WO2024054236A1 - Protéases immobilisées pour l'activation de la forme zymogène de la transglutaminase - Google Patents

Protéases immobilisées pour l'activation de la forme zymogène de la transglutaminase Download PDF

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WO2024054236A1
WO2024054236A1 PCT/US2022/076202 US2022076202W WO2024054236A1 WO 2024054236 A1 WO2024054236 A1 WO 2024054236A1 US 2022076202 W US2022076202 W US 2022076202W WO 2024054236 A1 WO2024054236 A1 WO 2024054236A1
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tamep
tap
immobilized
transglutaminase
tgase
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PCT/US2022/076202
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English (en)
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Erika M. MILCZEK
Robert Dicosimo
Steven Walsh
Malcolm MCCRAY
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Curie Co. Inc.
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Priority to PCT/US2022/076202 priority Critical patent/WO2024054236A1/fr
Publication of WO2024054236A1 publication Critical patent/WO2024054236A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y203/00—Acyltransferases (2.3)
    • C12Y203/02—Aminoacyltransferases (2.3.2)
    • C12Y203/02013—Protein-glutamine gamma-glutamyltransferase (2.3.2.13), i.e. transglutaminase or factor XIII
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1025—Acyltransferases (2.3)
    • C12N9/104—Aminoacyltransferases (2.3.2)
    • C12N9/1044—Protein-glutamine gamma-glutamyltransferase (2.3.2.13), i.e. transglutaminase or factor XIII
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/465—Streptomyces

Definitions

  • the field pertains to immobilized proteases and, in particular, to immobilized proteases for activation of the zymogen form of transglutaminase.
  • Transglutaminases are a family of enzymes that catalyze cross-linking between the gamma-carboxamide group in glutamine residues (acyl donors) and a variety of primary amines (acyl acceptors), including the amino group of lysine.
  • Tgases can be found throughout all groups of organisms including prokaryotes, eukaryotes, and plants. Tgases in animals, for example, include blood coagulation factor XIII, which is a multi-domain protein and depends on calcium for regulation of enzyme function. Microbial transglutaminases, on the other hand, have only a single domain and do not depend on calcium for activity, i.e., Tgases of microbial origin are calcium-independent. Thus, microbial Tgases represent a major advantage for their practical use.
  • Tgase is produced by fermentation of Streptomyces mobaraensis. Tgase is expressed as an inactive zymogen having a pro-peptide sequence at the N-terminus of the mature domain. The active enzyme is produced by removing the pro-peptide by proteolytic processing in solution to afford the mature domain.
  • TAMEP- activated Tgase has a N-terminal tetrapeptide (Phe-Arg-Ala-Pro), i.e., FRAP, which can be removed by a transglutaminase activating tripeptidyl aminopeptidase (TAP) from Streptomyces mobaraensis (SM-TAP) also in soluble form.
  • TAP transglutaminase activating tripeptidyl aminopeptidase
  • SM-TAP Streptomyces mobaraensis
  • SM-TAP cleaves between the Pro45 and Asp46 (FRAP-DSDD cleavage site, see SEQ ID NO:1) to afford mature, catalytically active Tgase, with FRAP removed, i.e., in a mature, catalytically active form.
  • FRAP-DSDD cleavage site see SEQ ID NO:1
  • Use of soluble enzymes during post-translational processing of the inactive zymogen of Tgase leads to complications in purification as this necessitates separation of multiple enzymes from a fermentation broth. Often this requires use of chromatographic purification resulting in increased manufacturing cost and complexity.
  • immobilization of enzymes can be useful in improving the catalytic performance of enzymes and can aid in simplifying downstream processing. Immobilization facilitates re-use of enzymes, allows for an easy and more simplified recovery of both enzymes and products, allows continuous operations of enzymatic processes, rapid termination of reactions, and greater variety of bioreactor designs.
  • TAMEP and a TAP such as SM-TAP used to activate Tgase in the zymogen form are deactivated by the catalytically active, mature Tgase and/or a catalytically active, not mature Tgase (i.e., FRAP-Tgase or Tgase having FRAP at its N-terminus).
  • Both TAMEP and SM-TAP are potentially substrates for mature, catalytically active Tgase or catalytically active, not mature Tgase, as they contain multiple glutamine and lysine residues, which may react with mature Tgase to form crosslinked products (Gln-Lys) or result in deamination of glutamine to glutamic acid (hydrolysis of Gin to Glu), thereby inactivating TAMEP and SM-TAP.
  • TAMEP and SM-TAP This deactivation of TAMEP and SM-TAP leads to the incomplete conversion of the zymogen form of Tgase to mature, catalytically active Tgase by TAMEP and SM-TAP and further increases the cost and complexity in the purification and isolation of recombinantly expressed, mature, catalytically active Tgase or catalytically active, not mature Tgase
  • TAMEP transglutaminase-activating M4 metalloprotease
  • TEP transglutaminase-activating tripeptidyl aminopeptidase
  • TAMEP transglutaminase-activating M4 metalloprotease
  • TAP tripeptidyl aminopeptidase
  • a method for activating a zymogen form of a transglutaminase or a variant thereof to produce a mature, catalytically active form of the transglutaminase comprising a) immobilizing at least one transglutaminase-activating M4 metalloprotease (TAMEP) and/or at least one tripeptidyl aminopeptidase (TAP), provided that if both TAMEP and TAP are immobilized, the TAMEP and TAP are separately immobilized on the same or different porous solid support; and b) contacting the zymogen form of 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 then TAMEP is contacted first.
  • TAMEP transglutaminase-activating M4 metalloprotease
  • TAP tripeptidyl aminopeptida
  • step a) may comprise providing at least one TAMEP and/or at least one TAP immobilized on a porous solid support.
