Immobilized proteases for activation of proenzyme-form transglutaminase.

Abstract

Immobilized proteases for the activation of the proenzyme form of transglutaminase are disclosed.

Description

[Technical Field]

[0001] Incorporation by Reference The sequence listing provided in the file named SequenceListing.xml, created on September 6, 2022, and submitted herewith, is 8,587 bytes in size and is incorporated by reference in its entirety.

[0002] The field relates to immobilized proteases, and in particular to immobilized proteases for the activation of the proenzyme form of transglutaminase. [Background technology]

[0003] Transglutaminases (Tgase, EC 2.3.2.13) are a family of enzymes that catalyze cross-linking between the gamma-carboxamide group of glutamine residues (acyl donors) and various primary amines (acyl acceptors), including the amino group of lysine. Tgase can be found in all groups of organisms, including prokaryotes, eukaryotes, and plants. For example, animal Tgase includes blood coagulation factor XIII, a multidomain protein that depends on calcium for enzyme function. In contrast, microbial transglutaminases have only a single domain and are calcium-independent. Therefore, microbial Tgase offers significant advantages for practical use.

[0004] Commercially available Tgase is produced by fermentation of Streptomyces mobaraensis. Tgase is expressed as an inactive proenzyme with a propeptide sequence at the N-terminus of the mature domain. The active enzyme is produced in solution by proteolytic treatment to remove the propeptide and generate the mature domain.

[0005] Zotsel, et al., Eur. J. Biochem., 270, 3214-3222 (2003) describe the activation of Tgase from Streptomyces mobaraensis by a soluble form of the Tgase-activating M4 metalloprotease (TAMEP), which cleaves the first 41 amino acids of the proenzyme form of Tgase, i.e., pro-Tgase, between Ser41 and Phe42 (GPS-FRAP cleavage site, see SEQ ID NO: 1).

[0006] Zotsel et al., Eur. J. Biochem., 270, 4149-4155 (2003) further reported that TAMEP-activated Tgase contains a tetrapeptide (Phe-Arg-Ala-Pro) at its N-terminus, i.e., FRAP, which can be removed by a soluble form of transglutaminase-activated tripeptidyl aminopeptidase (TAP) from Streptomyces mobaraensis (SM-TAP). SM-TAP cleaves between Pro45 and Asp46 (the FRAP-DSDD cleavage site, see SEQ ID NO: 1) to generate the mature, catalytically active form of Tgase without FRAP.

[0007] The use of soluble enzymes during post-translational processing of the inactive zymogen of Tgase leads to purification complications due to the need to separate multiple enzymes from the fermentation broth, which often necessitates the use of chromatographic purification, thereby increasing production costs and complexity.

[0008] In contrast, enzyme immobilization is useful for improving the catalytic performance of enzymes and can help simplify downstream processing: immobilization facilitates enzyme reuse, allows for easier and more simplified recovery of both enzyme and product, allows for continuous operation of enzymatic processes, rapid termination of reactions, and greater versatility in bioreactor design.

[0009] Unpublished studies have shown that commercially available immobilized proteases are not suitable for producing mature, catalytically active Tgase, i.e., Tgase from which the entire prosequence (including FRAP) has been removed. These commercially available immobilized proteases either cleave upstream of the desired amino acid position and / or overdigest the mature Tgase. Therefore, there is a need for an immobilized protease that generates mature, catalytically active Tgase from the proenzyme form by cleaving at the correct amino acid position after Pro45 without overdigesting the mature enzyme.

[0010] An additional factor complicating the post-translational processing of the zymogen form of Tgase to the mature, catalytically active Tgase is that TAPs, such as TAMEP and SM-TAP, which are natural proteases used to activate the zymogen form of Tgase, are inactivated by catalytically active mature Tgase and / or catalytically active immature Tgase (i.e., FRAP-Tgase or Tgase with a FRAP at its N-terminus).

[0011] Both TAMEP and SM-TAP contain multiple glutamine and lysine residues and are therefore potential substrates for mature, catalytically active Tgase or catalytically active but immature Tgase, which can react with mature Tgase to form cross-linked products (Gln-Lys) or cause deamination of glutamine to glutamic acid (hydrolysis of Gln to Glu), thereby inactivating TAMEP and SM-TAP. This inactivation of TAMEP and SM-TAP leads to incomplete conversion of the proenzyme form of Tgase to mature, catalytically active Tgase, further increasing the cost and complexity of purifying and isolating recombinantly expressed mature, catalytically active Tgase or catalytically active but immature Tgase.

[0012] Surprisingly, as disclosed herein, it has been found that immobilization of TAMEP and TAP, either separately or together via co-protease immobilization, protects both proteases from catalytic inactivation of Tgase, greatly simplifying downstream processing to obtain mature, catalytically active Tgase and facilitating recombinant expression and purification of both wild-type and variant forms of Tgase.

[0013] Thus, there remains a need to immobilize the proteases necessary to generate mature, catalytically active Tgase from its proenzyme form. The present disclosure addresses a long-standing unmet need for immobilized proteases that can facilitate the expression and purification of recombinantly expressed, mature, catalytically active Tgase. Summary of the Invention

[0014] In a first embodiment, a transglutaminase-activated M4 metalloprotease (TAMEP) immobilized on a porous solid support is disclosed.

[0015] In a second embodiment, a transglutaminase-activated tripeptidyl aminopeptidase (TAP) immobilized on a porous solid support is disclosed.

[0016] In a third embodiment, a transglutaminase-activated M4 metalloprotease (TAMEP) and tripeptidyl aminopeptidase (TAP) co-immobilized on a porous solid support is disclosed.

[0017] In a fourth embodiment, there is provided a method for activating a proenzyme form of transglutaminase or a variant thereof to produce a mature, catalytically active form of transglutaminase, comprising: a) immobilizing at least one transglutaminase-activated M4 metalloprotease (TAMEP) and / or at least one tripeptidyl aminopeptidase (TAP), provided that when both TAMEP and TAP are immobilized, TAMEP and TAP are separately immobilized on the same or different porous solid supports; and b) contacting the proenzyme form of transglutaminase with TAMEP and TAP to produce a mature, catalytically active form of transglutaminase, wherein at least one of TAMEP or TAP is immobilized and, if the contacting is sequential, TAMEP is contacted first. A method is disclosed that includes:

[0018] Alternatively, step a) may comprise providing at least one TAMEP and / or at least one TAP immobilized on a porous solid support.

[0019] In a fifth embodiment, there is provided a method for activating a proenzyme form of transglutaminase or a variant thereof to produce a mature, catalytically active form of transglutaminase, comprising: a) co-immobilizing at least one transglutaminase-activated M4 metalloprotease (TAMEP) and at least one tripeptidyl aminopeptidase (TAP) on the same porous solid support; and b) contacting the proenzyme form of transglutaminase with co-immobilized TAMEP and TAP to produce the mature, catalytically active form of transglutaminase A method is disclosed that includes:

[0020] In a sixth embodiment, there is provided a method for activating a proenzyme form of transglutaminase or a variant thereof to produce a catalytically active immature form of transglutaminase, comprising: a) immobilizing at least one transglutaminase-activated M4 metalloprotease (TAMEP) on a porous solid support; and b) contacting the proenzyme form of transglutaminase with at least one immobilized TAMEP to produce a catalytically active immature form of transglutaminase A method is disclosed that includes:

[0021] Optionally, the catalytically active immature transglutaminase can be separated from at least one of the immobilized or co-immobilized proteases.

[0022] In yet another aspect, for any embodiment disclosed herein, preferably the TAMEP is from a Streptomyces sp., and most preferably the TAMEP is from Streptomyces mobaraensis, and preferably the TAP is from a Streptomyces sp., and most preferably the TAP is from Streptomyces mobaraensis (SM-TAP). [Brief explanation of the drawings]

[0023] [Figure 1] Figure 1 shows an SDS-PAGE analysis of the S. mobaraensis Tgase variants described in Example 1. Lane 1 - mature Tgase obtained from a commercial source; Lane 2 - zymogen (pro-Tgase variant); Lane 3 - clarified lysate containing zymogen (crude pro-Tgase variant); Lane 4 - clarified lysate treated with immobilized protease for 60 minutes; Lane L - protein ladder. The expected molecular weight of the zymogen (pro-Tgase variant) is 43.6 kDa, and that of the mature Tgase variant is 38.9 kDa. [Figure 2]Figure 2 shows the activity of TAMEP and SM-TAP co-immobilized on the same porous solid support, TAMEP and SM-TAP immobilized on different porous solid supports, and TAMEP (soluble) and SM-TAP (soluble). DETAILED DESCRIPTION OF THE INVENTION

[0024] The following sequences comply with 37 CFR Sections 1.821-1.825 ("Requirements for Patent Applications Containing Nucleotide and / or Amino Acid Sequence Disclosures - Sequence Rules") and correspond to the sequence description requirements of World Intellectual Property Organization (WIPO) Standard ST.26(2021), European Patent Convention (EPC) and Patent Cooperation Treaty (PCT) Rules 5.2 and 49.5(a-bis), and Administrative Instructions Section 208 and Annex C. The symbols and formatting used for nucleotide and amino acid sequence data comply with the rules set forth in 37 CFR Section 1.822.

[0025] SEQ ID NO: 1 corresponds to the wild-type proenzyme form of Tgase (pro-Tgase) from Streptomyces mobaraensis. The leader sequence (pro-) is shown in bold underlined text.

[0026] SEQ ID NO:2 corresponds to a thermostable variant of the proenzyme form of Streptomyces mobaraensis Tgase (pro-Tgase variant) that has two additional methionine residues - one at the N-terminus of the pro-sequence and a second methionine between the pro-sequence and the N-terminus of the mature domain. The leader sequence (pro-) is shown in bold underlined text.

[0027] SEQ ID NO:3 corresponds to the wild-type proenzyme transglutaminase-activated M4 metalloprotease (TAMEP) from Streptomyces mobaraensis, lacking the native signal peptide and including a leading methionine to facilitate recombinant expression. The predicted leader sequence (pro-) is shown in bold underlined text.

