompass wild-type caspase-2 sequences from different species and functionally active variants thereof. The wild-type caspase-2 described herein may contain one or more amino acid substitutions, deletions, and / or insertions that are conservative modifications and do not alter the protease function of the enzyme. The wild-type caspase-2 and wild-type cp caspase-2 described herein do not contain amino acid substitutions that increase P1' tolerance.

[0106] As used herein, the terms "caspase-2 variant" and "cp caspase-2 variant" refer to a variant of wild-type caspase-2 or wild-type cp caspase-2 that has increased proteolytic activity, particularly increased P1' tolerance, and contains specific amino acid substitutions as described herein. The terms "caspase-2" and "cpcaspase-2", unless otherwise specified, encompass both wild-type versions of the enzyme, either circularly permuted or not, and variant versions of the enzyme, either circularly permuted or not, that contain increased P1' tolerance as described herein.

[0107] The boundaries between the small and large subunits and other domains can be determined experimentally by amino acid sequence analysis of mature caspases or by structural homology (e.g., the conserved Asp-X cleavage site in humans, e.g., Asp in SEQ ID NO: 2). 14or Asp of SEQ ID NO: 11 347 ) by examination of the CARD, large subunit (LS), intervening sequence (small subunit propeptide), and small subunit (SS) domains of caspase-2 in various species. The amino acid positions in the table below refer to amino acid positions in SEQ ID NOs: 11, 89, 92, 95, 98, 101, 104, 107, 110, 113, and 116.

[0108] [Table 1]

[0109] The caspases described herein comprise at least a portion of a caspase-2 small subunit and at least a portion of a caspase-2 large subunit. In a preferred embodiment, a propeptide of the small caspase-2 subunit is also present. The prodomain (CARD) is generally not required for enzymatic activity and is usually free in vivo. Caspases of the present invention may have a prodomain or a portion thereof. The propeptide of the small subunit is optionally included in cp-caspase-2. Preferably, both subunits are derived from the same species, although combinations of subunits from different species may be used. As mentioned above, portions of the large subunit and portions of the small subunit may be used in the caspases described herein, although the active site is preferably not deleted when designing the caspase-2 and / or cpcaspase-2 described herein.

[0110] Caspase-2 is unique in the caspase family in that it contains the following consensus sequence (SEQ ID NO: 277): QXXRXCSSPRXCALVXSXVTXDPXXADPLDHXKXGEXXEEVXXKVXTEXDFVXSVHRXXXAQAMRXCIEQFCQLPXHRTADGXVXXXXXXXVDXAVYSXDXELLQXDWVFEAXDNSHXPLXQNXXXXXFVXXXXXEXM XXXVVQDTXPERTGSPSXEQRDAGREGEGDPGSRRPVSLGRPRIXLXQRSXMICGFASLKXQRLSTAAMXXTXRXXXXVXEXNEAXRLRSRDTHLADXXVQXXARIKXRXGXAPGTPHXRCXEMSEFTXSXCNDXFLF Caspase-2 is the initiator caspase, while caspase-3 and caspase-6 are effector caspases. The structure of caspase-2 is stabilized by disulfide bonds, and wt-caspase-2 is the only caspase with a recognition site containing five amino acid residues (Grinshpon et al., AC. Biochem J. 2019; 476(22):3475-3492).

[0111] In preferred embodiments, the non-circularly permuted caspase-2 variants described herein comprise at least a portion of the small caspase-2 subunit and at least a portion of the large caspase-2 subunit, and an amino acid substitution at any one or more of positions 212, 431, 213, 323, 266, 409, 226, 296, or 326 of SEQ ID NO: 11, or positions functionally equivalent to positions 212, 431, 213, 323, 266, 409, 226, 296, or 326 of SEQ ID NO: 11. Specifically, the caspase-2 variants comprise improved P1' tolerance, particularly for amino acids other than glycine at the P1' position, compared to the respective wild-type caspase-2.

[0112] Those skilled in the art will readily understand that each wild-type caspase-2 is a protein comprising the amino acid sequence of the caspase-2 from which the caspase-2 variant is derived. For example, the caspase-2 variants described herein that are of human origin comprise improved P1' tolerance compared to human wild-type caspase-2 comprising SEQ ID NO: 11. According to a further specific example, the caspase-2 variants described herein that are of chimaera origin and comprise amino acid substitutions at any one or more of positions 369, 391, 174, 187, 227, 257, 284, or 287 of SEQ ID NO: 113 comprise improved P1' tolerance compared to chimaera wild-type caspase-2 comprising SEQ ID NO: 113. According to further specific examples, caspase-2 variants described herein that are of Tasmanian devil origin and comprise amino acid substitutions at any one or more of positions 386, 408, 189, 190, 203, 243, 273, 300, or 303 of SEQ ID NO: 95 comprise improved P1' tolerance compared to Tasmanian devil wild-type caspase-2 comprising SEQ ID NO: 95.

[0113] Additionally, the caspase-2 variants described herein can include the amino acid sequence of homologous wild-type caspase-2 from several different species, including but not limited to, mouse, sheep, Tasmanian devil, chicken, anole, alligator, Xenopus, danio, chimaera, sea squirt, or any other species, as shown in SEQ ID NOs: 11, 89, 92, 95, 98, 101, 104, 107, 110, 113, 116, and in FIG. 6. Circular permutation (CP) was first discovered in natural proteins in 1979. Circularly permuted (CP) proteins arise through the covalent attachment of native N- and C-termini and the introduction of new termini through cleavage elsewhere in the protein. In nature, this occurs either through duplication / deletion or fission / fusion events at the gene level. The new variants have an altered amino acid order but maintain the same tertiary structure. Despite one published variant, uncleavable reverse caspase-3, all described reverse variants still cleave themselves at the intersubunit linker, making their structure more similar to the wild-type variant. Circularly permuted, constitutively active forms of caspases-7 and -14 have been published, but all cleave their intersubunit linkers. It was not anticipated that circular permutation of caspase-2 would be successful. Because the structure of the N-terminus of the large subunit has not been fully determined, it was unclear whether the two subunits of caspase-2 could be linked. It was therefore surprising that the circularly permuted variants of caspase-2 provided herein were found to be active, and even more surprising that they exhibited significantly improved characteristics, in particular higher P1' tolerance, higher specificity, higher catalytic efficiency, increased thermal tolerance and tolerance to chaotropic conditions, and significantly improved manufacturability over wild-type caspase-2.

[0114] As used herein, the term "circularly permuted caspase-2" or "cp caspase-2" refers to an engineered caspase-2 variant containing an altered amino acid order, specifically, the amino acid order is altered compared to that of wild-type caspase-2. The cp caspase-2 referred to herein is a protease in which the small caspase-2 subunit is N-terminal to the large caspase-2 subunit. Specifically, the amino acid order is altered by linking the native N-terminus of LS to the C-terminus of SS and introducing a new terminus by cleavage elsewhere in the protease. The cp caspase-2 provided herein comprises, from N to C-terminus, the following structure: a small caspase-2 subunit or a functionally active variant thereof, covalently linked to the large caspase-2 subunit or a functionally active variant thereof, either directly or via a linker. The structure of the cp caspase-2 described herein is illustrated in Figures 2B, 2C, and 2D. Optionally, the two subunits are linked via a linker sequence of up to 12 or even more amino acids, so long as the remaining cp caspase 2 is still a functionally active variant of caspase 2 or cp caspase 2.

[0115] Therefore, a caspase-2 was engineered whose scaffold was altered by circular permutation, i.e., covalent ligation of the wild-type N- and C-termini and creation of new N- and C-termini by intramolecular cleavage of the protein backbone at different positions, resulting in an exchange of the arrangement of swapped domains, i.e., small and large caspase subunits, and which is active without the need for a processing step like wild-type executioner or apoptotic caspases.

[0116] The circularly permuted caspase-2 described herein includes wild-type caspase-2 sequences that do not have the amino acid alterations but have an altered order, and circularly permuted variants of wild-type caspase-2 that differ from the wild-type caspase-2 sequence by one or more amino acid substitutions, deletions, additions, etc., and that include increased proteolytic activity, particularly increased P1' tolerance.

[0117] Variants of the caspase and cp-caspase genes provided herein can be engineered, synthesized, or constructed from natural variants (e.g., polymorphisms, splice variants, mutants). Many methods have been developed for generating mutants (see generally Sambrook et al., supra; Ausubel, et al., supra). Briefly, a preferred method for generating several nucleotide substitutions utilizes an oligonucleotide spanning the mutated base or bases and containing the mutated base or bases. The oligonucleotide is hybridized with a complementary single-stranded nucleic acid, and second-strand synthesis is primed from the oligonucleotide. The double-stranded nucleic acid is prepared for transformation into a host cell, typically E. coli, although other prokaryotes, yeast, or other eukaryotes can alternatively be used. Standard screening and vector growth protocols are used to identify mutant sequences and obtain high yields. Similarly, deletions and / or insertions in the caspase-2 or cp-caspase-2 gene can be constructed by any of a variety of known methods, such as those described herein. For example, gene can be digested with restriction enzyme and re-ligated to delete sequence, or re-ligated with additional sequence to make insertion or large substitution.Other means of generating variant sequence can be used together with methods known in the art.Verification of variant sequence is typically achieved by restriction enzyme mapping, sequence analysis or probe hybridization.

[0118] Specifically, the cp caspases of the present invention are produced by rearranging the gene sequence of the caspase-2 gene so that the nucleic acid sequence encoding the small subunit precedes (is 5' to) the nucleic acid sequence encoding the large subunit. In particular, the wild-type cp caspase-2 or cp caspase-2 variants described herein are of animal origin, in particular mammalian, reptilian, or fish origin, in particular derived from caspase-2 of human (SEQ ID NO: 11), mouse (SEQ ID NO: 89), sheep (SEQ ID NO: 92), Tasmanian devil (SEQ ID NO: 95), chicken (SEQ ID NO: 98), anole (SEQ ID NO: 101), alligator (SEQ ID NO: 104), Xenopus (SEQ ID NO: 107), Danio (SEQ ID NO: 110), Chimaera (SEQ ID NO: 113), or Ascidian (SEQ ID NO: 116). Preferably, the cp caspase-2 described herein is derived from caspase-2 of human, marsupial, iguana, Tasmanian devil, chimaera, or cartilaginous fish.

[0119] According to certain embodiments, the wild-type cp caspase-2 or cp caspase-2 variants described herein comprise sequences that share greater than 80 or 90%, specifically at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%, sequence identity with the active site, such as sequences including SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, and SEQ ID NO: 50. Preferably, the cp caspase-2 described herein shares at least 90, 95, or more sequence identity with SEQ ID NOs: 46-50.

[0120] According to further specific embodiments, the wild-type cp caspase-2 or cp caspase-2 variant described herein comprises at least 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity to SEQ ID NO: 64 (Tasmanian devil, Tasmanian devil), SEQ ID NO: 66 (Anolis carolinensis), or SEQ ID NO: 68 (elephant shark, chimaera). According to a preferred embodiment, the wild-type cp caspase-2 or cp caspase-2 variant described herein comprises the amino acid sequence of SEQ ID NO: 9 or a functional variant thereof having at least 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity to SEQ ID NO: 9. Specifically, the cp caspase-2 described herein has the amino acid sequence of SEQ ID NO: 9 or a functionally active variant thereof comprising one or more amino acid substitutions or deletions, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, additions, or deletions.

[0121] According to a further preferred embodiment, the wild-type cp caspase-2 or cp caspase-2 variant described herein comprises the amino acid sequence of SEQ ID NO: 6 or a functional variant thereof having at least 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity to SEQ ID NO: 6. Specifically, the cp caspase-2 described herein has the amino acid sequence of SEQ ID NO: 6 or a functionally active variant thereof comprising one or more amino acid substitutions or deletions, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, additions, or deletions.

[0122] According to a further preferred embodiment, the wild-type cp caspase-2 or cp caspase-2 variant described herein comprises the amino acid sequence of SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76 or SEQ ID NO:77, or a functional variant thereof having at least 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity to SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76 or SEQ ID NO:77. Specifically, the cp caspase-2 described herein has the amino acid sequence of SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, or SEQ ID NO:77, or a functionally active variant thereof containing one or more amino acid substitutions or deletions, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, additions, or deletions, etc.

[0123] According to certain embodiments, the caspase-2 variants described herein comprise one or more amino acid substitutions at a position functionally equivalent to any of positions 171, 105, 172, 282, 225, 83, 185, 255, or 285 of SEQ ID NO:6, such as positions 171, 105, 172, 282, 225, 83, 185, 255, or 285 of SEQ ID NO:6, or positions 409, 431, 212, 213, 266, 226, 296, 323, or 326 of SEQ ID NO:11. The term "functionally equivalent" as used herein with respect to amino acid substitutions refers to amino acids at positions corresponding to positions in the sequence of caspase-2 from different species. Specifically, "functionally equivalent" means that the variants of caspase-2 or cpcaspase-2 described herein contain amino acid substitutions at positions numbered with respect to SEQ ID NO: 6 that are considered to correspond to the substitutions described herein and that have the same functional role in the variant. Specifically, amino acid substitutions at positions functionally equivalent to the amino acid substitutions described herein confer improved P1' tolerance to the variant.

[0124] Generally, functionally equivalent substitution mutations occur at homologous amino acid positions in the amino acid sequence of caspase-2. Thus, the term "functionally equivalent" as used herein encompasses "positionally equivalent" or "homologous" mutations to a given mutation, regardless of whether the specific function of the mutated amino acid is known. Positionally equivalent or homologous amino acid residues can be identified based on sequence alignment and / or molecular modeling.

[0125] By way of example, the residues shown in Table 63 below have been identified as positionally equivalent and / or functionally equivalent to positions 171, 105, 172, 282, 225, 83, 185, 255, and 285 of SEQ ID NO: 6. One of skill in the art would readily understand how to identify positionally equivalent and / or functionally equivalent positions in caspase-2 sequences of other species for the amino acid substitutions described herein.

[0126] [Table 2]

[0127] According to specific examples, the caspase-2 and cp-caspase-2 homologs described herein were constructed similarly to caspase-2 or cp-caspase-2 of human origin. For example, the wild-type sequences of caspase-2 in each species, such as Tasmanian devil caspase-2 (Tasmanian devil, UniProtKB14 ID G3VQP7) and chimaera caspase-2 (elephant shark, UniProtKB14 ID V9KZT1), were used to determine the caspase-2 subunits (see Figure 6: alignment: domain start region). Depending on the desired caspase structure, the order of the large and small subunits can be swapped to create a constitutively active circularly permuted caspase. Specifically, to ensure expression as a single-chain protein, in cases where the caspase is a cp-caspase containing a small subunit propeptide, the aspartic acid in the propeptide of the small subunit (Asp in the wild-type sequence of human caspase-2) is replaced with an aspartic acid in the propeptide of the small subunit (Asp in the wild-type sequence of human caspase-2). 343 (corresponding to ) is mutated, for example, to alanine to avoid cleavage of the propeptide. Additionally, the protein sequence may be codon-optimized for expression in a desired prokaryotic host, such as E. coli, and linker and / or tag sequences may be added. Exemplary variants resulting are Tasmanian devil cp caspase-2 (SEQ ID NO: 64) and elephant shark cp caspase-2 (SEQ ID NO: 68). In this specific example, residue Glu in cp caspase-2 (SEQ ID NO: 6) 105 and Glu 172 was inserted into Tasmanian devil cp caspase-2 to generate variant Tasmanian devil cp caspase-2 E105V, E172V (SEQ ID NO: 78). In a further specific example, Glu in cp caspase-2 (SEQ ID NO: 6) 105 and Gly 171 Mutations at the corresponding positions were inserted into elephant shark cp caspase-2 to generate variant elephant shark cp caspase-2 E105V, G171D (SEQ ID NO: 79).

[0128] Surprisingly, the amino acid substitutions described herein confer improved P1' tolerance to the caspase-2 and cpcaspase-2 described herein. As used herein, the term "improved P1' tolerance" refers to increased proteolytic activity of a caspase with respect to one or more P1' residues. For example, while wild-type human caspase-2 prefers glycine at the P1' position, the caspases and cpcaspases described herein can perform proteolytic cleavage at cleavage sites containing P1' residues other than glycine with increased activity compared to wild-type caspase-2 or cpcaspase-2 not containing the amino acid substitutions described herein. Specifically, the caspase-2 variants described herein contain improved P1' tolerance compared to the respective wild-type caspase-2. Those skilled in the art will readily understand that the respective wild-type caspase-2 is a protein containing the amino acid sequence of the caspase-2 from which the caspase-2 variants are derived. For example, caspase-2 variants described herein that are of human origin, particularly cp caspase-2s described herein, such as cp caspase-2s comprising SEQ ID NO: 70, comprise improved P1' tolerance compared to human cp caspase-2s comprising SEQ ID NO: 6. Specifically, caspases of the invention comprise at least a 5, 10, 25, 50, 75, or 100%, or greater, increase in proteolytic activity for at least one amino acid residue at the P1' position compared to cp caspase-2s that do not comprise the amino acid substitutions described herein.

[0129] As noted above, the cp caspase-2 and caspase-2 provided herein include at least the small caspase-2 subunit and the large caspase-2 subunit. As used herein, the term "small caspase-2 subunit" refers to a small subunit derived from caspase-2 that is covalently linked to a large caspase-2 subunit also derived from caspase-2, optionally with the two subunits linked via a linker sequence comprising one or more, up to 12 or more amino acids, so long as the remaining cp caspase-2 remains caspase-2 or a functionally active variant of cp caspase-2. According to a specific example, the small subunit of cp caspase-2 is derived from wild-type caspase-2 spanning amino acid residues 348 to 452 of the amino acid sequence of wild-type caspase-2 (SEQ ID NO: 11). Specifically, the small caspase-2 subunit comprises the amino acid sequence of SEQ ID NO:3, or a variant thereof having at least 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity to SEQ ID NO:3.

[0130] Specifically, a variant of the small caspase-2 subunit described herein is functionally active when direct or indirect fusion or combination with the large caspase-2 subunit or a combination with a variant thereof results in a functionally active caspase-2 variant. Specifically, the small subunit of cp caspase-2 described herein comprises the amino acid sequence 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:112, SEQ ID NO:115, SEQ ID NO:118, or a functionally active variant thereof comprising at least 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, or 98% sequence identity.

[0131] As used herein, the term "modified small caspase-2 subunit propeptide" refers to a propeptide of the small subunit of caspase-2 that has been modified at its C-terminus. According to a specific example, the propeptide of the small subunit of cp caspase-2 is derived from wild-type caspase-2 spanning amino acid residues 334-347 of the amino acid sequence of wild-type caspase-2 (SEQ ID NO: 11), including an amino acid substitution or deletion of at least one residue at its C-terminus. Specifically, the propeptide of the small subunit described herein comprises the amino acid sequence of SEQ ID NO: 2, where X can be any amino acid, preferably not D, preferably not E, and even more preferably A, or a variant thereof having one, two, or three amino acid substitutions or one, two, or three amino acid deletions or additions.

[0132] As used herein, the term "large caspase-2 subunit" refers to a large subunit derived from caspase-2, which is covalently linked to a small caspase-2 subunit also derived from caspase-2, optionally linked via a linker sequence. According to a specific example, the cp caspase-2 large subunit is derived from wild-type caspase-2 spanning amino acid residues 170-333 of the amino acid sequence of wild-type caspase-2 (SEQ ID NO: 11). Specifically, the large caspase-2 subunit comprises the amino acid sequence of SEQ ID NO: 4, or a variant thereof having at least 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity to SEQ ID NO: 4. Specifically, a variant of the large caspase-2 subunit described herein is functionally active when direct or indirect fusion or combination with a small caspase-2 subunit or combination with a variant thereof results in a functionally active caspase-2 variant.

[0133] Specifically, the large subunit of cp caspase-2 described herein comprises the amino acid sequence of SEQ ID NO:4, SEQ ID NO:90, SEQ ID NO:93, SEQ ID NO:96, SEQ ID NO:99, SEQ ID NO:102, SEQ ID NO:105, SEQ ID NO:108, SEQ ID NO:111, SEQ ID NO:114, SEQ ID NO:117, or a functionally active variant thereof comprising at least 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, or 98% sequence identity. Further provided herein are functionally active variants of the cp caspase-2 described herein that are essentially identical to the above-described cp caspase-2 but differ from its polypeptide or nucleotide sequence, respectively, in that they are derived from homologous sequences in different species.

[0134] As used herein, the term "catalytically active" refers to the ability of a caspase described herein to catalyze the hydrolysis of a peptide bond in a substrate. Caspases are endopeptidases that can force the formation of a tetrahedral intermediate by promoting cysteine ​​residues to act as nucleophiles for cleaving their substrates. Specifically, the cp caspase-2 described herein is catalytically active and can specifically cleave its substrate at the caspase recognition site described herein. Specifically, the cp caspase-2 described herein is catalytically active when dimerized, comprising two single-chain cp caspase-2 units described herein. Specifically, the cp caspase-2 described herein is catalytically active regardless of proteolytic cleavage of its subunits or propeptides; more specifically, the small caspase-2 subunit propeptide is not cleaved at its C-terminus. Thus, the cp caspase-2 described herein is not a zymogen because it does not require activation through cleavage, either through an activating enzyme or through autocatalytic cleavage.

[0135] Activation of wild-type caspase-2 requires cleavage at the C-terminus of the large subunit and the resulting separation of the small and large subunits. In mature wild-type caspase-2, the propeptide of the small subunit is removed by cleavage at its C-terminus. Surprisingly, the cp caspase-2 described herein does not require intersubunit cleavage for activation. Despite modifications that prevent intersubunit cleavage and separation of the C-terminus of the propeptide of the small subunit, the single-chain cp caspase-2 described herein is catalytically active. Specifically, the two single-chain cp caspase-2s described herein dimerize via a covalent bond, specifically via one or more disulfide bonds, although dimerization can also be independent of disulfide bonds.

[0136] Specifically, the catalytic efficiency of a protease is defined as the rate of hydrolysis, which is expressed by the Michaelis-Menten equation (k cat / K M ) can be determined using the Michaelis constant K M is equal to the substrate concentration at which the enzyme converts substrate to product at half its maximum rate and is therefore related to the affinity of the substrate for the enzyme. The catalytic constant (k cat ) is the rate of product formation when the enzyme is saturated with substrate and therefore reflects the maximum rate of the enzyme. The rate of product formation depends both on how well the enzyme binds the substrate and how quickly the enzyme converts the substrate to product after binding. A low K M The equation with the value V is the reaction rate at which the reaction reaches its maximum rate of reaction, V. max It approaches the target more quickly and therefore exhibits a high binding affinity. M The equation with V is such that the enzyme does not bind the substrate very efficiently and V is only when the substrate concentration is high enough to saturate the enzyme. max This means that the catalytic rate constant (k cat ) measures the number of substrate molecules turned over by the enzyme per second. Then, k cat The reciprocal of k is the time it takes for the enzyme to turn over a substrate molecule. catThe higher the k, the more substrate is turned over per second. cat K M Dividing by gives a measure of enzyme efficiency, k cat has a high turnover and K M can be increased by being a small number.