  • a method for activating a zymogen form of a transglutaminase or a variant thereof to produce a mature, catalytically active form of the transglutaminase 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 transglutaminase with co-immobilized TAMEP and TAP to produce a mature, catalytically active form of the transglutaminase.
  • TAMEP transglutaminase-activating M4 metalloprotease
  • TAP tripeptidyl aminopeptidase
  • a method for activating a zymogen form of a transglutaminase or a variant thereof to produce a catalytically active, not mature form of the transglutaminase comprising a) immobilizing at least one transglutaminase-activating M4 metalloprotease s(TAMEP) on a porous solid support, and b) contacting the zymogen form of transglutaminase with the immobilized at least one TAMEP to produce a catalytically active, not mature form of the transglutaminase.
  • the catalytically active, not mature transglutaminase can be separated from at least one of the immobilized or co-immobilized proteases.
  • the TAMEP is from Streptomyces sp. and, most preferably, the TAMEP is from Streptomyces mobaraensis and, preferably, the TAP is from Streptomyces sp. and, most preferably, the TAP is from Streptomyces mobaraensis (SM-TAP).
  • SM-TAP Streptomyces mobaraensis
  • Fig. 1 shows SDS-PAGE analysis of S. mobaraensis Tgase variant as described in Example 1 .
  • Lane 1 - mature Tgase secured from commercial sources;
  • 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.
  • 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.
  • Fig. 2 shows the activity of 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 TAMEP (soluble) with SM-TAP (soluble).
  • nucleotide and amino acid sequence data comply with the rules set forth in 37 C.F.R. ⁇ 1.822.
  • SEQ ID NO:1 corresponds to the wild-type zymogen form of Tgase from Streptomyces mobaraensis (Pro-Tgase). Leader sequence (pro-) is denoted in bold, underlined text.
  • SEQ ID NO:2 corresponds to a thermostable variant of Streptomyces mobaraensis Tgase in zymogen form with two additional methionine residues - one methionine is located at the N-terminus of the Pro-sequence and the second methionine is located between the Pro-sequence and the N-terminus of the mature domain (Pro-Tgase variant). Leader sequence (pro-) is denoted in bold, underlined text.
  • SEQ ID NO:3 corresponds to wild-type Streptomyces mobaraensis transglutaminase-activating M4 metalloprotease (TAMEP) in zymogen form with the native signal peptide deleted and a leading methionine included to facilitate recombinant expression.
  • Putative leader sequence (pro-) is denoted in bold, underlined text.
  • SEQ ID NO: 4 corresponds to the wild-type Streptomyces mobaraensis transglutaminase-activating tripeptidyl aminopeptidase (SM-TAP) in zymogen form with the native signal peptide deleted and a leading methionine included to facilitate recombinant expression.
  • Putative leader sequence (pro-) is denoted in bold, underlined text.
  • SEQ ID NO: 5 corresponds to a thermostable variant of Streptomyces mobaraensis Tgase having a FRAP tetrapeptide at the N-terminal end with 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).
  • Leader sequence (pro-) is denoted in bold, underlined text.
  • SEQ ID NO: 6 corresponds to a thermostable variant of Streptomyces mobaraensis Tgase with the FRAP tetrapeptide removed from the N-terminus of the mature domain of the thermostable Tgase variant and a leading methionine amino acid residue is located at the N- terminus of the mature domain of the thermostable Tgase variant (thermostable Tgase variant).
  • a compound or “at least one compound” may include a plurality of compounds, including mixtures thereof.
  • the terms “a,” “an,” “the,” “one or more,” and “at least one,” for example, can be used interchangeably herein.
  • the term “about” as used herein can allow for a degree of variability in a value or range, for example, within 10%, within 5%, or within 1% of a stated value or of a stated limit of a range.
  • the term “and/or” as used in a phrase 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).
  • compositions, formulation, or method includes any listed component, ingredient 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.
  • immobilized means a technical process in which 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 binding/attachment, ionic bonding, entrapment, or adsorption.
  • co-immobilized means that at least two distinct molecules, such as at least two different enzymes, are fixed to or within the same solid support.
  • solid support refers to a range of materials, either biological, non- biological, organic, inorganic, or a combination of any of these.
  • a solid support may be of any suitable composition to which the molecule to be attached may be applied.
  • porous solid support means simply any solid containing void space(s), i.e., space not occupied by the main framework of atoms that make up the structure of the solid.
  • supports include, but are not limited to, polyacrylate, polymethacrylate, polystyrene, etc. that can be modified with a functional group.
  • polymethacrylate can be modified with epoxide functional groups or primary amine groups capable of being activated by a crosslinking agent.
  • functional groups include but are not limited to, epoxide, alkyl, phenyl, sulphonic, amines (primary, secondary, tertiary or quaternary), etc.
  • porous solid supports include, but are not limited to, aminopropylsilated controlled pore glass (“CPG”), diatomaceous earth or metal-organic frameworks (“MOFS”).
  • CPG aminopropylsilated controlled pore glass
  • MOFS metal-organic frameworks
  • the porous solid support is selected from any of the supports set forth in Table 2 in the Examples below.
  • 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.
  • covalently bound refers to a chemical bond involving the sharing of electron pairs between atoms. These electron pairs are known as shared pairs or bonding pairs, and the stable balance of attractive and repulsive forces between atoms, when they share electrons is known as covalent bonding.
  • the pro and mature domains may be covalently bound via a peptide bond.
  • the term “ionic bond” may also be referred to as an electrovalent bond. It is a type of linkage formed from the electrostatic attraction between oppositely charged ions in a chemical compound. Such a bond forms when the valence (outermost) electrons of one atom are transferred permanently to another atom. The atom that loses the electrons becomes a positively charged ion, i.e., a cation, while the one that gains the electrons becomes a negatively charged ion, i.e., an anion.
  • An ionic bond is one example of a non- covalent bond.
