WO2018004014A1 - Recombinant protein having transglutaminase activity - Google Patents

Abstract

The invention provides an active microbial transglutaminase (MTG) that does not require protease treatment in a protein expression system using a universal host such as Escherichia coli, that is, an MTG mutant having transglutaminase activity, and a method for producing the same. The invention is a protein obtained by mutating an amino acid in the amino acid sequence of the propeptide moiety in wild-type microbial transglutaminase, wherein the protein is characterized by having transglutaminase activity.

Description

Recombinant protein with transglutaminase activity

The present invention relates to a recombinant protein having transglutaminase activity. Specifically, the present invention relates to the above recombinant protein in which the amino acid of the propeptide portion of the microorganism-derived transglutaminase is mutated.

Transglutaminase (TGase) is an acyl group transfer between a specific glutamine (Q) side chain γ-carboxyamide group and a primary amine including an amine group of lysine (K) side chain, or water. A post-translational modification enzyme that catalyzes the reaction and forms an isopeptide bond. In particular, among TGases, microtransglutaminase (MTG) derived from Streptomyces mobaraensis does not require a cofactor for the expression of catalytic activity, and is easily expressed in large quantities by a microbial host, and has a cross-linking activity (that is, the above isoforms). Since it has industrially superior features such as high activity to form peptide bonds, industrial use has been actively performed mainly in the food industry. On the other hand, recently, application of MTG to the pharmaceutical field has been attempted, and in particular, attention has been focused on the preparation of antibody-drug conjugate (ADC) by site-specific modification of antibodies (Non-Patent Documents 1 and 2). reference). Thus, the application range of MTG is expanding, and further application can be expected by improving its function. However, in the E. coli expression system, which is a general-purpose host for gene recombination, it is difficult to express the mature form of MTG itself, and it has been reported that it is insolubilized (Patent Document 1). MTG has a partial structure called a propeptide and a structure called a precursor fused to the N-terminal side of mature MTG. This propeptide is an intramolecular chaperone that leads to the correct folding of MTG. It can be said that it is indispensable to acquire. Therefore, in a state where a propeptide is added, expression in E. coli is possible (see Non-Patent Document 3). On the other hand, the MTG variant expressed with the propeptide added does not show activity due to the presence of the propeptide covering the active site. Therefore, it has been difficult to prepare an active recombinant MTG using E. coli as a host. Also, in the secretory expression system using other hosts, in order to obtain active MTG, it is necessary to finally cleave the propeptide site with an appropriate protease (Patent Document 2).
In previous studies, in order to solve the above-mentioned problems, the MTG precursor and the protease are co-expressed in the E. coli cytoplasm, thereby succeeding in obtaining active MTG simultaneously with the expression. In addition, a search for a more highly active MTG in which a propeptide is easily removed after protease treatment by screening a plurality of mutants by introducing a mutation into an amino acid in the propeptide and studying an expression process has been carried out ( Non-patent document 2).
On the other hand, with regard to the improvement of MTG, there are many that modify the MTG body, and there are few studies that attempt to improve the properties of MTG by modifying the propeptide itself. Among them, in one of the previous studies that modified the propeptide itself, the MTG precursor was regarded as a fusion protein with the C-terminus of the propeptide as a linker, and the linker was linked to the C-terminus of the propeptide against MTG derived from Streptomyces hygroscopicus We are proposing a new approach to improve the properties of TGase by inserting peptides. This linker peptide plays an essential role in satisfying the harmony of interaction between domains (propeptide and matured body). A linker peptide is a peptide that does not have a fixed secondary structure and has some flexibility to satisfy biological conditions. When polyglycine, GS, and PT (PTPPTTPT) were used as linker peptides, the specific activity increased in all mutants compared to the wild type. In particular, those with GS and PT linker inserted showed high specific activity. On the other hand, it is considered that the catalytic efficiency of the enzyme increased because the value of k _cat / K _m increased in the mutant with the linker inserted. This is presumably because the linker peptide is located in the vicinity of the active site, and the catalytic efficiency is increased by the interaction between the linker peptide and the catalytic region. In this way, the activity of MTG has been successfully improved by adding a specific sequence to the propeptide (see Non-Patent Document 4).
In recent years, it has been shown that this problem can be cleared by co-expressing a recombinant MTG and a protease in an E. coli host (see Non-Patent Document 2). However, in a system using a protease, there is a concern that MTG undergoes partial hydrolysis, leading to inactivation. Moreover, if the propeptide cleaved with the protease is not completely removed by purification, the reaction efficiency of MTG is lowered, which may affect the subsequent reaction. In any case, a protease treatment and a subsequent purification process are necessary, and an increase in costs associated with this may be considered.

JP-A-6-30771 JP 2005-229807 A

Therefore, the problem to be solved by the present invention is an active MTG (MTG variant having transglutaminase activity) that does not require protease treatment in a protein expression system using a general-purpose host such as Escherichia coli, a method for producing the same, etc. Is to provide.
The present inventor has intensively studied to solve the above problems. As a result, a mutant that expresses transglutaminase activity (crosslinking activity) in the state of an MTG precursor, that is, a state having a propeptide was searched. Specifically, the above activity is spontaneously expressed even in a state having a propeptide by modifying the propeptide possessed by the MTG precursor by genetic engineering and regulating the interaction between the active site and the propeptide. The present invention was completed by finding an MTG variant.
That is, the present invention is as follows.
(1) A protein obtained by mutating at least an amino acid in the amino acid sequence of a propeptide portion in a wild-type microorganism-derived transglutaminase, wherein the protein has transglutaminase activity.
(2) The protein according to (1) above, which has the activity in a state having the propeptide portion after the mutation.
(3) The protein according to (1) or (2) above, wherein the mutation is substitution with another amino acid.
(4) The protein according to any one of (1) to (3) above, wherein the amino acid in the amino acid sequence of the propeptide portion to be mutated contains lysine and / or at least one tyrosine.
(5) The protein according to (4) above, wherein the other amino acid obtained by mutating the at least one tyrosine is at least one selected from the group consisting of alanine, glutamine and histidine.
(6) Other amino acids obtained by mutating the lysine are arginine, aspartic acid, glutamic acid, alanine, cysteine, phenylalanine, glycine, histidine, isoleucine, leucine, methionine, asparagine, proline, glutamine, serine, threonine, valine, tryptophan. And the protein according to (4), which is at least one selected from the group consisting of tyrosine.
(7) An amino acid sequence in which at least one amino acid selected from the group consisting of the 10th, 12th and 16th amino acids in the amino acid sequence of wild-type microorganism-derived transglutaminase is substituted with another amino acid, or the substitution A protein comprising an amino acid sequence in which one or several amino acids excluding the amino acid at the substitution site are deleted, substituted, or added, and has transglutaminase activity.
(8) The protein according to (7), wherein the other amino acid obtained by mutating the 12th and / or 16th amino acid is at least one selected from the group consisting of alanine, glutamine and histidine.
(9) Other amino acids obtained by mutating the tenth amino acid are arginine, aspartic acid, glutamic acid, alanine, cysteine, phenylalanine, glycine, histidine, isoleucine, leucine, methionine, asparagine, proline, glutamine, serine, threonine , The protein according to (7), which is at least one selected from the group consisting of valine, tryptophan and tyrosine.
(10) The following protein (a) or (b):
(A) an amino acid sequence in which at least one amino acid selected from the group consisting of the 10th, 12th and 16th amino acids is substituted with another amino acid in the amino acid sequence shown in SEQ ID NO: 2 (b) (a) A protein having a transglutaminase activity, comprising an amino acid sequence in which one or several amino acids excluding the amino acid at the substitution site in the amino acid sequence are deleted, substituted or added, and having the transglutaminase activity (11) The protein according to (10) above, wherein the other amino acid obtained by mutating the second amino acid is at least one selected from the group consisting of alanine, glutamine and histidine.
(12) Other amino acids obtained by mutating the tenth amino acid are arginine, aspartic acid, glutamic acid, alanine, cysteine, phenylalanine, glycine, histidine, isoleucine, leucine, methionine, asparagine, proline, glutamine, serine, threonine The protein according to (10) above, which is at least one selected from the group consisting of valine, tryptophan and tyrosine.
(13) The transglutaminase activity according to any one of (1) to (12), wherein the transglutaminase activity has an activity level substantially equivalent to the transglutaminase activity of a wild-type microorganism-derived transglutaminase. protein.
(14) A gene encoding the protein according to any one of (1) to (13) above.
(15) A gene comprising the following DNA (a) or (b):
(A) In the base sequence shown in SEQ ID NO: 1, the 28th to 30th bases have been replaced with bases indicating codons of amino acids other than lysine, and / or the 34th to 36th and / or Alternatively, DNA consisting of a base sequence in which the 46th to 48th bases are replaced with bases indicating codons of amino acids other than tyrosine.
(B) a DNA that hybridizes under stringent conditions with a DNA comprising a base sequence complementary to the DNA of (a), wherein the base corresponding to the base of the substitution site is the base of the substitution site; DNA encoding the same protein having transglutaminase activity
(16) The gene according to (15), wherein the amino acid other than tyrosine is at least one selected from the group consisting of alanine, glutamine, and histidine.
(17) Amino acids other than lysine are composed of arginine, aspartic acid, glutamic acid, alanine, cysteine, phenylalanine, glycine, histidine, isoleucine, leucine, methionine, asparagine, proline, glutamine, serine, threonine, valine, tryptophan and tyrosine. The gene according to (15) above, which is at least one selected from the group.
(18) A recombinant vector comprising the gene according to any one of (14) to (17) above.
(19) A transformant comprising the recombinant vector according to (18) above.
(20) The transformant according to (19) above, wherein the transformant is transformed Escherichia coli.
(21) A method for producing the protein, comprising the step of culturing the transformant according to (19) or (20) above, and the step of collecting a protein having transglutaminase activity from the obtained culture.
(22) The method according to (21) above, wherein the collecting step comprises a protein purification step.
EFFECT OF THE INVENTION According to the present invention, an MTG variant that can be easily produced (purified) in an expression system using a microorganism such as Escherichia coli as a host, that is, an active MTG having transglutaminase activity is provided. be able to. If it is the said MTG variant, the protease process at the time of expression refinement | purification becomes unnecessary, and the effort and cost concerning removal of a propeptide and protease can be reduced significantly.

