JP2001321192A - Method for preparing protein sample for structural analysis - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for preparing a protein sample for the structural analysis suitable for the structural analysis of a protein. SOLUTION: This method for preparing a protein sample for the structural analysis comprises (i) reacting a protein with a compound containing a primary amino group by the action of a transglutaminase and/or (ii) reacting a protein with a peptide containing a glutamine residue by the action of a transglutaminase.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a method for preparing a protein sample for structural analysis. More specifically, the present invention relates to a method for preparing a protein sample suitable for analyzing a three-dimensional structure by improving the physical properties of a protein.

[0002]

2. Description of the Related Art X-rays and NMR are often used for structural analysis of proteins, but the former analyzes the solubility and crystallinity of the sample protein, while the latter analyzes the solubility of the protein. And high stability. For the purpose of preparing a protein sample suitable for performing such a structural analysis, there have been reported several methods for gently improving the physical properties of a protein. For example, in crystal structure analysis, a method of binding an antibody or incorporating a lipid to avoid association between hydrophobic surfaces of a membrane protein (Iwata, S. et al. (1995) Nature 376, 660. Shin
zawa-Itoh, K. etal. (1995) J. Mol. Biol. 246, 57
2.) and the like. In the NMR analysis, a method for preventing protein association using a solvent containing a surfactant or a salt (Anglister, J. et al. (1993) J. Biomol. NMR 3, 12
1. Zhang, O. et al. (1995) Biochemistry 34, 678.
4.) and so on. However, since these conditions are different from physiological conditions, there is a problem that the structure in a living body may not be reflected. Further, it is necessary to examine the dissolution conditions and crystallization conditions by trial and error depending on the properties of the protein, and there has been a demand for the development of a method having a wide range of application to save labor and time. At present, when protein engineering or drug design is performed based on structural analysis, it is significant to prepare a protein sample for structural analysis with improved physical properties as described above.

One method for improving the physical properties of a protein for the purpose of preparing a protein sample for structural analysis includes site-specific mutation. However, this method has a problem that it is necessary to construct an expression system using individual proteins, and that only a carboxamide group or a hydroxyl group, which is a functional group unique to an amino acid residue, can be introduced. For example, solubility can be expected to be improved by introduction of a sulfonyl group or a phosphate group, but these functional groups cannot be introduced by site-specific mutation. It is possible to introduce a functional group other than the amino acid residue by modification by a chemical reaction.However, in this case, it is necessary to avoid protein denaturation and side reactions, and the types of functional groups that can be introduced are limited. Have been.

[0004] On the other hand, transglutaminase is an enzyme that catalyzes an acyl transfer reaction of a γ-carboxamide group in a peptide chain of a protein. When this enzyme is allowed to act on a protein, an ε- (γ-Glu) -Lys crosslink formation reaction,
A substitution reaction with a glutamic acid residue by deamidation of the glutamine residue may occur. Transglutaminase is
It is widely used for the production of foods such as gelatin, cheese, yogurt, tofu, kamaboko, ham, sausage, noodles, etc. and for improving meat quality (Japanese Patent Laid-Open No. 64-27471). In addition, it is known as an enzyme having high industrial utility, such as being used for the production of heat-stable microcapsule materials and carriers for immobilized enzymes. Nevertheless, the industrial application of transglutaminase has been limited to the fields of gelation associated with protein cross-linking and suppression of the associated metabolism and degradation. For research applications, transglutaminase activity is evaluated by introducing a fluorescent or chelating reagent, or a carrier is bound to a column resin, and is used only for very limited purposes. Was.

[0005]

SUMMARY OF THE INVENTION An object of the present invention is to provide a method for preparing a protein sample for structural analysis suitable for structural analysis of a protein.

[0006]

Means for Solving the Problems The present inventors can change the physical properties of a protein without significantly changing its structure by modifying a glutamine residue and / or a lysine residue in a protein molecule with a specific compound. Focusing on that, furthermore, as a result of intensive study to solve the above problems,
When the glutamine residue of a protein is modified with a compound containing a primary amino group using a catalytic reaction by transglutaminase and / or the lysine residue is modified with a peptide containing a glutamine residue, the physical properties of the protein are structurally analyzed. The present invention has been found that it can be improved to a suitable one and that a functional group containing a stable isotope can be introduced. That is, the present invention
(I) reacting a protein with a compound containing a primary amino group by the action of transglutaminase, and / or (ii) reacting the protein with a peptide containing a glutamine residue by the action of transglutaminase. This is a method for preparing a protein sample for structural analysis.