  • non-covalently bound differs from covalently bound in that non-covalent binding does not involve the sharing of electrons between atoms. Thus, non-covalent binding can occur by completely exchanging electrons between atoms or by not exchanging 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.
  • Adsorption refers to a process that involves the accumulation of a substance or molecular species in higher concentrations on a surface., i.e., the adhesion of atoms, ions, or molecules from a gas, liquid, or dissolved solid to a surface. Adsorption can include, but is not limited to interactions such a hydrophobic or hydrophilic interactions.
  • zymogen and “proenzyme” are used interchangeably herein and refer to an inactive precursor of an enzyme, which may be converted into an active or mature enzyme by post-translational modification, for example, by catalytic action, such as via proteolytic cleavage of a pro-peptide sequence.
  • pro-peptide “pro-domain,” “pro-sequence,” and “pro-region” are used interchangeably herein and refer to a N-terminal peptide leader sequence (including the FRAP tetrapeptide) that, when fully cleaved, produces a mature, catalytically active Tgase. However, if the full pro-peptide is not cleaved such that it does not include FRAP (i.e., FRAPIess pro-peptide) then a catalytically active, not mature Tgase is produced.
  • FRAP i.e., FRAPIess pro-peptide
  • pro-peptide can be regarded as serving a regulatory function while the mature domain serves a catalytic function.
  • Pro-peptides generally are recognized to have four major functions: 1) pro-peptides can function as intramolecular chaperones or folding assistants by determining the three-dimensional structure of a protein; 2) pro-peptides can function as inhibitors or activation peptides; 3) pro-peptides can direct protein sorting into specific cellular compartments or extra-cellular space and 4) pro-peptides can mediate the precursor interaction with other molecules (such as peptides, proteins, and polysaccharides) or supramolecular structures (e.g., cell walls). A single pro-peptide can perform several or even all these functions.
  • transglutaminase (T gase, EC2.3.2.13) refers to a family of enzymes that catalyze the formation of an isopeptide bond between a primary amine, for example, the E- amine of a lysine molecule, and the acyl group of a protein- or peptide-bound glutamine. Transglutaminases may catalyze a transamidation reaction between glutamyl and lysyl side chains of target proteins. Proteins possessing Tgase activity have been found in microorganisms, plants, and animals. Tgases are widely distributed in various organs, tissues, and bodily fluids.
  • Tgases also form extensively cross-linked, generally insoluble, protein biopolymers that are needed for an organism to create barriers and stable structures.
  • Tgases of microbial origin unlike eukaryotic Tgases, are calcium-independent, which represents a major advantage for their practical use.
  • Microbial transglutaminase is one of the most extensively studied industrial enzymes for protein functionalization and protein cross-linking because of its ability to polymerize or functionalize proteins through the formation of a stable E-(y-glutamyl)lysine isopeptide bond without the constraint of a consensus sequence or additional cofactors.
  • Microbial Tgase is a subset of Tgases.
  • Tgase The most commonly used Tgase is microbial transglutaminase from Streptomyces mobaraensis, the wild-type zymogen form (Pro-Tgase) having the amino acid sequence corresponding to SEQ ID NO:1.
  • Pro-Tgase the wild-type zymogen form
  • zymogen form of Tgase It has been known that Tgase from Streptomyces mobaraensis is secreted in a zymogen form, i.e., Pro-Tgase. Activation of the zymogen form of Tgase (Pro-Tgase) occurs in two steps.
  • the pro-peptide is cleaved from the N-terminal end of the Pro-Tgase by a transglutaminase-activating M4 metalloprotease (“TAMEP”).
  • TAMEPs produce a catalytically active, not mature Tgase leaving a FRAP tetrapeptide at the N-terminal end of Tgase (i.e., FRAP-Tgase).
  • Some TAMEPs produce catalytically active, not mature Tgase having a partial FRAP peptide such as RAP, AP, or P attached to the N- terminal end of the Tgase.
  • the resulting pro-peptide does not contain the FRAP-tetrapeptide, i.e., it is a FRAPIess pro-peptide.
  • the N-terminal FRAP tetrapeptide on the T gase, or what remains of it, can then be subsequently cleaved by using a transglutaminase tripeptidyl aminopeptidase (“TAP”) to produce a mature, catalytically active, Tgase having no FRAP.
  • TAP transglutaminase tripeptidyl aminopeptidase
  • Transglutaminase variants and methods of producing such variants are disclosed, for example, in PCT Publication Numbers WO 2016/170447, published on October 27, 2016, and WO 2019/094301 , published on May 16, 2019.
  • a “protease” (also called a peptidase or proteinase) refers to enzymes capable of cleaving peptide bonds.
  • Proteases are any of various enzymes, such as endopeptidases and exopeptidases, that catalyze the hydrolytic breakdown of proteins into peptides and amino acids.
  • Proteases can be classified into seven broad groups: serine proteases, cysteine proteases, threonine proteases, aspartic proteases, glutamic proteases, metalloproteases, and asparagine peptide lyases.
  • Proteases can be found in animals, plants, bacteria, fungi, archaea, and viruses.
  • 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 .
  • peptides are used interchangeably herein and refer to a polymer of amino acids joined together by peptide bonds.
  • a “protein” or “polypeptide” comprises a polymeric sequence of amino acid residues.
  • the single and 3- letter codes for amino acids as defined in conformity with the IUPAC-IUB Joint Commission on Biochemical Nomenclature (JCBN) are used throughout this disclosure.
  • the single letter X refers to any of the twenty amino acids. It is also understood that a polypeptide may be coded for by more than one nucleotide sequence due to the degeneracy of the genetic code.
  • Mutations can be named by the one letter code for the parent amino acid, followed by a position number and then the one letter code for the variant amino acid.
  • mutating glycine (G) at position 87 to serine (S) is represented as “G087S” or “G87S.”