It is a figure which shows the measurement result (pH 6.0) of the recombinant MTG enzyme activity by the hydroxamate method. It is a figure which shows the measurement result (pH 7.4) of the recombinant MTG enzyme activity by the hydroxamate method. It is an experiment schematic diagram of the activity evaluation of the MTG variant by FRET. It is a figure which shows the FRET measurement result of a wild-type MTG precursor (Y12 MTG). (Left: Change in fluorescence spectrum over time; Right: Progress of crosslinking reaction based on maximum fluorescence intensity ratio) It is a figure which shows the FRET measurement result of a Y12A MTG variant. (Left: Change in fluorescence spectrum over time; Right: Progress of crosslinking reaction based on maximum fluorescence intensity ratio) It is a figure which shows the FRET measurement result of a Y12Q MTG variant. (Left: Change in fluorescence spectrum over time; Right: Progress of crosslinking reaction based on maximum fluorescence intensity ratio) It is a figure which shows the FRET measurement result of Y16A MTG variant. (Left: Change in fluorescence spectrum over time; Right: Progress of crosslinking reaction based on maximum fluorescence intensity ratio) It is a figure which shows the FRET measurement result of a Y16Q MTG variant. (Left: Change in fluorescence spectrum over time; Right: Progress of crosslinking reaction based on maximum fluorescence intensity ratio) It is a figure which shows the FRET measurement result of K10R / Y12A MTG variant. (Left: Change in fluorescence spectrum over time; Right: Progress of crosslinking reaction based on maximum fluorescence intensity ratio) It is a figure which shows the FRET measurement result of commercial MTG (made by Zedira). (Left: Change in fluorescence spectrum over time; Right: Progress of crosslinking reaction based on maximum fluorescence intensity ratio) It is a figure which shows the comparison result of the progress of the crosslinking reaction of commercial MTG and each MTG variant. It is a figure which shows the FRET measurement result of a Y12A MTG variant. (Left: Change in fluorescence spectrum over time; Right: Progress of crosslinking reaction based on maximum fluorescence intensity ratio) It is a figure which shows the FRET measurement result of K10R / Y12A MTG variant. (Left: Change in fluorescence spectrum over time; Right: Progress of crosslinking reaction based on maximum fluorescence intensity ratio) It is a figure which shows the FRET measurement result of commercial MTG (made by Zedira). (Left: Change in fluorescence spectrum over time; Right: Progress of crosslinking reaction based on maximum fluorescence intensity ratio) It is a figure which shows the comparison result of the progress of the crosslinking reaction of commercial MTG and each MTG variant. It is a figure which shows the FRET measurement result of a Y12A MTG variant. (Left: Change in fluorescence spectrum over time; Right: Progress of crosslinking reaction based on maximum fluorescence intensity ratio) It is a figure which shows the FRET measurement result of K10R / Y12A MTG variant. (Left: Change in fluorescence spectrum over time; Right: Progress of crosslinking reaction based on maximum fluorescence intensity ratio) It is a figure which shows the FRET measurement result of commercial MTG. (Left: Change in fluorescence spectrum over time; Right: Progress of crosslinking reaction based on maximum fluorescence intensity ratio) It is a figure which shows the comparison result of the progress of the crosslinking reaction of commercial MTG and each MTG variant. It is a figure which shows the result of the fluorescent dye modification with respect to a CQ-tag EGFP recombinant. It is a figure which shows the fluorescent dye modification result with respect to NK-tag EGFP recombinant. (Upper) Y12A MTG variant, Y12Q MTG variant; (Middle) Y16A MTG variant; (Lower) K10R / Y12A MTG variant It is a figure which shows the measurement result (pH 6.0) of the recombinant MTG enzyme activity by the hydroxamate method. It is a figure which shows the measurement result (pH 6.0) of the recombinant MTG enzyme activity by the hydroxamate method. It is a figure which shows the measurement result (pH 6.0) of the recombinant MTG enzyme activity by the hydroxamate method. It is a figure which shows the measurement result (pH 6.0) of the recombinant MTG enzyme activity by the hydroxamate method. It is a figure which shows the comparison result of the progress of the crosslinking reaction of commercial MTG and each MTG variant. It is a figure which shows the comparison result of the progress of the crosslinking reaction of commercial MTG and each MTG variant. It is a figure which shows the comparison result of the progress of the crosslinking reaction of commercial MTG and each MTG variant. It is a figure which shows the fluorescent dye modification result with respect to K-tag EGFP recombinant (EGFP-MRHKGS) of each MTG variant. It is a figure which shows the fluorescent dye modification result with respect to K-tag EGFP recombinant (MKHKGS-EGFP) of each MTG variant. It is a figure which shows the fluorescent dye modification result with respect to K-tag EGFP recombinant (MKHKGS-EGFP) of each MTG variant. It is a figure which shows the fluorescent dye modification result with respect to K-tag EGFP recombinant (EGFP-MRHKGS) of each MTG variant. It is a figure which shows the fluorescent dye modification result with respect to K-tag EGFP recombinant (EGFP-MRHKGS) of each MTG variant. It is a figure which shows the fluorescent dye modification result with respect to K-tag EGFP recombinant (EGFP-MRHKGS) of each MTG variant.

