Immobilized proteases for activating plasminogen form of transglutaminase

Abstract

The present invention discloses an immobilized protease for activating a transglutaminase in the zymogen form.

Description

[0001] Incorporated by Reference

[0002] The Sequence Listing is provided in a file entitled SequenceListing.xml, 8,587 bytes in size, created on September 6, 2022 and submitted herewith, the entire contents of which are incorporated herein by reference. Technical Field

[0003] The field is that of immobilized proteases, and in particular of immobilized proteases for activating transglutaminases in the zymogen form. Background Art

[0004] Transglutaminase (Tgase, EC2.3.2.13) is a family of enzymes that catalyzes the cross-linking between the γ-carboxamide group in glutamine residues (acyl donors) and various primary amines (acyl acceptors) including the amino group of lysine. Tgase can be present in all groups of organisms, including prokaryotes, eukaryotes and plants. For example, Tgase in animals includes coagulation factor XIII, which is a multidomain protein and relies on calcium for the regulation of enzyme function. On the other hand, microbial transglutaminase has only a single domain and does not rely on calcium for activity, i.e., Tgase of microbial origin is calcium-independent. Therefore, microbial Tgase shows great advantages in its practical application.

[0005] Commercially available Tgase is produced by fermentation of Streptomyces mobaraensis. Tgase is expressed as an inactive zymogen with a pro-peptide sequence at the N-terminus of the mature domain. The pro-peptide is removed by proteolytic treatment in solution to provide the mature domain, thereby producing an active enzyme.

[0006] Zotsel et al., Eur. J. Biochem., 270, 3214-3222 (2003) described that Tgase from Streptomyces mobara is activated by a soluble form of the Tgase-activating M4 metalloproteinase (TAMEP), which cleaves the first 41 amino acids of the zymogen form of Tgase (i.e., pro-Tgase) between Ser41 and Phe42 (GPS-FRAP cleavage site, see SEQ ID NO: 1).

[0007] Zotsel et al., Eur. J. Biochem., 270, 4149-4155 (2003) further reported that TAMEP activated Tgase has an N-terminal tetrapeptide (Phe-Arg-Ala-Pro), i.e. FRAP, which can be removed by a transglutaminase activating tripeptidyl aminopeptidase (TAP) (SM-TAP) from Streptomyces mobara, which is also a soluble form. SM-TAP is cut between Pro45 and Asp46 (FRAP-DSDD cleavage site, see SEQ ID NO: 1) to provide a mature, catalytically active Tgase with FRAP removal, i.e., a mature, catalytically active form.

[0008] The use of soluble enzymes during post-translational processing of the inactive proenzyme of Tgase complicates purification because it requires separation of multiple enzymes from the fermentation broth. This typically requires the use of chromatographic purification, resulting in increased manufacturing costs and complexity.

[0009] In contrast, enzyme immobilization can be used to improve the catalytic performance of the enzyme and can help simplify downstream processing. Immobilization facilitates enzyme reuse, allows easy and more simplified recovery of both enzyme and product, allows continuous operation of enzymatic processes, rapid termination of reactions, and a wider variety of bioreactor designs.

[0010] Unpublished studies have shown that commercially available immobilized proteases are not suitable for producing mature, catalytically active Tgase, i.e., Tgase with the entire pro sequence (including FRAP) removed. These commercially available immobilized proteases cleave upstream of the desired amino acid position, and / or over-digest mature Tgase. Therefore, there is a need for an immobilized protease that will produce mature, catalytically active Tgase from the zymogen form by cleaving at the correct amino acid position after Pro45, while not over-digesting the mature enzyme.

[0011] An additional factor complicating the post-translational processing of the zymogen form of Tgase to the mature, catalytically active Tgase form is that the native proteases TAMEP and TAP (such as SM-TAP) used to activate the zymogen form of Tgase are inactivated by the catalytically active, mature Tgase and / or the catalytically active, immature Tgase (i.e., FRAP-Tgase or Tgase with FRAP at its N-terminus).

[0012] Both TAMEP and SM-TAP are potential substrates of mature catalytically active Tgase or catalytically active immature Tgase, because they contain multiple glutamine and lysine residues, which can react with mature Tgase to form cross-linked products (Gln-Lys) or cause the deamination of glutamine to glutamate (hydrolysis of Gln to Glu), thereby inactivating TAMEP and SM-TAP. This deactivation of TAMEP and SM-TAP causes TAMEP and SM-TAP to incompletely convert Tgase in the form of zymogen into mature, catalytically active Tgase, and further increases the cost and complexity of purification and separation of recombinantly expressed, mature, catalytically active Tgase or catalytically active, immature Tgase.

[0013] Surprisingly, it was found that TAMEP and TAP were immobilized together as disclosed herein, either separately or by co-protease immobilization, and both proteases were found to be protected from the deactivation of Tgase catalysis. This greatly simplifies downstream processing to obtain mature, catalytically active Tgase, and is conducive to the recombinant expression and purification of both Tgase wild-type and variants.

[0014] Therefore, there exists a need for immobilizing the protease required to generate mature, catalytically active Tgase from the zymogen form of Tgase.The disclosure herein addresses the long-standing unmet need for an immobilized protease that can facilitate the expression and purification of recombinantly expressed mature, catalytically active Tgase. Summary of the invention

[0015] In a first embodiment, a transglutaminase-activating M4 metalloprotease (TAMEP) immobilized on a porous solid support is disclosed.

[0016] In a second embodiment, a transglutaminase-activating tripeptidyl aminopeptidase (TAP) immobilized on a porous solid support is disclosed.

[0017] In a third embodiment, a transglutaminase-activating M4 metalloprotease (TAMEP) and a tripeptidyl aminopeptidase (TAP) co-immobilized on a porous solid support is disclosed.

[0018] In a fourth embodiment, a method for activating a zymogen form of a transglutaminase or a variant thereof to produce a mature, catalytically active form of a transglutaminase is disclosed, the method comprising

[0019] a) immobilizing at least one transglutaminase-activating M4 metalloprotease (TAMEP) and / or at least one tripeptidyl aminopeptidase (TAP), with the proviso that if both TAEMP and TAP are immobilized, TAMEP and TAP are immobilized on the same or different porous solid supports, respectively; and

[0020] b) contacting a zymogen form of the transglutaminase with TAMEP and TAP, wherein at least one of TAMEP or TAP is immobilized, to produce a mature, catalytically active form of the transglutaminase, and further wherein if the contacting is sequential, TAMEP is contacted first.

[0021] Alternatively, step a) may comprise providing at least one TAMEP and / or at least one TAP immobilized on a porous solid support.

[0022] In a fifth embodiment, a method for activating a zymogen form of a transglutaminase or a variant thereof to produce a mature, catalytically active form of a transglutaminase is disclosed, the method comprising

[0023] a) co-immobilizing at least one transglutaminase-activating M4 metalloprotease (TAMEP) and at least one tripeptidyl aminopeptidase (TAP) on the same porous solid support; and

[0024] b) contacting the zymogen form of transglutaminase with co-immobilized TAMEP and TAP to produce the mature, catalytically active form of transglutaminase.

[0025] In a sixth embodiment, a method for activating a zymogen form of a transglutaminase or a variant thereof to produce a catalytically active, immature form of a transglutaminase is disclosed, the method comprising

[0026] a) immobilizing at least one transglutaminase-activating M4 metalloprotease (TAMEP) on a porous solid support; and

[0027] b) contacting the zymogen form of the transglutaminase with the immobilized at least one TAMEP to produce a catalytically active, immature form of the transglutaminase.

[0028] Optionally, the catalytically active, immature transglutaminase may be separated from at least one immobilized or co-immobilized protease.

[0029] In yet another aspect, for all embodiments disclosed herein, preferably TAMEP is from a Streptomyces species, and most preferably TAMEP is from S. mobara, and preferably TAP is from a Streptomyces species, and most preferably TAP is from S. mobara (SM-TAP).

[0030] BRIEF DESCRIPTION OF THE DRAWINGS AND SEQUENCES

[0031] Figure 1 SDS-PAGE analysis of the S. mobaraensis Tgase variants as described in Example 1 is shown. 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 the expected molecular weight of the mature Tgase variant is 38.9 kDa.

[0032] Figure 2 TAMEP and SM-TAP co-immobilized on the same porous solid support, TAMEP and SM-TAP each immobilized on a different porous solid support, and the activities of TAMEP (soluble) and SM-TAP (soluble) are shown.

[0033] The following sequences comply with 37 C.FR §§1.821-1.825 (“Requirements for Patent Applications Containing Nucleotide Sequence and / or Amino Acid Sequence Disclosures—Sequence Rule”) and conform to the sequence listing requirements of World Intellectual Property Organization (WIPO) Standard ST.26 (2021) and Rules 5.2 and 49.5(a-bis) of the European Patent Convention (EPC) and the Patent Cooperation Treaty (PCT), and Section 208 and Annex C of the Executive Order. The symbols and formats used for nucleotide and amino acid sequence data conform to the rules set forth in 37 C.FR §1.822.