  • G mutating glycine
  • S serine
  • G mutating glycine
  • a position followed by amino acids listed in parentheses indicates a list of modifications at that position by any of the listed amino acids.
  • 6 (L, I) means position 6 can be substituted with a leucine or isoleucine.
  • a slash (/) is used to define modifications, e.g., FA/, indicates that the position may have a phenylalanine or valine at that position.
  • mutant refers to a change introduced into a parental sequence, including, but not limited to, modifications such as insertions or deletions (including truncations), thereby producing a “variant.”
  • modifications such as insertions or deletions (including truncations)
  • the consequences of a mutation include, but are not limited to, the creation of a new character, property, function, phenotype, or trait not found in the protein encoded by the parental sequence.
  • variant proteins encompass “variant,” “mutant,” or “modified” proteins, which terms are used interchangeably herein.
  • Variant (i.e., mutant or modified) proteins differ from another (i.e., parental) protein or from one another due to modifications in one or more amino acid residues.
  • a variant may include one or more amino acid modifications such as one or more amino acid deletions/truncations, insertions, or substitutions as compared to the parental protein from which it is derived.
  • variants may have a specified degree of sequence identity with a reference protein or nucleic acid, e.g., as determined using a sequence alignment tool, such as BLAST, ALIGN, and CLUSTAL.
  • variant proteins or nucleic acids 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 percentage therebetween.
  • amino acid residue positions “corresponding to,” “corresponds substantially to,” “corresponding substantially to,” “correspond to,” or “corresponds” refers to an amino acid residue at the enumerated position in a protein or peptide, or an amino acid residue that is analogous, homologous, or equivalent to an enumerated residue in a protein or peptide.
  • corresponding region generally refers to an analogous position in a related protein or a reference protein.
  • variant proteins encompass “variant” or “mutant” proteins, which terms are used interchangeably herein.
  • Variant proteins differ from another (i.e., parental) protein and/or from one another by a small number of amino acid residues.
  • a variant may include one or more amino acid mutations (e.g., amino acid deletion, insertion or substitution) as compared to the parental protein from which it is derived.
  • variants may have a specified degree of sequence identity with a reference protein or nucleic acid, e.g., as determined using a sequence alignment tool, such as BLAST, ALIGN, and CLUSTAL.
  • variant proteins or nucleic acid may 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 with a reference sequence and integer percentage therebetween
  • wild-type in reference to an amino acid sequence or nucleic acid sequence indicates that the amino acid sequence or nucleic acid sequence is a native or naturally-occurring sequence.
  • naturally-occurring refers to anything (e.g., proteins, amino acids, or nucleic acid sequences) that is found in nature.
  • non-naturally occurring refers to anything that is not found in nature (e.g., recombinant/engineered nucleic acids and protein sequences produced in the laboratory or modification of the wild-type sequence).
  • derived from encompasses the terms “originated from,” “obtained from,” “obtainable from,” “isolated from,” “purified from,” and “created from,” and generally indicates that one specified material finds its origin in another specified material or has features that can be described with reference to another specified material.
  • isolated refers 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.
  • these terms may refer to a material which is substantially or essentially free from components which normally accompany it as found in its native state, such as, for example, an intact biological system.
  • An isolated nucleic acid molecule includes a nucleic acid molecule contained in cells that ordinarily express 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.
  • TAMEP transglutaminase-activating M4 metalloprotease
  • the preferred solid support is a porous solid support as described above. Any porous solid support to which TAMEP can be immobilized can be used. It may be pretreated or functionalized prior to application of the TAMEP to facilitate binding, or for any other desired purpose, such as fostering 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 selection of a suitable treatment and/or functionalization will depend upon the TAMEP and upon the attendant conditions and desired activity. Examples of such supports include, but are not limited to, polyacrylate, polymethacrylate, polystyrene, etc. that can be modified with a functional group.
  • polymethacrylate can be modified with epoxide functional groups or primary amine groups capable of being activated by a crosslinking agent.
  • epoxide functional groups include but are not limited to, epoxide, alkyl, phenyl, sulphonic, amines (primary, secondary, tertiary or quaternary), etc.
  • porous solid supports include, but are not limited to, aminopropylsilated controlled pore glass (“CPG”), diatomaceous earth or metal-organic frameworks (“MOFS”).
  • CPG aminopropylsilated controlled pore glass
  • MOFS metal-organic frameworks
  • the porous solid support is selected from any of the supports set forth in Table 2 in the Examples.
  • 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.
  • active TAMEP and “TAMEP” are used interchangeably herein.
  • TAMEP is made in a zymogen form
  • active TAMEP is made by removal of that portion of the N-terminus from the zymogen form needed to produce active enzyme.
  • TAMEP can be immobilized using any of methods of attachment discussed above and, in the Examples below, namely, by means of covalent bonds, ionic bonds, or adsorption.
  • TAMEP is immobilized using covalent bonds.
  • it is believed that TAMEP in a zymogen form may also be immobilized and subsequently activated.
  • 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.
  • TAMEP is obtained from Streptomyces mobaraensis.
  • SEQ ID NO:3 corresponds to the wild-type Streptomyces mobaraensis transglutaminase-activating M4 metalloprotease (TAMEP) in zymogen form with the native signal peptide deleted and a leading methionine included to facilitate recombinant expression.
  • the putative leader sequence (pro-) is denoted in bold, underlined text and provided as amino acids 2 to 197 of SEQ ID NO: 3.
  • Active TAMEP is produced by removal (such as by endogenous protease activity) of that portion of the N-terminus of the zymogen form needed to produce active enzyme. In the case of TAMEP, it is believed that the portion of the N-terminus of the zymogen form needed to produce active enzyme might involve removal of the identified putative pro region or a variant thereof from SEQ ID NO:3 to produce active TAMEP.
  • TAMEP from Streptomyces mobaraensis 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 (without the putative leader sequence or a variant thereof) of the amino acid sequence set forth in SEQ ID NO:3.