Hereinafter, the present invention will be described in detail. The scope of the present invention is not limited to these descriptions, and other than the following examples, the scope of the present invention can be appropriately changed and implemented without departing from the spirit of the present invention.
In addition, this specification includes the whole of Japanese Patent Application No. 2006-131883 specification (application on July 1, 2016) used as the foundation of this application priority claim. In addition, all publications cited in the present specification, for example, prior art documents, and publications, patent publications and other patent documents are incorporated herein by reference.
In the present specification, “MTG” means a microtransglutaminase derived from Streptomyces mobileaensis as described above. In addition, “MTG precursor” means MTG having a propeptide portion, and “MTG mutant” means MTG obtained by mutating (substituting) an amino acid in the MTG precursor.
1. protein
The protein of the present invention is a mutant protein of transglutaminase (MTG) derived from a microorganism (Streptomyces mobaaraensis). Specifically, the protein of the present invention is a protein obtained by mutating at least an amino acid in the amino acid sequence of the propeptide portion in wild-type MTG, and having transglutaminase activity. It has the activity in a state having a mutated propeptide portion (a state in which the mutated propeptide portion is present in a protein molecule).
Usually, in wild-type MTG, a partial structure called a propeptide (propeptide portion) is present in the form of being fused to the N-terminal side of mature MTG. In this specification, the so-called fusion protein of mature MTG and the propeptide portion is referred to as an MTG precursor. In MTG, the propeptide portion serves as an inhibitor and has a role for expressing transglutaminase activity (crosslinking activity) at an appropriate location. This cross-linking activity is a post-translational modification between a primary amine including a specific glutamine (Q) side chain γ-carboxyamide group and a lysine (K) side chain amine group, or water. The activity of catalyzing the acyl transfer reaction and forming an isopeptide bond. The propeptide portion in MTG is an intramolecular chaperone that leads to correct folding of the protein, and is indispensable for obtaining active MTG, that is, MTG having the above-mentioned crosslinking activity. In actinomycetes, which is the original host of MTG, the MTG precursor is first expressed, and then the propeptide portion is removed by a unique protease secreted by the host to become mature MTG. However, in the E. coli expression system, which is a general-purpose host for gene recombination, the expression of mature MTG itself is difficult (is insolubilized). In addition, those expressed in a state having a propeptide portion also have a problem that they do not show activity due to the presence of the propeptide portion covering the active site of MTG. Therefore, it has been conventionally difficult to prepare an active recombinant using Escherichia coli as a host. On the other hand, in recent years, it has been shown that the above problem can be solved by co-expressing a recombinant MTG and a protease in an E. coli host. However, in a system using a protease, there is a concern that MTG may be partially hydrolyzed, leading to inactivation. Further, if the protease and the cleaved propeptide are not completely removed by purification, the reaction efficiency of MTG decreases. Therefore, there is a concern that it affects the subsequent reaction, and there is a problem that the cost for purification increases. On the other hand, the present invention provides active MTG without the protease treatment, and can solve the problems in the prior art at once.
The protein of the present invention is a protein in which at least an amino acid in the amino acid sequence of the propeptide portion is mutated in a wild-type microorganism-derived transglutaminase and has transglutaminase activity. Preferably, those having the activity in the state having the propeptide portion after the mutation, and the mutation being a substitution with another amino acid. The amino acid to be substituted preferably contains at least one tyrosine. In this case, the other amino acid after substitution is preferably at least one selected from the group consisting of alanine, glutamine and histidine. The amino acid to be substituted preferably contains lysine, and in this case, other amino acids after substitution include arginine, aspartic acid, glutamic acid, glycine, asparagine, alanine, cysteine, glutamine, serine, threonine (threonine ), Preferably at least one selected from the group consisting of tryptophan, phenylalanine, isoleucine, leucine, methionine, proline, valine, tyrosine and histidine, more preferably at least one selected from the group consisting of aspartic acid and glutamic acid It is.
As the protein of the present invention, for example, in the amino acid sequence of wild-type microorganism-derived transglutaminase, at least one amino acid selected from the group consisting of the 10th, 12th and 16th amino acids is substituted with another amino acid. It comprises an amino acid sequence or an amino acid sequence in which one or several amino acids excluding the amino acid at the substitution site in the substituted amino acid sequence are deleted, substituted or added, and has transglutaminase activity Protein is preferred. In the present specification, the amino acid sequence of wild-type microorganism-derived transglutaminase is an amino acid sequence excluding the signal peptide part of wild-type microorganism-derived transglutaminase, and a methionine residue (M) is added to the N-terminus thereof. It means the amino acid sequence. The same applies to the amino acid sequence of SEQ ID NO: 2 described later. The same applies to the base sequence of SEQ ID NO: 1.
Here, the “amino acid sequence in which one or several amino acids are deleted, substituted or added” is, for example, about 1 to 10, preferably 1 to several, 1 to 5, 1 to 4 It is preferable that the amino acid sequence has one, three, one, two, or one amino acid deleted, substituted or added. The introduction of mutation such as deletion, substitution or addition may be carried out by a mutation introduction kit utilizing site-directed mutagenesis, for example, GeneTailor. ^TM Site-Directed Mutagenesis System (Invitrogen), and TaKaRa Site-Directed Mutagenesis System (Prime STAR (registered trademark) Mutagenesis Basal kit, Muta (registered trademark)-Super Ex. Can do. Whether or not the above deletion, substitution or addition mutation has been introduced can be confirmed using various amino acid sequencing methods and structural analysis methods such as X-ray and NMR.
The other amino acid is not particularly limited as far as the 12th and / or 16th amino acid residue is other than tyrosine, but for example, at least one selected from the group consisting of alanine, glutamine and histidine. Species can preferably be mentioned. The tenth amino acid residue is not particularly limited as long as it is other than lysine. For example, arginine, aspartic acid, glutamic acid, glycine, asparagine, alanine, cysteine, glutamine, serine, threonine (threonine), Preferred is at least one selected from the group consisting of tryptophan, phenylalanine, isoleucine, leucine, methionine, proline, valine, tyrosine and histidine, more preferably at least one selected from the group consisting of aspartic acid and glutamic acid. . By substituting the 10th amino acid, for example, the production of a self-crosslinked product of MTG itself can be suppressed while maintaining the crosslinking activity of MTG.
The protein of the present invention is also preferably the following protein (a) or (b).
(A) a protein comprising an amino acid sequence in which at least one amino acid selected from the group consisting of the 10th, 12th and 16th amino acids is substituted with another amino acid in the amino acid sequence shown in SEQ ID NO: 2
(B) a transglutaminase activity comprising an amino acid sequence in which one or several amino acids except the amino acid at the substitution site are deleted, substituted or added in the amino acid sequence of the above (a) (the amino acid sequence after the substitution) Protein with
As described above, the amino acid sequence shown in SEQ ID NO: 2 is an amino acid sequence obtained by adding a methionine residue (M) to the N-terminus of the amino acid sequence excluding the signal peptide portion of the wild-type microorganism-derived transglutaminase, It is an amino acid sequence consisting of a total of 376 amino acids.
As for the “amino acid sequence in which at least one amino acid selected from the group consisting of the 10th, 12th and 16th amino acids is substituted with another amino acid” in the protein (a) above, Applicable as appropriate.
The protein of (b) is one or several (for example, about 1 to 10, preferably 1 to several, except for the amino acid at the substitution site in the amino acid sequence contained in the protein of (a). Any protein may be used as long as it has a transglutaminase activity and has an amino acid sequence in which 5 to 1, 1 to 4, 1 to 3, 1 to 2, or 1 amino acid is deleted, substituted or added. There is no limitation. The method for introducing a mutation such as deletion, substitution or addition and the confirmation of whether or not a mutation has been introduced are the same as described above.
Moreover, as a protein functionally equivalent to the protein of said (a), the protein of the following (c) other than the protein of said (b) is mentioned, for example.
(C) a protein comprising an amino acid sequence having 80% or more identity (homology) to the amino acid sequence of (a) above (the amino acid sequence after the substitution) and having transglutaminase activity
As the protein of (c), those having the above identity of 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more are more preferable.
The proteins (b) and (c) (so-called mutant proteins) can also be prepared by genetic engineering using a gene encoding the amino acid sequence of the protein.
In the present invention, the transglutaminase activity can be evaluated and measured by, for example, a method by fluorescence resonance energy transfer (FRET) described in Examples described later.