[0007]

DESCRIPTION OF THE PREFERRED EMBODIMENTS Transglutaminase, an enzyme used in the present invention, is an enzyme that catalyzes an acyl transfer reaction of a γ-carboxamide group of a glutamine residue in a peptide chain of a protein. When this protein acts as an acyl receptor on the ε-amino group of a lysine residue in a protein, ε- (γ
-Glu) -Lys bond is formed.

The present invention includes a step of modifying a glutamine residue and / or a lysine residue of a protein by utilizing a transglutaminase-catalyzed reaction. FIG. 1 schematically shows an enzyme reaction mode. Formulas 1 and 3 show proteins containing a glutamine residue and a lysine residue, respectively, where R ₁ and R ₁ ′ are peptide chains, any of N-terminal amino acids or hydrogen, and R ₂ and R ₂ ′ are peptide chains. , C-terminal amino acid or hydroxyl group, and is not particularly limited as long as the protein can be a substrate for transglutaminase.
Formula 2 shows a compound containing a primary amino group on which transglutaminase acts, R ₃ is not limited, and specific examples of the compound containing a primary amino group shown in Formula 2 include hydroxylamine, glycine, Examples include lysine, aminoethyl hydrogen sulfate, aminoethyl phosphate, aminoethanol, and peptides containing lysine. Formula 4 is a peptide containing a glutamine residue on which transglutaminase acts, R ₃ ′ represents a peptide chain, any of an N-terminal amino acid or hydrogen, and R ₄ ′ represents a peptide chain, a C-terminal amino acid or a hydroxyl group, There is no particular limitation as long as the peptide serves as a substrate for transglutaminase. Equation 4
The peptide containing a glutamine residue shown in may be modified with a substituent such as a carbobenzoxy group, a sugar chain, a myristoyl group, or a phosphate group, and specific examples thereof include N-
Carbobenzoxy-L-glutamyl-glycine (N-carboben
zoxy-L-glutamyl-glycine (CBZ-Gln-Gly), N-carbobenzoxy-L-glutamyl-glutamyl-glycine (N-carbob
enzoxy-L-glutamyl-glutamyl-glycine) (CBZ-Gln-Gln-Gl
y), N-carbobenzoxy-L-glycyl-glycyl-glutamyl-glycine (N-carbobenzoxy-L-glycyl-glycyl-glu
tamyl-glycine) (CBZ-Gly-Gly-Gln-Gly).

The proteins to be modified in the method of the present invention include human plasma components such as albumin, immunoglobulin and blood coagulation factors; enzymes such as proteases and transferases; hormones such as growth hormone and erythropoietin; Cell growth factors; immune response regulators such as cell differentiation, induction and stimulation; cell-producing biologically active proteins such as monokines, cytokines and lymphokines; and the like. Regardless of their origin, these proteins may be of animal origin, plant origin, or microorganism origin. The protein may be a protein obtained by incorporating and expressing the genes of these proteins in Escherichia coli, yeast, animal cells, or the like, or a protein expressed by utilizing a cell-free protein synthesis system.

A protein modified by transglutaminase has at least one glutamine or lysine residue affected by transglutaminase in the molecule. Whether a glutamine residue or a lysine residue in a molecule is affected by transglutaminase can be confirmed, for example, by measuring the mass spectrum of the modified protein and detecting an increase in molecular weight accompanying the modification. Can be. When the protein does not have a glutamine residue or a lysine residue affected by transglutaminase, a glutamine residue or lysine residue affected by transglutaminase can be introduced by site-specific mutation.

There are two types of transglutaminase: calcium-independent and calcium-dependent, and any of them can be used in the present invention. Examples of the former include those derived from microorganisms such as actinomycetes and Bacillus subtilis (for example, see JP-A-64-27471). Examples of the latter include those derived from guinea pig liver (see, for example, Japanese Patent Publication No. 1-50382), human epidermal keratinocyte transglutaminase (Phillips, M. et al.
A. et al. (1990) Proc. Natl. Acad. Sci. USA 87,
9333.), human blood coagulation factor XIII (Ichinose, A. et a
l. (1990) Biochemistry 25, 6900.), those derived from microorganisms such as oomycetes, those derived from animals such as bovine blood and swine blood, those derived from fish such as salmon and red sea bream (for example, Journal of the Japanese Society of Fisheries Science, Vol. 56, pp. 125-132, 1990
Year), oysters, and the like. In addition, those produced by genetic recombination (for example, JP-A-1-300889, JP-A-6-225775)
See JP-A-7-23737. ) And the like.