[0137] Specifically, comparison of catalytic efficiency constants is used as a measure of an enzyme's preference for various substrates, i.e., substrate specificity: the higher the specificity constant, the more the enzyme "prefers" that substrate. Specifically, the catalytic activity of the caspase-2 or cpcaspase-2 described herein can be measured by examining the cleavage of a caspase substrate. Specifically, the cleavage activity of the caspases described herein can be examined by methods well known in the art. According to a specific example, but not limited to, the cleavage of a caspase substrate can be examined visually on an SDS-PAGE gel or by densitometric scanning. Specifically, the catalytic activity of the caspases described herein is analyzed using SDS-PAGE to separate the cleaved and uncleaved substrates from the caspase, and the band intensity of the cleaved substrate is determined to evaluate the percentage of cleavage product at a specific time point. Specifically, the caspase and substrate are mixed, samples are taken at regular intervals, and the reaction is stopped. Preferably, to standardize the process, only samples with approximately 50% of the substrate cleaved are used.

[0138] According to a further embodiment, the cleavage activity of the caspases described herein is determined using a Forster Resonance Energy Transfer (FRET) assay. In another embodiment, but not limited to, cleavage of a caspase substrate can be measured by measuring the increase in fluorescence when a peptide substrate comprising a caspase recognition sequence, a fluorophore, and a quencher as described herein is cleaved by the caspase. Specifically, the caspase and substrate are mixed at defined concentrations, and the increase in fluorescence is monitored for a period of time. This increase in fluorescence can be used to calculate the rate of product formation, which is then used to fit Michaelis-Menten kinetics. The resulting Michaelis-Menten parameter k cat and K. M can be used to define the catalytic efficiency of a caspase.

[0139] As used herein, the term "single-chain" refers to a polypeptide comprising a linear chain of amino acids. Proteins contain at least one long polypeptide, specifically a polypeptide comprising a linear chain of more than 100 amino acids. Short polypeptides, containing fewer than 20-30 residues, are generally referred to as peptides or oligopeptides. Individual amino acid residues are linked to each other by peptide bonds and adjacent amino acid residues. The sequence of amino acid residues in a protein is determined by the sequence of a gene, which is encoded by the genetic code. Specifically, as used herein, the term "single-chain" refers to a protein that is active regardless of proteolytic cleavage within its amino acid sequence.

[0140] In fully mature wild-type caspase-2, the small subunit is reduced to the p14 to p12 chains by cleavage after the recognition site CEESD (residues 343-347 of SEQ ID NO: 11, residues 17-21 of SEQ ID NO: 6). The propeptide of the small subunit of wild-type caspase-2 is thus separated from the small subunit by proteolytic cleavage after the recognition site CEESD. According to certain embodiments, the C-terminal amino acid of the small subunit propeptide of cp-caspase-2 is modified to prevent separation of the propeptide of the small subunit of cp-caspase-2 from the small subunit. Specifically, amino acid residue 21 of SEQ ID NO: 6 is substituted with any amino acid except aspartic acid (D) or glutamic acid (E). Specifically, the amino acid residues of SEQ ID NO: 6 are selected from the group consisting of alanine (A), arginine (R), asparagine (N), cysteine ​​(C), glutamine (Q), glycine (G), histidine (H), isoleucine (I), leucine (L), lysine (K), methionine (M), phenylalanine (F), proline (P), serine (S), threonine (T), tryptophan (W), tyrosine (Y), and valine (V). Specifically, the C-terminal aspartic acid of the propeptide of the small subunit of cp-caspase-2 is substituted with any amino acid residue other than aspartic acid or glutamic acid, preferably with alanine, to ensure expression of the cp-caspase-2 described herein as a single protein chain.

[0141] As described herein, the caspases of the present invention can be used to generate proteins of interest (POI) that contain an authentic N-terminus. As used herein, the term "authentic N-terminus" refers to the desired N-terminus of a protein generated using the means provided herein. In other words, a protein contains an authentic N-terminus if it contains an N-terminus designed to be generated by the methods of recombinant protein generation described herein. An authentic N-terminus may be the N-terminus naturally occurring in the protein to be generated, or the authentic N-terminus may be artificially designed, i.e., an N-terminus not naturally occurring in the protein. In a specific example, the P1' residue is the N-terminal amino acid of the POI, and cleavage by a caspase described herein generates an authentic N-terminus.

[0142] Low cleavage efficiency of substrates with suboptimal P1' residues or recognition sites can be problematic for applications, particularly large-scale applications, where authentic N-termini of the product are desired. Specifically, the caspases described herein have such high activity and efficiency that substrates with all P1' residues are still cleaved within a reasonable time frame, even in large-scale processes. For example, histidine is 50 times less tolerant than glycine, yet the cpCaspase-2 described herein can cleave 90% of substrates containing histidine at the P1' site within 2 hours at 25°C. In a more specific example, when the concentration of cpCaspase-2 is increased, even 50% of substrates with an isoleucine P1' residue can be cleaved within 2 hours.

[0143] By way of specific example, variants of caspase-2 containing improved P1' tolerance with increased specificity for the predetermined recognition sites described herein can be generated by screening for cp caspase-2 that can efficiently cleave substrates containing branched (Thr, Leu, Val, Ile) and acidic (Asp, Glu) residues as well as amino residues at their P1' site that are poorly tolerated by cp caspase-2, such as Gln and Pro.

[0144] For example, a circularly permuted catalytic subunit of aspartate transcarbamoylase (cpATCase), which possesses a new N-terminus on a beta-strand located within the protein, is used to select for variants of the caspase described herein that contain desired features, such as increased P1' tolerance or different or improved recognition site specificity. The respective E. coli gene is named pyrB, and its gene product forms a complex quaternary structure with the regular subunit pyrl with a stoichiometry of three regular subunit dimers and two catalytic subunit trimers. This cp enzyme is used to detect specific proteases by growth in E. coli. Because the enzyme can no longer fold correctly due to space restrictions within the protein, fusion of any stretch of amino acids toward this new N-terminus inactivates the enzyme. However, providing a protease capable of precisely cleaving off this additional stretch of amino acids reactivates the enzyme. Because this enzyme is essential for pyrimidine synthesis in E. coli, this reactivation can be used to exert strong selective pressure. E. coli mutants are provided that lack native ATCase (e.g., by deleting pyrB and pyrl) and carry a plasmid encoding cpATCase, e.g., cp-pyrB and pyrl, provided on a single vector, and are inhibited by an N-terminal fusion sequence carrying a protease recognition site. Thus, the E. coli mutant becomes a pyrimidine auxotroph, viable only on media supplemented with pyrimidines or when the cells are complemented with a vector encoding ATCase. cpATCase can be activated by catalytic (in vivo) cleavage of the N-terminal fusion sequence. E. coli can grow if the respective proteases are provided on additional plasmids. This allows for the selection of proteases that specifically recognize the recognition site in the N-terminal fusion and / or have increased tolerance for a particular P1' residue, e.g., proline (P).

[0145] According to certain embodiments, the caspase-2 or cp caspase-2 described herein comprises significantly improved specificity for recognition sites other than VDVAD compared to wild-type caspase-2.

[0146] As used herein, the term "linker" refers to any amino acid sequence that does not interfere with the function of the elements to which it is linked. A linker may link, for example, a nucleotide sequence or an amino acid sequence. A linker can be used between the small and large subunits of cpCaspase-2, or between caspase-2 or cpCaspase-2 and an N- or C-terminal tag, or between tag sequences. Linkers can also be used in the fusion proteins described herein. Linkers can be used to engineer an appropriate amount of flexibility. Preferably, linkers are short, e.g., 1 to 20 nucleotides or amino acids or many more, and are typically flexible. Commonly used amino acid linkers consist of several glycines, serine, and optionally alanine, in any order. Such linkers usually have a length of at least one of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 20 amino acids, as appropriate. Preferably, the linker contains 1 to 20 amino acid residues, and preferably is a short linker. Preferably, the linker is a GS, GGSGG (SEQ ID NO: 278), GSAGSAAGSG (SEQ ID NO: 279), (GS)n, GSGSGSG (SEQ ID NO: 280), GSG, or GGGGS (SEQ ID NO: 281) linker, or any combination thereof. In some embodiments, the linker comprises one or more units, repeats, or copies of a motif, such as, for example, GS, GSG, or G4S.

[0147] According to certain embodiments, the caspases and / or fusion proteins described herein comprise one or more N-terminal and / or C-terminal tag sequences. Such tag sequences can contain any number of amino acids, including more than 2, 5, or 10 amino acids, and 20, 50, 100, 200, or more amino acids. Specifically, the tag sequences used herein can be any tag sequence known to those skilled in the art. Specifically, the tag sequences used herein are selected from affinity tags, solubility-enhancing tags, or monitoring tags. Specifically, any tag with any function known in the art can be fused to caspase-2 or cpcaspase-2.

[0148] Affinity tags are, for example, amino acid sequences that can be used for the purification of proteins to which they are attached (e.g., fusion proteins with affinity tags at their N-terminus). These affinity tags have high affinity for suitable ligands of solid supports, such as chromatography resins, or directly for the resin. The selective binding of affinity-tagged fusion proteins to specific resins allows for highly efficient purification of the fusion protein and / or caspase (caspase-2, cpcaspase-2) in only a single chromatography step. According to certain embodiments, the affinity tag sequence used herein is selected from histidine (His) tags, particularly polyhistidine tags, arginine tags, particularly polyarginine tags, peptide substrates for antibodies, chitin-binding domains, RNAase S peptides, protein A, β-galactosidase, FLAG tags, Strep II tags, streptavidin-binding peptide (SBP) tags, calmodulin-binding peptide (CBP), glutathione S-transferase (GST), maltose-binding protein (MBP), S-tags, HA tags, c-Myc tags, or any other tags known to be useful for efficient purification of proteins fused to the tag. Preferably, the tag is a His tag containing one or more Hs, particularly a hexahistidine tag. Specifically, fusion proteins containing poly- or hexahistidine tags (His-tags) can be captured and purified by IMAC, preferably using Ni-NTA chromatography materials.

[0149] The solubility-enhancing tag can be fused C- or N-terminally to the POI and / or caspase (caspase-2, cpcaspase-2, wild-type or variant) described herein. The solubility-enhancing tag can increase the titer of the soluble fusion protein and / or caspase (caspase-2, cpcaspase-2) when expressed in a host cell, e.g., a bacterial cell, e.g., E. coli, particularly in the cytosol of E. coli, compared to expression of the untagged protein. According to further specific embodiments, the solubility-enhancing tag sequence used herein is selected from calmodulin-binding peptide (CBP), poly-Arg, poly-Lys, G B1 domain, protein D, the Z domain of Staphylococcus aureus protein A, and thioredoxin, or any other tag known to improve the solubility of a protein to which the tag is fused, e.g., during expression in a host cell. Preferably, the solubility tag is based on a highly charged peptide from a bacteriophage gene, such as, for example, the gene listed in US Pat. No. 8,535,908. Specifically, the solubility-enhancing tag sequence is selected from the group consisting of T7C, T7B, T7B1, T7B2, T7B3, T7B3, T7B4, T7B5, T7B6, T7B6, T7B7, T7B8, T7B9, T7B10, T7B11, T7B12, T7B13, T7A, T7A1, T7A2, T7A3, T7A4, T7A5, T7AC T3, N1, N2, N3, N4, N5, N6, N7, calmodulin-binding peptide (CBP), poly-Arg, poly-Lys, G B1 domain, protein D, the Z domain of staphylococcal protein A, DsbA, DsbC, and thioredoxin.

[0150] Preferably, the solubility-enhancing tag is selected from the group consisting of a T7A3 tag and a T7AC tag. According to certain embodiments, the tag is a modified T7A3 tag, herein referred to as T7AC (SEQ ID NO: 43). Preferably, one or more T7A3 (SEQ ID NO: 37) and / or T7AC (SEQ ID NO: 43) tags or functional variants thereof having 1 to 5 amino acid substitutions, additions, deletions, etc. are used.

[0151] According to further particular embodiments, the monitoring tag sequence used herein is m-Cherry, GFP or f-actin or any other tag useful for detection or quantification of caspases and / or fusion proteins during production, including fermentation, isolation and purification by simple in-situ, in-line online or at-line detectors such as UV, IR, Raman, fluorescence, etc.

[0152] The caspases and fusion proteins described herein may contain any number of tag sequences in any order and combination. Specifically, the caspases and fusion proteins described herein may contain one or more tag sequences of the same functionality, for example, more than one affinity tag, for example, two or more T7AC tags, or different functionality, for example, a T7AC affinity tag and an m-Cherry monitoring tag. Specifically, the caspases or fusion proteins described herein may contain an affinity tag, a solubility-enhancing tag, and a monitoring tag in any order, optionally separated by a linker sequence. For example, the caspases or fusion proteins described herein may contain an affinity tag and a solubility-enhancing tag, where the affinity tag is preferably a hexahistidine tag and the solubility-enhancing tag is preferably a T7AC tag. By way of further example, the tag sequences may be separated by a linker sequence described herein, which may contain a recognition site for specific cleavage by the caspases described herein.

[0153] The term "functional variant" or "functionally active variant" includes naturally occurring allelic variants as well as mutants or any other non-naturally occurring variants. As known in the art, an allelic variant, also called a homolog, is another form of a nucleic acid or peptide characterized by one or more nucleotide or amino acid substitutions, deletions, or additions that do not essentially alter the biological function of the nucleic acid or polypeptide. Specifically, a functional variant may include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid residue substitutions, deletions, and / or additions, or combinations thereof, where the substitutions, deletions, and / or additions are conservative modifications that do not alter the function of the enzyme. Specifically, functional variants described herein contain up to or including 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid substitutions, deletions, and / or additions, which are conservative modifications and do not alter the function of the enzyme. Specifically, functionally active variants described herein contain up to 15, preferably up to 10 or 5, amino acid substitutions, deletions, and / or additions, which are conservative modifications and do not alter the function of the enzyme.

[0154] Specifically, the functionally active variants described herein comprise at least 5%, or at least 10, 20, 30, 40, 50, 60, 70, 80, or 90% or more of the proteolytic activity of cp caspase-2 comprising SEQ ID NO:6 in the recognition site VDVAD, where glycine (G) is at the P1' position. Specifically, the functionally active variants described herein comprise at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or at least 90% or more of the proteolytic activity of cp caspase-2 comprising SEQ ID NO:6 in the recognition site VDVAD of the substrate VDVAD-E2 (SEQ ID NO:33). Specifically, the proteolytic activity is determined using a Forster resonance energy transfer (FRET) assay.

[0155] Functional variants may be obtained by sequence alterations in a polypeptide or nucleotide sequence, for example, by one or more point mutations, which, when used in conjunction with the present invention, retain or improve the function of the unaltered polypeptide or nucleotide sequence. Such sequence alterations include, but are not limited to, (conservative) substitutions, additions, deletions, mutations, and insertions. Conservative substitutions are substitutions that occur within a family of amino acids that are related in their side chains and chemical properties. Examples of such families are amino acids with basic side chains, acidic side chains, nonpolar aliphatic side chains, nonpolar aromatic side chains, uncharged polar side chains, small side chains, and large side chains.

[0156] Point mutation is specifically understood as the manipulation of a polynucleotide that results in the expression of an amino acid sequence that differs from the unmanipulated amino acid sequence by one or more single (non-conservative) or double substitutions or exchanges, deletions or insertions of amino acids for different amino acids. The term "sequence identity" as used herein is understood to refer to the relationship between two amino acid sequences or two nucleotide sequences, and is described by the degree of sequence identity or sequence complementarity. The sequence identity of a variant, homologue, or ortholog compared to a parent nucleotide or amino acid sequence indicates the degree of identity between two or more sequences. Two or more amino acid sequences may have, to a certain extent, up to 100%, the same or conserved amino acid residues at corresponding positions. Two or more nucleotide sequences may have, to a certain extent, up to 100%, the same or conserved base pairs at corresponding positions.

[0157] Sequence similarity searching is an effective and reliable strategy for identifying homologs with excessive (e.g., at least 50%) sequence identity. Commonly used sequence similarity searching tools include BLAST, FASTA, and HMMER. Sequence similarity searching can identify such homologous proteins or polynucleotides by detecting statistically significant similarities that reflect excess similarity and common ancestry. Homologues can encompass orthologs, which are understood herein as the same protein in different organisms, e.g., variants of such proteins in different organisms or species. To determine the % complementarity of two complementary sequences, one of the two sequences must be converted to its complementary sequence before the % complementarity can then be calculated as the % identity between the first sequence and the second converted sequence using the algorithm described above.

[0158] With respect to the amino acid sequences, homologs, and orthologs described herein, "percent (%) identity" is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in a particular polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and without considering any conservative substitutions as part of the sequence identity. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. For purposes described herein, sequence identity between two amino acid sequences can be determined using NCBI BLAST, specifically NCBI BLAST+ 2.9.0 program version (Apr-02-2019).

[0159] For example, " percent (%) identity " in the nucleotide sequence of nucleic acid molecule or its part, particularly in the coding DNA sequence, is defined as the percentage of nucleotides in the candidate DNA sequence that are identical to the nucleotides in the DNA sequence, after aligning the sequences and introducing gaps as necessary to achieve maximum percent sequence identity, and without considering any conservative substitutions as part of sequence identity.Alignment for determining percent nucleotide sequence identity can be achieved in various ways within the skill of the art, for example, by using publicly available computer software.Those skilled in the art can determine the appropriate parameters for measuring alignment, including any algorithms required to achieve maximum alignment over the entire length of the sequences being compared.

[0160] Optimal alignment may be determined using any suitable algorithm for aligning sequences, non-limiting examples of which include algorithms based on the Smith-Waterman algorithm, the Needleman-Wunsch algorithm, the Burrows-Wheeler Transform (e.g., Burrows Wheeler Aligner), ClustalW, Clustal X, BLAT, Novoalign (Novocraft Technologies; available at novocraft.com), ELAND (Illumina, San Diego, CA), SOAP (available at soap.genomies.org.cn), and Maq (available at maq.sourceforge.net).

[0161] According to certain embodiments, the caspases provided herein are used for the production of mature and / or functional proteins or polypeptides of interest. Specifically described herein is a process for producing a mature protein or polypeptide by producing the protein or polypeptide as a fusion protein comprising an N-terminal fusion sequence, the fusion sequence containing an engineered recognition site that is specifically recognized by a caspase described herein, such that cleavage of the protein by the caspase liberates the mature and / or functional protein of interest.

[0162] Fusion protein strategies to enhance expression levels, improve solubility, and facilitate target protein purification have existed since 1983 and even before. However, these strategies have not been widely used, and adapting fusion protein strategies for large-scale process development is challenging due to the specificity, activity, availability, and purity of the protease enzymes used. Specificity must be high enough to cleave only at engineered cleavage sites in the linker sequence connecting at least some of the proteins. Enzyme activity must be high enough to allow sufficient cleavage within a short period of time. This avoids hold-up times during production and minimizes target protein degradation during incubation. Proteases must be available at low cost, necessitating efficient expression systems and low-cost production methods. Proteases should also be sufficiently pure, free from even traces of nonspecific proteases, especially from the host organism. No protease meets these requirements for every conceivable target protein; therefore, a simple and effective method for adapting proteases to different POIs is needed.

[0163] Specifically described herein is a method for producing a POI having a predetermined N-terminal amino acid residue, comprising expressing the POI as a fusion protein in a host cell, wherein the N-terminus of the POI is fused to a fusion sequence containing a caspase recognition site, and the fusion protein is specifically cleavable by cpCaspase-2 described herein at the junction of the linker and the N-terminal amino acid residue of the POI. Specifically, the host cell does not express an endogenous functional protease capable of cleaving the fusion protein at the recognition site. According to the method specifically described herein, the fusion protein is isolated from the host cell and contacted with an extract containing cpCaspase-2 described herein, which cleaves the fusion protein precisely at the junction of the linker and the N-terminal amino acid residue of the POI, thereby producing a mature POI. Specifically, the extract containing the caspase is derived from cells that produce the caspase by recombinant DNA techniques.

[0164] There are no limitations regarding the protein or polypeptide of interest (POI). More specifically, the protein may be a polypeptide not naturally occurring in the host cell, i.e., a heterologous protein, or may be native to the host cell, i.e., a homologous protein to the host cell, but is produced, for example, by recombinantly integrating one or more copies of a nucleic acid sequence encoding the cognate POI into the genome or chromosome of the host cell, or by recombinantly modifying a promoter sequence controlling expression of the gene encoding the POI. By way of further example, the POI can also be expressed in a host using a vector, more specifically, a plasmid. The POI can be monomeric, dimeric, or multimeric, and can be homomeric or heteromeric. Examples of proteins that can be produced by the methods of the present invention include, but are not limited to, enzymes, regulatory proteins, receptors, growth factors, hormones, peptides, e.g., peptide hormones, cytokines, membrane, or transport proteins. The POI can also be an antigen, vaccine, antigen-binding protein, immunostimulatory protein, interleukin, interferon, allergen, full-length antibody or antibody fragment or derivative, or affinity scaffold used for vaccination. Antibody derivatives include, for example, single chain variable fragments (scFv), Fab fragments or single domain antibodies or camelid antibodies or heavy chain antibodies or derivatives thereof, such as V HH Fragments and the like, but are not limited to these.

[0165] As used herein, the term "fusion protein" refers to a POI that comprises an engineered fusion sequence at its N- or C-terminus, which comprises a caspase recognition site as described herein. Specifically, the fusion sequence described herein comprises at least one caspase recognition site, one or more tag sequences as described herein, and optionally one or more linker sequences as described herein. According to a specific example, the fusion protein comprises one or more tag sequences, one or more caspase recognition sites, and one or more POIs, which are optionally linked by a linker sequence.

[0166] According to certain embodiments, the fusion proteins provided herein comprise a first portion comprising one or more tag sequences, optionally linked by a linker sequence; a second portion comprising a recognition site for target-specific protein cleavage using cpCaspase-2 as described herein; and a third portion comprising a POI. Specifically, the fusion proteins described herein may comprise each portion more than once and in different orders. For example, the fusion proteins provided herein may comprise a first portion comprising a tag sequence, a second portion comprising a caspase recognition site, another first portion comprising the same or a different tag sequence, another second portion comprising the same or a different recognition site, and a third portion comprising a POI. By way of further example, the fusion proteins described herein may comprise more than one POI separated by one or more fusion sequences comprising one or more recognition sites. The cp caspase-2 or caspase-2 itself described herein can be part of a fusion protein or part of a fusion sequence, for example as a POI, to promote the production of the caspase itself.