[0034] SEQ ID NO: 1 corresponds to the wild-type zymogen form of Tgase from Streptomyces mobara (Pro-Tgase). The leader sequence (pro-) is indicated in bold, underlined text.

[0035] SEQ ID NO: 2 corresponds to a thermostable variant of the proenzyme form of Streptomyces mobara Tgase with two additional methionine residues - one methionine at the N-terminus of the Pro sequence and the second methionine between the Pro sequence and the N-terminus of the mature domain (Pro-Tgase variant). The leader sequence (pro-) is represented by bold, underlined text.

[0036] SEQ ID NO: 3 corresponds to the zymogen form of wild-type Streptomyces mobara transglutaminase-activating M4 metalloprotease (TAMEP) with the native signal peptide deleted and containing a leading methionine to facilitate recombinant expression. The putative leader sequence (pro-) is indicated in bold, underlined text.

[0037] SEQ ID NO: 4 corresponds to the zymogen form of wild-type Streptomyces mobara transglutaminase-activating tripeptidyl aminopeptidase (SM-TAP) with a native signal peptide deleted and containing a leading methionine to facilitate recombinant expression. The putative leader sequence (pro-) is indicated in bold, underlined text.

[0038] SEQ ID NO: 5 corresponds to a thermostable variant of Streptomyces mobaraensis Tgase having a FRAP tetrapeptide at the N-terminus and a methionine amino acid residue located between the FRAP tetrapeptide and the N-terminus of the mature domain of the thermostable Tgase variant (FRAP-Tgase variant). The leader sequence (pro-) is indicated in bold, underlined text.

[0039] SEQ ID NO:6 corresponds to a thermostable variant of S. mobara Tgase, wherein the FRAP tetrapeptide is removed from the N-terminus of the mature domain of the thermostable Tase variant and the leading methionine amino acid residue is located at the N-terminus of the mature domain of the thermostable Tgase variant (thermostable Tgase variant). DETAILED DESCRIPTION OF THE INVENTION

[0041] All patents, patent applications, and publications cited herein are incorporated by reference in their entirety.

[0042] Words using the singular include the plural and vice versa unless the context clearly indicates otherwise.

[0043] In this disclosure, a number of terms and abbreviations are used. Unless expressly stated otherwise, the following definitions apply.

[0044] As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly indicates otherwise. For example, the term "a compound" or "at least one compound" may include a plurality of compounds, including mixtures thereof. For example, the terms "a," "an," "the," "one or more," and "at least one" may be used interchangeably herein.

[0045] As used herein, the term "about" can allow for a certain degree of variability in values ​​or ranges, for example, within 10%, within 5%, or within 1% of a stated value or limit of a stated range.

[0046] The terms "and / or" and "or" are used interchangeably herein and refer to a specific disclosure of each of two specified features or components, either together or not together with the other. Thus, as used herein in phrases such as "A and / or B," the term "and / or" is intended to include "A and B," "A or B," "A" (alone), and "B" (alone). Similarly, as used in phrases such as "A, B, and / or C," the term "and / or" is intended to cover each of the following: "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).

[0047] The terms "comprises, comprising," "includes, including," "having," and their variations are used interchangeably and mean "including but not limited to." It should be understood that any aspect described herein with the language "comprising" also provides other similar aspects described with "consisting of" and / or "consisting essentially of."

[0048] The term "consisting of" means "including and limited to."

[0049] The term "consisting essentially of" means that the composition, formulation or method includes any listed components, ingredients or steps, and is open to unlisted components, ingredients or steps that do not materially affect the basic characteristics of the composition, formulation or method.

[0050] Throughout the application, various embodiments can be presented in range format.It should be understood that the description of range format is only for convenience and simplicity, and should not be interpreted as an inflexible limitation of the scope of the embodiments described herein.Therefore, the description of the scope should be considered to have specifically disclosed all possible subranges and each numerical value in the scope.For example, the description of the scope, such as from 1 to 6, should be considered to have subranges, such as from 1 to 2, from 1 to 3, from 1 to 4 and from 1 to 5, from 2 to 3, from 2 to 4, from 2 to 5, from 2 to 6, from 3 to 4, from 3 to 5, from 3 to 6 etc., and each numeral in the scope, for example, 1,2,3,4,5 and 6.No matter how the range width, this is applicable.

[0051] “Optional” or “optionally” means that the subsequently described event, condition, or material may or may not occur or exist, and that the description includes instances where the event, condition, or material occurs or exists and instances where it does not.

[0052] As used herein, the term "immobilized" refers to the technical process of immobilizing a molecule, such as an enzyme, to or within a solid support. This can be achieved by a variety of techniques, including but not limited to covalent binding / attachment, ionic bonding, capture or adsorption.

[0053] The term "co-immobilization" refers to the immobilization of at least two different molecules, such as at least two different enzymes, to or within the same solid support.

[0054] The term "solid support" refers to a range of materials, which may be biological materials, non-biological materials, organic materials, inorganic materials or any combination of these materials. Thus, the solid support may be any suitable composition to which the molecules to be attached may be applied.

[0055] As used herein, the term "porous solid support" simply refers to any solid containing void space, i.e., space not occupied by the main skeleton of atoms that make up the solid structure. Examples of such supports include, but are not limited to, polyacrylates, polymethacrylates, polystyrenes, etc. that 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, sulfonic acids, amines (primary, secondary, tertiary or quaternary), etc.

[0056] Other examples of porous solid supports include, but are not limited to, aminopropylsiliconized controlled pore glass ("CPG"), diatomaceous earth, or metal organic frameworks ("MOFS").

[0057] Preferably, the porous solid support is selected from any of the supports shown in Table 2 of 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.

[0058] The term "covalently bound" or "covalent bond" refers to a chemical bond involving shared electron pairs between atoms. These electron pairs are called shared electron pairs or bonding pairs, and when they share electrons, the stable balance of attractive and repulsive forces between atoms is called covalent bonding. For example, the pro domain and the mature domain can be covalently bound by a peptide bond.

[0059] The term "ionic bond" may also be referred to as an electrovalent bond. It is a type of connection formed by electrostatic attraction between oppositely charged ions in a compound. This type of bond is formed when the valence (outermost) electrons of one atom are permanently transferred to another atom. The atom that loses the electron becomes a positively charged ion, or cation, while the atom that gains the electron becomes a negatively charged ion, or anion. An ionic bond is an example of a non-covalent bond.

[0060] The term "non-covalently bonded" differs from covalent bonding in that non-covalent bonding does not involve sharing of electrons between atoms. Thus, non-covalent bonding can occur by 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.

[0061] As used herein, the term "adsorption" refers to a process involving the accumulation of a substance or molecular species at a higher concentration on a surface, i.e., atoms, ions or molecules from a gas, liquid or dissolved solid adhere to the surface. Adsorption may include, but is not limited to, interactions such as hydrophobic or hydrophilic interactions.

[0062] The terms "zymogen" and "proenzyme" are used interchangeably herein and refer to an inactive precursor of an enzyme that can be converted to an active or mature enzyme by post-translational modification, e.g., by catalytic action, such as by proteolytic cleavage of a propeptide sequence.

[0063] The terms "pro-peptide", "pro-domain", "pro-sequence" and "pro-region" are used interchangeably herein and refer to an N-terminal peptide leader sequence (including the FRAP tetrapeptide) that, when fully cleaved, produces a mature, catalytically active Tgase. However, if the complete propeptide is not cleaved so that it does not include FRAP (i.e., a FRAP-free propeptide), a catalytically active but immature Tgase is produced.

[0064] The propeptide can be considered to have a regulatory function, while the mature domain has a catalytic function. It is generally believed that the propeptide has four main functions: 1) the propeptide can act as an intramolecular chaperone or folding aid by determining the three-dimensional structure of the protein; 2) the propeptide can act as an inhibitor or activating peptide; 3) the propeptide can guide the sorting of proteins to specific cellular compartments or extracellular spaces; and 4) the propeptide can mediate the interaction of the precursor with other molecules (such as peptides, proteins and polysaccharides) or supramolecular structures (such as cell walls). A single propeptide can perform multiple or even all of the above functions.

[0065] The term "transglutaminase" (Tgase, EC2.3.2.13) refers to a family of enzymes that catalyze the formation of isopeptide bonds between primary amines (e.g., the ε-amine of a lysine molecule) and the acyl group of a protein or peptide-bound glutamine. Transglutaminases can catalyze the transamidation reaction between the glutamyl and lysyl side chains of a target protein. 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, usually insoluble protein biopolymers, which are required by organisms for creating barriers and stable structures.

