JPH0827180A - Separation of amino-terminated peptide of protein - Google Patents

Abstract

PURPOSE:To separate an N-terminated peptide of protein in a versatile way with a simple operation, by fragmenting and acetylating a protein, subjecting the fragments before and after the acetylation to reversed phase chromatography, and by recovering the fragments invariant in the eluting posi tion. CONSTITUTION:In separating the amino-terminated peptide' of a protein (e.g. calmodulin), the epsilon-amino group of the lysine in this protein is succinylated and protected, and the protein is subjected to an endopeptidase (e.g. alpha- chymotrypsin) reaction into fragments, which are then acetylated; the fragments, before and after the acetylation, are subjected to reversed phase chromatography, and the fragments invariant in the eluting position in the chromatography are recovered, thus efficiently separating the amino-terminated peptide of the protein with the amino terminal blocked, in a versatile way in a simple operation by such a means as to be applicable to various areas such as the genetic engineering requiring protein's partial structure determination.

Description

Detailed Description of the Invention

[0001]

The present invention relates to an amino terminal (hereinafter,
(Sometimes referred to as “N-terminal”), a method for separating an amino-terminal peptide of a blocked protein. INDUSTRIAL APPLICABILITY The present invention can be used in various fields, such as genetic engineering, in which partial structure determination of proteins is required.

[0002]

2. Description of the Related Art Determining the N-terminal amino acid peripheral sequence of a protein has a very important meaning at present. The Edman degradation method has already been established and widely used as an amino acid sequence analysis method from the N-terminus. However, it is said that 80% or more of the soluble proteins derived from eukaryotic cells have the N-terminal α-amino group blocked by an acetyl group, a formyl group or other groups. It is not possible to analyze end block proteins.

Groups that block the N-terminal of proteins include formyl group, acetyl group, myristoyl group,
Dimethyl group, glucuronyl group, glycosyl group, pyroglutamyl group, trimethyl group, pyrrolidone carboxyl group and the like have been reported. Acyl amino acid releasing enzyme (Acila amino acid free enzyme (Acila
mino acid releasing enzyme) and DIT
A method using C-glass is known.

Among these, the method of Hirano et al. (Hirano, H., Ko
matsu, S., Takakura, H., Sakiyama, F., Tunazawa, S. (1992)
J. Biochem. 111, 754-757), a protein having an N-terminal blocked was immobilized on a membrane, an acid and an enzyme were sequentially acted on to remove the blocking group, and the amino acid sequence was analyzed from the N-terminal by the Edman degradation method. . The N-terminal blocking group removed is identified by thin layer chromatography. According to this method, only three types of the N-terminal blocking groups, the formyl group, the pyroglutamyl group, and the acetyl group, can be removed, and there is a drawback that the versatility is lacking. In addition, the enzyme used in this method has a problem that it may not act as a protein as it is.

In the method using an acylamino acid-releasing enzyme, the N-terminal blocked protein is fragmented by enzymatic digestion. An acylamino acid-releasing enzyme was allowed to act on this, and the pattern before and after the enzyme treatment was applied to reverse phase HP.
Compare by LC. Only the N-terminal fragment from which the N-terminal blocking group has been removed changes the elution position.
Identification of the terminal block peptide is performed. Since this method uses an acylamino acid-releasing enzyme that acts specifically on an acylamino group, other N-terminal blocked proteins cannot be analyzed, and they have the drawback of lacking versatility.

In the method using DITC glass (Kenichi Mitsunaga et al. (1993), The 44th Annual Meeting of the Protein Structure Discussion Group), the protein blocked at the N-terminus was fragmented with lysyl endopeptidase, and then carboxy was added thereto. Act on peptidase B to remove the lysine at the carboxy terminus. This step can also be replaced by a method in which the ε-amino group of lysine is modified and fragmented with an enzyme. This is treated with DITC glass, fragments other than the N-terminal block peptide are adsorbed on the DITC glass, and the N-terminal block peptide is recovered. However, peptides other than the N-terminal blocked peptide were completely
Since it is not adsorbed on ITC glass, peptides other than the N-terminal blocking peptide are also recovered. Therefore, it is necessary to biotinize the recovered peptide and screen the N-terminal fragment again with an avidin-immobilized column.
The N-terminal fragment isolated by this work is analyzed by MS or the like. This method has versatility, but has a problem that the number of steps is large and the operation is complicated.

[0007]

As described above, the method for analyzing the amino acid sequence of a protein in which the N-terminal is blocked has problems that it is not versatile, the number of steps is large, and the operation is complicated.

Accordingly, the object of the present invention is to have general versatility,
N of the N-terminal blocked protein is easy to operate
It is to provide a method for separating a terminal peptide.