The proteins (a) to (c) in the present invention may be in the state where the propeptide portion is fused (bound) to the N-terminal side of the mature MTG as in the case of the wild-type MTG. However, as another embodiment, those having a linker sequence between the propeptide portion and the N-terminal side of mature MTG are also preferred. The linker sequence is not limited, but may be an amino acid sequence of about 1 to 30 residues, and specific examples thereof include GGGSLVPRGSGGS (thrombin linker sequence; SEQ ID NO: 10). In the amino acid sequence of wild-type MTG (SEQ ID NO: 2), the amino acid sequence from the N-terminal side to the 46th amino acid (proline) is the propeptide portion, and the 47th amino acid (aspartic acid) side to the C-terminal side. Up to here is the amino acid sequence of mature MTG.
The proteins (a) to (c) referred to in the present invention may be peptides derived from natural products, or may be obtained by artificial chemical synthesis, and are not limited.
A protein derived from a natural product may be obtained directly from a natural product by known recovery and purification methods, or a gene encoding the protein is incorporated into various expression vectors by a known gene recombination technique. After being introduced into cells and expressed, they may be obtained by known recovery and purification methods. Alternatively, a commercially available kit such as a reagent kit PROTEIOS ^TM (Toyobo), TNT ^TM System (Promega), PG-Mate of synthesizer ^TM The protein may be produced by a cell-free protein synthesis system using Toyobo and RTS (Roche Diagnostics), and may be obtained by a known recovery method and purification method, and is not limited.
Chemically synthesized proteins can be obtained using known protein synthesis methods. Examples of the synthesis method include an azide method, an acid chloride method, an acid anhydride method, a mixed acid anhydride method, a DCC method, an active ester method, a carboimidazole method, and a redox method. In addition, the solid phase synthesis method and the liquid phase synthesis method can be applied to the synthesis. A commercially available protein synthesizer may be used. After the synthesis reaction, the protein can be purified by combining known purification methods such as chromatography.
In the present invention, a derivative of the protein can be included together with or instead of the proteins (a) to (c). The derivative is meant to include all those that can be prepared from the protein. For example, a derivative in which a part of the constituent amino acid is replaced with a non-natural amino acid, or a constituent amino acid (mainly its side chain). And those having a part thereof chemically modified.
In the present invention, the protein (a) to (c) and / or a derivative of the protein can be included together with or in place of the protein and / or a salt of the derivative. The salt is preferably a physiologically acceptable acid addition salt or basic salt. Acid addition salts include, for example, salts with inorganic acids such as hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid, or acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, succinic acid, tartaric acid, citric acid, apple Examples thereof include salts with organic acids such as acid, oxalic acid, benzoic acid, methanesulfonic acid, and benzenesulfonic acid. Examples of basic salts include salts with inorganic bases such as sodium hydroxide, potassium hydroxide, ammonium hydroxide and magnesium hydroxide, and salts with organic bases such as caffeine, piperidine, trimethylamine and pyridine. .
The salt can be prepared using a suitable acid such as hydrochloric acid or a suitable base such as sodium hydroxide. For example, it can be prepared by treatment using standard protocols in water or in a liquid containing an inert water-miscible organic solvent such as methanol, ethanol or dioxane.
2. Recombinant gene
Although it does not limit as a gene which codes the protein of this invention mentioned above, The gene containing DNA of the following (a) or (b) is mentioned preferably. The following DNAs (a) and (b) are preferably structural genes of the protein of the present invention, but the gene containing these DNAs may be composed only of these DNAs. In addition, these DNAs may be included in part, and may also include known base sequences (transcription promoter, SD sequence, Kozak sequence, terminator, etc.) necessary for gene expression, and is not limited.
(A) In the base sequence shown in SEQ ID NO: 1, the 28th to 30th bases have been replaced with bases indicating codons of amino acids other than lysine, and / or the 34th to 36th and / or Alternatively, DNA consisting of a base sequence in which the 46th to 48th bases are replaced with bases indicating codons of amino acids other than tyrosine.
(B) a DNA that hybridizes under stringent conditions with a DNA comprising a base sequence complementary to the DNA of (a), wherein the base corresponding to the base of the substitution site is the base of the substitution site; DNA encoding the same protein having transglutaminase activity
In the present invention, the term “codon” means not only a three-base chain (triplet) on the RNA sequence after transcription but also a three-base chain on the DNA sequence. Therefore, the notation of the codon on the DNA sequence is performed using thymine (T) instead of uracil (U).
The base sequence shown in SEQ ID NO: 1 is a base sequence consisting of 1131 bases encoding wild-type microorganism-derived transglutaminase. As described above, in the present specification, the nucleotide sequence of SEQ ID NO: 1 is an amino acid sequence obtained by adding a methionine residue (M) to the N-terminus of the amino acid sequence excluding the signal peptide portion of wild-type microorganism-derived transglutaminase. This is a base sequence encoding the sequence.
In addition, the DNA of (a) above is preferably a DNA in which the base showing the codons of amino acids other than tyrosine is a base showing at least one codon selected from the group consisting of alanine, glutamine and histidine. .
Furthermore, as the DNA of the above (a), the bases indicating the codons of amino acids other than lysine are arginine, aspartic acid, glutamic acid, glycine, asparagine, alanine, cysteine, glutamine, serine, threonine (threonine), tryptophan, phenylalanine. Preferably a base showing at least one codon selected from the group consisting of isoleucine, leucine, methionine, proline, valine, tyrosine and histidine, more preferably at least one selected from the group consisting of aspartic acid and glutamic acid Preferred is DNA in the case of a base representing a codon. .
Such mutation-substituted DNAs are described in, for example, Molecular Cloning, A Laboratory Manual 4th ed. , Cold Spring Harbor Press (1989), Current Protocols in Molecular Biology, John Wiley & Sons (1987-1997) and the like. Specifically, it can be prepared using a mutation introduction kit using site-directed mutagenesis by a known method such as the Kunkel method or Gapped duplex method, and examples of the kit include QuickChange. ^TM Site-Directed Mutagenesis Kit (Stratagene), GeneTailor ^TM Preferred examples include Site-Directed Mutagenesis System (manufactured by Invitrogen), TaKaRa Site-Directed Mutagenesis System (Mutan-K, Mutan-Super Express Km, etc .: manufactured by Takara Bio Inc.) and the like.
In addition, using PCR primers designed so that a missense mutation is introduced so as to be a base indicating a codon of a desired amino acid, using a DNA containing a base sequence encoding wild-type MTG as a template under appropriate conditions It can also be prepared by performing PCR. The DNA polymerase used for PCR is not limited, but is preferably a highly accurate DNA polymerase. For example, Pwo DNA (Polymerase Roche Diagnostics), Pfu DNA polymerase (Promega), Platinum Pfx DNA polymerase ( Invitrogen), KOD DNA polymerase (Toyobo), KOD-plus-polymerase (Toyobo) and the like are preferable. The PCR reaction conditions may be appropriately set depending on the optimum temperature of the DNA polymerase to be used, the length and type of the DNA to be synthesized, etc. For example, in the case of cycle conditions, “90-98 ° C. for 5-30 seconds (thermal denaturation, Dissociation) → 50 to 65 ° C. for 5 to 30 seconds (annealing) → 65 to 80 ° C. for 30 to 1200 seconds (synthesis / elongation) ”is preferably performed under a total of 20 to 200 cycles.
The DNA of the above (b) uses the DNA of the above (a) or a DNA comprising a complementary base sequence, or a fragment thereof, as a probe, such as colony hybridization, plaque hybridization, Southern blot, etc. It can be obtained from a cDNA library or a genomic library by performing a known hybridization method. A library prepared by a known method may be used, or a commercially available cDNA library or genomic library may be used, and is not limited.
For details of the hybridization procedure, see Molecular Cloning, A Laboratory Manual 3rd ed. (Cold Spring Harbor Laboratory Press (1989) and the like can be appropriately referred to.
“Stringent conditions” in carrying out the hybridization method are conditions at the time of washing after hybridization, wherein the buffer salt concentration is 15 to 330 mM, the temperature is 25 to 65 ° C., preferably the salt concentration is 15 to It means a condition of 150 mM and a temperature of 45 to 55 ° C. Specifically, for example, conditions such as 50 mM at 80 mM can be exemplified. Furthermore, in addition to the conditions such as salt concentration and temperature, various conditions such as probe concentration, probe length, reaction time, etc. are taken into account, and the conditions for obtaining the DNA of (b) above are appropriately set. Can do.
The hybridizing DNA is preferably a base sequence having at least 40% homology (identity) to the DNA base sequence of (a) above, more preferably 60%, 80% or more, Examples include base sequences having 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity.
In the DNA of (b), the base corresponding to the base at the substitution site is the same as the base at the substitution site.
The “substitution site” referred to here is a site of base substitution made in the base sequence contained in the DNA of (a) above. Specifically, a base (triplet) indicating a modified codon generated by the base substitution. Means the site of