[0012] In the present invention, any transglutaminase can be used, and there is no limitation on the source and the production method. However, some proteins whose properties are to be improved are not suitable for an enzymatic reaction in a solvent containing calcium. Therefore, it is preferable to use calcium-independent transglutaminase for such proteins. For example, transglutaminase derived from the above microorganism (see, for example, JP-A-64-27471) satisfies all of the conditions, and can be said to be optimal at present. For example, Streptoverticillium glyceocarneum (Streptoverticill
ium griseocarneum) IFO 12776, Streptoverticillium cinnamonium
eum subsp. cinnamoneum) IFO 12852, Streptoverticillium mo
baraense) transglutaminase derived from IFO 13819 and the like. The activity unit of the transglutaminase used in the present invention is measured and defined as follows. That is, a reaction is carried out using benzyloxycarbonyl-L-glutaminylglycine and hydroxylamine as substrates, and the generated hydroxamic acid is converted into an iron complex in the presence of trichloroacetic acid, and the amount is measured by absorbance at 525 nm. In this way, a calibration curve is created from the amount of hydroxamic acid, and the amount of enzyme that produces 1 μmol of hydroxamate per minute is defined as one unit of transglutaminase activity. The details of this measurement method have already been reported (for example, see JP-A-64-27471).

For structural analysis of proteins, X-rays, neutrons, or NMR are often used. High solubility and crystallinity are required for X-ray and neutron analysis, and high solubility and stability are required for NMR analysis. For those that do not satisfy these conditions, it is necessary to prepare an appropriate protein sample by improving the solubility, stability and crystallinity of the protein to be analyzed. Improving the solubility of a protein refers to increasing the number of moles of the protein that can be dissolved in the solvent and avoiding protein association. Crystallization necessary for X-ray or neutron crystallography is difficult with a low-concentration protein solution, and sufficient signal intensity cannot be obtained in NMR measurement. Furthermore, protein association causes broadening of the NMR signal, which hinders structural analysis. In crystal structure analysis and NMR analysis, it is desirable to prepare a protein solution having a concentration of 0.1 mM or more, more preferably a protein solution having a concentration of 0.5 mM or more. According to the present invention, a protein sample suitable for structural analysis can be prepared even from a protein having low solubility in a natural state.

[0014] The improvement of protein stability means that degradation and denaturation hardly occur, and various changes including association and exchange reactions accompanying changes in temperature, pH, salt concentration, and the like are hardly caused. . In NMR analysis, it is known that signal relaxation is reduced with an increase in rotation and translational motion of a protein molecule, and a spectrum that can be easily analyzed is obtained. Therefore, in order to increase the rotation and translational movement, there is a tendency to measure at a temperature as high as possible within a range where the protein is kept stable. Therefore, proteins that are stable to high temperatures are suitable for NMR analysis and can be targeted for structural analysis. When assigning the NMR signal of a protein, the amide proton signal is often used as a base axis. However, as the pH increases, the exchange rate with water increases, and the amide proton signal may not be observed. Therefore, for NMR structural analysis, it is desirable that the protein be stable in a wide pH range.

[0015] Improving the crystallinity of a protein means making it easier to crystallize the protein. The crystallinity of a protein depends on the distribution of charges on the surface of the protein molecule, the degree of exposure of the hydrophobic region, and the like. Therefore, it is possible to improve the crystallinity by changing the surface charge and the hydrophobicity, and use the crystal structure analysis. Further, in the structural analysis, it is preferable to use a compound containing a stable isotope as a modifying compound. For example, labeled compounds that modulate at ¹³ C, ¹³ can C edit or apply filters NMR experiments, it is possible to observe separate the NMR signal which has been duplicated. As a result, the structure information is more accurately extracted, which leads to a precise three-dimensional structure determination. The stable isotope introduced into the compound to be modified is ² H or ³ H for ¹ H, ¹³ C for ¹² C, ¹⁵ N for ¹⁴ N, and ¹⁷ O for ¹⁶ O. Or
¹⁸ O and the like. ¹ H, ¹² C, ^{14 for} natural proteins
Because of the high content of N and ¹⁶ O, these nuclides are often substituted with isotopes, but this method is not limited to isotope labeling of hydrogen, carbon, nitrogen and oxygen atoms. Absent.