[0028] SEQ ID NO:4 corresponds to the wild-type proenzyme transglutaminase-activating tripeptidyl aminopeptidase (SM-TAP) from Streptomyces mobaraensis, lacking the native signal peptide and including a leading methionine to facilitate recombinant expression. The predicted leader sequence (pro-) is shown in bold underlined text.

[0029] SEQ ID NO: 5 corresponds to a thermostable variant of Streptomyces mobaraensis Tgase (FRAP-Tgase variant) having an N-terminal FRAP tetrapeptide with a methionine amino acid residue located between the FRAP tetrapeptide and the N-terminus of the mature domain of the thermostable Tgase variant. The leader sequence (pro-) is shown in bold underlined text.

[0030] SEQ ID NO: 6 corresponds to a thermostable variant of Streptomyces mobaraensis Tgase (thermostable Tgase variant) in which the FRAP tetrapeptide has been removed from the N-terminus of the mature domain of the thermostable Tgase variant and the first methionine amino acid residue is located at the N-terminus of the mature domain of the thermostable Tgase variant.

[0031] Detailed Description All patents, patent applications, and publications cited herein are incorporated by reference in their entirety.

[0032] Words using the singular include the plural and vice versa unless the context clearly dictates otherwise.

[0033] In this disclosure, a number of terms and abbreviations are used. Unless otherwise stated, the following definitions apply:

[0034] As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. For example, the terms "a compound" or "at least one compound" can include multiple compounds, including mixtures thereof. As used herein, the terms "a," "an," "the," "one or more," and "at least one," for example, can be used interchangeably.

[0035] As used herein, the term "about" may allow for some variation in value or range, such as, for example, within 10%, within 5%, or within 1% of the stated limits of the stated value or range.

[0036] The terms "and / or" and "or" are used interchangeably herein and refer to a specific disclosure in which, for each of two specified features or components, the other feature is present or absent. Thus, the term "and / or" used in phrases such as "A and / or B" is intended to include "A and B," "A or B," "A (alone)," and "B (alone)." Similarly, the term "and / or" used in phrases such as "A, B, and / or C" is intended to encompass each of the following aspects: "A, B, and C," "A, B, or C," "A or C," "A or B," "B or C," "A and C," "A and B," "B and C," "A" (alone), "B" (alone), and "C" (alone).

[0037] The terms "comprises," "comprising," "includes," "including," and "having," and conjugations thereof, are used interchangeably and mean "including, but not limited to." Whenever an embodiment is described herein in terms of "comprising," it is understood that other similar embodiments described in terms of "consisting of" and / or "consisting essentially of" are also provided.

[0038] The term "consisting of" means "including and limited to."

[0039] The term "consisting essentially of" means that the composition, formulation, or method includes any recited ingredients, components, or steps, and is open to unrecited ingredients, components, or steps that do not materially affect the essential characteristics of the composition, formulation, or method.

[0040] Throughout this application, various embodiments may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the embodiments described herein. Thus, the description of a range should be considered to specifically disclose not only the individual numerical values ​​within that range, but also all possible subranges. For example, a description of a range such as 1 to 6 should be considered to have subranges such as 1 to 2, 1 to 3, 1 to 4, 1 to 5, 2 to 3, 2 to 4, 2 to 5, 2 to 6, 3 to 4, 3 to 5, 3 to 6, etc., and individual numbers within that range, e.g., 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.

[0041] "Optionally" or "optionally" means that the subsequently described event, circumstance, or material may or may not occur or exist, and that the description includes instances in which the event, circumstance, or material occurs or exists, as well as instances in which it does not occur or exist.

[0042] As used herein, the term "immobilization" refers to a technological process by which a molecule, such as an enzyme, is fixed to or within a solid support. This can be achieved by a variety of techniques, including but not limited to covalent bonding / attachment, ionic bonding, entanglement, or adsorption.

[0043] The term "co-immobilized" means that at least two different molecules, such as at least two different enzymes, are immobilized on or within the same solid support.

[0044] The term "solid support" refers to a variety of biological, non-biological, organic, inorganic materials, or any combination thereof. Thus, a solid support can be of any suitable composition to which the attached molecules can be applied.

[0045] As used herein, the term "porous solid support" simply refers to any solid that contains void space, i.e., space not occupied by the main backbone of atoms that make up the structure of the solid. Examples of such supports include, but are not limited to, polyacrylates, polymethacrylates, polystyrenes, and the like, which can be modified with functional groups. For example, polymethacrylates can be modified with epoxide functional groups or primary amine groups that can be activated by crosslinking agents. Examples of such functional groups include, but are not limited to, epoxides, alkyls, phenyls, sulfones, amines (primary, secondary, tertiary, or quaternary), and the like.

[0046] Other examples of porous solid supports include, but are not limited to, aminopropylsilylated controlled pore glass ("CPG"), diatomaceous earth, or metal organic frameworks ("MOFS").

[0047] Preferably, the porous solid support is selected from any of the supports listed in Table 2 in the Examples below. Examples of suitable porous solid supports include, but are not limited to, IB-COV-1, IB-COV-2, IB-COV-3, IB-ADS-1, IB-ADS-2, IB-ADS-3, IB-ADS-4, IB-CAT-1, IB-ANI-1, IB-ANI-2, IB-ANI-3, IB-ANI-4, ECR8204F, ECR8209F, and ECR8215F.

[0048] The terms "covalently bonded" or "covalent bond" refer to a chemical bond involving the sharing of electron pairs between atoms. These electron pairs are known as covalent pairs or bond pairs, and the stable balance of attractive and repulsive forces between atoms when they share electrons is known as a covalent bond. As an example, a pro domain and a mature domain may be covalently linked via a peptide bond.

[0049] The term "ionic bond" is sometimes also called an ionic valence bond. It is a type of link formed from electrostatic attraction between oppositely charged ions in a chemical compound. Such a bond is formed when the valence electrons (outermost electrons) of one atom are permanently transferred to another atom. The atom that loses electrons becomes a positively charged ion, or cation, and the atom that gains electrons becomes a negatively charged ion, or anion. Ionic bonds are an example of noncovalent bonds.

[0050] The term "non-covalent bond" differs from a covalent bond in that a non-covalent bond does not involve the sharing of electrons between atoms. Thus, a non-covalent bond can occur by a complete exchange of electrons between atoms or by no exchange of electrons at all. Non-covalent bonds tend to be weaker than covalent bonds. Types of non-covalent bonds include, but are not limited to, ionic bonds, hydrogen bonds, and van der Waals interactions.

[0051] As used herein, the term "adsorption" refers to the process by which a substance or molecular species accumulates in high concentration on a surface, i.e., the process by which atoms, ions, or molecules from a gas, liquid, or dissolved solid attach to a surface. Adsorption includes interactions such as, but is not limited to, hydrophobic or hydrophilic interactions.

[0052] The terms "enzyme precursor" and "proenzyme" are used interchangeably herein and refer to an inactive precursor of an enzyme, which can be converted into an active or mature enzyme by post-translational modification, for example, by catalysis, such as through proteolytic cleavage of a propeptide sequence.

[0053] The terms "propeptide," "prodomain," "prosequence," and "proregion" are used interchangeably herein to refer to an N-terminal peptide leader sequence (including the FRAP tetrapeptide) that, when fully cleaved, generates a mature, catalytically active Tgase. However, if the full-length propeptide is not cleaved to not include FRAP (i.e., a FRAP-less propeptide), a catalytically active, immature Tgase is generated.

[0054] While the mature domain performs catalytic functions, the propeptide can be considered to perform regulatory functions. Propeptides are generally recognized to have four major functions: 1) they can act as intramolecular chaperones or folding assistants by determining the three-dimensional structure of the protein; 2) they can function as inhibitor or activator peptides; 3) they can direct protein sorting to specific cellular compartments or the extracellular space; and 4) they can mediate interactions of precursors with other molecules (such as peptides, proteins, and polysaccharides) or supramolecular structures (e.g., cell walls). A single propeptide may perform several, or even all, of these functions.

[0055] The term "transglutaminase" (Tgase, EC 2.3.2.13) refers to a family of enzymes that catalyze the formation of isopeptide bonds between primary amines, such as the ε-amine of lysine molecules, and the acyl group of glutamine attached to proteins or peptides. Transglutaminases can catalyze transamidation reactions between glutamyl and lysyl side chains of target proteins. Proteins with Tgase activity have been found in microorganisms, plants, and animals. Tgase is widely distributed in various organs, tissues, and body fluids. Tgase also forms extensively cross-linked, generally insoluble protein biopolymers necessary for organisms to create barriers and stable structures.

[0056] Unlike eukaryotic Tgases, microbial Tgases are calcium-independent, which is a major advantage for practical use. Microbial transglutaminases are one of the most widely studied industrial enzymes for protein functionalization and cross-linking due to their ability to polymerize or functionalize proteins by forming stable ε-(γ-glutamyl)lysine isopeptide bonds without the constraints of consensus sequences or additional cofactors. Microbial Tgases are a subset of Tgases.

[0057] The most commonly used Tgase is a microbial transglutaminase derived from Streptomyces mobaraensis, whose wild-type pro-Tgase has an amino acid sequence corresponding to SEQ ID NO: 1. The terms "pro-Tgase" and "pro-Tgase" are used interchangeably herein. Streptomyces mobaraensis Tgase is known to be secreted in its pro-Tgase form. Activation of the pro-Tgase occurs in two steps. First, the propeptide is cleaved from the N-terminus of pro-Tgase by a transglutaminase-activating M4 metalloprotease ("TAMEP"). Some TAMEPs leave a FRAP tetrapeptide at the N-terminus of Tgase, generating a catalytically active, immature Tgase (i.e., FRAP-Tgase). Some TAMEPs generate catalytically active, immature Tgase with a partial FRAP peptide, such as RAP, AP, or P, attached to the N-terminus of Tgase. In other words, the resulting propeptide does not contain the FRAP tetrapeptide, i.e., a FRAP-less propeptide. The N-terminal FRAP tetrapeptide on Tgase, or its remnants, can then be cleaved using transglutaminase tripeptidyl aminopeptidase ("TAP") to generate a mature, catalytically active Tgase lacking FRAP. For clarity, the terms "mature, catalytically active" and "catalytically active, mature" are used interchangeably herein. Similarly, the terms "not mature, catalytically active" and "catalytically active, not mature" are used interchangeably herein.