[0167] The fusion proteins described herein are encoded by heterologous genes that have been engineered so that the gene is translated into a protein by the host organism. Any living cell or organism can be used as the host organism. The living cell or organism can be prokaryotic or eukaryotic. Typical host cells for recombinant gene expression include, for example, Escherichia coli, Bacillus species, Streptomyces species, yeasts such as Saccharomyces, Schizosaccharomyces, Pichia, Kluyveromyces, or Hansenula species, insect cells, mammalian cell lines, and plant cells. Expression hosts can also be multicellular organisms such as transgenic plants, sheep, goats, cows, chickens, and rabbits, allowing products to be isolated from organs or body fluids such as milk, blood, or eggs. Alternatively, genes can be translated into proteins using cell-free translation systems, possibly coupled with an in vitro transcription system. These systems provide all the steps necessary to obtain proteins from DNA by supplying the necessary enzymes and substrates in an in vitro reaction. In principle, any living cell or organism can provide the enzymes necessary for this process, and extraction protocols for obtaining such enzyme systems are known in the art. Common systems used for in vitro transcription / translation are extracts or lysates from reticulocytes, wheat germ, or E. coli.

[0168] According to certain embodiments, the fusion protein is isolated and purified before cleavage with cpCaspase-2 as described herein. The physicochemical characteristics of the fusion sequence, including one or more tag sequences, can be used for homogeneous, efficient, and highly specific purification of the fusion protein. The characteristics of the fusion sequence should be considered for adsorption chromatography media or specific affinity purification methods. For example, tag sequences that increase binding to ion exchange columns (e.g., polyarginine), hydrophobic interaction columns (e.g., polyphenylalanine), or immobilized metal chelation columns (e.g., polyhistidine) can be included. Other non-limiting examples include fusion proteins or domains with affinity for substrates or ligands, such as maltose-binding protein MBP, glutathione S-transferase GST, protein A, biotinylated peptides or domains, and chitin-binding domain CBD. Further non-limiting examples include the use of tag sequences that increase solubility at elevated temperatures (e.g., thioredoxin) or that reversibly precipitate under certain conditions. Purification strategies based on the properties of the fusion sequence will likely be applicable to the entire fusion protein. Such combinations of specific purification methods can be used when the fusion sequences contain different tag sequences with different functionalities or when the fusion sequences exhibit different selective behavior on different chromatographic media.

[0169] According to certain embodiments, the number of steps required for maturation of the fusion protein, subsequent removal of the enzyme, and removal of the fusion sequence cleaved from the fusion protein can be reduced. If an affinity tag is incorporated into the fusion sequence, the same affinity tag can be fused to a caspase, for example, by recombinant DNA technology. Using this strategy, the fusion protein can be captured on a solid support, for example, a chromatography column, and then incubated with the cpCaspase-2 described herein fused to an affinity tag that exhibits affinity for the same solid support. After a suitable incubation period, the liquid phase of the reaction vessel will contain the protein of interest, and both the fusion moiety and the enzyme will be adsorbed to the solid phase.

[0170] According to further specific embodiments, cleavage of the fusion protein is induced in vivo. Cleavage in the cell has the advantage of not requiring post-production processing. However, the advantage of specific affinity purification based on the properties of the fusion moiety is lost in this case. Specifically, two alternative strategies are applicable. First, the caspase can be induced simultaneously with the fusion protein, for example, by using an expression cassette containing both the caspase and the fusion protein, or by engineering a fusion protein containing the caspase as part of the fusion protein, or by using an expression vector containing the caspase and the fusion protein under separate promoters that are simultaneously induced. The latter can be achieved by using the same promoter in the two transcription cassettes, or by using two promoters that are induced with the same inducer (e.g., IPTG / lactose), or by using two promoters that are inducible with different agents and to which both agents are added simultaneously. Alternatively, the caspase enzyme can be induced at a time different from the initiation of fusion protein production. The caspase can be produced before or, more preferably, after the initiation of fusion protein production. In the latter case, the target protein is more likely to fold into a soluble, active protein.

[0171] The term "mature form" or "mature protein" of interest refers to a polypeptide of interest in its desired form, free of prepeptides, leader sequences, or fusion sequences. Preferably, in its mature form, the protein begins with the amino-terminal amino acid or ends with the carboxy-terminal amino acid of the POI present in its biologically active or functional form. Specifically, the mature protein includes an authentic N- or C-terminus, which is the desired N- or C-terminus.

[0172] Further provided herein are methods for producing the cp caspase-2 or fusion proteins described herein. The cp caspase-2 produced by the methods described herein may comprise SEQ ID NO: 6 or contain amino acid substitutions related to SEQ ID NO: 6. Specifically, the cp caspase-2 may be derived from wild-type caspase-2.

[0173] Specifically, the fusion protein comprises a POI, which may be caspase-2 as described herein, and a protein tag as described herein. The use of a protein tag as described herein significantly increases expression of the fusion protein and improves production of the POI. Specifically, the methods for producing cp caspase-2 described herein allow for more efficient production of caspases, and the caspases produced by the methods include improved characteristics, such as, for example, improved P1' tolerance or improved target specificity.

[0174] By way of specific example, but not limitation, the wild-type or mutant caspase-2 or cp caspase-2 or fusion protein described herein is produced in a fermentation process that includes two steps: i. Biomass production: For biomass production up to a certain concentration of biomass, the first fed-batch stage is 0.05-0.5 hours. -1 or 0.05 to 0.4 o'clock -1 The specific growth rate (μ) is preferably 0.07 to 0.3 hr. -1 or 0.1 to 0.3 o'clock -1 Or 0.1 to 0.2 o'clock -1 and more preferably 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19 or 0.20. -1 Preferably, the μ is about 0.13 to 0.21 hr. -1 , and more preferably about 0.16 to 0.18 hours -1 and most preferably, the specific growth rate is about 0.17 hr -1In addition, any other feeding mode suitable for the formation of a certain amount of biomass can be applied, such as, but not limited to, step feeding, linear increase feeding, or constant feeding. The substrate feeding is performed by the exponential growth algorithm, X = X0, using a superposition feedback control of the mass loss in the substrate tank. * The substrate supply can be controlled by increasing the pump rate according to eμt. Specifically, the substrate supply contains glucose or glycerol or any other carbon source, and optionally contains Ca. 2+ , Mg 2+ and / or trace elements. In a preferred embodiment, the first fed-batch stage is carried out for 0.5 to 2.5 generations, more preferably for 0.7 to 2.3 generations. ii. A second fed-batch stage using exponential feeding (exponential substrate feeding) at a specific growth rate (μ) was performed at an even lower growth rate, 0.01–0.1 h . -1 or 0.01 to 0.07 o'clock -1 μ is preferably 0.01 to 0.03 o'clock -1 or 0.01 to 0.05 o'clock -1 or 0.02 to 0.05 o'clock -1 or 0.03 to 0.05 o'clock -1 or 0.03 to 0.07 o'clock -1 , 0.05~0.07 hours -1 μ of about 0.03, 0.05 or 0.07 hr -1A μ of about 0.10, 0.15, 0.20, 0.25, 0.30, 0.35, 0.40, 0.45, or 0.50 generations can be applied. For adaptation to low growth conditions, cells can initially be grown at low μ without induction, for example, for about 0.10, 0.15, 0.20, 0.25, 0.30, 0.35, 0.40, 0.45, or 0.50 generations. Subsequently, an inducer, e.g., IPTG for the T7 promoter / operator system, can be added. Isopropyl-β-d-1-thiogalactopyranoside (IPTG) is a molecular biology reagent. This compound is a molecular mimic of allolactose, a lactose metabolite that triggers transcription of the lac operon, and is used to induce protein expression whose genes are under the control of the lac operator. Induction can be carried out for one or two or more generations at different or varying IPTG concentrations ranging from 0.01 to 1.5 or 0.1 to 1.5 μmol / g CDM (cell dry mass), more preferably 0.1 to 1.3 or 0.2 to 1.3 or 0.3 to 1.3 or 0.5 to 1.3 μmol / g CDM, and more preferably about 0.5 to 0.9 μmol / g CDM or about 0.9 to 1.3 μmol / g actual CDM, preferably about 0.5, 0.9 or about 1.3 μmol / g CDM. Specifically, the fed-batch stage is carried out at 30°C.

[0175] Therefore, induction can be performed as follows: Induction is started in the fed-batch phase by adding a feed medium containing IPTG (so-called "overfed" induction, Table 20), reaching the final IPTG concentrations mentioned above in μmol IPTG / g theoretical CDM at the end of the fermentation. In another embodiment, the IPTG (µmol / g CDM) corresponding to the CDM at induction can be injected into the reaction vessel, and then the IPTG calculated for the actual CDM can be fed into the feed medium in the fermenter. To that end, the required IPTG can be transferred into a feed bottle calculated for the required IPTG up to the theoretical CDM at the end of the fermentation. Thus, the IPTG concentration relative to the theoretical CDM remains constant throughout the entire fermentation.

[0176] The resulting caspase or fusion protein can be isolated by cell disruption, for example, by high-pressure homogenization, centrifugation of cell debris, and concentration of the supernatant by tangential flow ultrafiltration. Further purification can be achieved by chromatography, such as ion exchange chromatography, hydrophobic interaction chromatography, size exclusion chromatography, isoelectric focusing, mixed-mode chromatography, reversed-phase high-performance chromatography, tangential flow ultrafiltration, depth filtration, sulfate, chloride, or ammonium citrate precipitation, heat precipitation, solubilization, crystallization, centrifugation, and the like. Specifically, when cp caspase-2 contains an affinity tag, it can be highly effectively purified by only one chromatography step, which is an affinity chromatography step. Preferably, the affinity tag is a 6His tag and the affinity chromatography is IMAC, more specifically, Ni-NTA chromatography.

[0177] Specifically, the methods can be used to produce cp caspase-2 with or without the tags and / or linkers described herein. Specifically, cp caspase-2 produced by the methods described herein contains significantly improved specificity for the recognition site VDVAD (SEQ ID NO:45) compared to wild-type caspase-2. Specifically, cp caspase-2 containing the exemplary amino acid sequences SEQ ID NO:6, SEQ ID NO:9, SEQ ID NO:13, SEQ ID NO:35, SEQ ID NO:39, or SEQ ID NO:41 recognizes and cleaves substrates containing the recognition site VDVAD with significantly improved specificity compared to wild-type caspase-2. Such increased specificity has the distinct advantage that it significantly reduces off-target effects and avoids proteolytic cleavage of target substrates or other proteins at sites other than the recognition site within the host. Specifically, the cp caspase-2 described herein is at least two-fold, and preferably at least three-fold, more specific for the recognition site VDVAD than wild-type caspase-2.

[0178] Further provided herein is a method for producing POI using the protein tag described herein.Specifically, POI is fused to the protein tag and cloned into an expression vector under operable linkage with a promoter, and the promoter can be an inducible promoter.The expression vector is incorporated into host cells, and the host cells are cultured under conditions that allow the expression of fusion protein, optionally followed by a growth step for the accumulation of biomass before recombinant protein is expressed.POI can be produced using the fed-batch process described herein, including the expression step described herein and optionally the growth step described herein.

[0179] According to certain embodiments of the methods for producing a POI described herein, the fusion protein is contacted with caspase-2 or cpcaspase-2 described herein after expression to produce a POI comprising a desired N-terminus, i.e., a native or engineered N-terminus free of any undesirable tags. Specifically, the fusion protein is contacted with a caspase enzyme after isolating the fusion protein from a host cell culture. Following production of the POI by the methods described herein, the POI may be further modified, purified and / or formulated.

[0180] The methods described herein specifically refer to the production of heterologous compounds. When used in reference to a nucleotide or amino acid sequence or protein, such terms refer to compounds that are foreign, i.e., "exogenous," to a given host cell, e.g., not naturally found, or that are naturally found in a given host cell, e.g., "endogenous," but that are therefore "non-naturally occurring" in the context of, for example, a heterologous construct using heterologous nucleic acid. Endogenously found heterologous nucleotide sequences may be produced in cells in unnatural, e.g., greater amounts than expected or found in nature. A heterologous nucleotide sequence, or a nucleic acid containing a heterologous nucleotide sequence, may differ in sequence from the endogenous nucleotide sequence but encode the same protein as found endogenously. Specifically, a heterologous nucleotide sequence is one that is not naturally found in the same relationship (i.e., "not naturally associated") in a host cell. Any recombinant or artificial nucleotide sequence is understood to be heterologous.

[0181] As used herein, the term "host cell" refers to one or more cells that can be used in the methods described herein. Typically, this term refers to a living cell capable of growth in cell culture into which a heterologous nucleic acid or amino acid sequence has been introduced. Specifically, the host cell is selected from the group consisting of bacterial cells, yeast cells, insect cells, mammalian cells, and plant cells. Mammalian cells used in accordance with the present disclosure are typically human or rodent cells, such as mouse, rat, or hamster cells, e.g., Chinese hamster ovary (CHO) cells. Preferably, the host cell is a bacterial or yeast cell selected from the group consisting of Escherichia coli, Pseudomonas species, Bacillus species, Streptomyces species, Saccharomyces species, Schizosaccharomyces species, Pichia species, Kluyveromyces species, and Hansenula species.

[0182] The term "expression" is understood as follows: For example, nucleic acid molecules containing the desired coding sequence of an expression product, such as the fusion protein described herein or the cp caspase-2 described herein, can be used for expression purposes. Hosts transformed or transfected with these sequences can produce the encoded protein. To effect transformation, the expression system can be included in a vector, but the associated DNA can also be integrated into the host chromosome. Specifically, the term refers to a host cell and a compatible vector under appropriate conditions for expression of a protein encoded by, for example, foreign DNA carried by the vector and introduced into the host cell.

[0183] Coding DNA is a DNA sequence that encodes a specific amino acid sequence for a specific polypeptide or protein. Promoter DNA is a DNA sequence that initiates, regulates, or otherwise mediates or controls the expression of coding DNA. Promoter DNA and coding DNA may be from the same gene or different genes, and may be from the same or different organisms. Recombinant cloning vectors often contain one or more replication systems for cloning or expression, one or more markers for selection in the host, e.g., antibiotic resistance, one or more nuclear localization signals (NLS), and one or more expression cassettes.

[0184] As used herein, "expression vector" or "vector" is defined as a DNA sequence required for the transcription of a cloned recombinant nucleotide sequence, i.e., the transcription of a recombinant gene and the translation of its mRNA, in a suitable host organism. To achieve expression, a sequence encoding a desired expression product, such as the fusion protein described herein or the cp caspase-2 described herein, is typically cloned into an expression vector containing a promoter that directs transcription. Suitable bacterial and eukaryotic promoters are well known in the art. The promoter used to direct the expression of a nucleic acid depends on the specific application. For example, strong constitutive promoters are typically used for the expression and purification of fusion proteins. In contrast, when the expression product is to be administered in vivo for gene regulation, constitutive or inducible promoters can be used, depending on the specific use of the expression product. Furthermore, a weak promoter may be preferred for administration. The promoter may also contain elements that respond to transactivation, such as a hypoxia response element, a Gal4 response element, and a lac repressor response element. An expression vector comprises an expression cassette, and typically further comprises an origin of autonomous replication in a host cell or a site for genomic integration, one or more selectable markers (e.g., amino acid synthesis genes or genes conferring antibiotic resistance such as zeocin, kanamycin, G418 or hygromycin), several restriction enzyme cleavage sites, a suitable promoter sequence and a transcription terminator, the components of which are operably linked to each other.

[0185] An "expression cassette" is a DNA coding sequence or segment of DNA encoding an expression product that can be inserted into a vector at defined restriction sites. The cassette restriction sites are designed to ensure that the cassette is inserted in the proper reading frame. Generally, foreign DNA is inserted at one or more restriction sites in the vector DNA and then carried by the vector into a host cell along with the transmissible vector DNA. A segment or sequence of DNA with inserted or added DNA, such as an expression vector, can also be called a "DNA construct."

[0186] As used herein, the term "vector" includes autonomously replicating nucleotide sequences as well as genome-integrating nucleotide sequences. A common type of vector is a "plasmid," which is generally a self-contained molecule of double-stranded DNA that can easily accept additional (exogenous) DNA and can be easily introduced into a suitable host cell. Plasmid vectors often contain coding DNA and promoter DNA and have one or more restriction sites suitable for inserting exogenous DNA. Specifically, the term "vector" or "plasmid" refers to a vehicle that can introduce a DNA or RNA sequence (e.g., an exogenous gene) into a host cell so as to transform the host and promote expression (e.g., transcription and translation) of the introduced sequence. Expression products such as caspase-2 or cp caspase-2 described herein can be expressed from an autonomously replicating nucleotide sequence or from a nucleotide sequence that is stably integrated into the genome of the host cell.

[0187] Any of the known procedures for introducing foreign nucleotide sequences into host cells may be used, including the use of calcium phosphate transfection, polybrene, protoplast fusion, electroporation, nucleofection, liposomes, microinjection, naked DNA, plasmid vectors, viral vectors, both episomal and integrative, and any of the other well-known methods for introducing cloned genomic DNA, cDNA, synthetic DNA, or other foreign genetic material into host cells (see, e.g., Sambrook et al.).

[0188] According to certain embodiments, the fusion proteins or cp-caspase-2 described herein are expressed as inclusion bodies. Methods for purifying recombinant proteins expressed as inclusion bodies are well known in the art. Typically, 70-80% of recombinant proteins expressed in bacteria, such as E. coli, are contained in inclusion bodies. Specifically, purification of expressed proteins from inclusion bodies requires two major steps: extraction of inclusion bodies from bacteria, e.g., via cell lysis, followed by affinity purification, followed by solubilization and optional refolding of the purified inclusion bodies. Pharmaceutical compositions comprising the cp caspase-2 or caspase-2 provided herein are further described herein. According to certain embodiments, such pharmaceutical compositions comprising the cp caspase-2 or variants thereof described herein are used, for example, to treat cancer, Alzheimer's disease, Parkinson's disease, or inflammatory diseases. Specifically, when used for diagnosis or treatment, the pharmaceutical compositions described herein further comprise a pharmaceutically acceptable carrier or excipient, such as a bulking agent. These pharmaceutical compositions can be administered in accordance with the present invention as a bolus injection or infusion or by continuous infusion. Suitable pharmaceutical carriers for facilitating such administration are well known in the art.

[0189] Pharmaceutically acceptable carriers generally include any and all suitable solvents, dispersion media, coatings, isotonic and absorption delaying agents, etc. that are physiologically compatible with the caspases provided by the present invention. Further examples of pharmaceutically acceptable carriers include sterile water, saline, phosphate buffered saline, dextrose, glycerol, ethanol, etc., and any combination thereof. Additional pharmaceutically acceptable carriers are known in the art and are described, for example, in Remington's Pharmaceutical Sciences (Gennaro, A.R., ed., Mack Publishing Co, 1985). Liquid formulations may be solutions, emulsions or suspensions and may include excipients such as suspending agents, solubilizers, surfactants, preservatives, and chelating agents. Exemplary formulations to be used for parenteral administration include, for example, formulations suitable for subcutaneous, intramuscular, or intravenous injection as a solution, emulsion, or suspension.

[0190] Specifically, the caspase-2 or cp caspase-2 described herein is administered in a therapeutically effective amount, which means a sufficient amount or activity to produce beneficial or desired results, including clinical results, when administered to a subject, for example, a patient suffering from cancer. Thus, the effective amount or its equivalent amount depends on the context in which the amount is applied. An effective amount is intended to mean the amount of compound that is sufficient to treat, prevent, or inhibit such a disease or disorder. The amount of compound that corresponds to such an effective amount will vary depending on a variety of factors, such as the given drug or compound, pharmaceutical formulation, route of administration, type of disease or disorder, and identity of the subject or host being treated, but can nevertheless be routinely determined by one of ordinary skill in the art.

[0191] The caspase-2 and its dimers described herein are particularly provided in isolated form, meaning that they are substantially pure, free of other proteins or enzymes. Nevertheless, such isolated enzymes may be included in a combined preparation containing, for example, a combination of isolated cp caspase-2 with at least one other enzyme or protein or an antibody, such as a monoclonal antibody or antibody fragment. As used herein, the terms "substantially pure" or "purified" refer to a preparation containing at least 50% (w / w), preferably at least 60%, 70%, 80%, 90%, or 95% of a compound, such as a caspase or POI. Purity is measured by methods appropriate for the compound (e.g., chromatography, polyacrylamide gel electrophoresis, HPLC analysis, etc.).

[0192] The following are specific embodiments described herein. 1. The following structure from N to C terminus: i. the small subunit of caspase-2, or a functionally active variant thereof, and ii. The large subunit of caspase-2 or a functionally active variant thereof A single-chain circularly permuted caspase-2 (cp caspase-2) comprising: A cp caspase-2 comprising one or more amino acid substitutions that increase P1' tolerance of said cp caspase-2 compared to a cp caspase-2 that does not have the amino acid substitutions.

[0193] 2. The cp caspase-2 of paragraph 1, comprising one or more amino acid substitutions at positions 171, 105, 172, 282, 225, 83, 185, 255, or 285 of SEQ ID NO:6, or at a position functionally equivalent to any of positions 171, 105, 172, 282, 225, 83, 185, 255, or 285 of SEQ ID NO:6, or any combination thereof. 3. A cp caspase-2 according to paragraphs 1 or 2, comprising the propeptide of the small caspase-2 subunit (SS propeptide) fused to the N-terminus of the small subunit. 4. The cp caspase-2 according to paragraph 3, wherein the SS propeptide comprises one or more amino acid substitutions at its C-terminus.

[0194] 5. The SS propeptide is at position Asp in SEQ ID NO: 2 14 or Asp of SEQ ID NO: 11 347 5. The cp caspase-2 according to paragraph 3 or 4, which comprises an amino acid substitution at a functionally equivalent position to: 6. The cp caspase-2 according to any one of paragraphs 1 to 5, further comprising one or more linker sequences, in particular linker sequences consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 amino acid residues. 7. The linker sequence contains glycine and / or serine residues, more specifically, the linker is selected from the group consisting of GS, GGSGG, GSAGSAAGSG, (GS) n , GSG, or G4S. 8. The cp caspase-2 according to paragraph 6 or 7, wherein the linker sequence is a subunit linker sequence between the small and large subunits. 9. The cp caspase-2 according to any one of paragraphs 1 to 8, comprising one or more C-terminal or N-terminal tags, in particular tags selected from the group consisting of affinity tags, solubility-enhancing tags and monitoring tags.