[0066] Unlike eukaryotic Tgase, Tgase of microbial origin is not calcium dependent, which represents its major advantage in practical applications. "Microbial transglutaminases" are among the most extensively studied industrial enzymes for protein functionalization and protein cross-linking due to their ability to polymerize or functionalize proteins by forming stable ε-(γ-glutamyl)lysine isopeptide bonds without the restriction of consensus sequences or other cofactors. Microbial Tgase is a subset of Tgase.

[0067] The most commonly used Tgase is a microbial transglutaminase from Streptomyces mobara, and its wild-type zymogen form (Pro-Tgase) has an amino acid sequence corresponding to SEQ ID NO: 1. The terms "Pro-Tgase" and "zymogen form of Tgase" are used interchangeably herein. It is known that Tgase from Streptomyces mobara is secreted in zymogen form, i.e. Pro-Tgase. The activation of the zymogen form of Tgase (Pro-Tgase) is carried out in two steps. First, the propeptide is cut from the N-terminus of Pro-Tgase by transglutaminase activating M4 metalloproteinase ("TAMEP") . Some TAMEPs produce catalytically active, immature Tgase, which has a FRAP tetrapeptide (i.e. FRAP-Tgase) at the N-terminus of Tgase. Some TAMEPs produce catalytically active, immature Tgase, which has a partial FRAP peptide, such as RAP, AP or P connected to the N-terminus of Tgase. In other words, the resulting propeptide does not contain a FRAP-tetrapeptide, i.e., it is a propeptide without FRAP. The N-terminal FRAP tetrapeptide or the remainder thereof on Tgase can then be cleaved by using a transglutaminase tripeptidyl aminopeptidase ("TAP") to produce a mature, catalytically active Tgase without FRAP. For clarity, the terms "mature, catalytically active" and "catalytically active, mature" are used interchangeably herein. Similarly, the terms "immature, catalytically active" and "catalytically active, immature" are used interchangeably herein.

[0068] Transglutaminase variants and methods of producing such variants are disclosed in, for example, PCT Publication Nos. WO2016 / 170447, published on October 27, 2016, and WO2019 / 094301, published on May 16, 2019.

[0069] "Protease" (also called peptidase or proteinase) refers to an enzyme that can cut peptide bonds. Protease is any of a variety of enzymes, such as endopeptidases and exopeptidases, that catalyze the hydrolysis of proteins into peptides and amino acids. Proteases can be divided into seven major categories: serine proteases, cysteine ​​proteases, threonine proteases, aspartic proteases, glutamic proteases, metalloproteases, and asparagine peptide lyases. Proteases are found in animals, plants, bacteria, fungi, archaea, and viruses. The terms "protease", "peptidase", and "proteinase" are used interchangeably herein.

[0070] The term "amino acid" refers to the basic chemical building block of a protein, peptide or polypeptide. The following abbreviations used herein to identify specific amino acids can be found in Table 1.

[0071] Table 1. One-letter and three-letter amino acid abbreviations

[0072]

[0073]

[0074] The terms "peptide", "protein" and "polypeptide" are used interchangeably herein and refer to amino acid polymers linked together by peptide bonds. "Protein" or "polypeptide" comprises a polymeric sequence of amino acid residues. The disclosure herein uses single-letter and three-letter codes for amino acids that conform to the standard definition of IUPAC-IUB Joint Commission on Biochemical Nomenclature (JCBN) throughout. The single letter X refers to any of the twenty amino acids. It should also be understood that due to the degeneracy of the genetic code, a polypeptide can be encoded by more than one nucleotide sequence. Mutations can be named by the single-letter code of the parent amino acid, followed by the position number and the single-letter code of the variant amino acid. For example, mutation of glycine (G) at position 87 to serine (S) is represented as "G087S" or "G87S". When describing a modification, the position is followed by a list of amino acids listed in brackets, indicating a list modified by any one of the listed amino acids at that position. For example, 6 (L, I) means that position 6 can be substituted by leucine or isoleucine. Sometimes, in a sequence, a slash ( / ) is used to define a modification, such as F / V, indicating that position may have either phenylalanine or valine.

[0075] It will be appreciated by those of ordinary skill in the art that the amino acid sequences disclosed herein may be modified while retaining the functions associated with the disclosed amino acid sequences. For example, it is well known in the art that changes at a given site in a gene result in the production of chemically equivalent amino acids but do not affect the functional properties of the encoded protein, which is common.

[0076] The term "mutation" herein refers to a change introduced into a parent sequence, including but not limited to modifications, such as insertions or deletions (including truncations), thereby producing a "variant". The consequences of a mutation include but are not limited to the creation of new features, properties, functions, phenotypes or traits not found in the protein encoded by the parent sequence.

[0077] Related (and derived) proteins encompass "variants," "mutants," or "modified" proteins, which terms are used interchangeably herein. Variant (i.e., mutant or modified) proteins differ from another (i.e., parent) protein, or differ from each other by modification of one or more amino acid residues. For example, a variant may include one or more amino acid modifications, such as one or more amino acid deletions / truncations, insertions, or substitutions, compared to the parent protein from which it is derived.

[0078] Alternatively or additionally, variants can have a sequence identity to a particular degree with a reference protein or nucleic acid, for example, 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%, 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 and integer percentages therebetween with a reference sequence.

[0079] As used herein, "corresponding to or corresponds to," "corresponds substantially to or corresponding substantially to," or "corresponds to," with respect to amino acid residue positions, refers to the amino acid residue at the enumerated position in a protein or peptide, or an amino acid residue that is similar, homologous, or equivalent to the enumerated residue in a protein or peptide. As used herein, a "corresponding region" generally refers to an analogous position in a related protein or reference protein.

[0080] It will be appreciated by those of ordinary skill in the art that the amino acid sequences disclosed herein may be modified while retaining the functions 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 chemically equivalent amino acids at a given site but do not affect the functional properties of the encoded protein are common.

[0081] Related (and derived) proteins encompass "variants" or "mutant" proteins, which terms are used interchangeably herein. Variant proteins differ from another (i.e., parent) protein and / or from each other in a few amino acid residues. Variants may include one or more amino acid mutations (e.g., amino acid deletions, insertions, or substitutions) compared to the parent protein from which they are derived. Alternatively or additionally, variants may have a specific degree of sequence identity to a reference protein or nucleic acid, for example, 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 the reference sequence and integer percentages in between.

[0082] The term "wild-type" in relation to an amino acid sequence or a nucleic acid sequence means 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 any substance (e.g., a protein, amino acid or nucleic acid sequence) found in nature. Conversely, the term "non-naturally occurring" refers to any substance 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).

[0083] The term "derived from" encompasses the terms "originating from," "obtained from," "obtainable from," "isolated from," "purified from," and "created from," and generally indicates that one particular material finds its source in, or has characteristics that can be described with reference to, another particular material.

[0084] 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 a material that is substantially or essentially free of components that normally accompany it in its natural state, such as, for example, an intact biological system. An isolated nucleic acid molecule includes a nucleic acid molecule contained in a cell that normally expresses the nucleic acid molecule, but the nucleic acid molecule is present extrachromosomally or at a chromosomal location that is different from its natural chromosomal location.

[0085] In a first embodiment, a transglutaminase-activating M4 metalloprotease ("TAMEP") immobilized on a porous solid support is disclosed.

[0086] For immobilized TAMEP, preferred solid support is porous solid support as described above. Any porous solid support that can immobilize TAMEP can be used. It can be pre-treated or functionalized before using TAMEP, to promote combination, or for any other desired purpose, such as cultivating conditions that are conducive to activity or any other desired characteristics, or avoid undesirable interaction with other entities. Many such surface treatments and / or functionalizations are known in the art, and the selection of suitable treatment and / or functionalization will depend on TAMEP and the activity of conditions and expectations therewith. The example of such support includes but is not limited to polyacrylate, polymethacrylate, polystyrene etc. that can be modified with functional groups. For example, polymethacrylate can be modified with epoxide functional groups or primary amine groups that can be activated by crosslinking agents. The example of such functional groups includes but is not limited to epoxide, alkyl, phenyl, sulfonic acid, amine (primary, secondary, tertiary or quarterly) etc.

[0087] Other examples of porous solid supports include, but are not limited to, aminopropylsiliconized controlled pore glass ("CPG"), diatomaceous earth, or metal organic frameworks ("MOFS").

[0088] Preferably, the porous solid support is selected from any of the supports shown in Table 2 of 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.

[0089] The term "active TAMEP" and "TAMEP" can be used interchangeably in this article. If TAMEP is prepared in zymogen form, active TAMEP is prepared by removing the N-terminal part from the zymogen form required for producing active enzyme. Any attachment method discussed above and in the following examples can be used, i.e., immobilized TAMEP by means of covalent bonds, ionic bonds or adsorption. Preferably, covalent bond immobilization TAMEP is used. Alternatively, it is believed that the TAMEP in zymogen form also can be immobilized and activated subsequently.