[0058] Transglutaminase variants and methods for producing such variants are disclosed, for example, in PCT Publication No. WO 2016 / 170447, published October 27, 2016, and PCT Publication No. WO 2019 / 094301, published May 16, 2019.

[0059] "Protease" (also called peptidase or proteinase) refers to an enzyme capable of cleaving peptide bonds. Proteases refer to various enzymes, such as endopeptidases and exopeptidases, that catalyze the hydrolysis of proteins into peptides and amino acids. Proteases can be broadly divided into seven groups: serine proteases, cysteine ​​proteases, threonine proteases, aspartic acid proteases, glutamic acid proteases, metalloproteases, and asparagine peptide lyases. Proteases can be found in animals, plants, bacteria, fungi, archaea, and viruses. The terms "protease," "peptidase," and "proteinase" are used interchangeably herein.

[0060] The term "amino acid" refers to the basic chemical structural unit of a protein, peptide, or polypeptide. The following abbreviations used herein to identify specific amino acids can be found in Table 1.

[0061] [Table 1]

[0062] The terms "peptide," "protein," and "polypeptide" are used interchangeably herein and refer to polymers of amino acids linked together by peptide bonds. A "protein" or "polypeptide" comprises a polymeric sequence of amino acid residues. Throughout this disclosure, one-letter and three-letter codes for amino acids are used, as defined in accordance with the IUPAC-IUB Joint Commission on Biochemical Nomenclature (JCBN). The single letter X refers to any of the 20 amino acids. It is also understood that due to the degeneracy of the genetic code, a polypeptide may be encoded by multiple nucleotide sequences. Mutations may be designated by the one-letter code for the parent amino acid, followed by the position number, and then the one-letter code for the variant amino acid. For example, a mutation of glycine (G) at position 87 to serine (S) would be designated "G087S" or "G87S." When describing modifications, a position followed by an amino acid listed in parentheses indicates a list of modifications at that position with any of the listed amino acids. For example, 6(L,I) means that position 6 may be substituted with leucine or isoleucine. In some cases, a slash ( / ) is used within the sequence to define a modification, for example, F / V indicates that there is a phenylalanine or a valine at that position.

[0063] Those skilled in the art will understand that modifications of the amino acid sequences disclosed herein can be made while retaining the function associated with the disclosed amino acid sequences. For example, it is well known in the art that alterations of a gene at a given site that result in the production of a chemically equivalent amino acid but do not affect the functional properties of the encoded protein are common.

[0064] As used herein, the term "mutation" refers to a change introduced into a parent sequence, including but not limited to modifications such as insertions or deletions (including truncations), thereby generating a "variant." The results of a mutation include, but are not limited to, the creation of new properties, properties, functions, phenotypes, or traits not found in the protein encoded by the parent sequence.

[0065] Related (and derived) proteins include "variant," "mutant," or "modified" proteins, and these terms are used interchangeably herein. Variant (i.e., mutant or modified) proteins differ from another (i.e., parent) protein, or from each other, by modifications of one or more amino acid residues. For example, a variant may contain one or more amino acid modifications, such as deletion / truncations, insertions, or substitutions of one or more amino acids, compared to the parent protein from which the variant is derived.

[0066] Alternatively, or in addition, a variant may have a particular degree of sequence identity with a reference protein or nucleic acid, as determined using sequence alignment tools such as, for example, BLAST, ALIGN, and CLUSTAL. For example, a variant protein or nucleic acid may have at least about 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or even 99.5% amino acid or nucleic acid sequence identity with a reference sequence, and integer percentages therebetween.

[0067] As used herein, with respect to amino acid residue positions, "corresponding to," "corresponds substantially to," "corresponding substantially to," "corresponding to," or "corresponds" refers to the amino acid residue at the recited position in a protein or peptide, or an amino acid residue that is similar, homologous, or equivalent to the recited residue in a protein or peptide. As used herein, a "corresponding region" generally refers to an analogous position in a related or reference protein.

[0068] Those skilled in the art will understand that modifications of the amino acid sequences disclosed herein can be made while retaining the function associated with the disclosed amino acid sequences. For example, it is well known in the art that genetic alterations that result in the production of a chemically equivalent amino acid at a given site but do not affect the functional properties of the encoded protein are common.

[0069] Related (and derived) proteins include "variant" or "mutant" proteins, and these terms are used interchangeably herein. Variant proteins differ from another (i.e., parent) protein and / or from each other by a small number of amino acid residues. A variant may contain one or more amino acid mutations (e.g., amino acid deletions, insertions, or substitutions) compared to the parent protein from which it is derived. Alternatively, or in addition, a variant may have a certain degree of sequence identity with a reference protein or nucleic acid, as determined using sequence alignment tools such as BLAST, ALIGN, and CLUSTAL. For example, a variant protein or nucleic acid can have at least about 35%, 40%, 45%, 50%, 55%, 60%, 65%, 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%, 99%, or even 99.5% amino acid sequence or nucleic acid identity to a reference sequence, and integer percentages therebetween.

[0070] The term "wild-type" with respect to an amino acid sequence or a nucleic acid sequence indicates that the amino acid sequence or nucleic acid sequence is a natural or naturally occurring sequence. As used herein, the term "naturally occurring" refers to something found in nature (e.g., a protein, amino acid, or nucleic acid sequence). Conversely, the term "non-naturally occurring" refers to something not found in nature (e.g., a recombinant / engineered nucleic acid and protein sequence produced in a laboratory, or a modification of a wild-type sequence).

[0071] The term "derived from" encompasses the terms "originating from," "obtained from," "obtainable from," "isolated from," "purified from," and "created from," and generally indicates that a particular material has characteristics that can be found originating from, or described with reference to, another particular material.

[0072] As used herein, the terms "isolated," "purified," "separated," and "recovered" refer to a material (e.g., a protein, nucleic acid, or cell) that is removed from at least one component with which it is naturally associated. For example, these terms can refer to material that is substantially or essentially free from components that normally accompany it as found in its natural state, such as, for example, an intact biological system. Isolated nucleic acid molecules include nucleic acid molecules that are normally contained within cells that express the nucleic acid molecule, but where the nucleic acid molecule is present extrachromosomally or at a chromosomal location that is different from its natural chromosomal location.

[0073] In a first embodiment, a transglutaminase-activated M4 metalloprotease ("TAMEP") immobilized on a porous solid support is disclosed.

[0074] For immobilized TAMEP, a preferred solid support is a porous solid support as described above. Any porous solid support to which TAMEP can be immobilized can be used. Prior to application of TAMEP, the solid support may be pretreated or functionalized for other desired purposes, such as to promote binding, to create conditions favorable for activity or other desired properties, or to avoid undesired interactions with other entities. Many such surface treatments and / or functionalizations are known in the art, and the selection of an appropriate treatment and / or functionalization will depend on the TAMEP, the associated conditions, and the desired activity. Examples of such supports include, but are not limited to, polyacrylates, polymethacrylates, polystyrenes, and the like, which can be modified with functional groups. For example, polymethacrylates can be modified with epoxide functional groups or primary amine groups that can be activated by a crosslinking agent. Examples of such functional groups include, but are not limited to, epoxides, alkyls, phenyls, sulfones, amines (primary, secondary, tertiary, or quaternary), and the like.

[0075] Other examples of porous solid supports include, but are not limited to, aminopropylsilylated controlled pore glass ("CPG"), diatomaceous earth, or metal organic frameworks ("MOFS").

[0076] Preferably, the porous solid support is selected from any of the supports listed in Table 2 in the Examples. Examples of suitable porous solid supports include, but are not limited to, IB-COV-1, IB-COV-2, IB-COV-3, IB-ADS-1, IB-ADS-2, IB-ADS-3, IB-ADS-4, IB-CAT-1, IB-ANI-1, IB-ANI-2, IB-ANI-3, IB-ANI-4, ECR8204F, ECR8209F, and ECR8215F.

[0077] The terms "active TAMEP" and "TAMEP" are used interchangeably herein. When TAMEP is made in its zymogen form, active TAMEP is made by removing that portion of the N-terminus of the zymogen form necessary to produce the active enzyme. TAMEP can be immobilized using any of the attachment methods described above, in the examples below, i.e., by covalent bonding, ionic bonding, or adsorption. Preferably, TAMEP is immobilized using covalent bonding. Alternatively, TAMEP in its zymogen form may be activated after immobilization.

[0078] TAMEP can be obtained from a variety of microbial sources, including, but not limited to, Streptomyces sp.Non-limiting examples include Streptomyces mobaraensis, Streptomyces huiliensis, Streptomyces sp. TYQ1024, Streptomyces abikoensis, Streptomyces hiroshimensis, Streptomyces albireticuli, Streptomyces triculaminicus, Streptomyces olivoverticillatus, Streptomyces luteoverticillatus, Streptomyces luteoverticillatus, Streptomyces cinnamoneus, Streptomyces caatingaensis, Streptomyces roseoverticillatus, Streptomyces griseocarneus, Streptomyces rectiverticillatus, Streptomyces roseifaciens, Streptomyces eurocidicus, Streptomyces klenkii, Streptomyces netropsis, Streptomyces hygroscopicus hygroscopicus, Streptomyces varsoviensis, and the like.Preferably, TAMEP is obtained from Streptomyces mobaraensis.