[0195] 10. The cp caspase-2 according to item 9, wherein the affinity tag is selected from the group consisting of a polyhistidine tag, a polyarginine tag, a peptide substrate for an antibody, a 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, Escherichia coli thioredoxin, NusA, a chitin-binding domain CBD, chloramphenicol acetyltransferase CAT, LysRS, ubiquitin, calmodulin, and lambda gpV, in particular the tag is a His tag comprising one or more His, more particularly a hexahistidine tag.

[0196] 11. The cp caspase-2 according to paragraph 9, wherein the solubility-enhancing tag is selected from the group consisting of T7C, T7B, T7B1, T7B2, T7B3, T7B3, T7B4, T7B5, T7B6, 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, the Z domain of staphylococcal protein A, and thioredoxin.

[0197] 12. The cp caspase-2 according to paragraph 9, wherein the monitoring tag is selected from the group consisting of m-Cherry, GFP, and f-actin. 13. A cp caspase-2 according to any one of clauses 9 to 12, comprising multiple tags, in particular an affinity tag and a solubility enhancing tag. 14. The cp caspase-2 according to paragraph 13, wherein the affinity tag is a hexahistidine tag and the solubility enhancing tag is a T7AC or T7A3 tag. 15. cp caspase-2 according to any one of paragraphs 6 to 14, wherein the linker sequence is a tag-linker sequence linking two tags or linking a tag to the small subunit, the large subunit or the SS propeptide of cp caspase-2.

[0198] 16. The cp caspase-2 according to any one of paragraphs 1 to 15, comprising one or more N-terminal tags, and optionally one or more tag-linker sequences between the tags or between the tag and the N-terminus of the small subunit or SS propeptide. 17. The cp caspase-2 according to any one of paragraphs 1 to 16, comprising one or more C-terminal tags and optionally one or more tag-linker sequences which are linker sequences between the tags or between the tag and the C-terminus of the large subunit.

[0199] 18. i. The small subunit of caspase-2 a) a first conserved region of the active center having at least 37.5% amino acid sequence identity with SEQ ID NO: 177 (first consensus: AAMRN TKR) or 100% sequence identity with XXXRNTXX (SEQ ID NO: 200), where X is any amino acid; and b) a second conserved region of the active center having at least 61.5% amino acid sequence identity with SEQ ID NO: 178 (second consensus: EGYAPGTEFHRCK) or 100% sequence identity with EGXXPGXXXHRCK (SEQ ID NO: 194), where X is any amino acid; Including, ii. The large subunit of caspase-2 is a) a third conserved region of the active center having at least 25.0% amino acid sequence identity with SEQ ID NO: 174 (third consensus: G-EKDLEFRSGGDVDH) or 100% sequence identity with X-XXXLXXRXGXXXDX (SEQ ID NO: 195), where X is any amino acid; b) a fourth conserved region of the active center having at least 53.3% amino acid sequence identity with SEQ ID NO: 175 (fourth consensus: LLSHGVEGGXYGVDG) or 100% sequence identity with XSSHGXXGXXYGXDG (SEQ ID NO: 196), where X is any amino acid; and c) a fifth conserved region of the active center having at least 50.0% amino acid sequence identity with SEQ ID NO: 176 (fifth consensus: QACRGDET) or 100% sequence identity with QACXGXXX (SEQ ID NO: 197), where X is any amino acid; 18. A functionally active variant of cp caspase-2 according to any one of items 1 to 17, comprising:

[0200] 19. A functionally active variant of cp caspase-2 comprising at least 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, or 98% sequence identity with the cp caspase-2 of any one of clauses 1-18. 20. A functionally active variant of paragraph 19, comprising at least 70, 75, 80, 85, 90, 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, 64, 66, 68, 73, 74, 75, 76, 77, 81, 82, 83, 84, or 85.

[0201] twenty one. 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:112, SEQ ID NO:115, SEQ ID NO:118, or a functionally active variant thereof having at least 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, or 98% sequence identity; and / or ii. The cp caspase-2 of any one of clauses 1 to 20, wherein the large subunit is selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 90, SEQ ID NO: 93, SEQ ID NO: 96, SEQ ID NO: 99, SEQ ID NO: 102, SEQ ID NO: 105, SEQ ID NO: 108, SEQ ID NO: 111, SEQ ID NO: 114, SEQ ID NO: 117, or a functionally active variant thereof having at least 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, or 98% sequence identity. twenty two. i. N- and / or C-terminal truncations, and / or ii. N-terminal and / or C-terminal extensions 22. The cp caspase-2 according to any one of items 1 to 21, comprising:

[0202] 23. Based on the position of SEQ ID NO: 6 or a position functionally equivalent to the position of SEQ ID NO: 6, iD, or Gly substituted with an amino acid selected from the group consisting of R, K, E, Q, N, A, S, T, P, H, and Y 171 , ii. Glu substituted with an amino acid selected from the group consisting of V, C, L, I, M, F, W, R, K, D, Q, and N 105 , iii. Glu substituted with an amino acid selected from the group consisting of V, C, L, I, M, F, W, R, K, D, Q, and N 172 , iv. Asp substituted with E or T, or an amino acid selected from the group consisting of R, K, Q, N, G, A, S, P, H, Y. 282 , vG, or Val substituted with an amino acid selected from the group consisting of A, S, T, P, H, Y, C, L, I, M, F, and W 225 , vi. Lys substituted with an amino acid selected from the group consisting of E, R, D, Q, and N 83 , vii. His substituted with an amino acid selected from the group consisting of A, G, S, T, P, and Y 185 , viii. Val substituted with an amino acid selected from the group consisting of M, C, L, I, F, and W 255 and / or ix. Asp 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 285 23. The cp caspase-2 according to any one of items 1 to 22, comprising one or more amino acid substitutions selected from:

[0203] twenty four. i.His 185 and Asp 282 , specifically those containing the substitutions H185A and D282T; ii.Glu 105 and Asp 285 , specifically including the replacement of E105V and D285E; iii.Glu 105 , Gly 171 , Val 225 , and Asp 282 , specifically those containing the substitutions E105V, G171D, V225G, and D282E; iv.Glu 105 , Gly 171 , Val 225 , Asp 282 , and Asp 285 , specifically those containing the substitutions E105V, G171D, V225G, D282E, and D285E; v.Lys 83 , Glu 105 , Glu 172 , Val 255 , and Asp 285 , specifically those containing the substitutions K83E, E105V, E172V, V255M, and D285Y; vi.Glu 105 and Gly 171 , specifically those containing E105V and G171D substitutions; vii.Glu 105 and Glu 172, specifically including replacements for E105V and E172V; and viii.Gly 171 and Glu 172 , specifically those containing the G171D and E172V substitutions and comprising an amino acid substitution at a position of SEQ ID NO: 6 or a functionally equivalent position to a position of SEQ ID NO: 6, selected from the group consisting of: The cp-caspase-2 has increased P1' tolerance compared to cp-caspase-2 without the respective amino acid substitution, and 14 or position Asp in SEQ ID NO: 11 347 23. The cp caspase-2 according to any one of items 1 to 22, which may comprise an SS propeptide containing an amino acid substitution of Ala at a position functionally equivalent to:

[0204] 25. The cp caspase-2 of any one of paragraphs 1 to 22, comprising SEQ ID NO:6 and one or more amino acid substitutions at positions 171, 105, 172, 282, 225, 83, 185, 255, or 285 of SEQ ID NO:6, or at positions functionally equivalent to positions 171, 105, 172, 282, 225, 83, 185, 255, or 285 of SEQ ID NO:6, or any combination thereof. 26. The cp caspase-2 according to paragraph 25, comprising any one or more of the amino acid substitutions G171D, E105V, E172V, D282E, D282T, V225G, K83E, H185A, V255M, D285Y, and D285E.

[0205] 27. An amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 13, 17, 18, 23, 24, 51, 52, 54, 70, 71, 72, 78, 79, 86, 87, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, and 192, or SEQ ID NOs: 1, 13, 17, 18, 23, 24, 51, 52, 54, 70, 71, 72, 78, 79, 86, 87, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191 and 192. 28. The cp caspase-2 according to any one of clauses 1 to 27, comprising a C-terminal tag and amino acid substitutions at positions 285 and 292 of SEQ ID NO: 6 or at positions functionally equivalent to positions 285 and 292 of SEQ ID NO: 6, in particular substitutions to Glu and Ser (D285E and D292S).

[0206] 29. The cp caspase-2 is recruited by a proteolytic cleavage recognition site comprising five amino acids of the sequence P5 P4 P3 P2 P1; P1 can be any amino acid, preferably D or E; P2 can be any amino acid, preferably A; P3 can be any amino acid, preferably V; P4 can be any amino acid, preferably D; P5 can be any amino acid, preferably V; Item 29. The cp caspase-2 according to any one of items 1 to 28.

[0207] 30. A method for producing circularly permuted caspase-2 (cp caspase-2), comprising: i. cloning a nucleotide sequence encoding caspase-2 into an expression vector under the control of a promoter; ii. transforming a host cell with the vector; iii. culturing the transformed host cells under conditions in which cp caspase-2 is expressed; iv. Optionally isolating cp caspase-2 from the host cell culture, optionally by disrupting the host cells; v. optionally purifying cp caspase-2; A method comprising: 31. The method according to claim 30, wherein the cp caspase-2 is a cp caspase-2 according to any one of claims 1 to 29.

[0208] 32. The method of paragraph 30 or 31, wherein the promoter is selected from the group consisting of a T7 promoter / operator, a XylS / Pm regulator / promoter or a variant of the Pm promoter, an araBAD promoter / operator, a T5, T7A1, T7A2, T7A3 promoter / operator, a phoA promoter / regulator, and a trp promoter / operator system. 33. The method of any one of clauses 30 to 32, wherein the caspase-2 comprises a solubility-enhancing tag selected from the group consisting of T7C, T7B, T7B1, T7B2, T7B3, T7B3, T7B4, T7B5, T7B6, T7B6, T7B7, T7B8, T7B9, T7B10, T7B11, T7B12, T7B13, T7A, T7A1, T7A2, T7A3, T7A4, T7A5, T7AC, T3, N1, N2, N3, N4, N5, N6, N7, calmodulin-binding peptide (CBP), DsbA, DsbC, poly-Arg, poly-Lys, G B1 domain, protein D, the Z domain of staphylococcal protein A, and a thioredoxin tag, and preferably comprises a T7AC or T7A3 tag.

[0209] 34. The method according to any one of clauses 30 to 33, wherein the cp caspase-2 comprises an affinity tag, preferably a His tag, even more preferably a 6-His tag. 35. The method of any one of clauses 30 to 34, wherein the host cell is a eukaryotic or prokaryotic host cell, preferably a yeast cell or a bacterial cell, even more preferably an E. coli cell. 36. The method of any one of clauses 30 to 35, wherein the cp caspase-2 comprises an N-terminal tag comprising an affinity tag, preferably a His tag, even more preferably a 6-His tag, and a solubility enhancing tag, preferably T7AC or T7A3. 37. The method of paragraph 36, wherein the cp caspase-2 further comprises a linker between the affinity tag and the solubility-enhancing tag.

[0210] 38. cp Caspase-2 has the following elements fused to its N-terminus in the order of N- to C-terminus: a. an affinity tag, preferably a 6-His tag; b. optionally a linker; c. a solubility-enhancing tag, preferably T7AC or T7A3; d. optionally a linker, and e.cp caspase-2 38. The method according to claim 36 or 37, comprising:

[0211] 39. cp Caspase-2 has the following elements fused to its N-terminus in the order of N- to C-terminus: a. a solubility-enhancing tag, preferably T7AC or T7A3; b. optionally a linker; c. an affinity tag, preferably a 6-His tag; d. optionally a linker, and e.cp caspase-2 38. The method according to claim 36 or 37, comprising:

[0212] 40. The culturing of step (iii) includes a fed-batch stage for expression of cp-caspase-2, and the fed-batch stage lasts for about 0.01 to 0.1 hours. -1 40. The method of any one of clauses 30 to 39, specifically comprising inducing expression of cp caspase-2 by addition of IPTG at a growth rate μ of about 0.01 to 1.5 μmol / g actual CDM (cell dry mass). 41. Growth rate μ is approximately 0.03~0.07 hours -1 Preferably, the temperature is about 0.05 to 0.07 hours. -1 or 0.03 to 0.05 o'clock -1 is preferably about 0.03, 0.05, or 0.07 hr -1 41. The method according to claim 40, wherein the method is any one of the following:

[0213] 42. The method according to item 40 or 41, wherein the IPTG concentration is about 0.5 to 1.3 μmol / g CDM, preferably about 0.5 to 0.9 μmol / g CDM or about 0.9 to 1.3 μmol / g CDM, preferably about 0.5, 0.9, or about 1.3 μmol / g CDM. 43. The culturing of step (iii) further comprises a first fed-batch step for generating biomass before the fed-batch step for expression of cp caspase-2, and the first fed-batch step lasts for about 0.07 to 0.3 hours. -1 43. The method according to any one of items 40 to 42, comprising a growth rate μ of 44. Growth rate μ is approximately 0.1~0.2 hours -1 , preferably about 0.13 to 0.21 hours -1 , and even more preferably about 0.16 to 0.18 hours -1 and most preferably about 0.17 hours -1 44. The method according to claim 43, wherein 45. The method according to any one of clauses 30 to 44, wherein the cp caspase-2 is purified using affinity chromatography, preferably IMAC.

[0214] 46. ​​cp caspase-2 obtained by the method according to any one of items 30 to 45. 47. A method for producing a protein of interest (POI) comprising an authentic N-terminus, comprising: providing a fusion protein comprising, from N- to C-terminus, one or more tags, optionally one or more tag-linker sequences, and a caspase recognition site fused at the N-terminus to a POI, wherein said caspase recognition site is specifically recognized by cpCaspase-2 according to any one of items 1 to 29; ii. contacting the fusion protein with the cp caspase-2 for a period of time sufficient for the cp caspase-2 to cleave the fusion protein; iii. optionally purifying the POI; A method comprising:

[0215] 48. A method for producing a protein of interest (POI) comprising an authentic N-terminus, comprising: expressing in the same host cell a fusion protein comprising from N to C-terminus optionally one or more tags, optionally one or more tag-linker sequences and a caspase recognition site fused at the N-terminus to a POI, wherein said caspase recognition site is specifically recognized by a cpCaspase-2 according to any one of paragraphs 1 to 29, and a cpCaspase-2 according to any one of paragraphs 1 to 29 which specifically recognizes the recognition site of the fusion protein; ii. optionally, the fusion protein and cp caspase-2 are under the control of the same promoter; iii. culturing the host cells, wherein the cp caspase-2 cleaves the fusion protein in the cells in vivo; iv. optionally isolating the POI from the cells and optionally purifying the POI; A method comprising:

[0216] 49. The method of item 47 or 48, wherein the fusion protein comprises a caspase recognition site comprising five amino acids of the sequence P5 P4 P3 P2 P1 and a cleavage site P1 / P1', where P1' is the N-terminal amino acid of the POI. 50. The method of paragraph 47 or 48, wherein the fusion protein and cp caspase-2 are under the transcriptional control of different promoters, and expression of cp caspase-2 is induced after expression of the fusion protein. 51. A fusion protein comprising a cp caspase-2 according to any one of paragraphs 1 to 29, in particular a fusion protein comprising a cp caspase-2 according to any one of paragraphs 1 to 29 at its N- or C-terminus, wherein the fusion protein has from N to C-terminus the following structure: i. one or more N-terminal tags; ii. optionally one or more tag-linker sequences, and iii. a caspase recognition site comprising five amino acids of the sequence P5 P4 P3 P2 P1; iv. Cleavage site P1 / P1', v.POI 51. The method according to any one of items 47 to 50, comprising: P1' being the N-terminal amino acid of said POI; and said cp caspase-2 specifically recognizing said recognition site.

[0217] 52. From N to C-terminus, the structure is: an N-terminal affinity tag, b. optionally a linker sequence; c. caspase recognition site, d. cleavage site P1 / P1', and e.POI wherein P1' is the N-terminal amino acid of the POI, and said recognition site is specifically recognized by the cp caspase-2 according to any one of items 1 to 29; ii. isolating the fusion protein; iii. purifying the fusion protein using an N-terminal affinity tag; iv. Providing a cp caspase-2 according to any one of items 1 to 29, which specifically recognizes the recognition site of the fusion protein; v. contacting the fusion protein with the cp caspase-2 for a period of time sufficient for the cp caspase-2 to cleave the fusion protein; vi. Optionally removing the cleaved affinity tag and, optionally, the uncleaved fusion protein using the affinity tag and cp Caspase-2; vii. Optionally, further purifying the POI; 52. The method according to any one of items 47 to 51, comprising:

[0218] 53. The method according to item 52, wherein the cp caspase-2 comprises an affinity tag at its N- or C-terminus identical to the affinity tag of the fusion protein, and the cp caspase-2 is removed in step vi. using said affinity tag.

[0219] 54. i. expressing in a host cell a fusion protein comprising one or more N-terminal affinity tags, optionally one or more tag-linker sequences, a caspase recognition site and a cleavage site P1 / P1′, where P1′ is the N-terminal amino acid of the POI, and a POI, wherein said recognition site is specifically recognized by cpCaspase-2 according to any one of items 1 to 29; ii. isolating the fusion protein and binding / capturing the fusion protein on a solid support using the affinity tag; iii. Providing a cp caspase-2 according to any one of items 1 to 29, which specifically recognizes the recognition site of the fusion protein; iv. contacting the cp caspase-2 with the bound / captured fusion protein for a period of time sufficient for the cp caspase-2 to cleave the fusion protein; v. Releasing the POI from the solid support; vi. isolating and optionally further purifying the POI; 54. The method of claim 52 or 53, comprising:

[0220] 55. The method according to paragraph 54, wherein the cp caspase-2 and the fusion protein contain the same affinity tag, allowing binding of the fusion protein and the caspase onto a solid support and release of the POI upon cleavage by the caspase. 56. The method according to item 54 or 55, wherein the solid support is a column, particularly a chromatography column, more particularly an immobilized metal affinity chromatography column (IMAC). 57. A method according to any one of paragraphs 47 to 56, using a flow reactor comprising immobilised cp caspase-2 according to any one of paragraphs 1 to 29. 58. An isolated nucleotide sequence encoding a cp caspase-2 according to any one of paragraphs 1 to 29.

[0221] 59. A vector, particularly a bacterial expression vector, comprising a nucleotide sequence according to paragraph 58. 60. An expression cassette comprising the nucleotide sequence of paragraph 58 operably linked to a regulatory element. 61. A host cell or host cell line expressing cp caspase-2 according to any one of paragraphs 1 to 29, wherein the host cell is selected from the group consisting of a bacterial cell, a yeast cell, an insect cell, a mammalian cell and a plant cell, preferably the host cell is a bacterial or yeast cell selected from the group consisting of Escherichia coli, Pseudomonas spp., Bacillus spp., Streptomyces spp., Saccharomyces spp., Schizosaccharomyces spp., Pichia spp., Kluyveromyces spp. and Hansenula spp.

[0222] 62. An expression system comprising a vector according to paragraph 59 or an expression cassette according to paragraph 60 and a host cell according to paragraph 61. 63. Use of cp caspase-2 according to any one of clauses 1 to 29 for the in vivo cleavage of a substrate in a non-human organism. 64. The use according to item 63, wherein the non-human organism is a prokaryote, in particular Escherichia coli. 65. Use of cp caspase-2 according to any one of paragraphs 1 to 29 for the production of a protein of interest (POI).

[0223] 66. The use according to paragraph 65, wherein the POI comprises an authentic N-terminus. 67. The following structure from N to C terminus: i. a tag sequence comprising a caspase recognition site comprising five amino acids of the sequence P5 P4 P3 P2 P1, which is specifically recognized by the cp caspase-2 according to any one of items 1 to 29; ii. a cleavage site P1 / P1', where P1' is the N-terminal amino acid of the protein of interest (POI), and iii. POI A fusion protein comprising: 68. The fusion protein of paragraph 67, wherein the tag sequence further comprises one or more tags selected from the group consisting of an affinity tag, a solubility-enhancing tag, and a monitoring tag.

[0224] 69. The fusion protein of paragraph 68, further comprising one or more tag-linker sequences. 70. i. Caspase-2 according to paragraph 74 or cp-caspase-2 according to any one of paragraphs 1 to 29, specifically for cleaving a fusion protein according to any one of paragraphs 67 to 69 or a fusion protein according to paragraphs 90 or 91. Kit including: 71. The kit according to paragraph 70, further comprising an expression vector comprising a polynucleotide encoding a protein tag according to paragraphs 75 to 89.

[0225] 72. The cp caspase-2 according to any one of paragraphs 1 to 29 for use in the treatment of a disease. 73. The cp caspase-2 according to any one of paragraphs 1 to 29 for use in the treatment of cancer, Alzheimer's disease, Parkinson's disease, or an inflammatory disease. 74. A caspase-2 comprising one or more amino acid substitutions at positions 409, 431, 212, 213, 266, 226, 296, 323, or 326 of SEQ ID NO: 11, or at positions functionally equivalent to any of positions 409, 431, 212, 213, 266, 226, 296, 323, or 326 of SEQ ID NO: 11, or a combination thereof, wherein the amino acid substitutions increase P1' tolerance compared to a caspase-2 comprising the same sequence but not comprising the amino acid substitutions. 75. A protein tag for enhancing expression of a POI, comprising a solubility-enhancing tag and the amino acid sequence VDVAD (SEQ ID NO: 45), wherein the sequence VDVAD is located at the C-terminus of the protein tag.

[0226] 76. The tag of paragraph 75, wherein the solubility-enhancing tag is selected from the group consisting of T7C, T7B, T7B1, T7B2, T7B3, T7B3, T7B4, T7B5, T7B6, T7B6, T7B7, T7B8, T7B9, T7B10, T7B11, T7B12, T7B13, T7A, T7A1, T7A2, T7A3, T7A4, T7A5, T3, N1, N2, N3, N4, N5, N6, N7, and T7AC. 77. The tag according to paragraph 76, wherein the solubility-enhancing tag is T7AC or T7A3. 78. The tag according to any one of clauses 75 to 77, further comprising a histidine tag sequence, preferably comprising 1 to 20 histidine residues, even more preferably a 3-His, 6-His, or 9-His tag sequence.

[0227] 79. The tag according to any one of clauses 75 to 78, wherein the solubility-enhancing tag is located at the N-terminus of the protein tag. 80. The tag of any one of clauses 78, wherein the histidine tag sequence is located at the N-terminus of the protein tag. 81. The tag according to any one of clauses 75 to 80, further comprising one or more linker sequences comprising one or more amino acid residues.