[0090] TAMEP can be obtained from a variety of microbial sources, including but not limited to Streptomyces species. Non-limiting examples include Streptomyces mobaraensis, Streptomyces huiliensis, Streptomyces sp. TYQ1024, Streptomyces abikoensis, Streptomyces hiroshimensis, Streptomyces albireticuli, Streptomyces triculaminicus, Streptomyces oliveoverticillatus, Streptomyces luteoverticillatus, Streptomyces cinnamoneus, Streptomyces caatingaensis, Streptomyces roseoverticillatus, Streptomyces grisea griseocarneus, Streptomyces rectiverticillatus, Streptomyces roseifaciens, Streptomyces eurocidicus, Streptomyces klenkii, Streptomyces netropsis, Streptomyces hygroscopicus, Streptomyces varsoviensis, etc. Preferably, TAMEP is obtained from Streptomyces mobaraensis.

[0091] SEQ ID NO:3 corresponds to the zymogen form of wild-type Streptomyces mobara transglutaminase activation property M4 metalloproteinase (TAMEP), wherein deleted natural signal peptide and comprise leading methionine to promote recombinant expression.The leader sequence (pro-) of inference is represented with bold underline text, and is provided as amino acid 2 to 197 of SEQ ID NO:3.Active TAMEP is produced by removing the N-terminal part of the zymogen form required for active enzyme (such as by endogenous protease activity).For TAMEP, it is believed that the N-terminal part of the zymogen form required for active enzyme may involve removing the inference pro region or its variant identified from SEQ ID NO:3 to produce active TAMEP.

[0092] Thus, it appears that there is an active TAMEP that can have at least 70% sequence identity with the active form of the amino acid sequence shown in SEQ ID NO:3. Even more preferably, the TAMEP from Streptomyces mobara comprises an active form of the amino acid sequence shown in SEQ ID NO:3 (without a putative leader sequence or variant thereof) with 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 a sequence with 99.5% amino acid sequence identity. Most preferably, the TAMEP from Streptomyces mobara comprises a sequence that is essentially composed of the active form of the amino acid sequence shown in SEQ ID NO:3.

[0093] The TAMEP sequence after removal of the putative leader sequence from the zymogen form corresponds to amino acids 198 to 728 of SEQ ID NO: 3. The TAMEP sequence after removal of the leader sequence from the zymogen form may correspond to one of the following amino acid ranges 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 a variant thereof. Alternatively, the TAMEP sequence after removal of the leader sequence from the zymogen form may correspond to one of the following amino acid ranges 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 a variant thereof.

[0094] The sequence of the active TAMEP may correspond to one of the following amino acid ranges 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 a variant thereof. Alternatively, the sequence of the active TAMEP may correspond to one of the following amino acid ranges 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 a variant thereof.

[0095] In a second embodiment, a transglutaminase-activating tripeptidyl aminopeptidase ("TAP") immobilized on a porous solid support is disclosed.

[0096] For immobilized TAP, preferred solid supports are porous solid supports as described above. Any porous solid supports that can immobilize TAP can be used. It can be pretreated or functionalized before applying TAP to promote binding, or for any other desired purpose, such as cultivating conditions that are conducive to activity or any other desired characteristics, or avoiding undesirable interactions with other entities. Many such surface treatments and / or functionalizations are known in the art, and the selection of suitable treatments and / or functionalizations will depend on TAP and the conditions and desired activity therewith. Examples of such supports include, but are not limited to, polyacrylates, polymethacrylates, polystyrenes, etc. that 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, sulfonic acid groups, amines (primary, secondary, tertiary or quaternary), etc.

[0097] Other examples of porous solid supports include, but are not limited to, aminopropylsiliconized controlled pore glass ("CPG"), diatomaceous earth, or metal organic frameworks ("MOFS").

[0098] Preferably, the porous solid support is selected from any of the supports shown in Table 2 of 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.

[0099] Any attachment method discussed in the examples above and below, i.e., by covalent bonding, ionic bonding, or adsorption, can be used to immobilize active TAP (i.e., not in zymogen form). Preferably, TAP is immobilized using covalent bonding. Alternatively, it is believed that zymogen-form TAP can also be immobilized and subsequently activated.

[0100] The terms "active TAP" and "TAP" are used interchangeably herein. If TAP is prepared in zymogen form, active TAP is prepared by removing the N-terminal portion of the zymogen form required to produce active enzyme, which can be obtained from a variety of microbial sources, including but not limited to Streptomyces species. Non-limiting examples include Streptomyces mobaraensis, Streptomyces huiliensis, Streptomyces caatingaensis, Streptomyces abikoensis, Streptomyces olivoverticillatus, Streptomyces luteoverticillatus, Streptomyces cinnamoneus, Streptomyces netropsis, Streptomyces eurucidicus, Streptomyces morookaense, Streptomyces hiroshimensis, Streptomyces roseifaciens, Streptomyces roseoverticillatus, Streptomyces hygroscopicus, and the like. Preferably, TAP is obtained from Streptomyces mobara. Preferably, TAP is obtained from Streptomyces mobara.

[0101] SEQ ID NO:4 corresponds to the zymogen form of wild-type Streptomyces mobara transglutaminase activation tripeptidyl aminopeptidase (SM-TAP), wherein the natural signal peptide is deleted and the leading methionine is included to promote recombinant expression. The presumed leader sequence (pro-) is represented by bold underline text, and is provided as amino acids 2 to 7 of SEQ ID NO:4. Active TAP is produced by removing the N-terminal portion of the zymogen form required for producing active enzyme. For TAP, it is believed that the N-terminal portion of the zymogen form required for producing active enzyme may involve removing the identified presumed pro region or its variant from SEQ ID NO:4 to produce active TAP.

[0102] Thus, it appears that there is an active TAP that may have at least 70% sequence identity with the active form of the amino acid sequence shown in SEQ ID NO: 4. Even more preferably, the TAP obtained from S. mobara 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 with the active form of the amino acid sequence shown in SEQ ID NO: 4 (without a putative leader sequence or variant thereof). Most preferably, the TAP from S. mobara comprises a sequence consisting essentially of the active form of the amino acid sequence shown in SEQ ID NO: 4.

[0103] The TAP sequence after removal of the putative leader sequence from the zymogen form corresponds to amino acids 8 to 451 of SEQ ID NO: 4. The TAP sequence after removal of the leader sequence from the zymogen form may correspond to one of the following amino acid ranges 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 variants thereof. Alternatively, the TAP sequence after removal of the leader sequence from the zymogen form may correspond to one of the following amino acid ranges 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 variants thereof.

[0104] The sequence of active TAP may correspond to one of the following amino acid ranges 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 variants thereof. Alternatively, the sequence of active TAP may correspond to one of the following amino acid ranges 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 variants thereof.

[0105] In the third embodiment, both TAMEP and TAP (active form of each protease) can be co-immobilized on any porous solid support disclosed herein. TAMEP and TAP have been discussed in more detail above. Any attachment method discussed above and in the following examples can be used, i.e. co-immobilization TAMEP and TAP by covalent bond, ionic bond or adsorption. Preferred co-immobilization mode is by covalent bond.

[0106] As shown in Example 8 below, co-immobilized TAMEP and SM-TAP and individually immobilized TAMEP and SM-TAP are superior to soluble TAMEP and soluble SM-TAP. In addition, surprisingly and unexpectedly found that, as disclosed herein, by individually immobilized TAMEP and SM-TAP or co-immobilized TAMEP and SM-TAP, these proteases seem to have been protected from being deactivated by mature catalytically active Tgase or catalytically active immature Tgase. This greatly simplifies downstream processing to obtain mature, catalytically active Tgase or catalytically active, immature Tgase, and is conducive to the recombinant expression and purification of Tgase wild type and variants.

[0107] In a fourth embodiment, a method for activating a zymogen form of a transglutaminase or a variant thereof to produce a mature, catalytically active form of a transglutaminase is disclosed, the method comprising

[0108] a) immobilizing at least one transglutaminase-activating M4 metalloprotease (TAMEP) and / or at least one tripeptidyl aminopeptidase (TAP), with the proviso that if both TAEMP and TAP are immobilized, TAMEP and TAP are immobilized on the same or different porous solid supports, respectively; and

[0109] b) contacting the zymogen form of the transglutaminase with TAMEP and TAP, wherein at least one of TAMEP or TAP is immobilized, to produce a mature, catalytically active form of the transglutaminase.

[0110] Alternatively, step a) may comprise providing at least one TAMEP and / or at least one TAP immobilized on a porous solid support.