The “corresponding base” of the “base corresponding to the base at the substitution site” means that when the DNA of (b) is hybridized with a complementary strand to the DNA of (a), It means a base (triplet) that is in a position opposite to the base complementary to the base at the site (triplet).
The DNA of (b) is, for example, a base that is not completely identical in terms of base sequence, but is completely identical in translated amino acid sequence compared to the DNA of (a) above. A DNA consisting of a sequence (that is, a DNA obtained by subjecting the DNA of (a) to a silent mutation) is particularly preferred.
As the gene encoding the protein of the present invention, codons corresponding to individual amino acids after translation are not particularly limited. Therefore, codons generally used in mammals such as humans after transcription (preferably frequency of use) DNA that shows a codon that is commonly used in microorganisms such as Escherichia coli and yeast, plants, etc. (preferably a codon that is frequently used). May be included.
Further, the gene encoding the protein of the present invention may include a DNA encoding the amino acid sequence of the linker sequence when the protein includes the linker sequence described above.
3. Recombinant vector and transformant
In order to express the protein of the present invention, it is necessary to first construct a recombinant vector by incorporating the above-described gene of the present invention into an expression vector. At this time, a transcription promoter, an SD sequence (when the host is a prokaryotic cell), and a Kozak sequence (when the host is a eukaryotic cell) are ligated upstream in advance as necessary. Alternatively, a terminator may be linked downstream, and an enhancer, splicing signal, poly A addition signal, selection marker, etc. may be linked. Each element necessary for gene expression such as the above transcription promoter may be included in the gene from the beginning, or may be used when originally included in the expression vector. A use aspect is not specifically limited.
As a method for incorporating the gene into the expression vector, various methods using known gene recombination techniques such as a method using a restriction enzyme and a method using topoisomerase can be employed. The expression vector is not limited as long as it can hold the gene encoding the protein of the present invention, such as plasmid DNA, bacteriophage DNA, retrotransposon DNA, retroviral vector, artificial chromosome DNA, and the like. A vector suitable for the host cell to be used can be appropriately selected and used.
Subsequently, the constructed recombinant vector is introduced into a host to obtain a transformant, which is cultured, whereby the protein of the present invention can be expressed. The “transformant” as used in the present invention means a gene into which a foreign gene has been introduced into the host, for example, a gene into which a foreign gene has been introduced by introducing plasmid DNA or the like into the host (transformation), Also included are those in which a foreign gene has been introduced by infecting a host with various viruses and phages (transduction).
The host is not limited as long as it can express the protein of the present invention after the introduction of the above recombinant vector, and can be selected as appropriate. For example, various animal cells such as humans and mice can be selected. And known hosts such as various plant cells, bacteria, yeast, and plant cells.
When animal cells are used as hosts, for example, human fibroblasts, CHO cells, monkey cells COS-7, Vero, mouse L cells, rat GH3, human FL cells and the like are used. Insect cells such as Sf9 cells and Sf21 cells can also be used.
When bacteria are used as hosts, for example, Escherichia coli, Bacillus subtilis and the like are used.
When yeast is used as a host, for example, Saccharomyces cerevisiae and Schizosaccharomyces pombe are used.
When plant cells are used as hosts, for example, tobacco BY-2 cells are used.
The method for obtaining the transformant is not limited and can be appropriately selected in consideration of the combination of the host and expression vector. For example, electroporation, lipofection, heat shock, PEG, Preferred examples include calcium phosphate method, DEAE dextran method, and methods of infecting various viruses such as DNA virus and RNA virus.
In the resulting transformant, the codon type of the gene contained in the recombinant vector may be the same as or different from the codon type of the host actually used, and is not limited.
4). Protein production
Specifically, the production of the protein of the present invention can be carried out by a method including a step of culturing the above-described transformant and a step of collecting a protein having transglutaminase activity from the obtained culture. Here, “cultured product” means any of culture supernatant, cultured cells, cultured cells, or disrupted cells or cells. The transformant can be cultured according to a usual method used for host culture. The protein of interest is accumulated in the culture. In the present invention, the collecting step may include a protein purification step.
As the medium used for the culture, any known natural medium and any known medium can be used as long as it contains a carbon source, a nitrogen source, inorganic salts, and the like that can be assimilated by the host, and can efficiently culture the transformant. Any synthetic medium may be used.
During the culture, the cells may be cultured under selective pressure in order to prevent the recombinant vector contained in the transformant from dropping and the gene encoding the target protein from dropping off. That is, when the selectable marker is a drug resistance gene, the corresponding drug can be added to the medium, and when the selectable marker is an auxotrophic complementary gene, the corresponding nutrient factor can be removed from the medium. it can.
When cultivating a transformant transformed with an expression vector using an inducible promoter as a promoter, a suitable inducer (for example, IPTG or the like) may be added to the medium as necessary.
The culture conditions of the transformant are not particularly limited as long as the productivity of the target protein and the growth of the host are not hindered, and are usually 10 to 40 ° C., preferably 20 to 37 ° C. and 5 to 100. Do time. The pH can be adjusted using an inorganic or organic acid, an alkaline solution, or the like. Examples of the culture method include solid culture, stationary culture, shaking culture, and aeration and agitation culture.
When the target protein is produced in the microbial cells or cells after culturing, the target protein can be collected by disrupting the microbial cells or cells. As a method for disrupting cells or cells, high-pressure treatment using a French press or homogenizer, ultrasonic treatment, grinding treatment using glass beads, enzyme treatment using lysozyme, cellulase, pectinase, etc., freeze-thawing treatment, hypotonic solution treatment, It is possible to use a lysis inducing treatment with a phage or the like. After crushing, the cells or cell crushing residues (including the cell extract insoluble fraction) can be removed as necessary. Examples of the method for removing the residue include centrifugation and filtration. If necessary, the residue removal efficiency can be increased by using a flocculant or a filter aid. The supernatant obtained after removing the residue is a cell extract soluble fraction and can be a crudely purified protein solution.
In addition, when the target protein is produced in the microbial cells or cells, the microbial cells and the cells themselves can be recovered by centrifugation, membrane separation, etc., and used without being crushed.
On the other hand, when the target protein is produced outside the cells or cells, the culture solution is used as it is, or the cells or cells are removed by centrifugation or filtration. Then, if necessary, the target protein is collected from the culture by extraction with ammonium sulfate precipitation, and further, if necessary, using dialysis and various chromatography (gel filtration, ion exchange chromatography, affinity chromatography, etc.) It can also be isolated and purified.
The production yield of the protein obtained by culturing the transformant is, for example, SDS-PAGE (polyacrylamide gel) in units such as per culture solution, per microbial wet weight or dry weight, or per crude enzyme solution protein. For example, electrophoresis).
In addition to the protein synthesis system using the transformant as described above, the target protein can also be produced using a cell-free protein synthesis system that does not use any living cells.
The cell-free protein synthesis system is a system that synthesizes a target protein in an artificial container such as a test tube using a cell extract. Cell-free protein synthesis systems that can be used also include cell-free transcription systems that synthesize RNA using DNA as a template.
In this case, the cell extract to be used is preferably derived from the aforementioned host cell. Examples of the cell extract include extracts derived from eukaryotic cells or prokaryotic cells, more specifically, CHO cells, rabbit reticulocytes, mouse L-cells, HeLa cells, wheat germ, budding yeast, E. coli, and the like. Liquid can be used. These cell extracts may be used after being concentrated or diluted, or may be used as they are, and are not limited.
The cell extract can be obtained by, for example, ultrafiltration, dialysis, polyethylene glycol (PEG) precipitation or the like.
Such cell-free protein synthesis can also be performed using a commercially available kit. For example, reagent kit PROTEIOS ^TM (Toyobo), TNT ^TM System (Promega), PG-Mate of synthesizer ^TM (Toyobo), RTS (Roche Diagnostics) and the like.
The target protein produced by cell-free protein synthesis can be purified by appropriately selecting means such as chromatography as described above.
Hereinafter, the present invention will be described more specifically with reference to examples. However, the present invention is not limited to these examples.