[0079] SEQ ID NO:3 corresponds to the wild-type zymogen-form transglutaminase-activating M4 metalloprotease (TAMEP) from Streptomyces mobaraensis, lacking its native signal peptide and containing a leading methionine to facilitate recombinant expression. The putative leader sequence (pro-) is shown in bold, underlined text and is listed as amino acids 2-197 of SEQ ID NO:3. Active TAMEP is generated by removal (e.g., by endogenous protease activity) of that portion of the N-terminus of the zymogen form necessary to generate the active enzyme. In the case of TAMEP, the portion of the N-terminus of the zymogen form necessary to generate the active enzyme is believed to be removed by removing the putative pro-region identified in SEQ ID NO:3 or a variant thereof to generate active TAMEP.

[0080] Thus, it appears that there may be an active TAMEP that shares at least 70% sequence identity with the active form of the amino acid sequence set forth in SEQ ID NO: 3. Even more preferably, the Streptomyces mobaraensis TAMEP comprises a sequence 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%, 99%, or even 99.5% amino acid sequence identity to the active form of the amino acid sequence set forth in SEQ ID NO: 3 (not including the putative leader sequence or a variant thereof). Most preferably, the Streptomyces mobaraensis TAMEP comprises a sequence consisting essentially of the active form of the amino acid sequence set forth in SEQ ID NO:3.

[0081] The sequence of TAMEP after the putative leader sequence has been removed from the zymogen form corresponds to amino acids 198 to 728 of SEQ ID NO: 3. The sequence of TAMEP after the leader sequence has been removed from the zymogen form can correspond to one of the following ranges of amino acids of SEQ ID NO: 3: 182 to 728; 183 to 728; 184 to 728; 185 to 728; 186 to 728; 187 to 728; 188 to 728; 189 to 728; 190 to 728; 191 to 728; 192 to 728; 193 to 728; 194 to 728; 195 to 728; 196 to 728; 197 to 728; 198 to 728; or variations thereof. Alternatively, the sequence of TAMEP after the leader sequence has been removed from the proenzyme form may correspond to one of the following ranges of amino acids of SEQ ID NO: 3: 199 to 728; 200 to 728; 201 to 728; 202 to 728; 203 to 728; 204 to 728; 205 to 728; 206 to 728; 207 to 728; 208 to 728; 209 to 728; 210 to 728; 211 to 728; 212 to 728; 213 to 728; 214 to 728; or variations thereof.

[0082] The sequence of active TAMEP may correspond to one of the following ranges of amino acids of SEQ ID NO: 3: 182 to 728; 183 to 728; 184 to 728; 185 to 728; 186 to 728; 187 to 728; 188 to 728; 189 to 728; 190 to 728; 191 to 728; 192 to 728; 193 to 728; 194 to 728; 195 to 728; 196 to 728; 197 to 728; 198 to 728; or variations thereof. Alternatively, the sequence of active TAMEP may correspond to one of the following ranges of amino acids of SEQ ID NO: 3: 199 to 728; 200 to 728; 201 to 728; 202 to 728; 203 to 728; 204 to 728; 205 to 728; 206 to 728; 207 to 728; 208 to 728; 209 to 728; 210 to 728; 211 to 728; 212 to 728; 213 to 728; 214 to 728; or variations thereof.

[0083] In a second embodiment, a transglutaminase-activated tripeptidyl aminopeptidase ("TAP") immobilized on a porous solid support is disclosed.

[0084] For immobilized TAP, a preferred solid support is a porous solid support as described above. Any porous solid support to which TAP can be immobilized can be used. Prior to application of TAP, the solid support may be pretreated or functionalized for other desired purposes, such as to promote binding, to create conditions favorable for activity or any other desired property, or to avoid undesired interactions with other entities. Many such surface treatments and / or functionalizations are known in the art, and the selection of an appropriate treatment and / or functionalization will depend on the TAP, as well as the associated conditions and desired activity. Examples of such supports include, but are not limited to, polyacrylates, polymethacrylates, polystyrenes, and the like, which can be modified with functional groups. For example, polymethacrylates can be modified with epoxide functional groups or primary amine groups that can be activated by a crosslinking agent. Examples of such functional groups include, but are not limited to, epoxides, alkyls, phenyls, sulfones, amines (primary, secondary, tertiary, or quaternary), and the like.

[0085] Other examples of porous solid supports include, but are not limited to, aminopropylsilylated controlled pore glass ("CPG"), diatomaceous earth, or metal organic frameworks ("MOFS").

[0086] Preferably, the porous solid support is selected from any of the supports listed in Table 2 in the Examples. Examples of suitable porous solid supports include, but are not limited to, IB-COV-1, IB-COV-2, IB-COV-3, IB-ADS-1, IB-ADS-2, IB-ADS-3, IB-ADS-4, IB-CAT-1, IB-ANI-1, IB-ANI-2, IB-ANI-3, IB-ANI-4, ECR8204F, ECR8209F, and ECR8215F.

[0087] Active TAP (i.e., not in its zymogen form) can be immobilized using any of the attachment methods described above, in the examples below, i.e., by covalent bonding, ionic bonding, or adsorption. Preferably, TAP is immobilized using covalent bonding. Alternatively, it is contemplated that TAP in its zymogen form may be activated after immobilization.

[0088] The terms "active TAP" and "TAP" are used interchangeably herein. Where TAP is made in its proenzyme form, active TAP is made by removing that portion of the N-terminus from the proenzyme form necessary to produce the active enzyme, and TAP can be obtained from a variety of microbial sources, including, but not limited to, Streptomyces sp. Non-limiting examples include Streptomyces mobaraensis, Streptomyces huiliensis, Streptomyces caatingaensis, Streptomyces abikoensis, Streptomyces olivoverticillatus, Streptomyces luteoverticillatus, Streptomyces cinnamoneus, Streptomyces netropsis, Streptomyces eurasidicus, Streptomyces Examples of suitable TAP include, but are not limited to, Streptomyces eurucidicus, Streptomyces morookaense, Streptomyces hiroshimensis, Streptomyces roseifaciens, Streptomyces roseoverticillatus, and Streptomyces hygroscopicus. Preferably, the TAP is obtained from Streptomyces mobaraensis. Preferably, the TAP is obtained from Streptomyces mobaraensis.

[0089] SEQ ID NO:4 corresponds to the wild-type zymogen form of transglutaminase-activating tripeptidyl aminopeptidase (SM-TAP) from Streptomyces mobaraensis, lacking its native signal peptide and including a leading methionine to facilitate recombinant expression. The predicted leader sequence (pro-) is shown in bold underlined text and is listed as amino acids 2-7 of SEQ ID NO:4. Active TAP is generated by removing that portion of the N-terminus of the zymogen form necessary to generate the active enzyme. In the case of TAP, the portion of the N-terminus of the zymogen form necessary to generate the active enzyme is believed to be removed by removing the putative pro-region identified in SEQ ID NO:4 or a variant thereof to generate active TAP.

[0090] Thus, it appears that there may be an active TAP that shares at least 70% sequence identity with the active form of the amino acid sequence set forth in SEQ ID NO: 4. Even more preferably, the TAP obtained from Streptomyces mobaraensis 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%, 99%, or even 99.5% amino acid sequence identity to the active form of the amino acid sequence set forth in SEQ ID NO: 4 (not including the putative leader sequence or a variant thereof). Most preferably, the Streptomyces mobaraensis TAP comprises a sequence consisting essentially of the active form of the amino acid sequence set forth in SEQ ID NO:4.

[0091] The sequence of TAP after the putative leader sequence has been removed from its zymogen form corresponds to amino acids 8 to 451 of SEQ ID NO: 4. The sequence of TAP after the leader sequence has been removed from its zymogen form can correspond to one of the following ranges of amino acids of SEQ ID NO: 4: 2 to 451; 3 to 451; 4 to 451; 5 to 451; 6 to 451; 7 to 451; or 8 to 451; or variations thereof. Alternatively, the sequence of TAP after the leader sequence has been removed from its zymogen form can correspond to one of the following ranges of amino acids of SEQ ID NO: 4: 9 to 451; 10 to 451; 11 to 451; 12 to 451; 13 to 451; 14 to 451; 15 to 451; 16 to 451; 17 to 451; 18 to 451; 19 to 451; or 20 to 451; or variations thereof.

[0092] The sequence of an active TAP may correspond to one of the following ranges of amino acids of SEQ ID NO: 4: 2 to 451; 3 to 451; 4 to 451; 5 to 451; 6 to 451; 7 to 451; or 8 to 451; or variations thereof. Alternatively, the sequence of an active TAP may correspond to one of the following ranges of amino acids of SEQ ID NO: 4: 9 to 451; 10 to 451; 11 to 451; 12 to 451; 13 to 451; 14 to 451; 15 to 451; 16 to 451; 17 to 451; 18 to 451; 19 to 451; or 20 to 451; or variations thereof.

[0093] In a third embodiment, both TAMEP and TAP (active forms of each protease) can be co-immobilized on any of the porous solid supports disclosed herein. TAMEP and TAP are described in more detail above. TAMEP and TAP can be co-immobilized using any of the attachment methods described above, in the examples below, i.e., by covalent bonding, ionic bonding, or adsorption. The preferred means of co-immobilization is by covalent bonding.

[0094] As shown in Example 8 below, co-immobilized TAMEP and SM-TAP, as well as separately immobilized TAMEP and SM-TAP, were superior to soluble TAMEP and soluble SM-TAP. Furthermore, surprisingly and unexpectedly, as disclosed herein, it was found that separately immobilizing TAMEP and SM-TAP or co-immobilizing TAMEP and SM-TAP appeared to protect these proteases from inactivation by mature, catalytically active Tgase or catalytically active immature Tgase. This significantly simplifies downstream processing to obtain mature, catalytically active Tgase or catalytically active immature Tgase, and facilitates recombinant expression and purification of wild-type and variant Tgase.

[0095] In a fourth embodiment, there is provided a method for activating a proenzyme form of transglutaminase or a variant thereof to produce a mature, catalytically active form of transglutaminase, comprising: a) immobilizing at least one transglutaminase-activated M4 metalloprotease (TAMEP) and / or at least one tripeptidyl aminopeptidase (TAP), provided that when both TAMEP and TAP are immobilized, TAMEP and TAP are separately immobilized on the same or different porous solid supports; and b) contacting the proenzyme form of transglutaminase with TAMEP and TAP to produce a mature, catalytically active form of transglutaminase, wherein at least one of TAMEP or TAP is immobilized; A method is disclosed that includes:

[0096] Alternatively, step a) may comprise providing at least one TAMEP and / or at least one TAP immobilized on a porous solid support.