  • TAMEP from Streptomyces mobaraensis comprises a sequence consisting essentially of the active form of the amino acid sequence set forth in SEQ ID NO:3.
  • the sequence of TAMEP after the putative leader sequence is 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 is removed from the zymogen form 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 a variation thereof.
  • sequence of TAMEP after the leader sequence is removed from the zymogen 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 a variation thereof.
  • 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 a variation thereof.
  • 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 a variation thereof.
  • TEP transglutaminase-activating tripeptidyl aminopeptidase
  • the preferred solid support is a porous solid support as described above.
  • Any porous solid support to which TAP can be immobilized can be used. It may be pretreated or functionalized prior to application of the TAP to facilitate binding, or for any other desired purpose, such as fostering 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 selection of a suitable treatment and/or functionalization will depend upon the TAP and upon the attendant conditions and desired activity. Examples of such supports include, but are not limited to, polyacrylate, polymethacrylate, polystyrene, etc. that can be modified with a functional group.
  • polymethacrylate can be modified with epoxide functional groups or primary amine groups capable of being activated by a crosslinking agent.
  • epoxide functional groups include but are not limited to, epoxide, alkyl, phenyl, sulphonic, amines (primary, secondary, tertiary or quaternary), etc.
  • porous solid supports include, but are not limited to, aminopropylsilated controlled pore glass (“CPG”), diatomaceous earth or metal-organic frameworks (“MOFS”).
  • CPG aminopropylsilated controlled pore glass
  • MOFS metal-organic frameworks
  • the porous solid support is selected from any of the supports set forth in Table 2 in the Examples.
  • 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.
  • Active TAP i.e., not in a zymogen form
  • TAP can be immobilized using any of methods of attachment discussed above and, in the Examples below, namely, by means of covalent bonds, ionic bonds, or adsorption.
  • TAP is immobilized using covalent bonds.
  • it is believed that TAP in a zymogen form may also be immobilized and subsequently activated.
  • active TAP and “TAP” are used interchangeably herein. If TAP is made in a zymogen form, then active TAP, is made by removal of that portion of the N- terminus from the zymogen form needed to produce active enzyme, TAP can be obtained from a variety of microbial sources, including, but not limited to, Streptomyces sp.
  • Nonlimiting 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.
  • TAP is obtained from Streptomyces mobaraensis.
  • TAP is obtained from Streptomyces mobaraensis.
  • SEQ ID NO: 4 corresponds to the wild-type Streptomyces mobaraensis transglutaminase-activating tripeptidyl aminopeptidase (SM-TAP) in zymogen form with the native signal peptide deleted and a leading methionine included to facilitate recombinant expression.
  • Putative leader sequence (pro-) is denoted in bold, underlined text and provided as amino acids 2 to 7 of SEQ ID NO: 4.
  • Active TAP is produced by removal of that portion of the N-terminus of the zymogen form needed to produce active enzyme. In the case of TAP, it is believed that the portion of the N-terminus of the zymogen form needed to produce active enzyme might involve removal of the identified putative pro region or a variant thereof from SEQ ID NO:4 to produce active TAP.
  • TAP that might share at least 70% sequence identity with an active form of the amino acid sequence set forth in SEQ ID NO:4.
  • TAP is obtained from Streptomyces mobaraensis 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 an active form (without the putative leader sequence or a variant thereof) of the amino acid sequence set forth in SEQ ID NO:4.
  • TAP from Streptomyces mobaraensis comprises a sequence consisting essentially of an active form of the amino acid sequence set forth in SEQ ID NO:4.
  • the sequence of TAP after the putative leader sequence is removed from the zymogen form corresponds to amino acids 8 to 451 of SEQ ID NO: 4.
  • the sequence of TAP after the leader sequence is removed from the zymogen form 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 a variation thereof.
  • sequence of TAP after the leader sequence is removed from the zymogen form 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 a variant thereof.
  • the sequence of 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 a variation thereof.
  • the sequence of 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 a variant thereof.
  • both TAMEP and TAP can be co-immobilized on any of the porous solid supports disclosed herein.
  • TAMEP and TAP are discussed in greater detail hereinabove.
  • TAMEP and TAP can be co-immobilized using any of methods of attachment discussed above and in the Examples below, namely, by means of covalent bonds, ionic bonds, or adsorption. The preferred means of co-immobilization is by covalent bonds.
  • a method for activating a zymogen form of a transglutaminase or a variant thereof to produce a mature, catalytically active form of the transglutaminase comprising a) immobilizing at least one transglutaminase-activating M4 metalloprotease (TAMEP) and/or at least one tripeptidyl aminopeptidase (TAP), provided that if both TAMEP and TAP are immobilized, the TAMEP and TAP are separately immobilized on the same or different porous solid support; and b) contacting the zymogen form of 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.
  • TAMEP transglutaminase-activating M4 metalloprotease
  • TAP tripeptidyl aminopeptidase
  • step a) may comprise providing at least one TAMEP and/or at least one TAP immobilized on a porous solid support.
  • the mature, catalytically active form of the transglutaminase is separated from the at least one of the immobilized proteases.
  • the at least one TAMEP is from Streptomyces sp. and the at least one TAP is from Streptomyces sp.
  • the at least one TAM EP is from Streptomyces mobaraensis and the at least one TAP is from Streptomyces mobaraensis.
  • a method for activating a zymogen form of a transglutaminase or a variant thereof to produce a mature, catalytically active form of the transglutaminase comprising a) co-immobilizing at least one transglutaminase-activating M4 metalloprotease (TAMEP) and at least one tripeptidyl aminopeptidase from (TAP) on the same porous solid support; and b) contacting the zymogen form of transglutaminase with co-immobilized TAMEP and TAP to produce a mature, catalytically active form of the transglutaminase.