In order to improve the solubility of the protein, it is desirable to modify the protein with a compound containing a hydrophilic functional group such as a carboxyl group, an amino group, a sulfonyl group or a phosphate group. As compounds of glycine, lysine, aminoethyl hydrogen sulfate, aminoethyl phosphate, amino group donors including polyethylene glycol and polysaccharides, lysine-containing peptides, etc., as the compound of formula 4, CBZ-Gln-Gly, CBZ-Gln -Gln-Gly, CBZ-Gly-Gl
y-Gln-Gly and the like. In order to improve the stability, for example, a compound containing a hydrophilic functional group such as a carboxyl group, an amino group, a sulfonyl group, a phosphate group or a compound containing a functional group serving as a donor or acceptor for hydrogen bonding It is desirable to modify proteins, and more specifically, compounds of formula 2 include hydroxylamine, glycine, lysine, aminoethyl hydrogen sulfate, aminoethyl phosphate, aminoethanol, peptides containing lysine, and the like.
Compounds of formula 4 include CBZ-Gln-Gly, CBZ-Gln-Gln-Gly, C
BZ-Gly-Gly-Gln-Gly and the like. Solubility and stability are not always achieved at the same time, but in general, stability often increases as solubility increases. More preferably, a compound is used which simultaneously improves both solubility and stability properties. In order to improve the crystallinity, it is desirable to change the surface charge and hydrophobicity of the protein to be modified.Examples of the compound of Formula 2 include hydroxyamine, glycine, an antibody, an antibody Fc fragment and a part thereof. No.

In order to react the protein to be modified with a compound containing a primary amino group or a peptide containing a glutamine residue in the presence of transglutaminase, the transglutaminase, the modification and the transglutaminase are used under the normal conditions of transglutaminase action. Protein and a compound containing a primary amino group or a peptide containing a glutamine residue may be allowed to coexist, for example, about pH 5.0 to about pH 9.0 in an aqueous solvent.
And more preferably in the range of about pH 6.0 to about pH 8.0,
Temperatures range from about 4 ° C to about 55 ° C, more preferably from about 25 ° C to
The protein to be modified, the compound containing a primary amino group or the peptide containing a glutamine residue, and the transglutaminase may be retained at about 40 ° C. Although the reaction time is not particularly limited, it is about 30 seconds to about 7 days, more preferably about 1 minute to about 2 days. In this reaction, the concentration of the protein to be modified is about 1 M to about 40 mM, and the concentration of the compound containing a primary amino group or the peptide containing a glutamine residue is about 10 M
The concentration is preferably in the range of μM to about 10 M, and the concentration of the compound containing a primary amino group or the peptide containing a glutamine residue is preferably at least 1 times, more preferably at least about 20 times, relative to the protein to be modified. . The amount of transglutaminase used is desirably in the range of about 10 nM to about 100 μM, which corresponds to about 0.01 to about 20 units per mmol of protein.

By causing transglutaminase to act on the protein, the protein is modified with a compound containing a primary amino group or a peptide containing a glutamine residue,
The physical properties of the protein are improved to those suitable for structural analysis, and as a result, a protein sample for structural analysis is prepared. The protein solubility and / or
Alternatively, structural analysis by NMR becomes possible with the improvement of stability, and furthermore, if the introduced compound is labeled with a stable isotope, it will lead to highly accurate structural analysis. On the other hand, improvement in solubility and / or crystallinity of a protein is useful for X-ray crystal structure analysis. The structural information obtained by both methods can be reflected in protein engineering or drug design.
For example, it becomes possible to solubilize a membrane protein such as a receptor in an aqueous solvent. An agonist or an antagonist can be designed based on the structural information of the solubilized receptor molecule for which NMR analysis and crystal structure analysis have become possible, and the drug can be developed into a novel drug. By using transglutaminase, both or one of glutamine residues and lysine residues can be modified, so that there is an advantage that various modified proteins can be prepared according to the sequence and shape of the protein. Furthermore, when a protein is modified with an enzyme, the functional group located on the surface of the protein is modified, so that it is considered that the three-dimensional structure is not easily damaged. This is a great advantage compared to a chemical modification method in which amino acid residues located inside the three-dimensional structure of a protein are modified. Hereinafter, the present invention will be described with reference to examples. Note that the present invention is not limited to the embodiments.