[0228] 82. The tag according to item 81, wherein the one or more linker sequences are located between the VDVAD sequence and the solubility-enhancing tag and / or histidine tag sequence. 83. The tag according to item 81 or 82, wherein one or more amino acid residues of the linker sequence are any of the naturally occurring amino acids or derivatives thereof, preferably selected from the group consisting of G, S, A, T, and N. 84. The tag of any one of clauses 81 to 83, wherein the linker sequence is GSG. 85. The tag of any one of clauses 81 to 83, wherein the linker sequence is GSGSGSG. 86. The tag according to any one of clauses 75 to 85, further comprising a signal peptide at the N-terminus of the protein tag. 87. The tag of paragraph 86, wherein the signal peptide is selected from the group consisting of ompA (outer membrane protein A), DsbA (thiol:disulfide exchange protein), MalE (maltose binding protein), PelB (pectate lyase B) from soft rot bacteria, PhoA (alkaline phosphatase), OmpC (outer membrane protein C), OmpF (outer membrane protein F), OmpT (protease VII), endoxylanase from Bacillus species, LamB (lambda receptor protein), Lpp (murein lipoprotein), LTB (heat-labile enterotoxin subunit B), PhoE (outer membrane pore protein E), and StII (heat-stable enterotoxin 2).

[0229] 88. The tag has the following structure from N- to C-terminus: a. T7AC-6-His-VDVAD; b. T7A3-6-His-VDVAD; c. T7AC-6-His-GSG-VDVAD; d. T7A3-6-His-GSG-VDVAD; e. T7AC-6-His-GSGSGSG-VDVAD; f. T7A3-6-His-GSGSGSG-VDVAD; g. 6-His-T7AC-VDVAD; h. 6-His-T7A3-VDVAD; i. 6-His-T7AC-GSG-VDVAD; j. 6-His-T7A3-GSG-VDVAD; k. 6-His-T7AC-GSGSGSG-VDVAD; l. 6-His-T7A3-GSGSGSG-VDVAD 88. The tag according to any one of items 75 to 87, comprising one of the following:

[0230] 89. The tag has the following structure from N- to C-terminus: a. ompA signal peptide-T7AC-6-His-VDVAD; b. ompA signal peptide-T7A3-6-His-VDVAD; c. ompA signal peptide-T7AC-6-His-GSG-VDVAD; d. ompA signal peptide-T7A3-6-His-GSG-VDVAD; e. ompA signal peptide-T7AC-6-His-GSGSGSG-VDVAD; f. ompA signal peptide-T7A3-6-His-GSGSGSG-VDVAD; g. ompA signal peptide-6-His-T7AC-VDVAD; h. ompA signal peptide-6-His-T7A3-VDVAD; i. ompA signal peptide-6-His-T7AC-GSG-VDVAD; j. ompA signal peptide-6-His-T7A3-GSG-VDVAD; k. ompA signal peptide-6-His-T7AC-GSGSGSG-VDVAD; l. ompA signal peptide-6-His-T7A3-GSGSGSG-VDVAD 88. A tag according to paragraph 86 or 87, comprising one of:

[0231] 90. A fusion protein comprising the protein tag according to any one of paragraphs 75 to 89 and a POI, wherein the N-terminus of the POI is fused to the C-terminus of the protein tag. 91. A fusion protein according to paragraph 90, wherein the N-terminus of the POI is directly fused to the C-terminus of a protein tag having the sequence VDVAD. 92. A method for generating a POI, comprising: i. providing a fusion protein according to paragraph 90 or 91 comprising a POI; ii. contacting the fusion protein with circularly permuted caspase-2 (cp caspase-2) for a period of time sufficient for the cp caspase-2 to cleave the fusion protein, thereby liberating the POI; iii. optionally purifying the POI; A method comprising:

[0232] 93. i. cloning a nucleotide sequence encoding the fusion protein of paragraph 90 or 91 into an expression vector under the control of a promoter; ii. transforming a host cell with the vector; iii. culturing the transformed host cells under conditions in which the fusion protein is expressed; iv. optionally isolating said fusion protein from the host cell culture, optionally by disrupting the host cells; v. purifying the fusion protein using IMAC chromatography; vi. contacting the fusion protein with circularly permuted caspase-2 (cp caspase-2) for a period of time sufficient for the cp caspase-2 to cleave the fusion protein, thereby liberating the POI; vii. Optionally, further purifying the POI; viii. Optionally modifying the POI; ix. optionally formulating the POI; 93. The method of paragraph 92, further comprising:

[0233] 94. The method of item 92 or 93, wherein the promoter is selected from the group consisting of the T7 promoter / operator, XylS / Pm regulator / promoter or a variant of the Pm promoter, the araBAD promoter / operator, the T5, T7A1, T7A2, T7A3 promoter / operator, the phoA promoter / regulator, and the trp promoter / operator system. 95. The method according to items 65-66, wherein the host cell is a eukaryotic or prokaryotic host cell, preferably a yeast or bacterial cell, preferably an E. coli cell.

[0234] The examples described herein are illustrative of the present invention and are not intended to be limitations thereon. Various embodiments of the present invention have been described in accordance with the present invention. Many modifications and variations can be made to the techniques described and illustrated herein without departing from the spirit and scope of the present invention. Therefore, it will be understood that the examples are illustrative only and do not limit the scope of the present invention. [Example]

[0235] Example 1 General Materials and Methods 1.1 E. coli strains E. coli BL21(DE3) was used for all standard protein expression. The E. coli strain NovaBlue (Novagen, Madison, WI, USA) was used as a host for plasmid extraction and for cloning experiments. 1.2 Culture medium TY (tryptone-yeast) medium (1% peptone, 0.7% yeast extract, 0.25% wt / vol NaCl). TB medium (1.2% peptone, 2.4% yeast extract, 0.4% glycerol, 17 mM KH2PO4, 72 mM K2HPO4). SOC (super optimal broth with catabolite repression) (2% wt / v tryptone, 0.5% wt / v yeast extract, 10 mM NaCl, 2.5 mM KCl, 10 mM MgCl2, and 20 mM glucose, pH 7.0).

[0236] Media for recovery of transformed cells. Optimized M9 minimal medium (50 mM NaHPO, 20 mM KHPO, 10 mM NaCl, 1 mM MgSO, 0.1 mM CaCl, 0.4% glucose, 20 mM NHCl, 0.5% w / v casamino acids, 10 μg / ml FeSO, and vitamins (0.001 mg / ml each of biotin, thiamine, riboflavin, pyridoxine, and niacinamide) was used). Induction was performed with 0.1–0.4 mM IPTG. 1.3 Recombinant protein expression Standard expression protocol: Substrate proteins were expressed in TY medium. Induction was carried out with 1 mM IPTG at OD600 1.0 at 37°C, 220 rpm for 4 hours. Expression protocol for caspases: Caspases were expressed in TB medium. Induction was with 0.4 mM IPTG at OD600 1.2 for 4 hours at 25°C.

[0237] 1.4 Cell lysis and protein purification Substrates and caspases were purified using immobilized metal affinity purification (IMAC). The collected cell pellet was suspended in Tris buffer (50 mM Tris, 50 mM NaCl, pH 7.5) and crushed in a French press. The clarified supernatant was applied to an IMAC column (HisTrap FF Crude, 1 ml, GE Healthcare). Washing was performed with running buffer (50 mM Tris / HCl, pH 7.4, 300 mM NaCl, 20 mM imidazole) for 5 column volumes, with the fifth wash fraction containing an increasing concentration of imidazole (40 mM). Elution was performed with buffer containing 250 mM imidazole for 5 column volumes. After affinity chromatography, imidazole and excess NaCl were exchanged into Tris buffer using a Sepharose column (HiTrap Desalting, 5 ml, GE Healthcare). All eluted fractions were pooled, the concentration was determined by BCA assay, and the protein was stored at -80°C in Tris buffer containing 2 mM DTT.

[0238] 1.5 Testing caspases - in vitro cleavage assay The activity of purified caspases was evaluated by in vitro cleavage assay. Samples were analyzed by SDS-PAGE to separate cleaved and unprocessed substrates. Band intensities were measured using ImageQuant TL 1D software, version 8.1 (GE Healthcare), and used for statistical analysis and calculation of cleavage efficiency. To standardize the process, samples with approximately 50% cleaved substrate were used for calculation. Standard conditions were defined as follows: an enzyme-to-substrate mass ratio of 1:100 (1 mg / ml substrate and 0.01 mg / ml caspase, molar ratio 1:170) in caspase assay buffer (20 mM PIPES, 100 mM NaCl, 10% sucrose, 0.1% CHAPS, 1 mM EDTA, 10 mM DTT, pH 7.2), incubation at 25° C. To allow the reaction to proceed slowly, the caspase concentration was increased to 0.1 mg / ml (enzyme-to-substrate mass ratio 1:10).

[0239] cp caspase-2 (0.01 mg / ml) (SEQ ID NO: 6) cleaved 50% of the P1' glycine-bearing substrate VDVAD-E2 (1 mg / ml) (SEQ ID NO: 33) within 1 minute in caspase assay buffer at 25°C (Figure 4), conditions that were defined as the standard activity against which all other reactions were compared. N-terminal Edman sequencing of the processed substrate demonstrated that cleavage occurred only between the VDVAD recognition site and the P1' glycine. Figure 4A shows a standard cleavage assay with cp caspase-2 (SEQ ID NO: 6) and VDVAD-E2 (SEQ ID NO: 33) with a P1' glycine. Cleavage of 1 mg / ml VDVAD-E2 by 0.01 mg / ml cp caspase-2 at 25°C is shown, with samples taken after 1.0, 2.5, and 5 minutes. After 2.5 minutes, 90% of the substrate was cleaved, and processing was complete in less than 5 minutes. For in vitro cleavage to compare activity with commercially available caspase-2, approximately 0.005 mg / ml of wt caspase-2 (caspase-2 (human), recombinant, active, Enzo Life Sciences Inc.; Farmingdale, NY, USA) was used to cleave 1 mg / ml VDVAD-E2 with a P1' glycine (mass ratio 1:200, molar ratio 1:340).

[0240] Example 2 Designed cp-caspase-2 constructs and substrates 2.1 Cloning of constructs Site-directed mutagenesis was performed to generate specific changes such as deletions, insertions, substitutions, or site mutations in the first protein, caspase-2, or cp caspase-2 (e.g., SEQ ID NO: 6) in the plasmid DNA. Specific primers were designed back-to-back and used for exponential amplification with high-fidelity DNA polymerases. After amplification, a KLD (kinase ligase DpnI) reaction was performed. In this process, the PCR product was incubated with kinase, ligase, and DpnI restriction enzyme to phosphorylate the PCR fragment and ligate it into a circular plasmid to remove the template DNA. The construct was transformed into NovaBlue heat-shocked cells, and an aliquot of the cell suspension was plated on TY agar containing the appropriate antibiotic. Successful cloning was confirmed by sequencing single colonies.

[0241] All substrates and caspases were expressed and purified as described in Example 1, sections 1.3 and 1.4. The protein and nucleotide sequences of all constructs are listed in FIG. 2.2 Caspase substrates Human ubiquitin-conjugating enzyme E2 L3 (E2; UniProt ID P6803612) as a fusion protein was used as a standard caspase substrate. A fusion protein (VDVAD-E2) was designed with an N-terminal His tag, a short GSG-linker, and the VDVAD caspase-2 recognition site. The first amino acid after the cleavage site (P1') was glycine (VDVAD-E2, SEQ ID NO: 33). The total protein has a size of 21.3 kDa, but once the tag is cleaved, the E2 protein itself has a size of 19.5 kDa. This difference is large enough to visualize the cleavage activity on SDS-PAGE.

[0242] Because the P1' site is known to affect cleavage activity, E2s with all possible 20 residues after the VDVAD cleavage site were expressed and purified. E2s with cleavage sites other than VDVAD were also cloned. All tested tag sequences fused to E2 proteins are listed in Table 1.

[0243] [Table 3] β-galactosidase was chosen as a model protein because its large size (116 kDa) makes it susceptible to nonspecific cleavage. An N-terminal His tag was added, as well as a GSG linker and the caspase-2 cleavage site VDVAD (SEQ ID NO: 34). Superoxide dismutase, SOD, was used as an additional model fusion protein with an N-terminal 6His tag and a recognition site, VDVAD, fused directly to the N-terminus of SOD (SEQ ID NO: 193). The effect of the His tag and VDVAD cleavage site on protein expression was evaluated using hFGF (human fibroblast growth factor). Three pET30a constructs (hFGF, 6H-hFGF, and 6H-VDVAD-hFGF) (SEQ ID NO: 32) were cloned.

[0244] Recombinant expression of wild-type (hFGF), His-tagged (6H-hFGF), and 6H-hFGF with the caspase-2 cleavage site VDVAD (6H-VDVAD-hFGF) was compared. Expression of both His-tagged variants was reduced. This effect was less pronounced for the 6H-VDVAD-hFGF variant. Interestingly, although total expression was significantly reduced, the amount of soluble protein remained the same for 6H-hFGF. Furthermore, for 6H-VDVAD-hFGF, it was even increased compared to wild-type hFGF.

[0245] It has been described that His-tags can affect both the rate of total and soluble production of recombinant proteins. An important result is that the VDVAD sequence itself does not appear to negatively affect the production or solubility of recombinant proteins. For proteins whose production levels are reduced by His-tags, caspase cleavage sites can be easily combined with other tags.

[0246] 2.3 Designed variants of circularly permuted caspase-2 Circularly permuted caspase-2: A circularly permuted caspase-2 variant (cp caspase-2) was designed. Based on the sequence of human caspase-2 (UniProtKB14 ID P42575, SEQ ID NO: 11), the N-terminal CARD was removed and the order of the large subunit (LS) and small subunit (SS) was swapped to generate a constitutively active caspase. The SS was linked to the N-terminus of the LS via a GS-linker. Optionally, the SS propeptide was linked to the N-terminus of the SS. In this case, to ensure expression as a single-chain protein, an aspartate (Asp in the wild-type sequence of caspase-2) was added. 343, Asp in cp caspase-2 21 ) was mutated to alanine to prevent cleavage of the small subunit from the p14 chain to the p12 chain. This resulted in the cp caspase-2 variants SEQ ID NO:9, SEQ ID NO:6, and SEQ ID NO:76. Both of the latter additionally contained an N-terminal 6His tag. The basic structures of these variants are shown in Figures 2B-2D and 3B-3D.

[0247] The protein sequence was codon-optimized for E. coli using the GeneArt™ online tool (Thermo Fisher Scientific). A glycine-serine linker, forming a BamHI restriction site, was added between the small and large subunits, allowing for separate cloning of the subunits and facilitating the creation of chimeras composed of subunits from various caspases. An N-terminal His tag allowed for IMAC purification. Figure 2 shows a schematic of the wild-type structure (SEQ ID NO: 11) and the cpCaspase-2 structure (e.g., SEQ ID NO: 9). Annotations are taken from the UniProtKB database (P42575). The structure of the active enzyme (caspase dimer) is shown in Figure 3. Figure 3 shows a schematic of the mature enzyme of the wild-type structure and the circularly permuted caspase-2 structure. Disulfide bonds between the small subunits, between the linkers, and between the N- and C-termini are shown. Mature wild-type caspase-2 consists of four protein chains, while cpCaspase-2 has only two.

[0248] All cp-caspase-2 variants described in section 2.3 were constructed based on SEQ ID NO: 6, unless otherwise stated. The amino acid positions of the mutations shown correspond to SEQ ID NO: 6, unless explicitly stated otherwise. All variants have a 6His tag, unless otherwise stated. cpCaspase-2Stop and cpCaspase-2 D285E: To test the effect of the propeptide annotated in UniProtKB14 (ID P42575) within the C-terminus of the large subunit, a truncated version was generated by deleting amino acids 286-292 within cpCaspase-2 of SEQ ID NO: 6, thereby generating a cpCaspase-2Stop variant (SEQ ID NO: 14) and a non-cleaved variant (cpCaspase-2 D285E) (SEQ ID NO: 13).

[0249] Cp Caspase-2 with a C-terminal Strep tag: A Strep tag was fused to the C-terminus to generate cp Caspase-2 Strep and cp Caspase-2 D285E Strep variants (SEQ ID NO: 15 and SEQ ID NO: 16, respectively). In SEQ ID NO: 15, a Strep tag was fused to the C-terminus of cp caspase 2 (SEQ ID NO: 6), which was mutated to VDQQS (substitution: D292S), because experiments have shown that VDQQE is recognized as the cleavage site. Despite the VDQQS mutation, the Strep tag was partially cleaved from the caspase. The cleavage product had the same size as the stop variant (31.9 kDa). This suggests that the DETD-R (Asp) site, rather than the VDQQS site, is involved. 285 and Arg 286 This indicates that the connection was cut off between

[0250] Therefore, a Strep tag was added to the C-terminus of cp caspase-2 with the D285E and E292S mutations. This variant (SEQ ID NO: 16) was expressed as a single chain of 33.9 kDa. Asp 285 Mutation of to Glu was shown to prevent cleavage. The C-terminal Strep tag did not affect the cleavage activity of this variant. Figure 5 shows a graphical representation of the C-terminal sequence of the cp caspase-2 variant. cp caspase-2 D282T and cp caspase-2 H185A D282T: Two cp caspase-2 variants were generated, the first with a D282T mutation and the second with an additional H185A mutation in cp caspase-2 (SEQ ID NO: 6), comprising SEQ ID NO: 17 and SEQ ID NO: 18, respectively.

[0251] cp caspase-2 G171D, cp caspase-2 V225G, and cp caspase-2 D282E: cp caspase-2 (SEQ ID NO: 6) is mutated at positions 171, 225, or 282, resulting in the amino acid exchange G171D, V225G, or D282E, respectively, resulting in variants with SEQ ID NO: 190, 192, and 191, respectively. Cp caspase-2 with various linkers between the small and large subunits: The GS linker between the small and large subunits of cp caspase-2 (SEQ ID NO: 6) was mutated. The resulting variants contained no linker (cp caspase-2 Δ linker, SEQ ID NO: 73), a GGSGG linker (cp caspase-2 5 aa linker, SEQ ID NO: 74), and a GSAGSAAGSG linker (cp caspase-2 10 aa linker, SEQ ID NO: 75).

[0252] Cp Caspase-2 with a Partial Small Subunit Propeptide and Cp Caspase-2 without the Small Subunit Propeptide: The propeptide of the small subunit of cp Caspase-2 (SEQ ID NO: 6) was mutated by site-directed mutagenesis. Deletion of residues 8-22 generated a variant without the propeptide (cp Caspase-2ΔSS Prop, SEQ ID NO: 76; see also Figures 2D and 3D), and deletion of residues 8-15 generated a variant with a partially deleted propeptide (cp Caspase-2 1 / 2ΔSS Prop, SEQ ID NO: 77). Circularly permutation-shifted cp caspase-2: A circularly permutation-shifted variant was generated using cp caspase-2ΔSS Prop (SEQ ID NO: 76). Three amino acids were deleted at the N-terminus of the small subunit and added to the C-terminus of the large subunit. Due to possible autocleavage detected when a Strep tag was added to the C-terminus of cp caspase-2, the mutations D267E and D274S according to SEQ ID NO: 76 were additionally inserted. The resulting variant cp caspase-2 C-term+3 (SEQ ID NO: 82) was expressed, purified, and tested as previously described.

[0253] Concurrently, a variant was generated from cpCaspase-2ΔSSProp (SEQ ID NO: 76) by deleting the 3 C-terminal residues of the large subunit and inserting them at the N-terminus of the small subunit. The resulting variant, cpCaspase-2 NTerm+3 (SEQ ID NO: 83), was expressed, purified, and tested as described in the standard protocol in Example 1. cpCaspase-2 C203S: A variant was generated by insertion of a C203S mutation into cpCaspase-2 (SEQ ID NO: 6) to give SEQ ID NO: 198. cp Caspase-2 S9 C203S: The substitution C203S was inserted into cp Caspase-2 S9 (SEQ ID NO:51) to generate SEQ ID NO:199. cp caspase-2 N85C and cp caspase-2 A86C: Variants were generated by insertion of the mutations N85C (SEQ ID NO: 80) and A86C (SEQ ID NO: 88) into cp caspase-2 (SEQ ID NO: 6).

[0254] Homolog cp caspase-2 variant: The cp caspase-2 variants from various species were constructed analogues to cp caspase-2 of human origin (SEQ ID NO: 6). Based on the sequences of Tasmanian devil caspase-2 (Tasmanian devil, UniProtKB14 ID G3VQP7, SEQ ID NO: 95) and chimaera caspase-2 (elephant shark, UniProtKB14 ID V9KZT1, SEQ ID NO: 113), the N-terminal CARD was removed and the order of the large and small subunits was swapped to generate a constitutively active caspase. SS was linked to the N-terminus of LS via a GS-linker. SS propeptide was linked to the N-terminus of SS. To ensure expression as a single-chain protein, an aspartate (Asp in the wild-type sequence of human caspase-2) was added. 343 , Asp in the cp protein 21 (corresponding to ) was mutated to alanine to avoid cleavage of the small subunit propeptide.

[0255] The protein sequence was codon-optimized for E. coli using the GeneArt™ online tool (Thermo Fisher Scientific). A glycine-serine linker that also created a BamHI restriction site between the small and large subunits was added, allowing for separate cloning of the subunits and facilitating the creation of chimeras composed of subunits from various caspases. An N-terminal His tag allowed for IMAC purification. The resulting variants are Tasmanian devil cp caspase-2 (SEQ ID NO: 64) and elephant shark cp caspase-2 (SEQ ID NO: 68). Residue Glu in cp caspase-2 (SEQ ID NO: 6) 105 and Glu 172 Mutations at positions corresponding to (and functionally equivalent to) were inserted into Tasmanian devil cp caspase-2 to generate variant Tasmanian devil cp caspase-2 E105V E172V (SEQ ID NO: 78).

[0256] Glu in cp caspase-2 (SEQ ID NO: 6) 105 and Gly 171Mutations at positions corresponding to were inserted into elephant shark cp caspase-2 to generate variant elephant shark cp caspase-2 E105V G171D (SEQ ID NO: 79). Additionally, variants containing an N-terminal T7AC tag (SEQ ID NOs: 84, 85, 86, 87) were cloned. Functionally equivalent positions are listed in Table 2.

[0257] [Table 4]

[0258] Figure 6 shows an alignment of the native sequences of caspase-2 homologs from various species. The unprocessed protein consists of a CARD domain, a large subunit (LS) containing two catalytic centers, a small subunit propeptide (SS Propeptide), and a small subunit (SS). Active sites 1-5 interact with the substrate. See Tables 3 and 4 for definitions of the subunits and active sites.