[0097] Optionally, the mature, catalytically active transglutaminase is separated from at least one of the immobilized proteases. Preferably, at least one TAMEP is from Streptomyces sp. and at least one TAP is from Streptomyces sp. Most preferably, at least one TAMEP is from Streptomyces mobaraensis and at least one TAP is from Streptomyces mobaraensis.

[0098] In a fifth embodiment, there is provided a method for activating a proenzyme form of transglutaminase or a variant thereof to produce a mature, catalytically active form of transglutaminase, comprising: a) co-immobilizing at least one transglutaminase-activated M4 metalloprotease (TAMEP) and at least one tripeptidyl aminopeptidase (TAP) on the same porous solid support; and b) contacting the proenzyme form of transglutaminase with co-immobilized TAMEP and TAP to produce the mature, catalytically active form of transglutaminase A method is disclosed that includes:

[0099] Optionally, the mature, catalytically active transglutaminase is separated from at least one of the immobilized proteases. Preferably, at least one TAMEP is from Streptomyces sp. and at least one TAP is from Streptomyces sp. Most preferably, at least one TAMEP is from Streptomyces mobaraensis and at least one TAP is from Streptomyces mobaraensis.

[0100] In a sixth embodiment, there is provided a method for activating a proenzyme form of transglutaminase or a variant thereof to produce a catalytically active immature form of transglutaminase, comprising: a) immobilizing at least one transglutaminase-activated M4 metalloprotease (TAMEP) on a porous solid support; and b) contacting the proenzyme form of transglutaminase with at least one immobilized TAMEP to produce a catalytically active immature form of transglutaminase.

[0101] Optionally, the catalytically active immature transglutaminase is separated from at least one of the immobilized TAMEPs. Preferably, the TAMEP is derived from Streptomyces sp. Most preferably, the TAMEP is derived from Streptomyces mobaraensis.

[0102] Any of the means of immobilization and co-immobilization discussed herein can be used to practice any of the embodiments disclosed herein. A preferred means of immobilization or co-immobilization is by covalent attachment.

[0103] TAMEP and TAP are described in detail above.

[0104] The foregoing non-limiting embodiments disclosed herein include: 1. Transglutaminase-activated M4 metalloprotease (TAMEP) immobilized on a porous solid support. 2. The immobilized TAMEP of embodiment 1, wherein the TAMEP is derived from Streptomyces sp. 3. The immobilized TAMEP of embodiment 1 or 2, wherein the TAMEP is derived from Streptomyces mobaraensis. 4. Transglutaminase-activated tripeptidyl aminopeptidase (TAP) immobilized on a porous solid support. 5. The immobilized TAP of embodiment 4, wherein the TAP is derived from Streptomyces sp. 6. The immobilized TAP of embodiment 4 or 5, wherein the TAP is derived from Streptomyces mobaraensis (SM-TAP). 7. Transglutaminase-activated M4 metalloprotease (TAMEP) and tripeptidyl aminopeptidase (TAP) co-immobilized on a porous solid support. 8. The co-immobilized TAMEP and TAP of embodiment 7, wherein TAMEP is from Streptomyces sp. and TAP is from Streptomyces sp. 9. The co-immobilized TAMEP and TAP of embodiment 7 or 8, wherein TAMEP is from Streptomyces mobaraensis and TAP is from Streptomyces mobaraensis. 10. A method for activating a proenzyme form of transglutaminase or a variant thereof to produce a mature, catalytically active form of transglutaminase, comprising: a) immobilizing at least one transglutaminase-activated M4 metalloprotease (TAMEP) and / or at least one tripeptidyl aminopeptidase (TAP), wherein when both TAMEP and TAP are immobilized, the TAMEP and TAP are separately immobilized on the same or different porous solid supports; or providing at least one TAMEP and / or at least one TAP immobilized on a porous solid support; and b) contacting the proenzyme form of transglutaminase with TAMEP and TAP to produce a mature, catalytically active form of transglutaminase, wherein at least one of TAMEP or TAP is immobilized; A method comprising: 11. The method of embodiment 10, wherein the mature, catalytically active form of the transglutaminase is separated from at least one of the immobilized proteases. 12. The method of embodiment 10 or 11, wherein TAMEP is from Streptomyces sp. and TAP is from Streptomyces sp. 13. The method of embodiment 10, 11 or 12, wherein TAMEP is from Streptomyces mobaraensis and TAP is from Streptomyces mobaraensis. 14. A method for activating a proenzyme form of transglutaminase or a variant thereof to produce a mature, catalytically active form of transglutaminase, comprising: a) co-immobilizing at least one transglutaminase-activated M4 metalloprotease (TAMEP) and at least one tripeptidyl aminopeptidase (TAP) on the same porous solid support; and b) contacting the proenzyme form of transglutaminase with co-immobilized TAMEP and TAP to produce the mature, catalytically active form of transglutaminase A method comprising: 15. The method of embodiment 14, wherein the mature, catalytically active transglutaminase is separated from at least one of the immobilized proteases. 16. The method of embodiment 14 or 15, wherein TAMEP is from Streptomyces sp. and TAP is from Streptomyces sp. 17. The method of embodiment 14, 15 or 16, wherein TAMEP is from Streptomyces mobaraensis and TAP is from Streptomyces mobaraensis. 18. A method for activating a proenzyme form of transglutaminase or a variant thereof to produce a catalytically active immature form of transglutaminase, comprising: a) immobilizing at least one transglutaminase-activated M4 metalloprotease (TAMEP) on the same porous solid support; and b) contacting the proenzyme form of transglutaminase with at least one immobilized TAMEP to produce a catalytically active immature form of transglutaminase A method comprising: 19. The method of embodiment 18, wherein the catalytically active, non-mature transglutaminase is separated from at least one of the immobilized TAMEPs. 20. The method of embodiment 18 or 19, wherein TAMEP is derived from Streptomyces sp. 21. The method of embodiment 18, 19 or 20, wherein TAMEP is derived from Streptomyces mobaraensis. 22. The immobilized and co-immobilized TAMEP and method according to embodiments 1 to 3 or 7 to 21, wherein TAMEP is an activated form of TAMEP having the sequence of SEQ ID NO: 3. 23. TAMEP is selected from the group consisting of amino acids 182 to 728, 183 to 728, 184 to 728, 185 to 728, 186 to 728, 187 to 728, 188 to 728, 189 to 728, 190 to 728, 191 to 728, 192 to 728, 193 to 728, 194 to 728, 195 to 728, 196 to 728, 197 to 728, 198 to 728, 199 to 728, 200 to 201 of SEQ ID NO: 3. 22. The immobilized and co-immobilized TAMEP and method according to any one of embodiments 1 to 3 or 7 to 21, wherein the TAMEP has a sequence comprising: 201 to 728, 202 to 728, 203 to 728, 204 to 728, 205 to 728, 206 to 728, 207 to 728, 208 to 728, 209 to 728, 210 to 728, 211 to 728, 212 to 728, 213 to 728, or 214 to 728. 24. Immobilized and co-immobilized TAP and methods according to embodiments 4 to 6 or 7 to 21, wherein the TAP is an activated form of TAP having the sequence of SEQ ID NO: 4. 25. Immobilized and co-immobilized TAP and methods according to embodiments 4 to 6 or 7 to 21, wherein the TAP has a sequence comprising amino acids 2 to 451, 3 to 451, 4 to 451, 5 to 451, 6 to 451, 7 to 451, or 8 to 451, 9 to 451, 10 to 451, 11 to 451, 12 to 451, 13 to 451, 14 to 451, 15 to 451, 16 to 451, 17 to 451, 18 to 451, 19 to 451, or 20 to 451 of SEQ ID NO: 4. [Example]

[0105] The following examples are intended to illustrate, but not limit, the present invention. Therefore, from the above discussion and examples, those skilled in the art can grasp the essential features of the present disclosure, and can make various changes and modifications to adapt to various applications and conditions without departing from the spirit and scope thereof.

[0106] Unless otherwise defined herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Singleton, et al., DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY, 2D ED., John Wiley and Sons, New York (1994), and Hale & Marham, THE HARPER COLLINS DICTIONARY OF BIOLOGY, Harper Perennial, NY (1991) provide those skilled in the art with a general dictionary of many of the terms used in this disclosure.

[0107] [Example 1] Expression of the pro-Tgase variant zymogen in Escherichia coli A. Construction of an expression plasmid for the expression of the proTgase variant (SEQ ID NO: 2) The gene encoding the pro-Tgase variant (i.e., the proenzyme form of Tgase variant) was codon-optimized for expression in Escherichia coli (E. coli) based on the published amino acid sequence (Kanaji, et al. (1993) J. Biol. Chem. 268 (16):11565-11572), synthesized with an additional C-terminal His tag, and cloned into a pET vector operably linked to a T7 promoter under the control of the lacI repressor. This expression vector also contains the pMB1 origin of replication and a kanamycin resistance gene. The resulting plasmid was first transformed into E. coli DH-10B using standard methods known in the art. Transformants were isolated by subjecting the cells to kanamycin selection, as known in the art (see, e.g., U.S. Pat. No. 8,383,346 and WO 2010 / 144103, both of which are incorporated by reference in their entireties), and the sequence of the pro-Tgase gene was confirmed by Sanger sequencing. Plasmids were recovered from positive clones using methods known in the art and transformed into E. coli BL21(DE3) for expression.