[0019]

EXAMPLES In the following examples, Streptov was used as the enzyme.
An attempt was made to improve the physical properties of proteins by using transglutaminase (hereinafter abbreviated as MTG) derived from erticillium, glycine as a primary amine, and introducing a charged carboxylic acid. Stable isotopes were also introduced by labeling the glycine nitrogen with ¹⁵ N (hereinafter referred to as ¹⁵ N-labeled glycine). FIG. 2 shows a schematic diagram of the reaction. Equation 5 in FIG. 2 shows a protein containing a glutamine residue, R
₁ is a peptide chain, either an N-terminal amino acid or hydrogen, R
₂ represents one of a peptide chain, a C-terminal amino acid and a hydroxyl group. The modification process or the ¹⁵ N labeling process by the action of transglutaminase is detected using NMR, and ¹ HN
MR measurement, ¹ H- ¹⁵ N HSQC measurement (Bodenhausen, G. & Ruben,
DJ (1980) Chem. Phys. Lett. 69, 185.) as needed. For each NMR measurement, for example, Bruker DMX-600
Using a spectrometer, 5% D ₂ O was added to the measurement sample for NMR lock to maintain the stability of the magnetic field. 20 mM sodium phosphate (pH 6) was used as the solvent, and the reaction temperature was 37 ° C.
And

Example 1 ( ¹⁵ N-labeled glycine of a model compound)
N-carboben as by modification) model Compounds of the protein to be modified down
zoxy-L-glutaminyl-glycine (hereinafter abbreviated as CBZ-Gln-Gly)
To follow the modification process by ¹⁵ N-labeled glycine. CBZ-Gln-Gly 2.0 mM, ¹⁵ N-labeled glycine 1
0.5 mM, the first ¹ H- ¹⁵ N HSQC spectra measured are mixed such that the MTG 4 [mu] M after 157 minutes, the same measurement was carried out every hour thereafter. Figure 3 shows the reaction time and spectrum. The time from mixing of each reagent to measurement corresponds to the transglutaminase reaction time. ^{For 1} H- ¹⁵ N HSQC measurement
^Since only a signal derived from ¹ H bound to ¹⁵ N is observed, the ¹⁵ N labeling process can be quantitatively followed.
However, it is difficult to completely eliminate the peaks derived from protons present at a high concentration.
The mark is added. A signal derived from ¹ H- ¹⁵ N was observed in the reaction product, indicating that the cross-linking reaction between the carboxamide nitrogen of the Gln residue and ¹⁵ N-labeled glycine was gradually progressing. From the above results, it was shown that modification with a compound containing a stable isotope is possible, and that the modification process can be traced using NMR. In addition, carboxylic acid is added to uncharged carboxamide by modifying glycine. The ability to add a newly charged functional group suggests that physical properties such as solubility and stability can be modified.

[0021] ¹⁵ N-labeled Example 2 (high molecular weight proteins
Bovine serum albumin ( modified with glycine) as a high molecular weight protein
bumin, bovine, hereinafter abbreviated as BSA) and modified with ¹⁵ N-labeled glycine. BSA was mixed at 2.4 mM, ¹⁵ N-labeled glycine at 127 mM, and MTG at 32 μM, incubated for 2 days, and then subjected to ¹ H- ¹⁵ N HSQC measurement. ¹ H- ¹⁵ N
Is observed one correlation peak on HSQC spectrum, less even if one ¹⁵ N-labeled glycine was found that bound to the glutamine residue of BSA (Figure 4).