[0259] UniProt IDs: Human (P42575), Mouse (P29594), Sheep (W5Q8H6), Tasmanian Devil (G3VQP7), Chicken (Q98943), Anole (Anolis) (H9GC58), Alligator (A0A1U8D1G6), Xenopus (F6RDY9), Danio (Q0PKX3), Chimaera (V9KZT1), Ascidian (A0A1W2WKB0) Figure 7 shows an alignment of the active sites of native sequences of caspase-2 from various species (see Table 24 for sequences and SEQ ID NOs). The active site interacts with the substrate and is relatively conserved. See Tables 3 and 4 for definitions of the subunits and active site. Numbers indicate the starting position of the first active site.

[0260] [Table 5]

[0261] [Table 6]

[0262] [Table 7]

[0263] Example 3 Selection of cp caspase-2 and all mutations found by selection Selection system for detecting variants with increased P1' tolerance A selection system was used to improve cp caspase-2. It is based on a circularly permuted ATCase (aspartate transcarbamoylase) catalytic subunit and a pyrimidine auxotroph. The pyrBI operon (encoding the regulatory pyrI and catalytic pyrB subunits of ATCase) was deleted in E. coli BL21(DE3), so this knockout strain can survive only in media containing pyrimidines or only if the cells are supplemented with a vector encoding ATCase. A specific protease is detected via growth in E. coli using the cp catalytic subunit of ATCase (cp-pyrB), which carries a new N-terminus within the protein. This is because fusion of any stretch of amino acids to its N-terminus renders the enzyme inactive because it can no longer fold properly due to space limitations within the protein. However, the enzyme becomes active again if a protease capable of precisely cleaving this additional stretch of amino acids is provided.

[0264] 3.1 Design of constructs and caspase mutant libraries Selection medium: optimized M9 medium (see Example 1, section 1.2) Strain: E. coli BL21(DE3) containing the pyrBI operon, exchanged for kanamycin resistance (i.e., pyrBI is deleted). Vectors: Expression of ATCase subunits, cp-pyrB, and pyrl from the pETDuet™-1 vector using the T7 promoter and ampicillin resistance as a selection marker; expression of various caspase variants from the pACYCDuet™-1 vector using the T7 promoter and chloramphenicol resistance marker. The selection protocol was carried out by co-transformation with ampicillin, kanamycin, and chloramphenicol, respectively, in the selection medium described above.

[0265] VDVAD-cpATCase The pETDuet-1 plasmid (substrate plasmid) used contained the pyrI gene (SEQ ID NO: 20) in MCSI and the cp-pyrB gene (SEQ ID NO: 21) in MCSII. In pyrI, a potential caspase cleavage site DQVD was inserted at the Asp 73 The cpATCase was altered to DQVE by mutation of . A 6His tag followed by a GSG linker and a caspase recognition site was fused to the N-terminus of cp pyrB c227

[25] . This prevents the enzyme from folding correctly, rendering it inactive; however, proteolytic cleavage of this tag can restore its function. The first Met residue in cp pyrB was deleted. The amino acid following Met is Thr. cpATCase is still active when this residue is substituted. Only mutations to His, Lys, Phe, Tyr, and Trp render it inactive. This allows for the selection of caspases with improved or altered recognition site specificity and / or improved P1' tolerance. A CpATCase construct with 6His-GSG-VDVAD-ΔM-X-pyrB (SEQ ID NO: 22) was used for in vivo selection of altered P1' tolerance.

[0266] Caspase mutant library construction—ep PCR and oe PCR A mutant gene library of various cp-caspase-2 variants was generated by error-prone (ep) PCR and overlap extension (oe) PCR of the vector and mutant caspase gene. Linear DNA fragments were ligated using T4 DNA ligase. The amount of mutations could be varied by changing the Mg(II) and Mn(II) ion concentrations in the PCR buffer. The concentrations used resulted in an average of one to three amino acid exchanges in the caspase. The cp-caspase-2 variants that constituted the mutant library are listed in the "Mutated Caspase" column of Table 5.

[0267] 3.2 Caspase library selection The caspase mutant library was transformed into E. coli BL21(DE3)ΔpyrBI electrocompetent cells already containing the cpATCase plasmid with the desired protease cleavage site and P1' residue. Selection was carried out in optimized M9 medium or on M9 agar plates at 30°C for 24-48 hours. Liquid culture was used to enrich for mutants with improved growth. IPTG concentrations of 0.025-1 mM were used in liquid culture and on agar plates.

[0268] A mutant library in E. coli BL21(DE3)ΔpyrBI cells was selected with VDVAD-cpATCase that varied in the P1' residue. Selections were performed with Pro, Met, Thr, and Val. Selection with P1'Met was performed with cp ATCase without the deletion of the natural methionine, and all other selections were performed with a construct containing SEQ ID NO: 22. Selection with Met, Thr, and Val as P1' led to hundreds of positive variants, so only the largest colonies were analyzed. All 77 clones with a total of 263 mutations were analyzed together from all combined selections. Some mutations were found multiple times in independent experiments. The resulting mutations of the variants compared to SEQ ID NO: 6 are shown in Table 5 below. The P1' amino acid used in the selection is shown under "P1'cpATCase".

[0269] Mutations of the variants were analyzed, and some were selected for expression and characterization by in vitro cleavage. Variants were selected if they were enriched in liquid culture or if they contained mutations that were found independently several times. A description of these variants can be found in Example 4.

[0270] [Table 8] JPEG2026031556000009.jpg250157 JPEG2026031556000010.jpg249157 JPEG2026031556000011.jpg139166

[0271] Example 4 Characterization of variants discovered by selection cpCaspase-2 S9 D285E and S9 D285: Selection of the cpCaspase-2 D285E (SEQ ID NO: 13) library containing approximately 5500 variants was performed with VDVAD-cpATCase containing methionine as P1' at an induction strength of 0.025 mM IPTG. The E105V mutation was repeatedly found among the 16 clones analyzed. Selected variants carrying this mutation (cpCaspase-2 S9 D285E, SEQ ID NO: 1) were expressed, purified, and tested as described in Example 1.

[0272] The selected cp caspase-2 S9 D285E was mutated to generate the cp caspase-2 S9 D285 variant (SEQ ID NO: 51). The variant was expressed, purified, and tested as previously described (Example 1). The cpCaspase-2 mS9 Pro D285E and cpCaspase-2 mS9 Pro D285:cpCaspase-2 S9 D285E (SEQ ID NO: 1) variants were used for further rounds of mutation due to their improved P1' tolerance. The new mutant library contained approximately 10,000 variants and was selected with VDVAD-ΔM-Pro-cpATCase. Liquid culture selection enriched for a variant with the mutations E105V, G171D, V225G, D282E, and D285E (mS9 Pro D285E, SEQ ID NO: 70). Caspases were expressed and purified as previously described.

[0273] The selected cpCaspase-2 mS9 Pro D285E (SEQ ID NO: 70) was mutated to generate the cpCaspase-2 mS9 Pro D285 variant (SEQ ID NO: 52). The variant was expressed, purified, and tested as previously described. Variants with cpCaspase-2 mS9 Thr 0.8:K83E, E105V, E172V, V255M, and D285Y mutations were selected from mutant cpCaspase-2 S9 D285 (SEQ ID NO: 51). New variants (SEQ ID NO: 53 and SEQ ID NO: 54) were enriched in liquid culture under selection with VDVAD-Thr-cpATCase and 0.8 mM IPTG. They were expressed, purified, and tested as described in Example 1.

[0274] cpCaspase-2 S17: mutant cpCaspase-2 D285E (SEQ ID NO: 13) VDVAD-cpATCase with Met as P1' and variants with E105V, C132R, E141G, H200R, and D285E mutations selected with 0.1 mM IPTG. The variants were never purified and tested in vitro, and mutations at positions 105, 132, and 105 were repeatedly found in various experiments. The variant with cp caspase-2 S20:C203Y and D285E mutations (SEQ ID NO: 26) was selected from the mutant cp caspase-2 D285E (SEQ ID NO: 13) with VDVAD-cpATCase with Met as P1' and 0.1 mM IPTG.

[0275] cpCaspase-2 D285E SV4:V201A and a variant with the D285E mutation (SEQ ID NO: 28) were selected from mutant cpCaspase-2 D285E (SEQ ID NO: 13) with VDVAD-Val-cpATCase and 0.1 mM IPTG. The V201A mutation was found several times independently. cpCaspase-2 SV19: cpCaspase-2 SV 19 (SEQ ID NO: 81) was selected from variants with a mutated C-terminus with VDVAD-Val-cpATCase and 0.1 mM IPTG. The sequence is equal to the consensus sequence of 13 active variants with mutated C-termini. The variant with the cpCaspase-2 D285E SV30:E174G and D285E mutations (SEQ ID NO: 30) was selected from the mutant cpCaspase-2 D285E (SEQ ID NO: 13) with VDVAD-Val-cpATCase and 0.1 mM IPTG. The variant was enriched in liquid culture.

[0276] Example 5 Cleavage activity of the generated caspases and their variants 5.1 β-galactosidase The model substrate β-galactosidase contains four DXXD sites and one DXXE site, three of which are on the surface and may be accessible to caspases. After incubation of 1 mg / ml of β-galactosidase fusion protein (the N-terminal tag contains the recognition site VDVAD) with 0.1 mg / ml of cp caspase-2 (SEQ ID NO: 6) for 24 hours, no nonspecific cleavage was observed. Correct cleavage of the His tag was confirmed by N-terminal protein sequencing.

[0277] 5.2 VDVAD-SOD cleavage Figure 4B shows cleavage of the substrate 6His-VDVAD-SOD (SEQ ID NO: 193) by cp caspase-2, SEQ ID NO: 6, within 1 hour: nearly 100% of the substrate was cleaved, whereas after 6 hours without cp caspase-2, no cleavage was observed. 5.3 VDVAD-Gly-E2 cleavage values ​​for all cp caspase-2 variants tested cp caspase-2 (0.01 mg / ml) (SEQ ID NO: 6) cleaved 50% of the P1' glycine-bearing substrate VDVAD-E2 (1 mg / ml) within 1 min at 25°C in caspase assay buffer, a condition defined as the standard activity against which all other reactions were compared (Figure 4A). Not all of the variants tested cleaved the standard substrate by 50% within 1 minute. A list of all cleavages with P1'Gly is provided in Table 6.

[0278] [Table 9]

[0279] 5.4 P1' Admissibility The cleavage site specificity and P1' tolerance of caspases have been studied using peptide substrates, degradome analysis, and phage libraries. Peptides are not ideal for this purpose because their structure influences cleavage activity. On the other hand, degradome studies are influenced by the sequences present in the cells being analyzed. To our knowledge, caspase specificity and P1' tolerance for protein substrates have not been systematically tested. Therefore, we substituted the P1' residue after the cleavage site in the fusion protein VDVAD-E2 (Example 2, section 2.2) to evaluate the P1' residue-dependent cleavage efficiency of cpCaspase-2. At the P1' position, glycine was highly favored, and cleavage before any other residue was at least 5-fold less effective. Moderately well-tolerated amino acid groups included small, basic, and aromatic residues, as well as Asn and Met.

[0280] Table 7 (Table 7.1 and Table 7.2) shows the cleavage of E2 substrates with the VDVAD recognition site and various P1' residues by cp-caspase-2 variants. The activity is listed as percent activity for each cp-caspase-2 variant for cleavage of VDVAD-E2 with a P1' glycine. Table 7 therefore shows the P1' tolerance of each cp-caspase-2 variant. All values ​​(mean ± standard deviation) were determined from at least three independent experiments performed with 1 mg / ml E2. For Asp-E2, Glu-E2, Ile-E2, Pro-E2, and Val-E2 cp-caspase-2, the concentration was 0.1 mg / ml, and for all others, it was 0.01 mg / ml. The values ​​listed already take these concentration differences into account. Table 8 (Table 8.1 and Table 8.2) further shows the % cleavage activity of all cp Caspase-2 variants for all P1' amino acids relative to the cleavage activity of standard cp Caspase-2 (SEQ ID NO: 6). Table 8 therefore shows the degree of increase (or decrease) in P1' tolerance.

[0281] [Table 10]

[0282] [Table 11] JPEG2026031556000015.jpg38170

[0283] [Table 12] JPEG2026031556000017.jpg93170

[0284] [Table 13]

[0285] Taken together, these data demonstrate that variants of cp caspase-2 containing amino acid substitutions at any one or more of positions 83, 105, 171, 172, 185, 225, 255, 282, 285 of SEQ ID NO: 6 exhibit significantly improved P1' tolerance for at least one amino acid. In most cases, these variants contain significantly improved P1' tolerance for multiple amino acids. Furthermore, these data indicate that amino acid substitutions at positions 85, 86, 132, 141, 174, 200, 201, and 203 of SEQ ID NO:6 do not significantly impair caspase activity, even if they do not improve P1' tolerance. Table 6 shows, for example, that variants containing amino acid substitutions at positions 85, 86, 132, 141, 174, 200, 201, or 203 of SEQ ID NO:6 still cleave approximately 50% of the substrate VDVAD-E2 within 2 or 3 minutes. These represent examples of functionally active variants of cp-caspase-2 of the present invention. Furthermore, all of the variants selected using the selection system described in Example 3 and shown in Table 24 are further examples of functionally active variants of cp-caspase-2 because all possess catalytic activity for cleavage of the VDVAD P1 motif (caspase-2 cleavage site). Otherwise, colonies / clones would not grow.

[0286] Example 6 cp caspase-2 variants that recognize a different recognition site from VDVAD 6.1 A system similar to 3.1 for in vivo selection of cp caspase-2 variants The selection system described in Example 3, section 3.1, is used to select for caspases that tolerate cleavage sites different from VDVAD. A gene library of the 6His-GSG-XDXXD-ΔM-Thr-pyrB (SEQ ID NO: 22) cpATCase construct was cloned with degenerate primers to insert random mutations into the caspase recognition sequence at positions P5, P3, and P2. E. coli BL21(DE3)ΔpyrBI cells were generated containing the cpCaspase-2 construct (SEQ ID NO: 7) in the pACYCDuet vector. After transformation of the cpATCase library into the cells, selection as described above was carried out in M9 medium or on M9 agar plates at 30°C for 24-48 hours. Several single colonies were sequenced to analyze the nucleotide sequence of cpATCase and detect alternative cleavage sites tolerated by cpcaspase-2. Alternative cleavage sites were cloned into the substrate protein and the activity of various cp caspase-2 variants was tested as described above in Example 1.

[0287] Example 7 residue Val 105 and Gly 171 Simultaneous mutation of 7.1 Construction Design and Selection Saturation mutagenesis with degenerate primers designed to generate all 19 possible amino acid substitutions in the protein was performed using cpCaspase-2 S9 (SEQ ID NO: 51) containing an additional G171D substitution as a template. A gene library containing all 400 variants with possible combinations of mutations at positions 105 and 171 was transformed into E. coli BL21(DE3)ΔpyrBI cells containing the VDVAD-cpATCase substrate (SEQ ID NO: 22) with a P1'Thr. Selection, as described in Example 3 above, was carried out in M9 medium or on M9 agar plates at 30°C for 24-48 hours. DNA from several single colonies was analyzed to detect mutation combinations in active variants. Combinatorial mutants were expressed, purified, and tested as described above in Example 1.

[0288] Example 8 Comparison of the generated variants with wild-type caspase-2 DEVD-E2 (SEQ ID NO: 57) DEVD is the preferred cleavage site for caspase-3 and caspase-7. DEVD-E2 was used to evaluate the influence of the P5 residue, because the effects of amino acids at the P2 and P3 positions on caspase-2 activity are not expected to be significant. The substrate was processed 140-fold slower by cpcaspase-2 (SEQ ID NO: 6) than by VDVAD-E2 (SEQ ID NO: 33). This indicates that recognition of the P5 residue is crucial for caspase-2 and cpcaspase-2. This is in accordance with the results from fluorescent peptides [26, 24] and confirms the initial assumption of this study that caspase-2 was more specific than other caspases due to its pentapeptide recognition site. This seems even more pronounced for circularly permuted variants, since the literature only describes a 35-fold increase in activity by VDVAD relative to DEVD

[26] .

[0289] 8.1 Comparison of specificity with wild-type caspase-2 The specificity of cp-caspase-2 (SEQ ID NO: 6) was compared with commercially available wild-type caspase-2 (human caspase-2, recombinant caspase-2, active caspase-2, Enzo Life Sciences, Farmingdale, NY, USA). 72 U / ml of wild-type caspase-2 was used in the cleavage reaction described herein. This is equivalent to approximately 0.005 mg / ml enzyme, half the concentration used in standard reactions with cp-caspase-2. However, wild-type caspase was 6-fold less active than cp-caspase-2 under the same conditions (1 mg / ml VDVAD-E2 was processed to 50% within 6 minutes).

[0290] While it can be difficult to compare the absolute activity of the enzymes, clear differences can be seen between their specificities due to different purity and concentrations. Wild-type caspase-2 cleaved DEVD-E2 only 44-fold slower than VDVAD-E2, while cp-caspase-2 is 140-fold more selective for VDVAD than DEVD. Thus, cp-caspase-2 is 3-fold more specific than the wild-type enzyme (Figure 9). Figure 9 shows the cleavage of DEVD-E2 by cp-caspase-2 (SEQ ID NO: 6) and wild-type caspase-2. Reduction of cleavage activity by the DEVD-E2 substrate is given as x-fold reduction compared to VDVAD-E2 processing. The graph shows the mean ± standard deviation of at least three independent experiments. ( * ) at the p level ≤ 0.05, ( ** ) at the level of p ≤ 0.01, and ( ***) indicates statistical significance at the level of p≦0.001. 8.2. Generation and characterization of wild-type caspase-2 Human caspase-2 was generated for comparison of wild-type caspase-2 with the cp-caspase-2 variant.

[0291] Generation of wt caspase-2: Production of wt caspase-2 was carried out in a 30 L (23 L net volume, 5 L batch volume) computer-controlled bioreactor (Bioengineering; Wald, Switzerland) equipped with a standard control unit (Siemens PS7, Intellution iFIX). pH was maintained at a set point of 7.0 ± 0.05 by addition of 25% w / w ammonia solution, and temperature was set at 37 °C ± 0.5 °C in the batch phase and 30 °C ± 0.5 °C in the fed-batch phase. To avoid oxygen limitation, DO levels were kept above 30% saturation by adjusting the stirrer speed and process air aeration rate. The maximum overpressure in the headspace was 1.1 bar. Inoculum precultures were grown in synthetic medium calculated to yield 3 g L. For incubation, 1 mL of deep-frozen MCB was aseptically transferred to 400 mL medium and grown in two 2000 mL shake flasks at 37°C and 180 rpm until an OD of approximately 4 was reached.

[0292] For the cultivation, a minimal medium calculated to produce 64 g of cell dry mass (CDM) during the batch phase and 890 g of CDM during the feeding phase was used. The batch medium was prepared volumetrically; the components were dissolved in 8 L RO-H2O. The feed medium was prepared gravimetrically; the final mass was 8.45 kg. All components for the feed medium were weighed and dissolved separately in RO-H2O. All components (obtained from MERCK) were added in relation to the theoretical grams of cell dry mass to be produced: the composition of the batch and feed medium was as follows: 94.1 mg / g KH2PO4, 31.8 mg / g H3PO4 (85%), 41.2 mg / g C6H5Na3O7*2H2O, 45.3 mg / g NH4SO4, 46.0 mg / g MgCl2*2H2O, 20.2 mg / g CaCl2*2H2O, 50 μL trace element solution, and 3.3 g / g C6H 12 O6*H2O. The trace element solution was prepared in 5N HCl and contained 40 g / L FeSO4·*7H2O, 10 g / L MnSO4·*H2O, 10 g / L AlCl3·*6H2O, 4 g / L CoCl2, 2 g / L ZnSO4·*7H2O, 2 g / L Na2MoO2·*2H2O, 1 g / L CuCl2·*2H2O, and 0.5 g / L H3BO3. Multi-ingredient yeast extract (150 mg / calculated CDMg) was added to the batch medium to promote initial population growth. Nitrogen levels were maintained by adding 25% wt / wt ammonium hydroxide solution for pH control. A total volume of 0.5 mL / L of antifoam agent (PPG 2000) was added at the start.

[0293] The fed-batch phase (29 h) was carried out at 30 °C using an exponential feeding strategy with a constant growth rate of μ = 0.1 h-1. Substrate feeding was controlled by increasing the pump rate according to the exponential growth algorithm, X = X0·eμt, using superimposed feedback control of mass loss in the substrate tank. Induction was initiated in the fed-batch phase by adding 0.5 μmol IPTG / g CDM directly to the feeding medium, and protein production was achieved within four generations. The IPTG concentration was calculated according to the theoretical final CDM.

[0294] Batch Media Components Ingredients Quantity KH2PO40.094g / g final CDM 85%H3PO40.032g / g final CDM Yeast extract 0.15g / CDMg (batch) C6H5Na3O 2H2O 0.25g / g Final CDM MgCl2·7H2O 0.1g / CDMg (batch) CaCl2·2H2O 0.02g / CDMg (batch) (NH4)2SO40.046g / g final CDM Trace element solution 50μL / CDMg (batch) C6H 12 O6·H2O 3.3g / CDMg (batch)

[0295] Fed-batch medium components Ingredients Quantity MgCl2·7H2O 0.1g / CDMg (fed batch) CaCl2·2H2O 0.02g / CDMg (fed batch) Trace element solution 50μL / CDMg (fed batch) C6H 12 O6·H2O 3.3g / CDMg (fed batch) In addition to standard online monitoring (pH, stirrer speed, temperature, and pO2), the concentrations of pO2 and O2 in the exhaust air were measured using a BlueSens gas analyzer. Standard offline process parameter sampling began after one generation in fed-batch mode. The first sample was collected from the bioreactor before induction. Optical density (OD600) was measured spectrophotometrically at a wavelength of λ = 600 nm. Samples were diluted in PBS to ensure measurements in the linear range of 0.1 to 0.8. Cell dry mass (CDM) was determined by centrifugation of 10 mL of cell suspension at 8500 rpm for 8 min. The supernatant was discarded, and the cells were resuspended in RO-HO and centrifuged. The water was discarded, and the cells were again resuspended in RO-HO. The cell suspension was transferred to a previously weighed beaker. The beaker was dried at 105 °C for at least 24 h and again weighed. The difference in mass accounts for the CDM.