Preparation of MTG variant DNA encoding MTG variant (specifically, MTG precursor variant) consisting of each amino acid sequence shown below is inserted between NdeI-XhoI of pET22b + to construct a recombinant expression plasmid vector did. In the amino acid sequences of the following MTG variants, the underlined amino acids are substitution-mutated amino acids.
Amino acid sequence of MTG variant (Y12A)

Amino acid sequence of MTG variant (Y12Q)

Amino acid sequence of MTG variant (Y16A)

Amino acid sequence of MTG variant (Y16Q)

Amino acid sequence of MTG variant (K10R / Y12A)

The obtained recombinant expression plasmid vector was transformed into E. coli BL21 (DE3) strain using a heat shock method, inoculated into an LB agar medium containing ampicillin sodium at 100 μg / mL, and allowed to stand at 37 ° C. overnight. A colony was obtained. The obtained Escherichia coli colonies were inoculated into 10 mL of LB medium (containing 100 μg / mL of ampicillin sodium) and cultured at 37 ° C. and 200 rpm for 4 hours. Inoculate in 500 mL of LB medium, and culture at 37 ° C. and 120 rpm. When OD ₆₀₀ = 0.6 is reached, Isopropyl β-D-1-thiogalactopylanoside is added at a final concentration of 0.5 mM, and the culture temperature is increased. The temperature was lowered to 15 ° C. and the culture was continued for another 16 hours. The cells were collected by centrifugation at 6000 g for 7 minutes. After washing 3 times with 1 × TBS Buffer (25 mM Tris-HCl, 150 mM NaCl, pH 7.4), all the supernatant was discarded and the pelleted cells were stored frozen at −80 ° C. The cryopreserved pellet is dissolved in 15 mL of 1 × TBS buffer, the cells are disrupted by sonication (Output 4, Duty 20, 12.5 min), and centrifuged (4 ° C., 18,000 × g, 20 min). The bacterial cells and the protein-containing solution were separated. The obtained solution was filtered through 0.45 μm and 0.22 μm PVDF membrane filters to remove insoluble fractions and bacterial cells. The resulting solution was purified with a hexahistidine tag (His Tag) introduced into the C-terminus of Y16A MTG using a HisTrap Excel column (1 mL). The fraction in which absorption at 280 nm derived from the absorbance of aromatic amino acids (tyrosine / tryptophan) in the protein was collected, and desalted with a PD-10 column. Next, purification by anion exchange chromatography was performed using a HiTrap Q column (1 mL). In the same manner as described above, a fraction in which absorption at 280 nm was confirmed was collected, and buffer exchange with 1 × PBS was performed using a PD-10 column. The purification conditions for each column are shown below.
・ HisTrap Excel (1 mL)
Buffer A (20 mM Tris-HCl, 0.5 M NaCl, 5 mM Imidazole, pH 7.4)
Buffer B (20 mM Tris-HCl, 0.5 M NaCl, 500 mM Imidazole pH 7.4)

・ HiTrap Q HP (1mL)
Buffer A (20 mM Tris-HCl)
Buffer B (20 mM Tris-HCl, 1M NaCl)

Evaluation of activity of recombinant MTG precursor by hydroxamate method (JE Folk & PW Cole, J. Biol. Chem., 1966, 241, 5518-5525) Z-Gln-Gly (Z-QG), hydroxylammonium chloride and reduced glutathione (GSH) mixed solution (substrate solution), trichloroacetic acid and iron chloride hexahydrate added with concentrated hydrochloric acid (reaction stopped) Solution), as a calibration curve, a dilution series of L-glutamic acid γ-monohydroxyamine salt and a dilution series of each MTG were prepared.
2. A blank 0.2 M Tris-HCl buffer (pH 7.4), a calibration curve dilution series, and 20 μL of each MTG dilution series were added to a 96-well plate.
3. 80 μL of substrate solution was added to each well and incubated at 37 ° C. for 10 min.
4). 100 μL of the reaction stop solution was added, and the absorption at 525 nm was immediately measured with a plate reader.
5. From the calibration curve, the amount of hydroxamic acid produced under each MTG condition was measured, and the specific activity (U / mg) was calculated. Here, the amount of enzyme that generates 1 μmol of hydroxamate per minute was defined as the TGase activity unit, 1 U (unit). Here, the final concentration of each reagent during the reaction is as follows. [Z-QG] = 30 mM, [hydroxylammonium chloride] = 0.1 M, [GSH] = 10 mM in 0.2 M Tris-Acetate (pH 6.0) or 0.2 M Tris-HCl (pH 7.4).

The enzyme activity measurement results under the conditions of pH 6.0 (FIG. 1) and pH 74 (FIG. 2) are shown below. From these results, Y12A MTG and Y12Q MTG showed no activity at pH 6.0, but Y12A MTG showed a little activity at pH 7.4. A slight improvement in activity was also observed for Y12Q MTG. However, compared to commercially available MTG, the enzyme activity in the hydroxamate method is remarkably low. From this result, it is considered that the Y12A MTG precursor and the Y12Q MTG precursor do not recognize either or both of Z-QG and hydroxylamine as a substrate, and the access between the MTG and the substrate is greatly reduced. It was strongly suggested.

Activity Evaluation of Recombinant MTG Mutants by Fluorescence Resonance Energy Transfer (FRET) Texas Red-QG (200 μM), CRK-tag EGFP Recombinant (having MRHKGS (SEQ ID NO: 8) sequence at C-terminus) (10 μM) and various sets The replacement MTG variant (1 μM) was mixed at 37 ° C. in a volume of 50 μL. 5 μL was sampled from the sample solution over time, mixed with 495 μL of 1 mM N-ethylmaleimide solution (in 10 mM Tris-HCl (pH 8.0)) to stop the crosslinking reaction, and the fluorometer (PerkinElmer, LS 55) The fluorescence spectrum was measured with (excitation light wavelength: 460 nm, fluorescence spectrum measurement range: 480 to 680 nm).
<Enzyme reaction conditions>
Lys peptide substrate fusion protein: CRK-EGFP (C-terminal MRHKGS) 10 μM
Small molecule Gln substrate: Texas Red-QG 200 μM
MTG 1μM
Fluorescence spectrum measurement conditions Excitation wavelength 460nm
Measurement wavelength 480 ~ 680nm
FIG. 3 shows an experimental schematic diagram for evaluating the activity of the MTG mutant by FRET.
FIGS. 4 to 11 show the analysis results by FRET and the change over time in the degree of progress of the crosslinking reaction of MTG based on the ratio of the maximum fluorescence intensity of EGFP and the maximum fluorescence intensity of Texas Red (I ₆₁₀ / I ₅₀₈ ).
From the above results, it was revealed that the Y12A MTG mutant and the commercially available MTG finally reached the same reaction rate, and thus the Y12A MTG mutant showed the same reactivity as the commercially available MTG. However, since the difference in activity differs greatly between the results of FRET and hydroxamate, it can be said that the difference in substrate recognition ability of the Y12A MTG mutant was reflected. Moreover, it was shown that activity is remarkably higher than a wild-type MTG precursor (Y12).
From the above results, it was shown that the Y12A MTG mutant is a mutant that expresses crosslinking activity without cleaving the propeptide. At the same time, it was shown that the initial activity was slightly lower than that of commercial MTG.