[0111] Optionally, a mature, catalytically active form of transglutaminase is separated from the at least one immobilized protease. Preferably, the at least one TAMEP is from a Streptomyces species, and the at least one TAP is from a Streptomyces species. Most preferably, the at least one TAMEP is from Streptomyces mobara and the at least one TAP is from Streptomyces mobara.

[0112] In a fifth embodiment, a method for activating a zymogen form of a transglutaminase or a variant thereof to produce a mature, catalytically active form of a transglutaminase is disclosed, the method comprising

[0113] a) co-immobilizing at least one transglutaminase-activating M4 metalloprotease (TAMEP) and at least one tripeptidyl aminopeptidase (TAP) on the same porous solid support; and

[0114] b) contacting the zymogen form of transglutaminase with co-immobilized TAMEP and TAP to produce the mature, catalytically active form of transglutaminase.

[0115] Optionally, a mature, catalytically active transglutaminase is separated from at least one immobilized protease. Preferably, the at least one TAMEP is from a Streptomyces species, and the at least one TAP is from a Streptomyces species. Most preferably, the at least one TAMEP is from Streptomyces mobara and the at least one TAP is from Streptomyces mobara.

[0116] In a sixth embodiment, a method for activating a zymogen form of a transglutaminase or a variant thereof to produce a catalytically active, immature form of a transglutaminase is disclosed, the method comprising

[0117] a) immobilizing at least one transglutaminase-activating M4 metalloprotease (TAMEP) on a porous solid support; and

[0118] b) contacting the zymogen form of the transglutaminase with the immobilized at least one TAMEP to produce a catalytically active, immature form of the transglutaminase.

[0119] Optionally, catalytically active, immature transglutaminase is separated from the at least one immobilized TAMEP. Preferably, the TAMEP is from a Streptomyces species. Most preferably, the TAMEP is from Streptomyces mobaraensis.

[0120] Any of the immobilization and co-immobilization means discussed herein can be used to practice any of the embodiments disclosed herein. A preferred immobilization or co-immobilization means is via a covalent bond.

[0121] TAMEP and TAP are discussed in detail above.

[0122] Non-limiting embodiments of the foregoing disclosed herein include:

[0123] 1. Transglutaminase-activating M4 metalloprotease (TAMEP) immobilized on a porous solid support.

[0124] 2. The immobilized TAMEP of embodiment 1, wherein the TAMEP is from a Streptomyces species.

[0125] 3. The immobilized TAMEP according to embodiment 1 or 2, wherein the TAMEP is derived from Streptomyces mobaraensis.

[0126] 4. Transglutaminase-activating tripeptidyl aminopeptidase (TAP) immobilized on a porous solid support.

[0127] 5. The immobilized TAP of embodiment 4, wherein the TAP is from a Streptomyces species.

[0128] 6. The immobilized TAP according to embodiment 4 or 5, wherein the TAP is from Streptomyces mobaraensis (SM-TAP).

[0129] 7. Transglutaminase-activating M4 metalloproteinase (TAMEP) and tripeptidyl aminopeptidase (TAP) co-immobilized on a porous solid support.

[0130] 8. The co-immobilized TAMEP and TAP of embodiment 7, wherein TAMEP is from a Streptomyces species and TAP is from a Streptomyces species.

[0131] 9. The co-immobilized TAMEP and TAP of embodiment 7 or 8, wherein TAMEP is from Streptomyces mobaraensis and TAP is from Streptomyces mobaraensis.

[0132] 10. A method for activating a zymogen form of a transglutaminase or a variant thereof to produce a mature, catalytically active form of a transglutaminase, the method comprising

[0133] a) immobilizing at least one transglutaminase-activating M4 metalloprotease (TAMEP) and / or at least one tripeptidyl aminopeptidase (TAP), provided that if both TAEMP and TAP are immobilized, TAMEP and TAP are immobilized on the same or different porous solid supports, respectively, or providing at least one TAMEP and / or at least one TAP immobilized on a porous solid support; and

[0134] b) contacting the zymogen form of the transglutaminase with TAMEP and TAP, wherein at least one of TAMEP or TAP is immobilized, to produce a mature, catalytically active form of the transglutaminase.

[0135] 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.

[0136] 12. The method of embodiment 10 or 11, wherein TAMEP is from a Streptomyces species and TAP is from a Streptomyces species.

[0137] 13. The method of embodiment 10, 11 or 12, wherein TAMEP is from Streptomyces mobaraensis and TAP is from Streptomyces mobaraensis.

[0138] 14. A method for activating a zymogen form of a transglutaminase or a variant thereof to produce a mature, catalytically active form of a transglutaminase, the method comprising

[0139] a) co-immobilizing at least one transglutaminase-activating M4 metalloprotease (TAMEP) and at least one tripeptidyl aminopeptidase (TAP) on the same porous solid support; and

[0140] b) contacting the zymogen form of transglutaminase with co-immobilized TAMEP and TAP to produce the mature, catalytically active form of transglutaminase.

[0141] 15. The method of embodiment 14, wherein mature, catalytically active transglutaminase is separated from the at least one immobilized protease.

[0142] 16. The method of embodiment 14 or 15, wherein TAMEP is from a Streptomyces species and TAP is from a Streptomyces species.

[0143] 17. The method of embodiment 14, 15 or 16, wherein TAMEP is from Streptomyces mobaraensis and TAP is from Streptomyces mobaraensis.

[0144] 18. A method for activating a zymogen form of a transglutaminase or a variant thereof to produce a catalytically active, immature form of a transglutaminase, the method comprising

[0145] a) immobilizing at least one transglutaminase-activating M4 metalloprotease (TAMEP) on the same porous solid support; and

[0146] b) contacting the zymogen form of the transglutaminase with the immobilized at least one TAMEP to produce a catalytically active, immature form of the transglutaminase.

[0147] 19. The method of embodiment 18, wherein catalytically active, immature transglutaminase is separated from the at least one immobilized TAMEP.

[0148] 20. The method of embodiment 18 or 19, wherein the TAMEP is from a Streptomyces species.

[0149] 21. The method of embodiment 18, 19 or 20, wherein TAMEP is from Streptomyces mobaraensis.

[0150] 22. The immobilized and co-immobilized TAMEP and method of embodiments 1-3 or 7-21, wherein the TAMEP is an active form of TAMEP having the sequence of SEQ ID NO: 3.

[0151] 23. The immobilized and co-immobilized TAMEP and methods of embodiments 1-3 or 7-21, wherein the TAMEP has a sequence comprising the following 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. 28, 198 to 728, 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 or 214 to 728.

[0152] 24. The immobilized and co-immobilized TAP and methods of embodiments 4-6 or 7-21, wherein the TAP is an active form of TAP having the sequence of SEQ ID NO: 4.

[0153] 25. The immobilized and co-immobilized TAP and methods of embodiments 4-6 or 7-21, wherein the TAP has a sequence comprising the following 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, 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. Example

[0154] The following examples are intended to illustrate the present invention, but not to limit the present invention. Therefore, from the above discussion and examples, those skilled in the art can determine the essential features of the present disclosure, and without departing from the spirit and scope of the present disclosure, various changes and modifications can be made to adapt to various uses and conditions.

[0155] 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 the present disclosure belongs. Singleton et al., DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY, 2D Edition, 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 the present disclosure.

[0156] Example 1. Expression of Pro-Tgase Variant in E. coli

[0157] A. Construction of expression plasmid for expressing Pro-Tgase variant (SEQ ID NO: 2)

[0158] According to the published amino acid sequence (Kanaji et al., (1993) J.Biol.Chem.268(16):11565-11572), the gene encoding the Pro-Tgase variant (i.e., the zymogen form of the Tgase variant) was codon optimized for expression in E. coli, the gene was synthesized together with an additional C-terminal His tag, and cloned into a pET vector, operably connected to a T7 promoter controlled by a lacI repressor. The expression vector also contains a 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. As known in the art (see, e.g., U.S. Patent No. 8,383,346 and WO2010 / 144103, both of which are incorporated herein by reference in their entirety), transformants were isolated by subjecting cells to kanamycin screening, and the sequence of the Pro-Tgase gene was verified by Sanger sequencing. Plasmids were recovered from positive clones using methods known in the art and transformed into E. coli BL21 (DE3) for expression.

[0159] B. Expression and isolation of recombinant Pro-Tgase variant (SEQ ID NO: 2) in E. coli

[0160] E. coli strain BL21 (DE3) containing the Pro-Tgase expression vector was cultured in Luria broth at 37°C overnight until the culture reached saturation. The next morning, the culture was inoculated into a shake flask containing a medium including glycerol, soy peptone, yeast extract, magnesium sulfate heptahydrate, and potassium dihydrogen phosphate, and shaken continuously at 30-34°C for up to 10 hours. Isopropyl β-d-1-thiogalactopyranoside (IPTG) was added to a final concentration of 0.1-1 mM and incubated at 20-25°C for up to 24 hours.