Example 3 (Solubility by protein modification)
And stability improvement) The sample described in Example 2 (hereinafter abbreviated as glycinated BSA) and BSA
Were compared. In order to make the target sample the same conditions except that MTG was not added, BSA 2.4 mM, ¹⁵ N
The one in which only labeled glycine 127 mM was incubated for 2 days was used. Because of the high solubility and stability of BSA at room temperature, the temperature was increased to create a state where BSA could easily associate. In NMR analysis, it is known that signal relaxation is reduced with an increase in rotation and translational movement of a protein molecule, and a spectrum that can be easily analyzed is obtained. Therefore, in order to increase the rotation and translational movement, there is a tendency to measure at a temperature as high as possible within a range where the protein is kept stable. Thus, the improved solubility and stability under high temperature conditions is due to NM
Preferable for R analysis and works favorably for structural analysis.

When 5 μl of both samples were incubated at 72 ° C. for 10 minutes, a white precipitate was formed. This was suspended in 995 μl of a phosphate buffer, centrifuged at 15,000 rpm for 3 minutes, and the absorbance of the supernatant was compared at ₂₈₀ nm (hereinafter abbreviated as OD ₂₈₀ ). The concentration values of higher OD _{28 0} is increased, which indicates that excellent solubility and stability. OD ₂₈₀ = 0.459 0.004 for glycinated BSA, OD ₂₈₀ = 0 for BSA.
The value was 366 0.006, indicating that the glycinated BSA retained a higher concentration, and that modification with glycine improved the solubility and stability. In addition,
As the measurement results, an average value and a standard deviation of two experiments are described.

Example 4 (Effects on the three-dimensional structure of proteins )
Influence of modification) In order to compare the steric structure of glycinated BSA described in Example 2 with that of BSA, ¹ H-NMR spectrum was measured (FIG. 5A,
B). The measurement temperature is 37 ° C. Peaks derived from unknown low molecular weight compounds contained in purchased BSA (Sigma product)
At 3.70 ppm, a peak derived from unreacted glycine alpha proton was observed at 3.85 ppm, a peak derived from water was observed at 4.70 ppm, and other signals were derived from glycinated BSA (FIG. 5A) or BSA (FIG. 5B). It is. Most of the signals, including the signal near 0 ppm characteristic of proteins having a higher-order structure, have not undergone a chemical shift change due to glycation. Therefore, BSA
It was found that glycine could be modified without losing the three-dimensional structure of.

[0025]

According to the present invention, any protein and 1
By acting transglutaminase on a peptide containing a secondary amine or a glutamine residue, the protein is modified without impairing its original three-dimensional structure, and a protein sample for structural analysis suitable for three-dimensional structure analysis is prepared. be able to. Furthermore, by using a compound containing a stable isotope as a compound to be modified, highly accurate structural analysis can be performed.

[Brief description of the drawings]

FIG. 1 shows a reaction mode in a modification step of a protein glutamine residue and a lysine residue.

FIG. 2 shows a reaction mode of a step of modifying ¹⁵ N glycine to a glutamine residue.

FIG. 3. N-Carbobenzoxy-L- with ¹⁵ N-labeled glycine
¹ H- ¹⁵ N HSQ in the modification process of glutaminyl Lou glycine
It shows the change with time of the C spectrum.

FIG. 4. ¹ H- of BSA modified with ¹⁵ N-labeled glycine
A ¹⁵ N HSQC spectrum.

FIG. 5 (A) is a ¹ H-NMR spectrum of BSA. (B)
^{1 is} a ¹ H-NMR spectrum of BSA modified with ¹⁵ N-labeled glycine.

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[Procedure amendment]

[Submission date] June 13, 2001 (2001.6.1
3)

[Procedure amendment 1]

[Document name to be amended] Statement

[Correction target item name] Claim 5

[Correction method] Change

[Correction contents]

[Procedure amendment 2]

[Document name to be amended] Statement

[Correction target item name] 0008

[Correction method] Change

[Correction contents]