  • TAMEP transglutaminase-activating M4 metalloprotease
  • TAP tripeptidyl aminopeptidase from
  • the mature, catalytically active transglutaminase is separated from the at least one of the immobilized proteases.
  • the at least one TAMEP is from Streptomyces sp. and the at least one TAP is from Streptomyces sp.
  • the at least one TAMEP is from Streptomyces mobaraensis and the at least one TAP is from Streptomyces mobaraensis.
  • a method for activating a zymogen form of a transglutaminase or a variant thereof to produce a catalytically active, not mature form of the transglutaminase comprising a) immobilizing at least one transglutaminase-activating M4 metalloprotease (TAMEP) on a porous solid support; and b) contacting the zymogen form of transglutaminase with immobilized at least one TAMEP to produce a catalytically active, not mature form of the transglutaminase.
  • TAMEP transglutaminase-activating M4 metalloprotease
  • the catalytically active, not mature transglutaminase is separated from the at least one of the immobilized TAMEP.
  • the TAMEP is from Streptomyces sp.
  • the TAMEP is from Streptomyces mobaraensis.
  • TAMEP and TAP are discussed in detail above.
  • Non-limiting embodiments of the foregoing disclosed herein include:
  • TAMEP transglutaminase-activating M4 metalloprotease
  • TAMEP The immobilized TAMEP of embodiments 1 or 2 wherein the TAMEP is from Streptomyces mobaraensis.
  • TAMEP transglutaminase-activating M4 metalloprotease
  • TAP tripeptidyl aminopeptidase
  • a method for activating a zymogen form of a transglutaminase or a variant thereof to produce a mature, catalytically active form of the transglutaminase comprising a) immobilizing at least one transglutaminase-activating M4 metalloprotease (TAMEP) and/or at least one tripeptidyl aminopeptidase from (TAP), provided that if both TAMEP and TAP are immobilized, the TAMEP and TAP are separately immobilized on the same or different porous solid support, or providing at least one TAMEP and/or at least one TAP immobilized on a porous solid support; and b) contacting the zymogen form of 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.
  • TAMEP transglutaminase-activating M4 metalloprotease
  • a method for activating a zymogen form of a transglutaminase or a variant thereof to produce a mature, catalytically active form of a transglutaminase comprising a) co-immobilizing at least one transglutaminase-activating M4 metalloprotease (TAMEP) and at least one tripeptidyl aminopeptidase from (TAP) on the same porous solid support; and b) contacting the zymogen form of transglutaminase with co-immobilized TAMEP and TAP to produce a mature, catalytically active form of the transglutaminase.
  • TAMEP transglutaminase-activating M4 metalloprotease
  • TAP tripeptidyl aminopeptidase from
  • TAMEP is from Streptomyces mobaraensis and the TAP is from Streptomyces mobaraensis.
  • a method for activating a zymogen form of a transglutaminase or a variant thereof to produce a catalytically active, not mature form of a transglutaminase comprising a) immobilizing at least one transglutaminase-activating M4 metalloprotease (TAMEP) on a same porous solid support; and b) contacting the zymogen form of transglutaminase with immobilized at least one TAMEP to produce a catalytically active, not mature form of the transglutaminase.
  • TAMEP transglutaminase-activating M4 metalloprotease
  • TAMEP is from Streptomyces mobaraensis.
  • TAMEP is the active form of TAMEP having the sequence in SEQ ID NO: 3.
  • TAMEP has a sequence comprising 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 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 of SEQ ID NO: 3.
  • TAP is the active form of TAP having the sequence in SEQ ID NO: 4.
  • 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 1 Expression of Zymogen of Tqase variant (Pro-Tqase variant) in Escherichia coli
  • A. Construction of expression plasmid for expression of a Pro-Tgase variant (SEQ ID NO:2) [00105]
  • the gene coding for the Pro-Tgase variant i.e., zymogen form of Tgase variant
  • the expression vector also contains the pMB1 origin of replication and a kanamycin resistance gene.
  • the resulting plasmid was transformed first into E. coli DH-10B, using standard methods known in the art.
  • the transformants were isolated by subjecting the cells to kanamycin selection, as known in the art (See, e.g., US Pat. No. 8,383,346 and W02010/144103, both of which are incorporated by reference herein, in their entirety), and the sequence of the Pro-Tgase gene was verified by Sanger sequencing.
  • the plasmid was recovered from a positive clone, using methods known in the art, and transformed into E. coll BL21 (DE3) for expression.
  • E. coll strain BL21 (DE3), containing the Pro-Tgase expression vector, was cultured overnight in Luria broth at 37 °C until the culture reached saturation. The following morning, the culture was used to inoculate a shake flask containing a medium including glycerol, soy peptone, yeast extract, magnesium sulfate heptahydrate, and potassium phosphate monobasic, at 30-34 °C for up to 10 hours with continuous shaking. Isopropyl
  • IPTG Isopropyl
  • the Pro-Tgase variant may be secreted, for example, from a microbial strain that is known to those skilled in the art to secrete Tgase such as Streptomyces mobaraensis or Bacillus subtilis.
  • Tgase such as Streptomyces mobaraensis or Bacillus subtilis.
  • the pellet is discarded and the supernatant is recovered and is assessed by SDS-PAGE as described in Example 1 D and by spectroscopy. Tgase activity is assessed using the Colorimetric Activity Assay described in Example 1C.
  • Tgase activity was measured herein 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, the hydroxylamine is incorporated to form Z-glutamylhydroxamate- glycine, which develops a colored complex with iron (III), detectable at 525 nm after incubation at 37 °C for 5-60 minutes.
  • ZQG N-benzyloxycarbonyl-L-glutaminyl-glycine
  • the calibration was performed using L-glutamic acid gamma-monohydroxamate (Millipore® Sigma®) as standard.