(1) Reaction between small molecule primary amine substrate and small molecule Gln substrate Based on the following enzyme reaction conditions, the cross-linking activity of the Y12A MTG mutant was evaluated using a fluorescent small molecule substrate. The results are shown in FIGS.
<Enzyme reaction conditions>
Small molecule primary amine substrate: FITC-cadaverine 10 μM
Small molecule Gln substrate: Texas Red-QG 200 μM
MTG 1μM
Fluorescence spectrum measurement conditions Excitation wavelength 460nm
Measurement wavelength 480 ~ 680nm
From the results shown in FIGS. 11 to 13, it is clear that the Y12A MTG mutant shows almost no activity compared to the commercially available MTG, which supports the measurement result of the recombinant MTG enzyme activity by the hydroxamate method in FIG. . The K10R / Y12A MTG mutant (double mutant) was suggested to have slightly higher activity than the Y12A mutant, but the activity was lower than that of the commercially available enzyme.
(2) Reaction between 6 amino acid Lys peptide substrate and small molecule Gln substrate Based on the following enzyme reaction conditions, the crosslinking activity of the Y12A MTG mutant was evaluated using a fluorescent small molecule substrate. The results are shown in FIGS.
<Enzyme reaction conditions>
Lys peptide substrate: FAM-MRHKGS (SEQ ID NO: 8) 10 μM
Small molecule Gln substrate: Texas Red-QG 200 μM
MTG 1μM
Fluorescence spectrum measurement conditions Excitation wavelength 480nm
Measurement wavelength 495-680nm
From the results of FIGS. 14 to 16, it was suggested that the Y12A MTG mutant had a slightly lower initial activity than the commercially available MTG, but the cross-linking efficiency when the enzyme reaction reached equilibrium was almost the same. The K10R / Y12A MTG mutant (double mutant) showed similar catalytic behavior to the Y12A mutant.
From the comparison with the result of (1) above, the Y12A MTG mutant and the K10R / Y12A MTG mutant (double mutant) are sufficient for the small molecule Gln substrate when the peptidic Lys substrate is used. It was shown to express crosslinking activity.
On the other hand, from the comparison of the horizontal axes of FIG. 11 and FIG. 19, the same Lys peptide substrate sequence (MRHKGS (SEQ ID NO: 8)) is reacted with the same small molecule Gln substrate (TexasRed-QG). Regardless, it was suggested that the proteinaceous substrate (CRK-EGFP) fused as an additional sequence to the protein reacts faster.
(3) Summary From the above results, an appropriate mutation (Y12X, X = A or Q; Y16X, X = A or Q; K10R / Y12A) was introduced into the propeptide sequence covering the active site of MTG, and It has been clarified that catalytic activity is expressed even in MTG mutants having a propeptide by using a highly reactive peptidic substrate.

Experiment of fluorescent dye modification to MTG-reactive peptide-tagged protein CQ-tag EGFP recombinant (having LLQG sequence at C-terminus), fluorescent dye FITC-cadaverine, and Y12A MTG mutant or Y12Q MTG mutant in PBS (pH 7. 4) The mixture was mixed and reacted at 37 ° C for 1 hour. Further, NK-tag EGFP recombinant (having MKHKGSGGGSGGGS (SEQ ID NO: 9) sequence at the N-terminus), fluorescent dye FITC-β-Ala-QG, and Y12A MTG mutant or Y12Q MTG mutant in PBS (pH 7.4) And mixed at 37 ° C. for 1 hour. Finally, NK-tag EGFP recombinant (MKHKGSGGGSGGGS (SEQ ID NO: 9) sequence at the N-terminus), fluorescent dye FITC-β-Ala-QG, and Y16A MTG variant were mixed in PBS (pH 7.4). And reacted at 37 ° C. for 1 hour.
After the reaction, SDS-PAGE was performed to obtain a FITC-derived fluorescent image labeled with the protein using a fluorescent imager (Bio-Rad, Molecular Imager FX Pro). In addition, proteins were stained with Coomassie Brilliant Blue (CBB). Here, the concentrations of EGFP to which the substrate peptide tag was added, fluorescent dyes, and various MTGs were set to 1 μM, 20 μM, and 1 μM, respectively. The results are shown in FIGS.
From the results of FIGS. 20 and 21,
-Between small molecule primary amine substrate and Gln substrate peptide added protein (FIG. 17)
Lys substrate peptide-added protein—small molecule Gln substrate (FIG. 18)
In any of the cases, since a fluorescent band was observed in the vicinity of the molecular weight of the EGFP recombinant, it became clear that the cross-linking reaction proceeded between the substrate peptide sequence added to the protein and the small molecule substrate. Of the MTG prosequence variant was also shown to have cross-linking activity.
In addition, in the Y12Q MTG mutant lane of FIG. 20, although a fluorescent band was confirmed in a portion corresponding to the MTG mutant, although it was not confirmed in the Y12A MTG mutant lane, the mutation was introduced. It was suggested that the primary amine substrate was labeled on the Gln residue.
On the other hand, in any of the MTG mutant lanes of Y12A, Y12Q, and Y16A in FIG. 21, a fluorescent band was confirmed at a position corresponding to the MTG mutant. Since fluorescence was not confirmed with commercially available MTG, the small molecule Gln substrate (FITC-β-Ala-QG) is labeled on the Lys residue (K10) in the propeptide sequence of these mutants. It was suggested. In the K10R / Y12A MTG mutant (double mutant) in which K10 was substituted with an Arg residue, the label product of MTG itself was not confirmed.

DNAs encoding various MTG mutants shown in Table 3 below (specifically, MTG precursor mutants) were prepared, and using the same method as in Example 1, construction of a recombinant expression plasmid vector, Expression and purification were performed to obtain various MTG mutant proteins. DNAs encoding various MTG mutants are No. 5 which are mutation modes shown in the “mutation introduction position” column of Table 3. DNA encoding 2 to 21 MTG variants. Specifically, in the same manner as the DNA encoding the MTG variant constructed in Example 1, an amino acid obtained by adding a peptide of “GSHHHHHH” (SEQ ID NO: 11) to the C-terminus of the amino acid sequence of wild-type MTG (SEQ ID NO: 2) Based on the sequence (amino acid sequence of wild-type (WT) MTG No. 1 in Table 3), the propeptide portion of the amino acid sequence was assigned No. 1 in Table 3. This is a DNA encoding an MTG variant into which an amino acid substitution mutation of 2 to 21 is introduced or a linker sequence (thrombin linker sequence: SEQ ID NO: 10) is inserted. In Table 3, No. For the 9 MTG variants (K10X (X ≠ K, R) / Y12A / TL), for the tenth K, various amino acids other than K and R (ie, X (X ≠ K, R)) It has been replaced (see FIGS. 24, 27, 28, 31).

Regarding the obtained wild type MTG (No. 1) and various MTG variants (No. 2 to 21), 1) a hydroxamate method using a small molecule substrate, and 2) a method using a peptide (protein) substrate 2-1) Peptide cross-linked FRET, 2-2) Protein label (SDS-PAGE)
The transglutaminase activity was measured and evaluated by each analysis method. The results are summarized in Table 4 below. In addition, in each evaluation result (+,-, etc.) in Table 4, a drawing number (FIGS. 22 to 34) as a basis of the evaluation is also shown.

Specifically, 1) The hydroxamate method using a small molecule substrate was carried out in the method of Example 2 described above except that it was performed only under the conditions of pH 6.0 among the conditions of pH 6.0 and pH 7.0. The specific activity (U / mg) was calculated in the same manner as this method. The results are shown in FIGS.
Further, 2) the method using a peptide (protein) substrate is as follows.
2-1) In the method of peptide cross-linking FRET, the enzyme activity was evaluated in the same manner as in the method of Example 3 described above. The results are shown in FIGS. However, for the MTG variants whose results are shown in FIGS. 27 and 28, the concentration used was 0.1 μM instead of 1 μM.
2-2) In the method of protein labeling (SDS-PAGE), K-tag EGFP recombinant (MKHKGS (SEQ ID NO: 12) consists of an amino acid sequence having an N-terminus, and MKHKGS-EGFP (SEQ ID NO: 14). (Also referred to as a fluorescent dye) FITC-β-Ala-QG and various MTG mutants were mixed in PBS (pH 7.4) and reacted at 37 ° C. for 1 hour. After the reaction, SDS-PAGE was performed to obtain a FITC-derived fluorescent image labeled with the protein using a fluorescent imager (Bio-Rad, Molecular Imager FX Pro). In addition, proteins were stained with Coomassie Brilliant Blue (CBB). Here, the concentrations of EGFP to which the substrate peptide tag was added, fluorescent dyes, and various MTGs were set to 1 μM, 20 μM, and 1 μM, respectively. The results are shown in FIGS.
Further, a K-tag EGFP recombinant different from the above (consisting of an amino acid sequence having the MRHKGS (SEQ ID NO: 13) sequence at the C-terminus, also referred to as EGFP-MRHKGS (SEQ ID NO: 15)), fluorescent dye FITC-β -Ala-QG and various MTG variants were mixed in PBS (pH 7.4) and reacted at 37 ° C for 1 hour. After the reaction, SDS-PAGE was performed to obtain a FITC-derived fluorescent image labeled with the protein using a fluorescent imager (Bio-Rad, Molecular Imager FX Pro). In addition, proteins were stained with Coomassie Brilliant Blue (CBB). Here, the concentrations of EGFP to which the substrate peptide tag was added, fluorescent dyes, and various MTGs were set to 1 μM, 20 μM, and 1 μM, respectively. The results are shown in FIGS. 29 and 32-34.