The present invention includes a step of modifying a glutamine residue and / or a lysine residue of a protein by utilizing a transglutaminase-catalyzed reaction. FIG. 1 schematically shows an enzyme reaction mode. Formulas 1 and 3 show proteins containing a glutamine residue and a lysine residue, respectively, where R ₁ and R ₁ ′ are peptide chains, any of N-terminal amino acids or hydrogen, and R ₂ and R ₂ ′ are peptide chains. , C-terminal amino acid or hydroxyl group, and is not particularly limited as long as the protein can be a substrate for transglutaminase.
Formula 2 shows a compound containing a primary amino group on which transglutaminase acts, R ₃ is not limited, and specific examples of the compound containing a primary amino group shown in Formula 2 include hydroxylamine, glycine, Examples include lysine, aminoethyl hydrogen sulfate, aminoethyl phosphate, aminoethanol, and peptides containing lysine. Formula 4 is a peptide containing a glutamine residue on which transglutaminase acts, R ₃ ′ represents a peptide chain, any of an N-terminal amino acid or hydrogen, and R ₄ ′ represents a peptide chain, a C-terminal amino acid or a hydroxyl group, There is no particular limitation as long as the peptide serves as a substrate for transglutaminase. Equation 4
The peptide containing a glutamine residue shown in may be modified with a substituent such as a carbobenzoxy group, a sugar chain, a myristoyl group, or a phosphate group, and specific examples thereof include N-
Carbobenzoxy-L- glutaminyl -glycine (N-carbob
enzoxy-L- glutaminyl- glycine) (CBZ-Gln-Gly), N-carbobenzoxy-L- glutaminyl- glycine (N-carbobenzoxy-L- glutaminyl-L-glutaminyl- glycin)
e) (CBZ-Gln-Gln-Gly), N-carbobenzoxy- glycyl-
Glycyl -L-glutaminyl -glycine (N-carbobenzoxy- gl
ycyl-glycyl-L-glutaminyl- glycine) (CBZ-Gly-Gly-Gln
-Gly). <Sequence list free text> SEQ ID NO: 1: Transglutaminase substrate

[Procedure amendment 3]

[Document name to be amended] Statement

[Correction target item name] 0025

[Correction method] Change

[Correction contents]

[0025]

According to the present invention, any protein and 1
By acting transglutaminase on a peptide containing a secondary amine or a glutamine residue, the protein is modified without impairing its original three-dimensional structure, and a protein sample for structural analysis suitable for three-dimensional structure analysis is prepared. be able to. Furthermore, by using a compound containing a stable isotope as a compound to be modified, highly accurate structural analysis can be performed.

[Sequence List] SEQUENCE LISTING <110> Ajinomoto CO., Inc. <120> A method of preparing proteins for structural analysis <130> Y1H0417 <140> 2000-141151 <141> 2000-05-15 <160> 1 <170 > PatentIn Ver. 2.1 <210> 1 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: substrate for transglutaminase <220> <221> MOD_RES <222> (1) < 223> Xaa is N-carbobenzoxy-glycyl residue <400> 1 Xaa Gly Gln Gly 1

 ────────────────────────────────────────────────── ─── Continued on the front page (72) Inventor Keiichi Yokoyama 1-1 Suzuki-cho, Kawasaki-ku, Kawasaki-shi, Kanagawa Prefecture Ajinomoto Co. Central Research Laboratory F-term (reference) 2G045 AA40 BB60 DA36 FA40 4B064 AG01 BH04 CA21 CB30 CD13 CD20 DA13 4H045 AA20 BA11 BA13 DA89 EA50 FA70

Claims (6)

[Claims] (I) reacting a protein with a compound containing a primary amino group by the action of transglutaminase; and / or (ii) reacting a protein with a peptide containing a glutamine residue by the action of transglutaminase. A method for preparing a protein sample for structural analysis, comprising: 2. The method according to claim 1, wherein the compound containing a primary amino group and / or the peptide containing a glutamine residue contains a stable isotope. 3. The compound according to claim 1, wherein the compound containing a primary amino group further contains a hydrophilic functional group or a functional group serving as a hydrogen bond donor or acceptor.
The method described in. 4. A compound containing a primary amino group comprising hydroxylamine, glycine, lysine, aminoethyl hydrogen sulfate,
The method according to any one of claims 1 to 3, wherein the method is selected from the group consisting of peptides containing aminoethyl phosphate, aminoethanol and lysine. 5. A peptide comprising a glutamine residue, wherein the peptide comprises N-carbobenzoxy-L-glutamyl-glycine, N-carbobenzoxy-L-glycyl-glycyl-glutamyl-glycine and
The method according to any one of claims 1 to 4, wherein the method is selected from the group consisting of N-carbobenzoxy-L-glycyl-glycyl-glutamyl-glycine. 6. The method according to claim 1, wherein the structural analysis is an NMR structural analysis or a crystal structure analysis.