[0161] The cells were harvested by centrifugation at 8000 x g for up to 60 minutes. The supernatant was discarded and the pellet was resuspended in 50 mM Tris (hydroxymethyl) aminomethane (Tris) HCl at pH 8 at a concentration of 20% w / v. The cells were lysed using a high pressure homogenizer at a pressure of 15000-20000 psi. The crude lysate was clarified by centrifugation at 15000 x g for up to 60 minutes. The lysate was purified by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE, Figure 1 ) and clarified lysates containing Pro-Tgase were evaluated spectroscopically and Tgase activity was evaluated using the colorimetric activity assay described in Example 1C.

[0162] Alternatively, Pro-Tgase variants can be secreted, for example, from microbial strains known to those skilled in the art that secrete Tgase, such as Streptomyces mobaraensis or Bacillus subtilis. The precipitate is discarded and the supernatant is recovered and evaluated by SDS-PAGE as described in Example 1D and by spectroscopy. Tgase activity is evaluated using a colorimetric activity assay as described in Example 1C.

[0163] C. Colorimetric Activity Assay

[0164] 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 a low molecular weight amine acceptor substrate and hydroxylamine as an 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) that can be detected at 525 nm after incubation at 37°C for 5-60 minutes. L-glutamic acid γ-monohydroxamate was used. The enzyme was used as a standard for calibration. The amount of the peptide derivative that catalyzes the formation of 1 μmol of γ-glutamylhydroxylamine per minute was defined as 1 unit of Tgase.

[0165] D. SDS-PAGE gel

[0166] This technique was used to evaluate the activation level of Pro-Tgase variant (SEQ ID NO: 2) by TAMEP (see Embodiment 3) and to analyze the molecular weight of the expressed protein. Mature Tgase was obtained from a commercial source (Moo Gloo TI preparation, ) and used as molecular weight standard for SDS-PAGE analysis ( Figure 1 , lane 1).

[0167] A 10 g aliquot of the proteolytic cleavage product or expressed protein was added to the Novex TM Bolt TM LDS sample buffer and Novex TM Bolt TM Samples were added to reducing agent, mixed as directed, heated at 95°C for 5 min, and then run on 4-12% SDS-PAGE gels (Invitrogen TM NuPAGE TM 4-12% Bis-Tris gel) at 170 volts for 30 minutes. TM Safe Stain (Invitrogen TM ) The gel was stained for 30-60 min, then destained in deionized water and photographed.

[0168] Example 2. Pro-Tgase variant (SEQ ID NO: 2) or FRAP-Tgase variant (SEQ ID NO: NO:5) purification

[0169] This method was used to purify Pro-Tgase variant (SEQ ID NO: 2) (prepared as in Example 1) or FRAP-Tgase variant (SEQ ID NO: 5). FRAP-Tgase variant (SEQ ID NO: 5) was prepared by incubating lysate containing Pro-Tgase variant (SEQ ID NO: 2) with supernatant containing TAMEP (Example 3, loaded at 1% vol) at 37°C for 60 minutes.

[0170] The pH of the lysate was lowered to pH 5-6 using 2-(N-morpholino)ethanesulfonic acid (MES). The mixture was then clarified by centrifugation at 4000 g for 10-20 minutes. The clarified lysate was then loaded onto an SP Sepharose column. On, according to the manufacturer's instructions, the column had previously been washed with water (10 column volumes) and then equilibrated with 25mM MES buffer (10 column volumes) at pH 5.5. The lysate was passed through the column at 1 mL / min and then washed with 25mM MES (10 column volumes) at pH 5.5 containing 50mM sodium chloride. The pro-Tgase variant (SEQ ID NO: 2) or the FRAP-Tgase variant (SEQ ID NO: 5) was then eluted with 25mM MES buffer (3 column volumes) at pH 5.5 containing 1M sodium chloride. The Tgase activity was evaluated using a colorimetric activity assay as described in Example 1C. In the colorimetric activity assay, the Pro-Tgase variant (SEQ ID NO: 2) did not show activity above baseline. Only activated forms of Tgase, such as the FRAP-Tgase variant (SEQ ID NO: 5), showed activity in this assay.

[0171] The N-terminal peptide sequence of the FRAP-Tgase variant was confirmed using N-terminal sequencing (SEQ ID NO: 5). N-terminal sequencing technology was used to evaluate the activation level of TAMEP on Pro-Tgase variant (SEQ ID NO: 2) or the activation level of TAP (such as SM-TAP) on FRAP-Tgase (SEQ ID NO: 5).

[0172] This technique is used to determine the N-terminal sequence of a protein. An aliquot of protein (10 g), FRAP-Tgase variant (SEQ ID NO: 5), was added to a commercial SDS-PAGE sample buffer (Novex TM Bolt TM LDS sample buffer and Novex TM Bolt TM Sample reducing agent, mixed as indicated) and heated at 95°C for 5 minutes. The samples were then subjected to 4-12% SDS-PAGE (Invitrogen TM NuPAGE TM 4-12% Bis-Tris Gel) gel was electroporated at 170 V for 30 min. The SDS-PAGE gel was then washed with water and analyzed using the Mini Blot Module (Invitrogen) according to the manufacturer's instructions. TM ) was run at 20 volts for 60 minutes and transferred to a 0.45um PVDF membrane. Then, the membrane was filtered with SimplyBlue TM Safe Stain (Invitrogen TM) The membrane was stained until a clear protein band was visible. After washing with deionized water, the protein band corresponding to the FRAP-Tgase variant (SEQ ID NO: 5) of the correct molecular weight was excised and sent to the protein facility of the Office of Biotechnology at Iowa State University for N-terminal sequencing analysis, where Edman degradation analysis was used to determine the first 4-5 amino acids of the protein.

[0173] Example 3. Expression of zymogen forms of TAMEP and SM-TAP and isolation of catalytically active forms

[0174] A. Expression of the zymogen forms of TAMEP (SEQ ID NO: 3) and SM-TAP (SEQ ID NO: 4) and isolation of mature catalytically active forms of TAMEP and SM-TAP

[0175] The genes encoding wild-type Streptomyces mobara Tgase protease, transglutaminase activating metalloproteinase (TAMEP) and Streptomyces mobara tripeptidyl aminopeptidase (SM-TAP) are synthesized by Integrated DNA Technologies (Coralville, IA). The expression constructs of TAMEP and SM-TAP are designed with N-terminal SacB signal sequence and 6 His tags, and cloned using methods well known in the art. The subtilis SCK6 delta-AlaR purchased from Bio-Technical Resources (Manitowoc, WI) is cultivated overnight at 37 ° C in 5mL LB medium supplemented with 40mg / mL D-alanine. The next day, the culture was diluted to OD600 of 1.0, and xylose was added to a final concentration of 1%. After 2 hours, 250 μL of glycerol and connected DNA were added, and the culture tube was returned to the incubator and cultured for another 90 minutes. After incubation, 10-1000 μL of culture was spread on LB agar plates. Flat plate is grown overnight at 37 ℃.On the second day, from each flat plate, select 2-8 bacterium colony and be inoculated in 3mLLB broth.Culture is hatched 48 hours at 37 ℃, and regularly collects supernatant sample.SDS-PAGE is used to confirm that the ripe, catalytically active form of enzyme is secreted into culture medium (determined according to molecular weight).The expected molecular weight of ripe, catalytically active TAMEP is about 56kDa, and the expected molecular weight of ripe, catalytically active SM-TAP is about 50kDa.It seems that the ripe, catalytically active form of every kind of protease is formed from zymogen form by the composition process such as endogenous protease activity.By centrifuging 10 minutes with 8000x g, separate two kinds of ripe, catalytically active protease TAMEP and SM-TAP from respective cell culture.Directly use after supernatant separation, without further purification.

[0176] B. TAMEP activity assay

[0177] The activity of TAMEP from Example 3A above was determined using the following assay. Soluble TAMEP was added to the purified Pro-Tgase variant at 1% vol (Example 2, 5 g / L, 500 μL). The reaction was then incubated at 37 ° C, 300 rpm, with sampling time points between 1-120 minutes. At each time point, a supernatant sample was taken and quenched with ethylenediaminetetraacetic acid (EDTA). The supernatant was then analyzed by ultra-high performance liquid chromatography coupled to mass spectrometry (UPLC-MS, Thermo Scientific TM Vanquish TM UPLCand Thermo Scientific TM ISQ TM The samples were analyzed by EM MS to determine the total ion counts of the propeptide cleaved from the Pro-Tgase variant (SEQ ID NO: 2) by TAMEP (i.e., the propeptide without the FRAP tetrapeptide). TM Vanquish TM C18 column (50 mm x 2.1 mm ID, 1.5 μm particle size, Thermo Scientific TM ) with a linear gradient from 5% to 90% in 0.1% formic acid in acetonitrile for 1.5 minutes. N-terminal peptide sequencing as described in Example 2 above confirmed that the FRAP amino acid sequence was present at the N-terminal end of the catalytically active, immature FRAP-Tgase variant (SEQ ID NO: 5).