[0296] To determine the content of cp caspase-2 and variants, an approximately 1.0 mg CDM aliquot of the sample was centrifuged (13,200 rpm for 10 minutes); the supernatant was discarded, the inside of the tube was carefully blotted dry, and the sample was stored at -20°C. The E. coli cell mass was harvested by centrifugation at 18,590 rcf for 15 minutes, and the supernatant was discarded. The E. coli cell harvest was solubilized using homogenization buffer (50 mM sodium phosphate, 300 mM NaCl, pH 8.0). The cells were resuspended at a concentration of 400 g wet cell mass per liter. Cell lysis was performed by high-pressure homogenization at 1400 bar / 140 bar with two passes using an in-line counterflow chiller set at 10°C. The homogenate was centrifuged at 18,590 rcf for 2.5 hours at 4°C. The pellet was discarded, and the supernatant was used. Before chromatography, the supernatant was filtered through a 0.22 μm membrane. Poly-His-tagged wt caspase-2 was captured using immobilized metal affinity chromatography (IMAC) using the following buffers: equilibration buffer: 50 mM sodium phosphate, 300 mM NaCl, 20 mM imidazole, pH 8.0; elution buffer: 50 mM sodium phosphate, 300 mM NaCl, 500 mM imidazole, pH 8.0.

[0297] Imidazole was added to the clarified supernatant to a final concentration of 20 mM imidazole prior to IMAC. 57 CV of the clarified supernatant was loaded onto an equilibrated Ni-Sepharose 6 Fast Flow column (50 × 18 mm, 35 mL). A 7-minute residence time was used during loading and a 3-minute residence time was used for subsequent steps. After loading was complete, the column was washed with equilibration buffer for 10 CV. Bound wt caspase 2 was eluted using a step gradient to 100% elution buffer for 10 CV. The eluted fractions were analyzed using SDS-PAGE, and all fractions containing wt caspase-2 were used in the next purification step. The captured eluate of wt caspase-2 was buffer exchanged prior to the polishing chromatography step. Tangential flow UF / DF with a 5 kDa cutoff membrane was used with a sample buffer of 50 mM sodium citrate, pH 5.0. A total of 5 volumes were exchanged.

[0298] The capture step employed cation exchange chromatography on SP Sepharose HP (5 x 24 mm, 0.5 mL) using the following buffers: Equilibration Buffer A: 50 mM sodium citrate, pH 5.0. Elution Buffer B: 50 mM sodium citrate, 1 M NaCl, pH 5.0. The buffer-exchanged capture eluate was loaded onto the equilibrated polishing column. The residence time was held constant at 5 min. 37 CV of the buffer-exchanged capture eluate was loaded onto the column. wt caspase-2 was eluted with a linear gradient of 0 to 100% B over 10 CV. Elution fractions were analyzed by Western blot and SDS-PAGE, and fractions positive for the small subunit of wt caspase-2 were combined and stored at -80°C. Oxidation-induced activity loss was reversed by incubating wt caspase-2 with 100 mM DTT for 15 min before performing enzyme kinetic measurements. Characterization of wt caspase-2 FRET assay Michaelis-Menten kinetics were determined for wt caspase-2 and cp caspase-2 toward the following substrates: VDVADFA, VDVADGA, VDVADQA, and VDVADVA. The P1' amino acid is indicated by bold and underlined font.

[0299] [Table 14]

[0300] The FRET results in Table 9 demonstrate significant differences between the two proteases. cp caspase-2 exhibits catalytic efficiency roughly one order of magnitude higher than wt caspase-2. While the Michaelis constant, KM, appears largely unaffected by the circular permutation, the turnover number, kcat, accounts for the complete difference in catalytic efficiency, kcat / KM, between wt caspase-2 and cp caspase-2. Purified wt caspase-2 appears to exhibit slightly better P1' tolerance compared to cp caspase-2 (neither contains the amino acid substitutions described herein for improved P1' tolerance). For example, F as P1' is cleaved with 2.5% catalytic efficiency in cp caspase-2 compared to 3.2% in wt caspase-2. This slight increase in P1' (1.3-2.3-fold increase) is overshadowed, on average, by the 11-fold lower catalytic efficiency and 8-fold lower turnover number of wt caspase-2. High temperature tolerance Cleavage of a thermostable model fusion tag protein, GFP, was used to quantify the tolerance of caspase-2 to high temperatures.

[0301] [Table 15]

[0302] The GFP cleavage results in Table 10 demonstrate comparable thermostability between the two proteases. The cleavage reaction with cp caspase-2 is 1.6-fold faster than with wt caspase-2 at 25°C. This difference increases to 2.3-fold at 50°C, indicating increased stability of cp caspase-2 at elevated temperatures. In general, the cleavage reaction at 50°C is 1.9-fold faster for wt caspase-2 and 2.8-fold faster for cp caspase-2. This is a clear advantage if a thermostable target protein must be processed. Tolerance to chaotropic conditions Cleavage of a model fusion tag protein, FGF2, which is stable in 4 M urea, was used to quantify the tolerance of caspase-2 to chaotropic conditions.

[0303] [Table 16]

[0304] The FGF2 cleavage results in Table 11 show comparable tolerance to chaotropic conditions between the two proteases. To quantify the cleavage products within the linear range, the reactions had to be stopped at different time points. Both proteases exhibited nearly identical behavior in the presence of 4 M urea, with the reaction rates reduced to 1.2% and 1.3% for wt caspase-2 and cp caspase-2, respectively. For this particular model protein, cp caspase-2 exhibited a 3.2-fold increase in reaction rate compared to wt caspase-2.

[0305] manufacturability Perhaps the greatest observable difference between the two proteases lies in ease of manufacture. To represent the difference in manufacturability between wt caspase-2 and cp caspase-2, we calculated the amount of dry cell mass required to produce 1 milligram of purified enzyme. This takes into account differences in specific protein content of E. coli fermentations and differences in downstream processing yields. This does not account for differences in biomass yield between fermentations. Producing 1 mg of wt caspase-2 required 70 g of cell dry mass (CDM). Producing cp caspase-2 required only 34 mg of CDM per milligram of pure enzyme. This represents a 2,033-fold difference in manufacturability. conclusion FRET assay results using four different P1' amino acids showed a general trend of 10-fold higher catalytic efficiency for cp-caspase-2 compared to wt-caspase-2. Cleavage of non-peptide substrates was 2- to 3-fold faster, depending on the protein substrate. Cyclic permutation of caspase-2 apparently led to increased thermostability, as indicated by a greater increase in turnover rate at 50°C. It also appeared to tolerate chaotropic conditions slightly better. The biggest difference between the wt and cp enzymes is their manufacturability. While the expression level of wt caspase-2 is very low (below the limit of quantification), cp caspase-2 reaches an expression level with a specific protein content of 80 mg / g CDM. This also results in significantly lower losses in DSP, and a process yield of approximately 35% can be achieved for cp caspase-2.

[0306] Example 9 cp Caspase-2 and variant generation process 9.1 Upstream processing of cp caspase-2 and variants For the production of cp-caspase-2 and variants with and without solubility tags, laboratory-scale fermentations were carried out as described below. Different expression clones were compared with respect to cell growth and soluble recombinant protein production. For final process optimization, a series of culture runs were performed following a design of experiments (DoE) approach.

[0307] 9.1.1 Bacterial strains, plasmids, and cp-caspase-2 and variants E. coli strain BL21(DE3) [F - , fhuA2, lon, ompT, gal, dcm, ΔhsdS λ DE3 [λ sBamHIo, ΔEcoRI-B int::(lacI::PlacUV5::T7 gene1)i21 Δnin5] was transformed with pET30a vectors carrying the genes for cp caspase-2 or variants with and without solubility tags under the T7 promoter / operator system. Expression systems cultivated in lab-scale bioreactors are listed in Table 12.

[0308] [Table 17]

[0309] 9.1.2 Laboratory-scale fermentation of cp caspase-2 and variants. 9.1.2.1 Fermentation medium For high cell density (HCD) cultivation experiments, a minimal medium calculated to produce 80 g cell dry mass (CDM) during the batch phase and 1450 g CDM during the feeding phase was used. The batch medium was prepared volumetrically; components were dissolved in 10 L RO-HO. The feed medium was prepared gravimetrically; the final mass was 10.1 kg. All components for the feed medium were weighed and dissolved separately in RO-HO. All components (obtained from MERCK) were added in relation to the theoretical grams of cell dry mass to be produced: the composition of the batch and feed medium was as follows: 94.1 mg / g KH2PO4, 31.8 mg / g H3PO4 (85%), 41.2 mg / g C6H5Na3O7*2H2O, 45.3 mg / g NH4SO4, 46.0 mg / g MgCl2*2H2O, 20.2 mg / g CaCl2*2H2O, 50 μL trace element solution, and 3.3 g / g C6H 12 O6*H2O. The trace element solution was prepared in 5N HCl and contained 40 g / L FeSO4·*7H2O, 10 g / L MnSO4·*H2O, 10 g / L AlCl3·*6H2O, 4 g / L CoCl2, 2 g / L ZnSO4·*7H2O, 2 g / L Na2MoO2·*2H2O, 1 g / L CuCl2·*2H2O, and 0.5 g / L H3BO3. Multi-component yeast extract (150 mg / calculated CDMg) was added to the batch medium to promote initial population growth. Nitrogen levels were maintained by adding 25% wt / wt ammonium hydroxide solution for pH control. A total volume of 0.5 mL / L of antifoam agent (PPG 2000) was added at the start. A preculture for inoculation was grown in a synthetic medium calculated to yield 3 g / L.

[0310] [Table 18]

[0311] [Table 19]

[0312] 9.1.2.2 Culture and induction conditions for standardized laboratory-scale fermentations All HCD fermentations were carried out in a 30 L (23 L net volume, 5 L batch volume) computer-controlled bioreactor (Bioengineering; Wald, Switzerland) equipped with a standard control unit (Siemens PS7, Intellution iFIX). pH was maintained at a set point of 7.0 ± 0.05 by adding 25% w / w ammonia solution, and temperature was set at 37 °C ± 0.5 °C during the batch phase and 30 °C ± 0.5 °C during the fed-batch phase. To avoid oxygen limitation, DO levels were maintained above 30% saturation by adjusting the stirrer speed and process air aeration rate. The maximum overpressure in the headspace was 1.1 bar. Foaming was suppressed by adding 0.5 mL / L antifoam (PPG 2000 Sigma Aldrich) to the batch medium and by pulse addition of antifoam during the fed-batch phase. Cultures were inoculated with overnight precultures. Precultures were prepared by inoculating 200 mL LB medium with 1 mL of deep-frozen WCB in a 2000 mL shake flask. Cells were incubated at OD on an orbital shaker at 180 rpm and 37°C. 600 The preculture was then grown in batches until it reached an initial OD of approximately 4. 600 The cells were inoculated with 1000 ml of ethanol and cultured at 37°C. At the end of the batch phase, exponential substrate feeding was initiated as soon as the cells entered the stationary growth phase. The fed-batch phase (29 hours) was carried out at 30°C with μ = 0.1 h -1 The exponential growth algorithm, X = X0·e, was implemented using a convolutional feedback control of mass loss in the substrate tank. μt Substrate feeding was controlled by increasing the pump rate according to the formula: Induction was started in the fed-batch phase by adding 0.5 μmol IPTG / g CDM directly to the feeding medium, and protein production was achieved within four generations. The IPTG concentration was calculated according to the theoretical final CDM.

[0313] 9.1.2.3 Culture and induction conditions for DoE approaches The pre-culture and batch stages were identical to the standardized fermentation described previously. The fed-batch stage was carried out at 30°C. For biomass production, the first fed-batch stage was carried out with exponential feeding (μ = 0.17 h ) for 1.72 generations. -1 ) As previously described, an exponential growth algorithm, X = X0, was used with superimposed feedback control of mass loss in the substrate tank. * e μt Substrate feeding was controlled by increasing the pump rate according to the following formula: In the second feeding stage, the substrate was fed at lower growth rates (0.03, 0.05, and 0.07 h ). -1 The fermentation time was adjusted to 60.5, 39, and 30 h, resulting in a total feed time of 60.5, 39, and 30 h. The calculated CDM was 70 g / L. To ensure adequate adaptation to the low growth conditions, cells were grown for 0.25 generations without induction. Subsequently, induction was performed with three different IPTG concentrations (0.5, 0.9, and 1.3 μmol / CDMg) for two generations. Nine DoE fermentations were performed.

[0314] 9.1.2.4 Fermentation Monitoring In addition to standard online monitoring (pH, stirrer speed, temperature, and pO2), the concentrations of pO2 and O2 in the exhaust air were measured by a BlueSens gas analyzer. Sampling of standard offline process parameters began after one generation in fed-batch mode. The first sample was collected from the bioreactor before induction. Optical density (OD 600 ) was measured spectrophotometrically at a wavelength of λ = 600 nm. Samples were diluted in PBS to ensure measurements in the linear range of 0.1 to 0.8. Cell dry mass (CDM) was determined by centrifugation of 10 mL of cell suspension at 8500 rpm for 8 minutes. The supernatant was discarded, and the cells were resuspended in RO-HO and centrifuged. The water was discarded, and the cells were again resuspended in RO-HO. The cell suspension was transferred into a previously weighed beaker. The beaker was dried at 105 °C for at least 24 hours and again weighed. The difference in mass accounts for the CDM. To determine the content of cp caspase-2 and variants, an approximately 1.0 mg CDM aliquot of the sample was centrifuged (10 min at 13200 rpm); the supernatant was discarded, the inside of the tube was carefully blotted dry, and the sample was stored at -20°C.

[0315] 9.1.2.5 Determination of cp-caspase-2 and variants in fermentation samples Cell disintegration, fractionation of soluble and insoluble recombinant proteins, and IB lysis: Cell disintegration was performed from fermentation samples containing approximately 1.0 mg of CDM. 200 μL of cell integration buffer was added to the cell pellet and vortexed until the pellet was completely resuspended. For cell disruption, 50 μL of lysozyme and 50 μL of benzonase were added and incubated with shaking at room temperature. 100 μL of Triton X-100 was added and the sample was again incubated with shaking. Subsequently, the sample was centrifuged at 13,000 rpm at 4°C to separate soluble proteins and inclusion bodies (IBs). The supernatant was transferred to a new reaction tube for direct analysis (SDS-PAGE) or stored at -20°C.

[0316] The remaining pellet (IBs and cell debris) was washed twice by resuspension in 1 mL Tris / HCl (100 mM). After resuspension, the pellet was centrifuged at 13,000 rpm for 10 minutes at 4°C. The supernatant was discarded. 400 μL IB solvent buffer was then added and incubated with shaking at room temperature for 30 minutes. Finally, the sample was again centrifuged, and the supernatant containing the dissolved IBs was used for analysis (SDS-PAGE) or stored at -20°C.

[0317] [Table 20]

[0318] [Table 21]

[0319] [Table 22]

[0320] SDS-PAGE Recombinant proteins were separated and analyzed using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Electrophoresis was performed using acrylamide gradient precast gels (NuPAGE 4-12% BisTris, Thermo Fisher Scientific, Waltham, MA, USA) and NuPAGE® MES SDS Running Buffer. Loading samples were prepared by mixing 13 μL of supernatant (soluble fraction) or IB supernatant (insoluble fraction) with 5 μL LDS sample buffer (4×) and 2 μL NuPAGE® Reducing Agent (10×) and incubating the mixture at 70°C for 10 minutes in a thermos mixer. Ready-to-use molecular weight markers (Mark12®, Unstained Standard, Invitrogen) were directly loaded as size markers. For quantification, purified T7AC_6H_cpCasp2 standards (75, 50, and 25 μg / mL) listed in Table 12, generated as described in Example 9 (see sections 9.1 and 9.2), were used. Electrophoresis settings were 200 V and 400 mA for 40-50 minutes in an XCell SureLock™ Electrophoresis Cell chamber (Thermo Fisher Scientific). After electrophoresis, the SDS gel was fixed in a fixative solution (40% ethanol; 50% d The gels were fixed in HO; 10% acetic acid for 30 min and then stained with Coomassie Brilliant Blue R250 staining solution for 30 min. Finally, the gels were destained in destaining solution (25% acetic acid; 8% ethanol; 67% dThe gels were destained in HO for at least 2 hours. The gels were transferred to water, scanned with a desktop scanner, converted to grayscale, and analyzed using the software ImageQuant TL (7.0). The concentrations of cp caspase-2 and variants were quantified via linear regression curves.

[0321] 9.1.2.6 When μ=0.1 -1 Comparison of the production of cp caspase-2 and variants with and without solubility tags in fermentations with and without an IPTG concentration of 0.5 μmol IPTG / CDMg during induction. While overexpression of cp-caspase-2 has been attempted in E. coli, the expression rate of soluble cp-caspase-2 was generally low. To increase fermentation titers, a solubility tag was added to the enzyme. The tag, T7A3 (SEQ ID NO: 37), is based on a highly negatively charged peptide derived from the T7 bacteriophage. For use with cp-caspase-2 variants, we modified the tag after using a cleavage site prediction algorithm to avoid autocatalytic cleavage. The modified solubility tag, T7AC (SEQ ID NO: 43), was engineered to double the expression level of soluble cp-caspase-2. For this soluble, His-tagged enzyme, we developed a downstream process based on an IMAC capture step and a cation exchange chromatography (CEX) polishing step. This downstream process allowed us to produce highly pure (>99% protein purity by reverse-phase HPLC) cp-caspase-2 at a scale of several hundred milligrams.

[0322] Standardized laboratory-scale fermentations were performed to evaluate the production of cp-caspase-2 and variants with and without a solubility tag (T7AC). Expression clones were compared with respect to cell growth and production of soluble and insoluble recombinant protein. Comparing the production of 6H-cpCasp2D and T7AC-6H-cpCasp2D in laboratory-scale fermentations, we observed that production of 6H-cpCasp2D without a solubility tag primarily led to inclusion body formation (Figure 10A). At the end of the culture, the calculated CDM was not reached due to excessively high expression levels (Figure 11). Addition of the T7AC solubility tag to the N-terminus of the caspase increased soluble expression (Figure 10B), resulting in slightly lower overall recombinant protein expression. Cell growth followed the calculated CDM (Figure 11). The final CDM was approximately 77.5 g / L, respectively, for a total of 1549 g. The solubility tag did not negatively affect subsequent metal affinity chromatography.

[0323] Figure 10 shows laboratory-scale fermentations of E. coli BL21(DE3)(pET30a_6H-cpCasp2D) (A, left two graphs) and BL21(DE3)(pET30a_T7AC-6H-cpCasp2D) (B, right two graphs): expression of soluble and insoluble 6H-cpCaspase-2D (cpCasp2) (A) and T7AC-6H-cpCaspase-2D (T7AC-6H-cpCasp2) (B) over time as specific yield [mg / g] and volumetric yield [g / L], with (T7AC_6H-cpCasp2, B) and without (cpCasp2, A) the solubility tag, T7AC. FIG. 11 shows the lab-scale fermentation:biomass process of E. coli BL21(DE3)(pET30a_6H-cpCasp2D) and BL21(DE3)(pET30a_T7AC-6H-cpCasp2D).

[0324] Comparing the production of three cp-caspase-2 variants (cp-caspase-2, mS9Pro E285, and mS9 Pro D285) with and without the T7AC solubility tag, we found that the variants themselves had no effect on performance, with no significant differences in cell growth or soluble cp-caspase-2 expression. The T7AC solubility tag significantly improved soluble expression of all three variants. Cell growth kinetics across all cultures was similar (VC<4%). Minor deviations were observed only at the end of fermentation (Figure 12). The fermentation strategy and low induction level (0.5 μmol IPTG / CDMg) did not strain the host metabolism. Addition of a T7AC solubility tag to the N-terminus of all cp caspase-2 variants increased soluble expression levels (Figure 13). Final soluble product titers were up to 1.2 g / L.

[0325] Figure 12 shows the biomass process of laboratory-scale fermentation of three cp caspase-2 variants (cp caspase-2 (cpCasp2D), mS9 Pro E285 (mS9ProE), and mS9 Pro D285 (mS9ProD)) with and without the T7AC solubility tag in E. coli BL21(DE3) with the pET30a vector; the mean and standard deviation for these six cultures are shown. Total CDM is shown as the average of all six fermentations, including the standard deviation, compared to expected growth (calculated CDM). Figure 13 shows the normalized soluble production of cp caspase-2 of three different cp caspase-2 variants (cp caspase-2 (cpCasp2D), mS9 Pro E285 (mS9ProE), and mS9 Pro D285 (mS9ProD)) with and without the T7AC solubility tag in E. coli BL21(DE3) harboring the pET30a vector.

[0326] 9.1.2.7 DoE Approach to Process Optimization For final process optimization, a series of cultivation runs were performed following the previously described design of experiments (DoE) approach. The production clone BL21(DE3) (pET30a-T7AC_6H_cpCasp2) was used. Various growth rates (μ = 0.03, 0.05, and 0.07 h ) were used. -1 The effects of induction (0.5, 0.9, and 1.3 μmol IPTG / CDMg) and induction strength (0.5, 0.9, and 1.3 μmol IPTG / CDMg) on ​​cell growth and production of soluble and insoluble recombinant proteins were investigated. The results are shown in Table 18.

[0327] [Table 23]

[0328] It was observed that the specific yield of soluble cp-caspase-2 was higher at low growth rates and IB formation was reduced. -1 At the end of the fermentation, the calculated CDM was not reached due to too high an expression level (Figure 14). Figure 14 shows the growth kinetics of E. coli BL21(DE3)(pET30a-T7AC_6H-cpCasp2) in a carbon-limited two-stage fed-batch culture (μ=0.17 followed by 0.03 h during induction) with three different IPTG induction intensities. Nevertheless, μ=0.03 -1 and 0.9 or 0.5 μmol IPTG / CDMg, the highest volumetric solubility yields were reached (FIG. 15).

[0329] Figure 15 shows E. coli BL21(DE3)(pET30a-T7AC_6H-cpCasp2) in a carbon-limited two-stage fed-batch culture (μ=0.17 followed by 0.03 h during induction) at three different IPTG induction intensities. -1 ) and induced with various IPTG levels to obtain volumetric titers of soluble cp-caspase-2 (soluble POI [g / L]). cp-caspase-2 was quantified by SDS-PAGE. The mean and standard deviation for each determination are shown (n=3). This process can be used for all cp caspase-2 variants, whether or not they contain mutations at positions that increase P1' permissiveness.

[0330] 9.2 Downstream processing of cp caspase-2 and variants 9.2.1 Downstream processing of cp-caspase-2 without a solubility tag The E. coli cell mass from the fermentation described under 10.1 was harvested by centrifugation at 18,590 rcf for 15 minutes and the supernatant discarded. The E. coli cell harvest was solubilized using homogenization buffer (50 mM sodium phosphate, 500 mM NaCl, 20 mM imidazole, pH 7.0). The cells were resuspended at a concentration of 150 g wet cell mass per liter. Cell lysis was carried out by high-pressure homogenization at 700 bar / 70 bar by two passes. The homogenate was centrifuged at 18,590 rcf for 2 hours. The pellet was discarded and the supernatant used. Prior to chromatography, the supernatant was filtered through a 0.22 μm membrane. Poly-His-tagged cp-caspase-2 was captured using immobilized metal affinity chromatography. The following buffers were used: equilibration buffer: 50 mM sodium phosphate, 500 mM NaCl, 20 mM imidazole, pH 7.0; wash buffer: 50 mM sodium phosphate, 500 mM NaCl, 20 mM imidazole, 30% isopropanol, pH 7.0; elution buffer: 50 mM sodium phosphate, 500 mM NaCl, 500 mM imidazole, pH 7.0.