[0108] B. Expression and isolation of recombinant pro-Tgase variant (SEQ ID NO: 2) in E. coli Escherichia coli (E. coli) BL21(DE3) strain containing the pro-Tgase expression vector was grown overnight in Luria broth at 37°C until the culture reached saturation. The next morning, the culture was used to inoculate a shake flask containing medium containing glycerol, soy peptone, yeast extract, magnesium sulfate heptahydrate, and monobasic potassium phosphate and incubated at 30–34°C for up to 10 hours with continuous shaking. Isopropyl β-d-1-thiogalactopyranoside (IPTG) was added to a final concentration of 0.1–1 mM, and incubation was continued at 20–25°C for up to 24 hours.

[0109] Cells were harvested by centrifugation at 8,000 × g for up to 60 minutes. The supernatant was discarded, and the pellet was resuspended in 20% w / v of 50 mM tris(hydroxymethyl)aminomethane (Tris) HCl, pH 8. Cells were lysed using a high-pressure homogenizer at 15,000–20,000 psi. The crude lysate was clarified by centrifugation at 15,000 × g for up to 60 minutes. The clarified lysate containing pro-Tgase was assessed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE, Figure 1) and spectroscopy as described in Example 1D, and Tgase activity was assessed using the colorimetric activity assay described in Example 1C.

[0110] Alternatively, pro-Tgase variants may be secreted from microbial strains known to those skilled in the art to secrete Tgase, such as Streptomyces mobaraensis or Bacillus subtilis. The pellet is discarded, and the supernatant is collected and evaluated by SDS-PAGE as described in Example 1D and by spectroscopy. Tgase activity is assessed using the colorimetric activity assay described in Example 1C.

[0111] C. Colorimetric Activity Assay Herein, Tgase activity was measured using a colorimetric hydroxamate activity assay (Folk and Cole (1965) J Biol Chemistry 240(7):2951-2960). Briefly, the hydroxamate assay uses N-benzyloxycarbonyl-L-glutaminyl-glycine (ZQG) as the low molecular weight amine acceptor substrate and hydroxylamine as the amine donor. In the presence of catalytically active Tgase, hydroxylamine is incorporated to form Z-glutamylhydroxamate-glycine, which forms a colored complex with iron(III) and is detected at 525 nm after incubation at 37 °C for 5–60 min. Calibration was performed using L-glutamic acid gamma-monohydroxamate (Millipore® Sigma®) as a standard. One unit of Tgase is defined as the amount of enzyme that catalyzes the production of 1 μmol of the peptide derivative of gamma-glutamylhydroxylamine per minute.

[0112] D. SDS-PAGE gel This technique was used to evaluate the activation level of the pro-Tgase variant (SEQ ID NO: 2) by TAMEP (see Example 3) and to analyze the molecular weight of the expressed protein. Mature Tgase was obtained from a commercial source (Moo Gloo TI Formula, Ajinomoto®) and used as a molecular weight standard for SDS-PAGE analysis (Figure 1, lane 1).

[0113] A 10 μg aliquot of proteolytic cleavage product or expressed protein was added to Novex™ Bolt™ LDS Sample Buffer and Novex™ Bolt™ Sample Reducing Agent, mixed as directed, heated to 95°C for 5 minutes, and run on a 4-12% SDS-PAGE gel (Invitrogen™ NuPAGE™ 4-12% Bis-Tris gel) at 170 volts for 30 minutes. The gel was stained with SimplyBlue™ Safe Stain (Invitrogen™) for 30-60 minutes, then destained with deionized water and photographed.

[0114] [Example 2] Purification of pro-Tgase variant (SEQ ID NO: 2) or FRAP-Tgase variant (SEQ ID NO: 5) This method was used to purify either the pro-Tgase variant (SEQ ID NO: 2) (prepared in Example 1) or the FRAP-Tgase variant (SEQ ID NO: 5), which was prepared by incubating a lysate containing the pro-Tgase variant (SEQ ID NO: 2) with a supernatant containing TAMEP (Example 3, added at 1% vol) at 37°C for 60 minutes.

[0115] The pH of the lysate was lowered to pH 5–6 using 2-(N-morpholino)ethanesulfonic acid (MES). The mixture was clarified by centrifugation at 4000 g for 10–20 minutes. The clarified lysate was then washed with water (10 column volumes) and then loaded onto an SP Sepharose column (Cytiva®) equilibrated with 25 mM MES buffer, pH 5.5 (10 column volumes) according to the manufacturer's specifications. The lysate was loaded onto the column at 1 mL / min and then washed with 25 mM MES, pH 5.5, containing 50 mM sodium chloride (10 column volumes). The pro-Tgase variant (SEQ ID NO: 2) or FRAP-Tgase variant (SEQ ID NO: 5) was then eluted with 25 mM MES buffer, pH 5.5, containing 1 M sodium chloride (3 column volumes). Tgase activity was assessed using the colorimetric activity assay described in Example 1C. The pro-Tgase variant (SEQ ID NO: 2) did not exhibit activity above baseline in the colorimetric activity assay. Only activated forms of Tgase, such as the FRAP-Tgase variant (SEQ ID NO: 5), exhibited activity in this assay.

[0116] The N-terminal peptide sequence of the FRAP-Tgase variant (SEQ ID NO: 5) was confirmed using N-terminal sequencing. To evaluate the activation level of FRAP-Tgase (SEQ ID NO: 5) by TAMEP, a pro-Tgase variant (SEQ ID NO: 2) or a TAP such as SM-TAP, N-terminal sequencing was used.

[0117] This technique was used to determine the N-terminal sequence of a protein. An aliquot of protein (10 μg), i.e., the FRAP-Tgase variant (SEQ ID NO: 5), was added to commercially available SDS-PAGE sample buffer (Novex™ Bolt™ LDS Sample Buffer and Novex™ Bolt™ Sample Reducing Agent, mixed as directed) and heated to 95°C for 5 minutes. The sample was then electroporated through a 4-12% SDS-PAGE (Invitrogen™ NuPAGE™ 4-12% Bis-Tris Gel) gel at 170 volts for 30 minutes. The SDS-PAGE gel was then washed with water and run at 20 volts for 60 minutes using a Mini Blot module (Invitrogen™) according to the manufacturer's instructions. It was then transferred to a 0.45 μm PVDF membrane. The membrane was stained with SimplyBlue™ Safe Stain (Invitrogen™) until clear protein bands were visible. After washing with deionized water, the protein band corresponding to the appropriate molecular weight of the FRAP-Tgase variant (SEQ ID NO: 5) was excised and sent to the Protein Facility at the Iowa State University Office of Biotechnology for N-terminal sequencing analysis, where Edman degradation analysis was used to determine the first 4–5 amino acids of the protein.

[0118] [Example 3] Expression of proenzyme forms of TAMEP and SM-TAP and isolation of catalytically active forms A. Expression of the zymogen form of TAMEP (SEQ ID NO: 3) and the zymogen form of SM-TAP (SEQ ID NO: 4) and isolation of the mature, catalytically active forms of TAMEP and SM-TAP Genes encoding wild-type S. mobaraensis Tgase protease, transglutaminase-activated metalloprotease (TAMEP), and S. mobaraensis tripeptidyl aminopeptidase (SM-TAP) were synthesized by Integrated DNA Technologies (Coralville, IA). Expression constructs for TAMEP and SM-TAP were designed with an N-terminal SacB signal sequence and a hexa-His tag and cloned using methods well known in the art. B. subtilis SCK6 delta-AlaR was purchased from Bio-Technical Resources (Manitowoc, WI) and grown overnight at 37°C in 5 mL of LB medium supplemented with 40 mg / mL D-alanine. The next day, the culture was diluted to an OD of 1.0, and xylose was added to a final concentration of 1%. After 2 hours, 250 μL of glycerol-ligated DNA was added, and the culture tube was returned to the incubator for an additional 90 minutes. After incubation, 10–1000 μL of the culture was spread onto LB agar plates. The plates were grown overnight at 37°C. The next day, 2–8 colonies were picked from each plate and inoculated into 3 mL of LB broth. The cultures were incubated at 37°C for 48 hours, with periodic supernatant samples taken. Using SDS-PAGE, the secretion of the mature, catalytically active enzyme into the medium was confirmed by molecular weight determination. The predicted molecular weight of mature, catalytically active TAMEP is approximately 56 kDa, and that of mature, catalytically active SM-TAP is approximately 50 kDa. The mature, catalytically active forms of each protease appear to be formed from the proenzyme form by a constitutive process, such as endogenous protease activity. The mature, catalytically active proteases, both TAMEP and SM-TAP, were isolated from the respective cell cultures by centrifugation at 8000 × g for 10 minutes, and the supernatants were used as isolated without further purification.

[0119] B. TAMEP activity assay The activity of TAMEP from Example 3A above was measured using the following assay. Soluble TAMEP was added to purified pro-Tgase variant (Example 2, 500 μL, 5 g / L) at 1% vol. The reaction was then incubated at 37°C and 300 rpm for time points between 1 and 120 minutes. At each time point, a sample of the supernatant was removed and quenched with ethylenediaminetetraacetic acid (EDTA). The sample was then analyzed by ultra-performance liquid chromatography coupled to mass spectrometry (UPLC-MS, Thermo Scientific™ Vanquish™ UPLC and Thermo Scientific™ ISQ™ EM MS) to determine the total ion counts of the pro-Tgase variant (SEQ ID NO: 2), i.e., the propeptide cleaved by TAMEP from the propeptide without the FRAP tetrapeptide. Samples were analyzed using an Accucore™ Vanquish™ C18 column (50 mm i.d. x 2.1 mm, 1.5 μm particle size, Thermo Scientific™) with a linear gradient of 5% to 90% 0.1% formic acid in acetonitrile in 1.5 min. N-terminal peptide sequencing, performed as described in Example 2 above, confirmed that the FRAP amino acid sequence was present at the N-terminus of the catalytically active immature FRAP-Tgase variant (SEQ ID NO: 5).