  • One unit of Tgase is defined as the amount of enzyme that catalyzes formation of 1 pmol of the peptide derivative of gamma-glutamylhydroxylamine per minute.
  • This technique is used to assess the level of activation of a Pro-Tgase variant (SEQ ID NO:2) by TAMEP (see Example 3) and to analyze the molecular weight of expressed proteins.
  • Mature Tgase was secured from commercial sources (Moo Gloo Tl formula, Ajinomoto®) and used as a molecular weight standard for SDS-PAGE analysis (Fig. 1. Lane 1).
  • Pro-Tgase Variant SEQ ID NO:2
  • FRAP-Tqase Variant SEQ ID NO:5
  • This method was used to purify either Pro-Tgase variant (SEQ ID NO:2) (as prepared 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, loading at 1 %vol) at 37 °C for 60 minutes.
  • 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 show activity above baseline in the Colorimetric Activity Assay. Only activated forms of Tgases, for example, FRAP-Tgase variant (SEQ ID NO:5), showed activity in this assay.
  • N-terminal peptide sequence of the FRAP-Tgase variant was confirmed using N-terminal sequencing.
  • the technique of N-terminal sequencing was used to assess the level of activation of Pro-Tgase variant (SEQ ID NO:2) by TAMEP or FRAP- Tgase (SEQ ID NO:5) by TAP, such as SM-TAP.
  • subtilis SCK6 delta-AlaR purchased from Bio-Technical Resources (Manitowoc, Wl) were grown overnight at 37 °C in 5 mL of LB medium supplemented with 40 mg/mL D-alanine. The following day, the culture was diluted to an OD600 of 1 .0 and xylose was added to a final concentration of 1%. After 2 hours, 250 pL of glycerol and ligated DNA was added and the culture tube was returned to the incubator for an additional 90 minutes. Following incubation, 10-1000 pL of culture was spread onto LB agar plates. Plates were grown at 37 °C overnight. The following day, 2-8 colonies were selected from each plate and inoculated into 3 mL of LB broth.
  • TAMEP from Example 3A above was determined using the following assay. Soluble TAMEP was added at 1 %vol to purified Pro-Tgase variant (Example 2, 500 pL at 5 g/L). The reactions were then incubated at 37 °C, 300 rpm, with time points being taken between 1-120 minutes. At each time point, a sample of the supernatant was removed and quenched with ethylenediaminetetraacetic acid (EDTA).
  • EDTA ethylenediaminetetraacetic acid
  • SM-TAP The activity of SM-TAP from Example 3A above was determined using the following assay. Soluble SM-TAP was added at 1 %vol to purified FRAP-Tgase variant (Example 2, 500 pL at 5 g/L). The reactions were then incubated at 37 °C, 300 rpm, with time points collected from 1-120 minutes. At each time point a sample of the supernatant was removed and quenched with phenylmethylsulfonyl fluoride (PMSF).
  • PMSF phenylmethylsulfonyl fluoride
  • Samples were analyzed by ultra-performance liquid chromatography coupled to mass spectrometry (UPLC- MS, Thermo ScientificTM VanquishTM UPLC and Thermo ScientificTM ISQTM EM MS) to determine the total area count of FRAP tetrapeptide produced by the cleavage of FRAP- Tgase variant (SEQ ID NO:5) by SM-TAP.
  • Samples were run on an AccucoreTM VanquishTM C18 column (50mm x 2.1 mm ID, 1.5 pm particle size, Thermo ScientificTM) on a linear gradient from 5 to 90% of 0.1% formic acid in acetonitrile over 1.5 minutes.
  • N-terminal peptide sequencing performed as described in Example 2 above, confirmed that the FRAP tetrapeptide had been removed from the N-terminal end of the FRAP-Tgase variant (SEQ ID NO:5) resulting in a mature, catalytically active Tgase variant (SEQ ID NO:6).
  • TAMEP Example 3A
  • SM-TAP Example 3A
  • IB-COV-1 IB-COV-2
  • IB-COV-3 IB-COV-3
  • ANI-1 modified for covalent binding
  • ECR8204F ECR8209F
  • ECR8215F ECR8215F
  • porous solid support was weighed into a separate glass vial then washed with water (10 ml_, 4x). The porous solid supports were then washed with 50 mM phosphate buffer adjusted to pH 8 (two porous solid support volumes, 4x).
  • ANI-1 porous solid support was modified for covalent binding by washing (4 porous solid support volumes, 1x) with a freshly prepared 1% glutaraldehyde solution in 50 mM phosphate buffer, pH 8, for 1 hour at 20 °C. The glutaraldehyde solution was then removed and the porous solid support was washed (two porous solid support volumes, 4x) with 50 mM phosphate buffer, pH 8. [00122] For all covalent porous solid supports, B.
  • subtilis supernatant(s) containing TAMEP and/or SM-TAP was then incubated on the porous solid support for 18-20 hours at 20 °C, 700 rpm at 4:1 supernatant volume to porous solid support mass. The agitation was halted, and the protease(s) incubated on the porous solid support for an additional 2 hours. The supernatant was then removed from the porous solid support and then the porous solid support was washed with 50 mM phosphate buffer adjusted to pH 8 (4 porous solid support volumes, 1x). The porous solid supports were then washed with 50 mM phosphate buffer, pH 8, containing 500 mM sodium chloride (4 porous solid support volumes, 2x).
  • Each porous solid support was weighed into a separate glass vial then washed with water (10 ml_, 4x). The porous solid supports were then washed with 50 mM phosphate buffer adjusted to pH 7 (two porous solid support volumes, 4x). B. subtilis supernatant containing TAMEP (0.2-0.5 g/L) was then incubated on the porous solid support for 18-20 hours at 20 °C, 700 rpm at 4:1 supernatant volume to porous solid support mass. The agitation was halted, and the supernatant incubated on the porous solid support for an additional 2 hours. The supernatant was then removed from the porous solid support and then the porous solid support was washed with 50 mM phosphate buffer adjusted to pH 7 (4 porous solid support volumes, 4x).