[0331] The clarified supernatant was loaded onto a Ni-Sepharose 6 Fast Flow column equilibrated to a capacity of approximately 40 mg / mL. A residence time of 3 to 5 minutes was used. After loading was complete, the column was washed with equilibration buffer for 5 column volumes (5 CV), wash buffer for 10 CV, and equilibration buffer for 5 CV. Bound cp-caspase-2 was eluted using a linear gradient of 0 to 100% elution buffer over 10 CV, with a 10 CV hold step to ensure complete elution of all protein. The eluted fractions were analyzed using SDS-PAGE and all fractions containing cp caspase-2 were used in the next purification step.

[0332] The captured eluate of cp caspase-2 was buffer exchanged prior to the polishing chromatography step. Tangential flow UF / DF with a 5 kDa cutoff membrane was used with a sample buffer of 50 mM sodium citrate, pH 5.0. A total of 5 volumes were exchanged. The capture step employed cation exchange chromatography on a SOURCE 30S using the following buffers: Equilibration Buffer A: 50 mM sodium citrate, pH 5.0. Elution Buffer B: 50 mM sodium citrate, 1 M NaCl, pH 5.0. The buffer-exchanged capture eluate was loaded onto the equilibrated polishing column. The residence time was held constant at 5 min. The column was loaded to a capacity of approximately 100 mg / ml. cpCaspase-2 was eluted with a linear gradient of 0 to 100% B over 20 CV. Elution fractions were analyzed using RP-HPLC as described under 10.3, and fractions showing approximately 99% purity were combined and stored at -80°C. 9.2.2 Downstream processing of solubility-tagged cp-caspase-2 Lysis was carried out as described in section 9.2.1, except that the cells were resuspended at a concentration of 200 g wet cell mass per liter.

[0333] IMAC was performed as previously described, except that the column was loaded to a capacity of approximately 30 mg / ml and a residence time of 2-3 minutes was selected. After loading was complete, the column was washed with equilibration buffer for 5 column volumes (5 CV), wash buffer for 5 CV, and equilibration buffer for 1 CV. Bound cp-caspase-2 was eluted using a linear gradient of 0-50% elution buffer over 5 CV, followed by a gradient of 50-100% B over 1 CV, followed by a 2 CV hold step at 100% to completely elute all protein. The elution peak fractions were used for the next purification step. Buffer exchange of the capture eluate was performed as previously described. The capture step employed cation exchange chromatography on SP Sepharose High Performance using the following buffers: Equilibration Buffer A: 50 mM sodium citrate, pH 5.0. Elution Buffer B: 50 mM sodium citrate, 1 M NaCl, pH 5.0. The buffer-exchanged capture eluate was loaded onto the equilibrated polishing column. The residence time was held constant at 1-2 min. The column was loaded to a capacity of approximately 50 mg / ml. The column was washed with 30% B for 5 column volumes (5 CV), and cp caspase-2 was eluted with 10 CV of 45% B. The column was stripped with 3 CV of 100% B. The eluted fractions were aliquoted and stored at -80°C. Purity was determined by RP-HPLC to be approximately 99% (98.6-99.4%).

[0334] 9.3 Characterization of cp caspase-2 and variants 9.3.1 Purity Determination of cp Caspase-2 and Variants (HPLC) Experiments were performed on a Tosoh TSKgel Protein C4-300, L x ID 5 cm x 4.6 mm, 3 μm column with a guard column on a Waters e2695 HPLC. Mobile phase A was water with 0.15% trifluoroacetic acid (TFA), and mobile phase B was acetonitrile with 0.15% TFA. The flow rate was 1 ml / min. The column oven temperature was 40°C, and the autosampler temperature was 10°C. The following gradient, shown in Table 19, was used:

[0335] [Table 24] 200 μL of purified cp-caspase-2 (or variant) sample (approximately 4 g / L) was diluted with 100 μL PBS and 100 μL 2 M dithiothreitol (DTT). 10 μL of 0.22 μm filtered sample was injected. The outlet was monitored at 214 nm and 280 nm. The HCP peak eluted between retention times of 3.8 and 9 minutes. The cp-caspase-2 peak eluted between 9.2 and 12.4 minutes. The peak area within the 214 nm signal was used to calculate the purity of the protein of interest.

[0336] 9.3.2 Quantification of released fusion tag by RP-HPLC A calibration curve was generated by mixing substrate proteins, such as human fibroblast growth factor 2 (hFGF-2) and cp caspase-2, in a 10:1 ratio (in triplicate) and incubating at 25°C for 4 hours with shaking. The reaction was stopped by adding formic acid to a final concentration of 0.3% or cystamine to a final concentration of 10 mM. Each triplet was diluted with PBS buffer to obtain six different concentrations (100 μM, 46 μM, 21 μM, 10 μM, 4 μM, and 2 μM). Ten μL of the 0.22 μm filtered sample was injected into a reversed-phase high-pressure liquid chromatograph (RP-HPLC) using the method outlined below. The outlet was monitored at 214 nm. The fusion tag peak eluted between retention times of 3.9 and 5.6 minutes. The peak area within the 214 nm signal was used to calculate the amount of fusion tag using a linear calibration function.

[0337] The experiments were performed on a Tosoh TSKgel Protein C4-300, L x ID 5 cm x 4.6 mm, 3 μm column with a guard column on a Waters e2695 HPLC. Mobile phase A was water with 0.15% trifluoroacetic acid (TFA), and mobile phase B was acetonitrile with 0.15% TFA. The flow rate was 1 mL / min. The column oven temperature was 40°C, and the autosampler temperature was 10°C. The following gradient was used (Table 20):

[0338] [Table 25]

[0339] 9.3.3 Determining Enzyme Activity by FRET Assay Forster resonance energy transfer (FRET) assays for the determination of Michaelis-Menten enzyme activity parameters were carried out as follows. Substrates were obtained from Bachem AG and were of the general structure Abz-VDVAD-XA-Dap (Dnp), with all 20 amino acids substituted for X (P1' position). All substrates were dissolved in 10 mM HEPES, pH 7.5, to a concentration of 750 μM. Abz stands for 2-aminobenzoyl, and Dap (Dnp) stands for α,β-diamino-propionic acid (2,4-dinitrophenyl). The assay buffer was 50 mM HEPES, 150 mM NaCl, pH 7.2. Calibration curves were generated by incubating various amounts of substrate (20 μM, 6.9 μM, 2.4 μM, 0.8 μM, 0.3 μM, 0.1 μM) with 72 μM cpCaspase-2 D285E in phosphate-buffered saline (PBS) for up to 24 h at room temperature. 100% conversion was assumed. Fluorescence was measured in black 96-well plates on a Tecan Infinite M200 Pro plate reader. The excitation wavelength was 320 nm, and the emission wavelength was 420 nm.

[0340] Michaelis-Menten kinetics were measured by varying substrate concentrations (200 μM, 100 μM, 50 μM, 20 μM, and 10 μM) at a constant enzyme concentration ([E] = 1 μM). Fluorescence was measured for 3–15 min (or 3–20 h for proline as P1'), and the initial slope was determined by calculating the slope of the first measurement in μM of product produced per second. Fluorescence was measured in a black 96-well plate on a Tecan Infinite M200 Pro plate reader. The excitation wavelength was 320 nm, and the emission wavelength was 420 nm. In the FRET assay, all substrates except proline as P1' showed excellent linearity for at least several minutes. Evaluation of the data was performed by fitting the data in TableCurve 2D v5 software to Michaelis-Menten kinetics: where v is the initial slope and V max is the maximum speed and K M is the Michaelis constant and [S] is the substrate concentration. The parameter V max and K. M V max Dividing by the enzyme concentration [E] gives k cat was calculated.

[0341] An example kinetic curve can be seen in FIG. The results are shown in Tables 21 and 22 and in Figure 8. Figure 8 shows exemplary Michaelis-Menten kinetics measured by FRET assay. The measured substrate was Abz-VDVADHA-Dap (Dnp) at the concentrations given on the x-axis. The y-axis gives the measured initial slope values. The shaded circles represent the measured data points, the solid line represents the model fit, and the dashed lines represent the 95% upper and lower confidence intervals of the model fit.

[0342] [Table 26] JPEG2026031556000032.jpg142170

[0343] [Table 27] JPEG2026031556000034.jpg141170

[0344] 9.3.4 Determination of enzyme activity with model proteins Purified model proteins with a fusion tag fused to the N-terminus of the POI, e.g., MHHHHHHGSGVDVAD (SEQ ID NO: 252), were used as substrates for kinetic assays. All model protein substrates were formulated in PBS. The model proteins used were human fibroblast growth factor 2 (FGF-2), produced as a soluble protein in the cytosol of E. coli; human tumor necrosis factor alpha (TNFα), produced as a soluble protein in the periplasm of E. coli; and a single-chain variable fragment, BIWA4 (scFv), produced as an inclusion body in the cytosol of E. coli. The digestion buffer was PBS. To determine Michaelis-Menten kinetics, six different concentrations of the model protein hFGF-2 (100 μM, 384 μM, 668 μM, 952 μM, 1236 μM, and 1520 μM) were incubated (in triplicate) with 1 μM of various cp-caspase-2 variants. The reaction was stopped after 45 s by adding formic acid to a final concentration of 0.1%.

[0345] To determine Michaelis-Menten kinetics, five different concentrations (50, 101, 135, 220, and 305 μM) of the model protein BIWA4 were incubated (in triplicate) with 10 μM mS9ProD / E. The reaction was stopped after 420 s by adding formic acid to a final concentration of 0.2%. To determine Michaelis-Menten kinetics, six different concentrations (100 μM, 299 μM, 498 μM, 697 μM, 896 μM, and 1093 μM) of the model protein tumor necrosis factor-α were incubated (in duplicate) with 10 μM of various cp-caspase-2 variants. The reaction was stopped after 420 s by adding formic acid to a final concentration of 0.1%. Product formation was determined using the RP-HPLC method outlined in Section 9.3.2. The initial velocity (v) for each concentration was calculated using the fusion tag peak area at the initial slope. This data was imported into TableCurve 2D and fitted with Michaelis-Menten kinetics to obtain V max and K. M The values ​​for were obtained.

[0346] [Table 28]

[0347] 9.3.5 Protein cleavage in solution The fusion proteins described in Section 9.3.4 were used as substrates for the kinetic assays. All model protein substrates were formulated in PBS. The buffer for digestion was PBS. Product formation was determined using the RP-HPLC method outlined in Section 9.3.2.

[0348] For fusion protein digestion, specific concentrations of fusion protein were incubated with defined concentrations of cpCaspase-2 at room temperature under stirring. For hFGF-2 digestion, 2.9 g / L of hFGF-2 fusion protein was incubated with 0.055 g / L of cpCaspase-2 or variants mS9 Pro D285E or mS9 Pro D. Cleavage of FGF-2 fusion protein was also performed with various concentrations of FGF-2 (2 g / L and 10 g / L) and cpCaspase-2 (0.02 g / L, 0.1 g / L), and product generation was determined over time. For TNF-alpha digestion, 2.4 g / L of TNF-alpha fusion protein was incubated with 0.046 g / L of cpCaspase-2 or variant mS9 Pro D285E. For digestion of GFP, 9.1 g / L of GFP fusion protein was incubated with 0.11 g / L of cp caspase-2 or variant mS9 Pro D285E.

[0349] Tag cleavage from FGF-2 by cpCaspase-2 and its variants demonstrated very rapid processing. Complete tag removal for hFGF-2 was measured after 15 minutes for mS9 Pro D285E and mS9 Pro D, and after 180 minutes for cpCaspase-2, as shown in Figure 17. Cleavage kinetics for 2.9 g / L hFGF-2 fusion proteins incubated with 0.055 g / L of T7AC_cpCasp2D (SEQ ID NO: 41), T7AC_mS9ProE (SEQ ID NO: 71), and T7AC_mS9ProD (SEQ ID NO: 72).

[0350] FIG. 18 shows the cleavage kinetics for hFGF-2 fusion proteins incubated with cp caspase-2 (cpCasp2, SEQ ID NO: 6) at various concentrations. Figure 18 shows the effect of fusion protein and enzyme concentration in the example of FGF-2 cleavage by cpCaspase-2. When the ratio of fusion protein to enzyme is kept constant, cleavage appears to be equally fast. At high substrate concentrations, i.e., high concentrations of fusion protein, the reaction remains fast even when cpCaspase-2 is used at a dilution of 1:500. TNF-alpha is a more difficult substrate due to its N-terminal valine. The cleavage reaction is slower compared to FGF-2, but high yields are still possible. TNF-alpha fusion proteins can be efficiently cleaved by cp caspase-2 or the mS9 Pro D285E variant, or variants, generating up to 98% of the desired cleaved protein (Figure 19). As shown in Figure 20, cleavage of the GFP fusion protein is slower, but is still able to process up to 60% of the GFP.

[0351] 9.3.6 Protein cleavage with immobilized enzymes Enzyme immobilization was performed by amine coupling. The primary amino group of a lysine residue on the enzyme was coupled to an activated NHS-group located on a spacer arm in the resin. Coupling forms a stable amide bond. cpCaspase-2 was immobilized at the following concentrations: 1 μM, 10 μM, 50 μM, and 100 μM. The enzyme was diluted in coupling buffer (0.2 M NaHCO3, 0.5 M NaCl, pH 8.3) to reach the desired concentration. For a 500 μl column, approximately 1.5–2 ml of resin slurry in 100% isopropanol was transferred to a 15 ml centrifuge tube. The first step was to wash the resin to remove the isopropanol. This was done with 10–15 resin volumes of cold 1 mM HCl. Immediately after the washing step, the resin and enzyme-coupling buffer were mixed using a vortex. The samp...

Claims

1. From N to the C-terminus, the structure is as follows: i. A small subunit of caspase-2 having a sequence selected from the group consisting of SEQ ID NOs. 91, 94, 100, 103, 106, 109, 112, or 118, which is covalently bound to the following ii via a linker or directly, or a functionally active variant thereof containing a sequence having at least 90% sequence identity with SEQ ID NOs. 91, 94, 100, 103, 106, 109, 112, or 118. ii. A large subunit of caspase-2 having a sequence selected from the group consisting of SEQ ID NOs: 90, 93, 99, 102, 105, 108, 111, or 117, or a functionally active variant thereof containing a sequence having at least 90% sequence identity with SEQ ID NOs: 90, 93, 99, 102, 105, 108, 111, or 117. A single-chain cyclically substituted caspase-2 (cp caspase-2) containing, The cp caspase-2 is a protease and comprises one or more amino acid substitutions at a position functionally equivalent to any of positions 171, 105, 172, 282, 225, 83, 185, or 255 of SEQ ID NO: 6, or at any combination thereof.

2. The cp caspase-2 according to claim 1, comprising a propeptide (SS propeptide) of a small caspase-2 subunit fused to the N-terminus of a small subunit.

3. SS propeptide is Asp 347 The cp caspase-2 according to claim 2, comprising an amino acid substitution in a functionally equivalent position.

4. The cp caspase-2 according to any one of claims 1 to 3, wherein the linker sequence consists of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 amino acid residues.

5. cp caspase-2 according to any one of claims 1 to 4, comprising one or more C-terminal or N-terminal tags selected from the group consisting of affinity tags, solubility enhancement tags, and monitoring tags.

6. The cp caspase-2 according to claim 5, comprising an affinity tag and a solubility-enhancing tag.

7. The cp caspase-2 according to claim 5 or 6, wherein the linker sequence is a tag-linker sequence that links two tags or links a tag to a small subunit, large subunit, or SS propeptide of cp caspase-2.

8. i. The small subunit of caspase-2, a) A first conserved region of the active site having at least 37.5% amino acid sequence identity with Sequence ID No. 177 (First Consensus: AAMRNTKR), or 100% sequence identity with XXXRNTXX (Sequence ID No. 200) [wherein X is any amino acid], and b) A second conserved region of the active site having at least 61.5% amino acid sequence identity with SEQ ID NO: 178 (second consensus: EGYAPGTEFHRCK), or 100% sequence identity with EGXXPGXXXHRCK (SEQ ID NO: 194) [wherein X is any amino acid]. Includes, ii. The large subunit of caspase-2, a) A third conserved region of the active site having at least 25.0% amino acid sequence identity with SEQ ID NO: 174 (third consensus: G-EKDLEFRSGGDVDH), or 100% sequence identity with X-XXXLXXRXGXXXDX (SEQ ID NO: 195) [wherein X is any amino acid], b) The fourth conserved region of the active site having at least 53.3% amino acid sequence identity with SEQ ID NO: 175 (fourth consensus: LLSHGVEGGXYGVDG), or 100% sequence identity with XXSHGXXGXXYGXDG (SEQ ID NO: 196) [wherein X is any amino acid], and c) A fifth conserved region of the active site having at least 50.0% amino acid sequence identity with SEQ ID NO: 176 (fifth consensus: QACRGDET), or 100% sequence identity with QACXGXXX (SEQ ID NO: 197) [wherein X is any amino acid]. cp caspase-2 according to any one of claims 1 to 7, comprising:

9. Based on the position functionally equivalent to the position of sequence number 6, i. Gly substituted with D, or with an amino acid selected from the group consisting of R, K, E, Q, N, A, S, T, P, H, Y 171 , ii. Glu that is substituted with V, or with an amino acid selected from the group consisting of C, L, I, M, F, W, R, K, D, Q, N 105 , iii. Glu that is substituted with V, or with an amino acid selected from the group consisting of C, L, I, M, F, W, R, K, D, Q, N 172 , iv. Asp substituted with E or T, or with an amino acid selected from the group consisting of R, K, Q, N, G, A, S, P, H, Y 282 , v. Val substituted with G, or with an amino acid selected from the group consisting of A, S, T, P, H, Y, C, L, I, M, F, W 225 , vi. Lys substituted with E, or with an amino acid selected from the group consisting of R, D, Q, and N 83 , vii. His substituted with A, or with an amino acid selected from the group consisting of G, S, T, P, and Y 185 , and / or Val substituted with M or substituted with an amino acid selected from the group consisting of C, L, I, F, W 255 , cp caspase-2 according to any one of claims 1 to 8, comprising one or more amino acid substitutions selected from.

10. i. His 185 and Asp 282 , ii. Glu 105 and Asp 285 , iii. Glu 105 Gly 171 Val 225 , and Asp 282 , iv. Glu 105 Gly 171 Val 225 Asp 282 , and Asp 285 , v. Lys 83 , Glu 105 , Glu 172 Val 255 , and Asp 285 , vi. Glu 105 and Gly 171 , vii. Glu 105 and Glu 172 , and viiii. Gly 171 and Glu 172 , A group consisting of the following, comprising an amino acid substitution at a position functionally equivalent to the position in SEQ ID NO: 6, cp caspase-2 according to any one of claims 1 to 9.

11. i. Replacement of H185A and D282T, ii. Replacement of E105V and D285E, iii. Replacement of E105V, G171D, V225G, and D282E, iv. Replacement of E105V, G171D, V225G, D282E, and D285E, v. Replacement of K83E, E105V, E172V, V255M, and D285Y, vi. Replacement of E105V and G171D, vii. Replacement of E105V and E172V, and viiii. Replacement of G171D and E172V cp caspase-2 according to any one of claims 1 to 10, comprising an amino acid substitution at a position functionally equivalent to the position of SEQ ID NO: 6, selected from the group consisting of the above.

12. An amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 17, 18, 23, 24, 51, 52, 54, 70, 71, 72, 78, 79, 86, 87, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, and 192, or SEQ ID NOs: 1, 17, 18, 23, 24, 51, 52, 54, 70, 71, 72, cp caspase-2 according to any one of claims 1 to 11, comprising an amino acid sequence having at least 90%, specifically at least 95%, specifically at least 99%, sequence identity with any one of 78, 79, 86, 87, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, and 192.

13. The cp caspase-2 is recruited by a recognition site for proteolytic cleavage containing five amino acids in the sequence P5 P4 P3 P2 P1, P1 is either D or E, P2 is A, P3 is V, P4 is D, and P5 is V. cp caspase-2 according to any one of claims 1 to 12.

14. A method for producing cyclically substituted caspase-2 (cp caspase-2) according to any one of claims 1 to 13, i. A step of cloning the nucleotide sequence encoding cp caspase-2 into an expression vector under the control of a promoter, ii. The step of transforming host cells with the vector, iii. The method comprising the step of culturing transformed host cells under conditions in which cp caspase-2 is expressed.

15. A method for producing cyclically substituted caspase-2 (cp caspase-2) according to any one of claims 1 to 13, i. A step of cloning the nucleotide sequence encoding cp caspase-2 into an expression vector under the control of a promoter, ii. The step of transforming host cells with the vector, iii. A step of culturing transformed host cells under conditions in which cp caspase-2 is expressed, iv. The step of isolating cp caspase-2 from the host cell culture, v. A step to purify cp caspase-2, The above method, including.

16. A method for producing a target protein (POI), i. A fusion protein comprising one or more tags extending from the N to the C-terminus and a caspase recognition site fused to a POI at the N-terminus, the caspase recognition site being specifically recognized by the cp caspase-2 described in any one of claims 1 to 13; ii. The step of bringing the fusion protein into contact with the cp caspase-2 for a period of time sufficient for the cp caspase-2 to cleave the fusion protein, The above method, including.

17. A method for producing a target protein (POI), i. A fusion protein comprising one or more tags extending from the N to the C-terminus and a caspase recognition site fused to a POI at the N-terminus, the caspase recognition site being specifically recognized by the cp caspase-2 described in any one of claims 1 to 13; ii. The step of bringing the fusion protein into contact with the cp caspase-2 for a period of time sufficient for the cp caspase-2 to cleave the fusion protein, iii. Steps for purifying POI, The above method, including.

18. An isolated nucleotide sequence encoding cp caspase-2 according to any one of claims 1 to 13, or a vector or expression cassette containing the nucleotide sequence.

19. The isolated nucleotide sequence or vector or expression cassette according to claim 18, wherein the vector is a bacterial expression vector, or the expression cassette comprises a sequence encoding cp caspase-2 operably linked to a regulatory element.

20. A host cell or host cell line expressing cp caspase-2 according to any one of claims 1 to 13, The host cell or host cell system is one in which the host cell is selected from the group consisting of bacterial cells, yeast cells, insect cells, mammalian cells, and plant cells.

21. Use of cp caspase-2 according to any one of claims 1 to 13 for in vivo cleavage of a substrate in a non-human organism.

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