<Consideration of this example>
Table 4 summarizes this example.
First, as shown in FIGS. 22 and 23, in the measurement of MTG activity by the hydroxamate method, the Y12A, Y12Q, Y16A, and Y16Q mutants did not show activity, but the double mutant in which both Y12 and Y16 were mutated to A was shown. A significant improvement in activity was observed. Subsequently, in FIG. 24, with regard to double mutants in which Y12 is fixed to A and K10 is substituted with various amino acids, significant activity improvement was observed in mutants substituted with acidic amino acids such as D and E. Next, as shown in FIG. 25, in the single or double mutant of Y12H and Y16H, significant activity improvement was observed in the MTG mutant having the mutation of Y12H, and further improvement in activity was observed in the double mutant of Y12H / Y16H. It was done. Further, no significant activity was observed in the K10D or K10E mutants that did not have the Y12A mutation.
Next, in FIG. 26, WT / TL having only the thrombin linker sequence does not show activity, whereas single or double mutants in which a mutation of A or Q is introduced into Y12 or Y16 show a significant improvement in activity. Indicated. In order to estimate the difference in the activity of each mutant, the same examination was performed with the MTG concentration set to 1/10. 27 and 28, each K10X / Y12A / TL mutant showed high crosslinking activity in the order of X = D> E, G> K (Y12A / TL)> A, C, N>M> H.
Finally, FIG. 29 shows that the activity is slightly expressed by insertion of the thrombin linker. Furthermore, from FIGS. 30 and 31, the MTG mutant having a mutation introduced into Y12 or Y16 showed a significant improvement in activity. Further, from FIGS. 32 and 33, the MTG mutant having the mutation of K10R / Y12H or K10R / Y12H / Y16H showed pH responsiveness and showed significantly higher activity at pH 6.0 than at pH 7.4. Finally, in FIG. 34, the expression of activity was confirmed for a single mutant having no mutation in Y12 or Y16 and having a mutation of K10D or K10E.

According to the present invention, it is possible to provide an MTG mutant that can be easily produced (purified) in an expression system using a microorganism such as Escherichia coli as a host, that is, an active MTG having transglutaminase activity. . If it is the said MTG variant, the protease process at the time of expression refinement | purification becomes unnecessary, and the effort and cost concerning removal of a propeptide and protease can be reduced significantly.
Furthermore, the MTG variant according to the present invention does not show a crosslinking activity with respect to a small molecule substrate having low reactivity, and is a nonspecific crosslinking that may cause a problem when MTG is used for site-specific modification of a protein. It can prevent reaction. That is, the MTG variant according to the present invention recognizes a substrate by the MTG variant having the propeptide only when a peptidic substrate having a higher affinity than the propeptide into which the mutation has been introduced is used. A peptide sequence-selective cross-linking reaction can be performed. The MTG mutant according to the present invention can also be used for site-specific modification of a target protein substrate in a host.

Sequence number 3: Recombinant protein Sequence number 4: Recombinant protein Sequence number 5: Recombinant protein Sequence number 6: Recombinant protein Sequence number 7: Recombinant protein Sequence number 8: Peptide Sequence number 9: Peptide Sequence number 10: Peptide Sequence number 11: Peptide Sequence number 12: Peptide Sequence number 13: Peptide Sequence number 14: Recombinant protein Sequence number 15: Recombinant protein

Claims (22)

1. A protein obtained by mutating at least an amino acid in the amino acid sequence of the propeptide portion of a wild-type microorganism-derived transglutaminase and having transglutaminase activity.
2. The protein according to claim 1, which has the activity in a state having the propeptide portion after the mutation.
3. The protein according to claim 1 or 2, wherein the mutation is substitution with another amino acid.
4. The protein according to any one of claims 1 to 3, wherein the amino acid in the amino acid sequence of the propeptide part to be mutated contains lysine and / or at least one tyrosine.
5. The protein according to claim 4, wherein the other amino acid obtained by mutating the at least one tyrosine is at least one selected from the group consisting of alanine, glutamine and histidine.
6. Other amino acids obtained by mutating the lysine are arginine, aspartic acid, glutamic acid, alanine, cysteine, phenylalanine, glycine, histidine, isoleucine, leucine, methionine, asparagine, proline, glutamine, serine, threonine, valine, tryptophan and tyrosine. The protein according to claim 4, which is at least one selected from the group consisting of:
7. Amino acid sequence in which at least one amino acid selected from the group consisting of the 10th, 12th and 16th amino acids in the amino acid sequence of wild-type microorganism-derived transglutaminase is substituted with another amino acid, or the substituted amino acid sequence A protein comprising an amino acid sequence in which one or several amino acids excluding the amino acid at the substitution site are deleted, substituted or added, and having transglutaminase activity.
8. The protein according to claim 7, wherein the other amino acid obtained by mutating the 12th and / or 16th amino acid is at least one selected from the group consisting of alanine, glutamine and histidine.
9. Other amino acids obtained by mutating the tenth amino acid are arginine, aspartic acid, glutamic acid, alanine, cysteine, phenylalanine, glycine, histidine, isoleucine, leucine, methionine, asparagine, proline, glutamine, serine, threonine, valine, The protein according to claim 7, which is at least one selected from the group consisting of tryptophan and tyrosine.
10. The following protein (a) or (b).
    (A) an amino acid sequence in which at least one amino acid selected from the group consisting of the 10th, 12th and 16th amino acids is substituted with another amino acid in the amino acid sequence shown in SEQ ID NO: 2 (b) (a) A protein having a transglutaminase activity, comprising an amino acid sequence in which one or several amino acids except the amino acid at the substitution site are deleted, substituted or added in the amino acid sequence of
11. The protein according to claim 10, wherein the other amino acid obtained by mutating the 12th and / or 16th amino acid is at least one selected from the group consisting of alanine, glutamine and histidine.
12. Other amino acids obtained by mutating the tenth amino acid are arginine, aspartic acid, glutamic acid, alanine, cysteine, phenylalanine, glycine, histidine, isoleucine, leucine, methionine, asparagine, proline, glutamine, serine, threonine, valine, The protein according to claim 10, which is at least one selected from the group consisting of tryptophan and tyrosine.
13. The protein according to any one of claims 1 to 12, wherein the transglutaminase activity has an activity level substantially equivalent to a transglutaminase activity of a wild-type microorganism-derived transglutaminase.
14. A gene encoding the protein according to any one of claims 1 to 13.
15. A gene comprising the following DNA (a) or (b):
    (A) In the base sequence shown in SEQ ID NO: 1, the 28th to 30th bases have been replaced with bases indicating codons of amino acids other than lysine, and / or the 34th to 36th and / or Alternatively, DNA consisting of a base sequence in which the 46th to 48th bases are replaced with bases indicating codons of amino acids other than tyrosine.
    (B) a DNA that hybridizes under stringent conditions with a DNA comprising a base sequence complementary to the DNA of (a), wherein the base corresponding to the base of the substitution site is the base of the substitution site; DNA encoding the same protein having transglutaminase activity
16. The gene according to claim 15, wherein the amino acid other than tyrosine is at least one selected from the group consisting of alanine, glutamine and histidine.
17. The amino acid other than lysine is selected from the group consisting of arginine, aspartic acid, glutamic acid, alanine, cysteine, phenylalanine, glycine, histidine, isoleucine, leucine, methionine, asparagine, proline, glutamine, serine, threonine, valine, tryptophan and tyrosine. The gene according to claim 15, which is at least one selected from the group consisting of
18. A recombinant vector comprising the gene according to any one of claims 14 to 17.
19. A transformant comprising the recombinant vector according to claim 18.
20. 20. The transformant according to claim 19, wherein the transformant is transformed Escherichia coli.
21. A method for producing the protein, comprising the step of culturing the transformant according to claim 19 or 20, and the step of collecting a protein having transglutaminase activity from the obtained culture.
22. The method according to claim 21, wherein the collecting step includes a protein purification step.

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