[0120] C. SM-TAP activity assay The activity of SM-TAP from Example 3A above was measured using the following assay. Soluble SM-TAP was added to purified FRAP-Tgase variant (Example 2, 500 μL at 5 g / L) at 1% vol. The reaction was then incubated at 37°C and 300 rpm, and time points were collected from 1 to 120 minutes. At each time point, samples of the supernatant were removed and quenched with phenylmethylsulfonyl fluoride (PMSF). Samples were analyzed by ultra-performance liquid chromatography coupled to mass spectrometry (UPLC-MS, Thermo Scientific™ Vanquish™ UPLC and Thermo Scientific™ ISQ™ EM MS) to determine the total area counts of the FRAP tetrapeptide generated by cleavage of the FRAP-Tgase variant (SEQ ID NO: 5) by SM-TAP. Samples were analyzed using an Accucore™ Vanquish™ C18 column (50 mm i.d. x 2.1 mm, 1.5 μm particle size, Thermo Scientific™) with a linear gradient of 5 to 90% 0.1% formic acid in acetonitrile in 1.5 min. N-terminal peptide sequencing, performed as described in Example 2 above, confirmed that the FRAP tetrapeptide had been removed from the N-terminus of the FRAP-Tgase variant (SEQ ID NO: 5), yielding the mature, catalytically active Tgase variant (SEQ ID NO: 6).

[0121] [Example 4] Immobilization of mature, catalytically active proteases on porous solid supports. A. Immobilization of proteases on covalent porous solid supports The following procedures describe the immobilization of TAMEP (Example 3A) and / or SM-TAP (Example 3A) to IB-COV-1, IB-COV-2, IB-COV-3, ANI-1 (modified for covalent attachment), ECR8204F, ECR8209F, and ECR8215F.

[0122] Each porous solid support was weighed into a separate glass vial and then washed with water (10 mL, 4x).The porous solid support was then washed with 50 mM phosphate buffer adjusted to pH 8 (2x the volume of the porous solid support, 4x).

[0123] The ANI-1 porous solid support was modified for covalent binding by washing with a freshly prepared 1% glutaraldehyde solution in 50 mM phosphate buffer (pH 8) for 1 hour at 20° C. (4 volumes of porous solid support, 1×). The glutaraldehyde solution was then removed, and the porous solid support was washed with 50 mM phosphate buffer (pH 8) (4 times the volume of the porous solid support, 4×).

[0124] For all covalently bonded porous solid supports, B. subtilis supernatant containing TAMEP and / or SM-TAP (0.2–0.5 g / L) was incubated on the porous solid support at a 4:1 ratio of supernatant volume to porous solid support mass at 20°C and 700 rpm for 18–20 hours. The agitation was stopped, and the protease was incubated on the porous solid support for an additional 2 hours. The supernatant was then removed from the porous solid support, and the porous solid support was washed with 50 mM phosphate buffer adjusted to pH 8 (4 times the volume of the porous solid support, 1x). The porous solid support was then washed with 50 mM phosphate buffer (pH 8) containing 500 mM sodium chloride (4 times the volume of the porous solid support, 2x).

[0125] B. Immobilization of TAMEP on an Ionic Porous Solid Support The following procedure describes the immobilization of TAMEP (Example 3A) to IB-CAT-1, IB-ANI-1 (unmodified), IB-ANI-2, IB-ANI-3, and IB-ANI-4.

[0126] Each porous solid support was weighed into a separate glass vial and then washed with water (10 mL, 4x). The porous solid support was then washed with 50 mM phosphate buffer adjusted to pH 7 (2x the volume of the porous solid support, 4x). Next, B. subtilis supernatant containing TAMEP (0.2–0.5 g / L) was incubated on the porous solid support at a 4:1 ratio of supernatant volume to porous solid support mass at 20°C and 700 rpm for 18–20 h. The stirring was stopped, and the supernatant was incubated on the porous solid support for an additional 2 h. The supernatant was then removed from the porous solid support, and the porous solid support was washed with 50 mM phosphate buffer adjusted to pH 7 (4x the volume of the porous solid support, 4x).

[0127] C. Immobilization of SM-TAP on an Ionic Porous Solid Support The following procedure describes the immobilization of SM-TAP (Example 3A) to IB-CAT-1, IB-ANI-1 (unmodified), IB-ANI-2, IB-ANI-3, and IB-ANI-4.

[0128] Each porous solid support was weighed into a separate glass vial and then washed with water (10 mL, 4x). The porous solid support was then washed with 50 mM carbonate buffer adjusted to pH 10.5 (2x the volume of the porous solid support, 4x). Next, B. subtilis supernatant containing TAMEP and / or SM-TAP (0.2–0.5 g / L) was incubated on the porous solid support at a 4:1 ratio of supernatant volume to porous solid support mass at 20°C and 700 rpm for 18–20 h. The stirring was stopped, and the protease was incubated on the porous solid support for an additional 2 h. The supernatant was then removed from the porous solid support, and the porous solid support was washed with 50 mM carbonate buffer adjusted to pH 10.5 (4x the volume of the porous solid support, 4x).

[0129] D. Co-immobilization of TAMEP and SM-TAP on an ionic porous solid support TAMEP and SM-TAP were co-immobilized on IB-CAT-1, IB-ANI-1 (unmodified), IB-ANI-2, IB-ANI-3, and IB-ANI-4 using the same protocol as described in Example 4C above.

[0130] E. Immobilization of Proteases on Adsorptive Porous Solid Supports The following procedures describe the immobilization of TAMEP (Example 3A) and / or SM-TAP (Example 3A) to IB-ADS-1, IB-ADS-2, IB-ADS-3, and IB-ADS-4.

[0131] Each porous solid support was weighed into a separate glass vial and then washed with water (10 mL, 4x). The porous solid support was then washed with 50 mM phosphate buffer adjusted to pH 8 (2x the volume of the porous solid support, 4x). Next, B. subtilis supernatant containing TAMEP and / or SM-TAP (0.2–0.5 g / L) was incubated on the porous solid support at a 4:1 ratio of supernatant volume to porous solid support mass at 20°C and 700 rpm for 18–20 h. The stirring was stopped, and the protease was incubated on the porous solid support for an additional 2 h. The supernatant was then removed from the porous solid support, and the porous solid support was washed with 50 mM phosphate buffer adjusted to pH 8 (4x the volume of the porous solid support, 4x).

[0132] [Table 2]

[0133] [Example 5] Detection of proteolytic digestion of immobilized TAMEP In this example, we demonstrate the activation of purified pro-Tgase variant (SEQ ID NO: 2), i.e., the proenzyme form of the Tgase variant. The purified pro-Tgase variant (SEQ ID NO: 2) and the clarified lysate containing pro-Tgase (SEQ ID NO: 2) showed similar results in all tests. For simplicity, we demonstrate the protease activation of purified pro-Tgase (SEQ ID NO: 2) in this study.

[0134] TAMEP (Example 3A) (20 mg, prepared in Example 4) immobilized on a porous solid support was added to 1.5 mL glass vials. Next, 200 μL of purified pro-Tgase variant (5 g / L, SEQ ID NO: 2, described in Example 2 above) was added to each vial. The vials were then incubated at 37°C and 700 rpm for time points between 1 and 120 minutes. At each time point, a sample of the supernatant was removed and quenched with EDTA. The samples were then analyzed as described in Example 3B. Tgase activity was assessed using the colorimetric activity assay described in Example 1C. N-terminal peptide sequencing, as performed in Example 2, confirmed the presence of the FRAP tetrapeptide in the catalytically active, immature FRAP-Tgase variant (SEQ ID NO: 5). Activity assay results indicated that TAMEP retained protease activity when immobilized on all porous solid supports shown in Table 2.

[0135] [Example 6] Detection of proteolytic digestion of immobilized SM-TAP In this example, we demonstrate the activation of purified FRAP-Tgase variant (SEQ ID NO: 5). Purified FRAP-Tgase variant (SEQ ID NO: 5) and clarified lysates containing FRAP-Tgase (SEQ ID NO: 5) showed similar results in all tests. For simplicity, we demonstrate the protease activation of purified FRAP-Tgase (SEQ ID NO: 5) herein.

[0136] SM-TAP (Example 3A) immobilized on a porous solid support (20 mg of porous solid support, prepared in Example 4) was added to 1.5 mL glass vials. Next, 200 μL of purified FRAP-Tgase variant (5 g / L, SEQ ID NO: 5, described in Example 2 above) was added to each vial. The vials were then incubated at 20°C and 700 rpm, and time points were collected from 1 to 120 minutes. At each time point, a sample of the supernatant was removed and quenched with PMSF. The samples were analyzed as described in Example 3C. Tgase activity was assessed using the colorimetric activity assay described in Example 1C above. N-terminal peptide sequencing, performed as described in Example 2 above, indicated that the FRAP tetrapeptide had been removed from the N-terminus of the FRAP-Tgase variant (SEQ ID NO: 5), resulting in a thermostable, mature, catalytically active Tgase variant (SEQ ID NO: 6). The results of the activity assays showed that SM-TAP retained protease activity when immobilized on all of the porous solid supports shown in Table 2.

[0137] [Example 7] Detection of proteolytic digestion by co-immobilized TAMEP and SM-TAP In this example, we demonstrate the activation of purified pro-Tgase variant (SEQ ID NO: 2), i.e., the proenzyme form of the Tgase variant. The purified pro-Tgase variant (SEQ ID NO: 2) and the clarified lysate containing pro-Tgase (SEQ ID NO: 2) showed similar results in all tests. For simplicity, we demonstrate the protease activation of purified pro-Tgase (SEQ ID NO: 2) in this study.

[0138] The porous solid supports showing top performance under the conditions described in Examples 5 and 6 above were selected for co-immobilization.