[0178] C. SM-TAP activity assay

[0179] The activity of SM-TAP from Example 3A above was determined using the following assay. Soluble SM-TAP was added to the purified PRAP-Tgase variant (Example 2, 5 g / L, 500 μL) at 1% vol. The reaction was then incubated at 37°C, 300 rpm, with time points collected from 1-120 minutes. Supernatant samples were taken at each time point and quenched with phenylmethylsulfonyl fluoride (PMSF). The supernatant was analyzed by ultra-high performance liquid chromatography coupled to mass spectrometry (UPLC-MS, Thermo Scientific TM Vanquish TM UPLC and Thermo Scientific TM ISQ TMThe samples were analyzed by EM MS to determine the total area counts of the FRAP tetrapeptide generated by SM-TAP cleavage of the FRAP-Tgase variant (SEQ ID NO: 5). TM Vanquish TM C18 column (50 mm x 2.1 mm ID, 1.5 μm particle size, Thermo Scientific TM ) with a linear gradient from 5% to 90% in 0.1% formic acid in acetonitrile for 1.5 minutes. N-terminal peptide sequencing 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), thereby generating a mature, catalytically active Tgase variant (SEQ ID NO: 6).

[0180] Example 4. Immobilization of mature, catalytically active proteases on porous solid supports

[0181] A. Immobilization of Proteases on Covalent Porous Solid Supports

[0182] The following procedure describes the immobilization of TAMEP (Example 3A) and / or SM-TAP (Example 3A) on IB-COV-1, IB-COV-2, IB-COV-3, ANI-1 (modified for covalent binding), ECR8204F, ECR8209F, and ECR8215F.

[0183] 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 (twice the volume of the porous solid support, 4x).

[0184] The ANI-1 porous solid support was modified for covalent binding by washing with a freshly prepared solution of 1% glutaraldehyde in 50 mM phosphate buffer at pH 8 for 1 hour at 20°C (4 times the volume of the porous solid support, 1x). The glutaraldehyde solution was then removed and the porous solid support was washed with 50 mM phosphate buffer at pH 8 (twice the volume of the porous solid support, 4x).

[0185] Then for all covalent porous solid supports, the subtilis supernatant containing TAMEP and / or SM-TAP (0.2-0.5g / L) is on porous solid support, under 20 ℃, 700rpm, supernatant volume and porous solid support quality are 4:1 conditions, hatch 18-20 hours.Stop stirring, and protease is hatched 2 hours again on porous solid support.Then remove supernatant from porous solid support, then wash porous solid support (4 times of porous solid support volume, 1x) with the 50mM phosphate buffer adjusted to pH 8.Then wash porous solid support with the 50mM phosphate buffer containing 500mM sodium chloride (4 times of porous solid support volume, 2x) of pH 8.

[0186] B. Immobilization of TAMEP on Ionically Porous Solid Supports

[0187] The following procedure describes the immobilization of TAMEP (Example 3A) on IB-CAT-1, IB-ANI-1 (unmodified), IB-ANI-2, IB-ANI-3, IB-ANI-4.

[0188] Each porous solid support is weighed and put into a separate glass bottle, then washed with water (10mL, 4x).Then wash the porous solid support (twice the volume of the porous solid support, 4x) with the 50mM phosphate buffer adjusted to pH 7. Then the subtilis supernatant containing TAMEP (0.2-0.5g / L) is on the porous solid support, in 20 ℃, 700rpm, supernatant volume and porous solid support quality are 4:1 conditions, hatch 18-20 hours. Stop stirring, and the supernatant is hatched again on the porous solid support for 2 hours. Then remove the supernatant from the porous solid support, then wash the porous solid support (4 times the volume of the porous solid support, 4x) with the 50mM phosphate buffer adjusted to pH 7.

[0189] C. Immobilization of SM-TAP on Ionically Porous Solid Supports

[0190] The following procedure describes the immobilization of SM-TAP (Example 3A) on IB-CAT-1, IB-ANI-1 (unmodified), IB-ANI-2, IB-ANI-3, IB-ANI-4.

[0191] Each porous solid support is weighed and put into a separate glass bottle, then washed with water (10mL, 4x).Then the porous solid support is washed with the 50mM carbonate buffer adjusted to pH 10.5 (twice the volume of the porous solid support, 4x).Then the subtilis supernatant containing TAMEP and / or SM-TAP (0.2-0.5g / L) is on the porous solid support, at 20 ℃, 700rpm, supernatant volume and porous solid support quality are 4:1 conditions, hatched 18-20 hours. Stop stirring, and protease is hatched on the porous solid support for another 2 hours.Then remove supernatant from the porous solid support, then wash the porous solid support with the 50mM carbonate buffer adjusted to pH 10.5 (4 times the volume of the porous solid support, 4x).

[0192] D. Co-immobilization of TAMEP and SM-TAP on ionically porous solid supports

[0193] TAMEP and SM-TAP were co-immobilized on IB-CAT-1, IB-ANI-1 (unmodified), IB-ANI-2, IB-ANI-3, IB-ANI-4 using the same protocol described in Example 4C above.

[0194] E. Immobilization of proteases on adsorbent porous solid supports

[0195] The following procedures describe the immobilization of TAMEP (Example 3A) and / or SM-TAP (Example 3A) on IB-ADS-1, IB-ADS-2, IB-ADS-3, and IB-ADS-4.

[0196] Each porous solid support is weighed and put into a separate glass bottle, then washed with water (10mL, 4x).Then wash the porous solid support (twice the volume of the porous solid support, 4x) with the 50mM phosphate buffer adjusted to pH 8.Then the subtilis supernatant containing TAMEP and / or SM-TAP (0.2-0.5g / L) is on the porous solid support, in 20 ℃, 700rpm, supernatant volume and porous solid support quality are 4:1 conditions, hatch 18-20 hours.Stop stirring, and protease is hatched on the porous solid support for another 2 hours.Then remove supernatant from the porous solid support, then wash the porous solid support (4 times the volume of the porous solid support, 4x) with the 50mM phosphate buffer adjusted to pH 8.

[0197] Table 2: Porous solid supports tested for the individual immobilization of TAMEP and SM-TAP. TAMEP and SM-TAP retained activity when immobilized on all supports listed.

[0198]

[0199] Example 5. Detection of proteolytic digestion of immobilized TAMEP

[0200] This example shows the activation of purified Pro-Tgase variant (SEQ ID NO: 2), i.e., the zymogen form of the Tgase variant. Both the purified Pro-Tgase variant (SEQ ID NO: 2) and the clarified lysate containing Pro-Tgase (SEQ ID NO: 2) performed similarly in all studies. For simplicity, the protease activation of purified Pro-Tgase (SEQ ID NO: 2) is presented herein.

[0201] TAMEP (Example 3A) immobilized on a porous solid support (20 mg, as prepared in Example 4) was added to a 1.5 mL glass vial. Then, purified Pro-Tgase variant (200 μL, 5 g / L, SEQ ID NO: 2, as described in Example 2 above) was added to each vial. The vial was then incubated at 37 ° C, 700 rpm, and the sampling time point was between 1-120 minutes. At each time point, the supernatant sample was taken out and quenched with EDTA. The sample was then analyzed as described in Example 3B. Tgase activity was evaluated using a colorimetric activity assay as described in Example 1C. N-terminal peptide sequencing as performed in Example 2 confirmed that the FRAP tetrapeptide was present in catalytically active, immature FRAP-Tgase variants (SEQ ID NO: 5). The activity assay results showed that TAMEP retained protease activity when immobilized on all porous solid supports shown in Table 2.

[0202] Example 6. Detection of proteolytic digestion of immobilized SM-TAP

[0203] This example shows the activation of purified FRAP-Tgase variant (SEQ ID NO: 5). Both purified FRAP-Tgase variant (SEQ ID NO: 5) and clarified lysate containing FRAP-Tgase (SEQ ID NO: 5) performed similarly in all studies. For simplicity, the protease activation of purified FRAP-Tgase (SEQ ID NO: 5) is presented herein.