  • Each porous solid support was weighed into a separate glass vial then washed with water (10 mL, 4x). The porous solid supports were then washed with 50 mM carbonate buffer adjusted to pH 10.5 (two porous solid support volumes, 4x). B. subtilis supernatant(s) containing TAMEP and/or SM-TAP (0.2-0.5 g/L) was then incubated on the porous solid support for 18-20 hours at 20 °C, 700 rpm at 4:1 supernatant volume to porous solid support mass. The agitation was halted, and the protease(s) incubated on the porous solid support for an additional 2 hours. The supernatant was then removed from the porous solid support and then the porous solid support was washed with 50 mM carbonate buffer adjusted to pH 10.5 (4 porous solid support volumes, 4x).
  • TAMEP and SM-TAP were co-immobilized onto 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.
  • porous solid support was weighed into a separate glass vial then washed with water (10 ml_, 4x). The porous solid supports were then washed with 50 mM phosphate buffer adjusted to pH 8 (two porous solid support volumes, 4x). B. subtilis supernatant(s) containing TAMEP and/or SM-TAP (0.2-0.5 g/L) was then incubated on the porous solid support for 18-20 hours at 20 °C, 700 rpm at 4:1 supernatant volume to porous solid support mass. The agitation was halted, and the protease(s) incubated on the porous solid support for an additional 2 hours. The supernatant was then removed from the porous solid support and then the porous solid support was washed with 50 mM phosphate buffer adjusted to pH 8 (4 porous solid support volumes, 4x).
  • Table 2 Porous solid supports tested for separate immobilization of TAMEP and SM- TAP. TAMEP and SM-TAP retained activity when immobilized onto all supports listed.
  • This example shows the activation of purified FRAP-Tgase variant, (SEQ ID NO:5).
  • purified FRAP-Tgase variant SEQ ID NO:5
  • clarified lysate containing FRAP- Tgase SEQ ID NO:5
  • protease activation of purified FRAP-Tgase SEQ ID NO:5 is presented herein.
  • 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 variant (200 pL at 5 g/L, SEQ ID NO:5, as described in Example 2 above) was added into each vial. The vials were then incubated at 20 °C, 700 rpm, with time points collected from 1-120 minutes. At each time point a sample of the supernatant was removed and quenched with PMSF. 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, showed that the FRAP tetrapeptide had been removed from the N-terminal end of the FRAP-Tgase variant (SEQ ID NO:5), resulting in the thermostable mature, catalytically active Tgase variant (SEQ ID NO:6).
  • Activity assay results showed that SM-TAP retained protease activity when immobilized on all the porous solid supports shown in Table 2.
  • FRAP tetrapeptide will only be produced if TAMEP first cleaves the pro-peptide from Pro-Tgase variant (SEQ ID NO:2) 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-terminal end of the FRAP-Tgase variant (SEQ ID NO:5) to produce 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 the porous solid supports set forth in Table 3 below.
  • Table 3 A selection of the porous solid supports from Table 2 were tested for coimmobilization of TAMEP and SM-TAP. TAMEP and SM-TAP retained activity on all the porous solid supports shown below.
  • TAMEP and SM-TAP concentrations are based on total protein.
  • Fig. 2 shows that the most FRAP was produced by the co-immobilized TAMEP and SM-TAP when compared to the amount of FRAP produced by either TAMEP and SM-TAP immobilized separately or TAMEP and SM-TAP both in soluble form i.e., not immobilized.
  • Fig. 2 also shows that more FRAP was produced by TAMEP and SM-TAP immobilized separately as compared to soluble TAMEP and SM-TAP over 120 minutes.
  • co-immobilized TAMEP and SM-TAP and separately immobilized TAMEP and SM-TAP outperformed soluble TAMEP and SM-TAP.
  • SEQ ID NO:2 A thermostable variant of Streptomyces mobaraensis Tgase in zymogen form with two methionine amino acid residues - one methionine is located at the
  • N-terminus of the Pro-sequence and the second methionine is located between the Prosequence and the N-terminus of the mature domain (Pro-Tgase variant).
  • Leader sequence (pro-) is denoted in bold, underlined text.
  • SEQ ID NO:3 Wild-type Streptomyces mobaraensis transglutaminase-activating M4 metalloprotease (TAMEP) in zymogen form with the native signal peptide deleted and a leading methionine included to facilitate recombinant expression.
  • Putative leader sequence (pro-) is denoted in bold, underlined text.
  • SEQ ID NO:4 Wild-type Streptomyces mobaraensis transglutaminase-activating tripeptidyl aminopeptidase (SM-TAP) in zymogen form with the native signal peptide deleted and a leading methionine included to facilitate recombinant expression.
  • Putative leader sequence (pro-) is denoted in bold, underlined text.
  • SEQ ID NO:5 A thermostable variant of Streptomyces mobaraensis Tgase having a FRAP tetrapeptide at the N-terminus of the mature domain with a methionine amino acid residue located between the FRAP tetrapeptide and N-terminus of the mature domain of the thermostable Tgase variant (FRAP-Tgase variant).
  • Leader sequence (pro-) is denoted in bold, underlined text.
  • thermostable variant of Streptomyces mobaraensis Tgase with the FRAP tetrapeptide 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.

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Abstract

L'invention concerne des protéases immobilisées pour l'activation de la forme zymogène de la transglutaminase.
PCT/US2022/076202 2022-09-09 2022-09-09 Protéases immobilisées pour l'activation de la forme zymogène de la transglutaminase WO2024054236A1 (fr)

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WO2010144103A1 (fr) 2009-06-11 2010-12-16 Codexis, Inc. Synthèse en parallèle automatisée combinée de variants polynucléotidique
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