[0139] The porous solid support co-immobilized with TAMEP and SM-TAP (20 mg) prepared in Example 4 above was added to a 1.5 mL glass vial. Next, 200 μL of purified pro-Tgase variant (5 g / L, SEQ ID NO: 2, described in Example 2 above) was added to the vial. The vial was then incubated at 20°C and 700 rpm, and time points were collected from 1 to 120 minutes. At each time point, a sample of the supernatant was removed and quenched with PMSF and EDTA. The sample was then analyzed as described in Example 3C. The FRAP tetrapeptide is only generated when TAMEP first cleaves the propeptide from the pro-Tgase variant (SEQ ID NO: 2), and then SM-TAP cleaves the FRAP tetrapeptide from the resulting FRAP-Tgase variant (SEQ ID NO: 5). Tgase activity was assessed using the colorimetric activity assay described in Example 1C. N-terminal peptide sequencing, performed as described in Example 2, confirmed that FRAP had been removed from the N-terminus of the FRAP-Tgase variant (SEQ ID NO: 5), generating a mature, catalytically active Tgase variant (SEQ ID NO: 6). Activity assay results showed that co-immobilized TAMEP and SM-TAP retained protease activity on all porous solid supports shown in Table 3 below.

[0140] [Table 3]

[0141] [Example 8] Measurement of TAMEP and TAP activity The same concentrations of TAMEP and SM-TAP were used in both soluble and immobilized forms.

[0142] Unless otherwise stated, all TAMEP and SM-TAP concentrations are based on total protein.

[0143] A. Measurement of the combined activity of soluble TAMEP (non-immobilized) and soluble SM-TAP (non-immobilized) Purified pro-Tgase variant (SEQ ID NO: 2, 250 μL at 5 g / L) was added to a 1.5 mL glass vial containing soluble TAMEP (0.1 mg, non-immobilized) and soluble SM-TAP (0.1 mg, non-immobilized). The sample was analyzed in the same manner as in Example 3C.

[0144] B. Measurement of the combined activity of soluble TAMEP (non-immobilized) and SM-TAP immobilized on IB-COV-3 Purified pro-Tgase variant (SEQ ID NO: 2, 250 μL at 5 g / L) was added to a 1.5 mL glass vial containing soluble TAMEP (0.1 mg, non-immobilized) and immobilized SM-TAP (10 mg porous solid support, 0.1 mg SM-TAP). Samples were analyzed in the same manner as described in Example 3C above.

[0145] C. Measurement of the combined activity of IB-COV-1-immobilized TAMEP and soluble SM-TAP (non-immobilized) Purified pro-Tgase variant (SEQ ID NO: 2, 250 μL at 5 g / L) was added to a 1.5 mL glass vial containing immobilized TAMEP (10 mg porous solid support, 0.1 mg TAMEP) and soluble SM-TAP (0.1 mg, non-immobilized). Samples were analyzed in the same manner as described in Example 3C above.

[0146] D. Measurement of the combined activity of TAMEP immobilized on IB-COV-1 and SM-TAP immobilized on IB-COV-3 Purified pro-Tgase variant (SEQ ID NO: 2, 250 μL at 5 g / L) was added to 1.5 mL glass vials containing separately immobilized TAMEP (10 mg porous solid support, 0.1 mg TAMEP) and SM-TAP (10 mg porous solid support, 0.1 mg SM-TAP). Samples were analyzed in the same manner as described in Example 3C above.

[0147] Measurement of the combined activity of TAMEP and SM-TAP co-immobilized on E.ECR8215F or IB-COV-3 Purified pro-Tgase variant (SEQ ID NO: 2, 250 μL at 5 g / L) was added to a 1.5 mL glass vial containing co-immobilized TAMEP and SM-TAP (20 mg of porous solid support, 0.2 mg of TAMEP and SM-TAP combined). Samples were analyzed in the same manner as described in Example 3C above.

[0148] Co-immobilized TAMEP and SM-TAP were found to be superior to soluble and separately immobilized TAMEP and SM-TAP. Specifically, when compared to soluble forms of the protease (non-immobilized), or when only TAMEP or TAP was immobilized, with the other in solution or immobilized on a separate porous solid support, the results showed an increase in FRAP total area counts by UPLC-MS (described in Example 3C). The results are shown in Table 4 and Figure 2.

[0149] Furthermore, Figure 2 shows that the co-immobilized TAMEP and SM-TAP produced the most FRAP compared to the amount of FRAP produced by either separately immobilized TAMEP and SM-TAP or the soluble forms, i.e., both non-immobilized TAMEP and SM-TAP. Figure 2 also shows that the separately immobilized TAMEP and SM-TAP produced more FRAP over 120 min than the soluble forms. In other words, the co-immobilized TAMEP and SM-TAP, as well as the separately immobilized TAMEP and SM-TAP, outperformed the soluble forms.

[0150] [Table 4]

[0151] Amino acid sequence SEQ ID NO: 1 Wild-type Streptomyces mobaraensis Tgase proenzyme (pro-Tgase). The leader sequence (pro-) is shown in bold underlined text.

[0152] [ka]

[0153] SEQ ID NO: 2: A thermostable variant of the proenzyme form of Streptomyces mobaraensis Tgase (pro-Tgase variant) that contains two methionine amino acid residues - one methionine located at the N-terminus of the pro-sequence and the second methionine located between the pro-sequence and the N-terminus of the mature domain. The leader sequence (pro-) is shown in bold underlined text.

[0154] [ka]

[0155] SEQ ID NO: 3: Wild-type proenzyme transglutaminase-activated M4 metalloprotease (TAMEP) from Streptomyces mobaraensis, deleted of its native signal peptide and containing a leading methionine to facilitate recombinant expression. The predicted leader sequence (pro-) is shown in bold underlined text.

[0156] [ka]

[0157] SEQ ID NO: 4: Wild-type proenzyme transglutaminase-activating tripeptidyl aminopeptidase (SM-TAP) from Streptomyces mobaraensis, deleted of its native signal peptide and containing a leading methionine to facilitate recombinant expression. The predicted leader sequence (pro-) is shown in bold underlined text.

[0158] [ka]

[0159] SEQ ID NO: 5: A thermostable variant of Streptomyces mobaraensis Tgase (FRAP-Tgase variant) having an N-terminal FRAP tetrapeptide with a methionine amino acid residue located between the FRAP tetrapeptide and the N-terminus of the mature domain of the thermostable Tgase variant. The leader sequence (pro-) is shown in bold underlined text.

[0160] [ka]

[0161] A thermostable variant of Streptomyces mobaraensis Tgase in which the SEQ ID NO:6 FRAP tetrapeptide has been removed from the N-terminus of the mature domain of the thermostable Tgase variant and an initial methionine amino acid residue is located at the N-terminus of the mature domain of the thermostable Tgase variant.

[0162] [ka]

Claims

1. Transglutaminase-activated M4 metalloprotease (TAMEP) immobilized on a porous solid support.

2. 2. The immobilized TAMEP of claim 1, wherein the TAMEP is derived from Streptomyces sp.

3. The immobilized TAMEP of claim 2, wherein the TAMEP is derived from Streptomyces mobaraensis.

4. Transglutaminase-activated tripeptidyl aminopeptidase (TAP) immobilized on a porous solid support.

5. The immobilized TAP of claim 4, wherein the TAP is derived from Streptomyces sp.

6. The immobilized TAP of claim 5, wherein the TAP is derived from Streptomyces mobaraensis.

7. Transglutaminase-activated M4 metalloprotease (TAMEP) and tripeptidyl aminopeptidase (TAP) co-immobilized on a porous solid support.

8. 8. The co-immobilized TAMEP and TAP of claim 7, wherein the TAMEP is derived from Streptomyces sp. and the TAP is derived from Streptomyces sp.

9. 9. The co-immobilized TAMEP and TAP of claim 8, wherein the TAMEP is derived from Streptomyces mobaraensis and the TAP is derived from Streptomyces mobaraensis.

10. 1. A method for activating a proenzyme form of transglutaminase or a variant thereof to produce a mature, catalytically active form of transglutaminase, comprising: a) immobilizing at least one transglutaminase-activated M4 metalloprotease (TAMEP) and / or at least one tripeptidyl aminopeptidase (TAP), with the proviso that when both TAMEP and TAP are immobilized, the TAMEP and TAP are separately immobilized on the same or different porous solid supports; and b) contacting the proenzyme form of transglutaminase with TAMEP and TAP to produce a mature, catalytically active form of transglutaminase, wherein at least one of TAMEP or TAP is immobilized. A method comprising:

11. 11. The method of claim 10, wherein the mature, catalytically active transglutaminase is separated from at least one of the immobilized proteases.

12. 12. The method of claim 10 or 11, wherein the TAMEP is derived from Streptomyces sp. and the TAP is derived from Streptomyces sp.

13. 13. The method of claim 12, wherein the TAMEP is derived from Streptomyces mobaraensis and the TAP is derived from Streptomyces mobaraensis.

14. 1. A method for activating a proenzyme form of transglutaminase or a variant thereof to produce a mature, catalytically active form of transglutaminase, comprising: a) co-immobilizing at least one transglutaminase-activated M4 metalloprotease (TAMEP) and at least one tripeptidyl aminopeptidase (TAP) on the same porous solid support; and b) contacting the proenzyme form of transglutaminase with co-immobilized TAMEP and TAP to produce the mature, catalytically active form of transglutaminase. A method comprising:

15. 15. The method of claim 14, wherein the mature, catalytically active transglutaminase is separated from the immobilized protease.

16. 16. The method of claim 14 or 15, wherein the TAMEP is derived from Streptomyces sp. and the TAP is derived from Streptomyces sp.

17. 17. The method of claim 16, wherein the TAMEP is derived from Streptomyces mobaraensis and the TAP is derived from Streptomyces mobaraensis.

18. 1. A method for activating a proenzyme form of transglutaminase or a variant thereof to produce a catalytically active immature form of transglutaminase, comprising: a) immobilizing at least one transglutaminase-activated M4 metalloprotease (TAMEP) on a porous solid support; and b) contacting the proenzyme form of transglutaminase with at least one immobilized TAMEP to produce a catalytically active immature form of transglutaminase; A method comprising:

19. 19. The method of claim 18, wherein the catalytically active immature form of transglutaminase is separated from at least one of the immobilized TAMEPs.

20. 20. The method of claim 18 or 19, wherein the TAMEP is derived from Streptomyces sp.

21. 21. The method of claim 20, wherein the TAMEP is derived from Streptomyces mobaraensis.

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