[0204] SM-TAP (Example 3A) immobilized on a porous solid support (20 mg porous solid support, as prepared in Example 4) was added to a 1.5 mL glass vial. Then, purified FRAP-Tgase variants (200 μL, 5 g / L, SEQ ID NO: 5, as described in Example 2 above) were added to each vial. The vials were then incubated at 20 ° C, 700 rpm, and time points of 1-120 minutes were collected. Supernatant samples were taken out at each time point and quenched with PMSF. Samples were analyzed as described in Example 3C. Tgase activity was evaluated using the colorimetric activity assay described in Example 1C above. N-terminal peptide sequencing performed as described in Example 2 above showed that the FRAP tetrapeptide had been removed from the N-terminus of the FRAP-Tgase variant (SEQ ID NO: 5), thereby producing a thermostable, mature, catalytically active Tgase variant (SEQ ID NO: 6). Activity assay results showed that SM-TAP retained protease activity when immobilized on all porous solid supports shown in Table 2.

[0205] Example 7. Detection of proteolytic digestion by co-immobilized TAMEP and SM-TAP

[0206] This example shows the activation of purified Pro-Tgase variant (SEQ ID NO: 2), i.e., the zymogen form of the Tgase variant. Purified Pro-Tgase variant (SEQ ID NO: 2) and clarified lysate containing Pro-Tgase (SEQ ID NO: 2) performed similarly in all studies. For simplicity, the protease activation of purified Pro-Tgase (SEQ ID NO: 2) is presented herein.

[0207] The porous solid support that performed best under the conditions described in Examples 5 and 6 above was selected for co-immobilization.

[0208] The porous solid support (20 mg) co-immobilized with TAMEP and SM-TAP prepared in Example 4 above is added to a 1.5 mL glass vial. Then, purified Pro-Tgase variant (200 μL, 5 g / L, SEQ ID NO: 2, as described in Example 2 above) is added to the vial. The vial is then incubated at 20 ° C, 700 rpm, and the time point of 1-120 minutes is collected. Supernatant samples are taken out at each time point and quenched with PMSF and EDTA. Samples are then analyzed as described in Example 3C. Only when TAMEP first cuts the propeptide from Pro-Tgase variant (SEQ ID NO: 2), and then SM-TAP cuts the FRAP tetrapeptide from the resulting FRAP-Tgase variant (SEQ ID NO: 5), FRAP tetrapeptide will be produced. Tgase activity is evaluated using the colorimetric activity assay as 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) to generate 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 listed in Table 3 below.

[0209] Table 3: Porous solid supports selected from Table 2 were tested for co-immobilization of TAMEP and SM-TAP. TAMEP and SM-TAP retained activity on all porous solid supports shown below.

[0210]

[0211]

[0212] Example 8. Determination of the activity of TAMEP and TAP

[0213] The same concentrations of TAMEP and SM-TAP were used in soluble and immobilized forms.

[0214] Unless otherwise stated, all TAMEP and SM-TAP concentrations are based on total protein.

[0215] A. Determination of the combined activity of soluble TAMEP (not immobilized) and soluble SM-TAP (not immobilized)

[0216] Purified Pro-Tgase variant (SEQ ID NO: 2, 250 μL, 5 g / L) was added to a 1.5 mL glass vial containing soluble TAMEP (0.1 mg, not immobilized) and soluble SM-TAP (0.1 mg, not immobilized). The samples were analyzed in the same manner as in Example 3C.

[0217] B. Determination of the combined activity of soluble TAMEP (unimmobilized) and SM-TAP immobilized on IB-COV-3

[0218] Purified Pro-Tgase variant (SEQ ID NO: 2, 250 μL, 5 g / L) was added to a 1.5 mL glass vial containing soluble TAMEP (0.1 mg, unimmobilized) and immobilized SM-TAP (10 mg porous solid support, 0.1 mg SM-TAP). The samples were analyzed in the same manner as described in Example 3C above.

[0219] C. Determination of the combined activity of TAMEP immobilized on IB-COV-1 and soluble SM-TAP (not immobilized)

[0220] Purified Pro-Tgase variant (SEQ ID NO: 2, 250 μL, 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, not immobilized). The samples were analyzed in the same manner as described in Example 3C above.

[0221] D. Determination of the combined activity of TAMEP immobilized on IB-COV-1 and SM-TAP immobilized on IB-COV-3

[0222] Purified Pro-Tgase variant (SEQ ID NO: 2, 250 μL, 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 SM-TAP (10 mg porous solid support, 0.1 mg SM-TAP), respectively. The samples were analyzed in the same manner as described in Example 3C above.

[0223] E. Determination of the combined activity of TAMEP and SM-TAP co-immobilized on ECR8215F or IB-COV-3

[0224] Purified Pro-Tgase variant (SEQ ID NO: 2, 250 μL, 5 g / L) was added to a 1.5 mL glass vial containing co-immobilized TAMEP and SM-TAP (20 mg porous solid support, 0.2 mg TAMEP and SM-TAP, combined). The samples were analyzed in the same manner as described in Example 3C above.

[0225] It was found that co-immobilized TAMEP and SM-TAP performed better than soluble and individually immobilized TAMEP and SM-TAP. Specifically, the results showed that co-immobilized TAMEP and SM-TAP increased the total area counts of FRAP measured by UPLC-MS (as described in Example 3C) compared to the protease in soluble form (unimmobilized), or only immobilizing one of TAMEP or TAP while the other was in solution, or TAMEP and TAP were immobilized on separate porous solid supports. The results are shown in Tables 4 and Figure 2 middle.

[0226] also, Figure 2 It was shown that most FRAP was produced by co-immobilized TAMEP and SM-TAP compared to the amount of FRAP produced by immobilized TAMEP and SM-TAP alone or both TAMEP and SM-TAP in soluble form (ie, not immobilized). Figure 2 It was also shown that TAMEP and SM-TAP immobilized alone produced more FRAPs than soluble TAMEP and SM-TAP within 120 minutes. In other words, co-immobilized TAMEP and SM-TAP and individually immobilized TAMEP and SM-TAP outperformed soluble TAMEP and SM-TAP.

[0227] Table 4. Determination of the combined activity of TAMEP and SM-TAP as described in Examples 8A-8E.

[0228]

[0229]

[0230] ++ indicates >2-fold increase in FRAP production compared to soluble (non-immobilized) TAMEP and SM-TAP,

[0231] + indicates a 1.5- to 2-fold increase in FRAP production, and

[0232] - indicates <1.5-fold increase in FRAP yield as determined by UPLC-MS total area counting (as performed in Example 3C).

[0233] Amino acid sequence

[0234] SEQ ID NO: 1 Wild-type Streptomyces mobara Tgase proenzyme (Pro-Tgase). The leader sequence (pro-) is indicated in bold, underlined text.

[0235]

[0236] SEQ ID NO:2 A thermostable variant of the proenzyme form of Streptomyces mobara Tgase having two methionine amino acid residues - one methionine at the N-terminus of the Pro sequence and the second methionine between the Pro sequence and the N-terminus of the mature domain (Pro-Tgase variant). The leader sequence (pro-) is indicated in bold, underlined text.

[0237]

[0238] SEQ ID NO:3 The zymogen form of wild-type Streptomyces mobara transglutaminase-activating M4 metalloprotease (TAMEP) with the native signal peptide deleted and including a leader methionine to facilitate recombinant expression. The putative leader sequence (pro-) is indicated in bold, underlined text.

[0239]

[0240]

[0241] SEQ ID NO: 4 The zymogen form of wild-type Streptomyces mobaraensis transglutaminase-activating tripeptidyl aminopeptidase (SM-TAP) with the native signal peptide deleted and containing a leading methionine to facilitate recombinant expression. The putative leader sequence (pro-) is indicated in bold, underlined text.

[0242]

[0243] SEQ ID NO: 5 A thermostable variant of Streptomyces mobaraensis Tgase having a FRAP tetrapeptide at the N-terminus of the mature domain and a methionine amino acid residue located between the FRAP tetrapeptide and the N-terminus of the mature domain of the thermostable Tgase variant (FRAP-Tgase variant). The leader sequence (pro-) is indicated in bold, underlined text.

[0244]

[0245] SEQ ID NO: 6 A thermostable variant of Streptomyces mobaraensis Tgase, wherein the FRAP tetrapeptide is removed from the N-terminus of the mature domain of the thermostable Tase variant and a leading methionine amino acid residue is located at the N-terminus of the mature domain of the thermostable Tgase variant.

[0246]

[0247]

Claims

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

2. The immobilized TAMEP according to claim 1, wherein the TAMEP is from Streptomyces sp.

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

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

5. The immobilized TAP according to claim 4, wherein the TAP is from a Streptomyces species. The immobilized TAP according to claim 5 , wherein the TAP is from Streptomyces mobaraensis.

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

8. The co-immobilized TAMEP and TAP according to claim 7, wherein the TAMEP is from a Streptomyces species and the TAP is from a Streptomyces species.

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

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

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

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

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

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

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

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

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

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

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. The method of claim 18 or 19, wherein the TAMEP is from a Streptomyces species.

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

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