[0009]

Means for Solving the Problems As a result of earnest research, the present inventors have found that, if necessary, after protecting the ε-amino group of lysine in a protein, the protein is fragmented and treated with acetylation. The present invention has been completed by finding that the above object can be achieved by utilizing the fact that the elution position on the reverse phase chromatogram does not change before and after the treatment with the N-terminal block peptide alone.

That is, according to the present invention, a step of fragmenting a protein, a step of acetylating the obtained fragment, and a fragment whose elution position in reverse phase chromatography does not change before and after the acetylation treatment are collected. A method for separating an amino-terminal peptide of a protein whose amino terminal is blocked is provided. Furthermore, the present invention provides a method which further comprises a step of protecting the ε-amino group of lysine in the protein prior to the step of fragmenting the protein in the above method.

The present invention will be described in detail below.

In the first step of the method of the present invention, the ε-amino group of lysine in the protein whose amino terminus is blocked is protected. The ε-amino group of lysine is preferably protected by succinylation, but is not limited thereto as long as it is a protection method that does not cause deprotection of the ε-amino group under acidic conditions. These methods are known per se, and specific methods for succinylation are described in detail in the Examples below. If it is known that the ε-amino group does not exist in the N-terminal fragment of the protein, the first
It is not necessary to perform the steps, and the following second step can be started.

In the second step of the method of the present invention, the protein in which the ε-amino group of lysine is protected in the first step is fragmented. The fragmentation method is not particularly limited, and may be a chemical or biological method. Among these, the method of treating with endopeptidase is preferable. Examples of preferable endopeptidases include, but are not limited to, α-chymotrypsin, trypsin, and Achromobacter protease I.

In the subsequent third step, part or all of the fragmented product obtained in the second step is acetylated. Since the ε-amino group of lysine is protected in the first step, the only remaining amino group is the N-terminal α-amino group of the internal peptide generated by fragmentation. Therefore, all fragments having an α-amino group at the N-terminus are acetylated,
Only the N-terminal fragment of the protein does not have an amino group because it is originally blocked at the N-terminal, and is not modified by this treatment. Thus, all but the N-terminal fragment of the protein is acetylated at its N-terminus and only the N-terminal fragment is unmodified.

Acetylation of the α-amino group at the N-terminal can be carried out using, for example, a mixture of acetic anhydride, pyridine and water. Specific examples of acetylation using a mixture of acetic anhydride, pyridine and water are described in the following examples.

In the third step, the fragments other than the N-terminal fragment of the protein are eluted on the reverse phase chromatogram before and after the acetylation treatment because the N-terminal α-amino group is acetylated to change the hydrophobicity. The position changes greatly. On the other hand, the N-terminal fragment of the protein is not modified and the elution position on the reverse phase chromatogram does not change before and after the acetylation treatment. From this, the identification and purification of the N-terminal fragment of the blocked protein can be easily performed.

Therefore, in the method of the present invention, the fragmented products before and after the acetylation treatment are subjected to reverse phase chromatography and the elution positions thereof are compared. This means that when only a part of the fragmentation product is acetylated in the third step,
It can be carried out by subjecting the fragmented product after the acetylation treatment and the fragmented product before the acetylation treatment to reverse phase chromatography and comparing the elution positions thereof. Also,
When acetylating the entire amount of the fragmentation product, reverse phase chromatography is performed prior to the acetylation treatment to check the elution position of each fragment, and then each fraction is acetylated and then subjected to the same conditions again. Reverse phase chromatography and compare the elution positions.

As described above, the method of the present invention comprises acetylating the α-amino group of a peptide fragment other than the N-terminal peptide to change its hydrophobicity and change the elution position on the reverse phase chromatogram. Since it is used, the conditions of the reverse phase chromatography are not limited at all, and any of the commonly used reverse phase chromatography can be adopted. For example, in the following Examples, ODS silica is used as a chromatography carrier and 0.1% trifluoroacetic acid aqueous solution-acetonitrile is used as a mobile phase, as widely used.
Needless to say, it is not limited to this.

The amino-terminal peptide can be recovered by recovering the fraction whose elution position on the reverse phase chromatogram did not change before and after the acetylation treatment.

The N-terminal fragment thus obtained can be analyzed by a conventional method to identify the N-terminal blocking group and determine the amino acid sequence. These are preferably carried out using MS, more preferably ESI / MS / MS.

[0021]

EFFECTS OF THE INVENTION According to the present invention, selective separation of an N-terminal fragment of a protein whose N-terminal is blocked can be carried out easily and reliably. Therefore, time and labor are reduced, and the N-terminal amino acid sequence analysis work becomes efficient. Also, the method of the present invention uses all blocked N
It is possible to separate the ends, and there is an advantage that it has versatility.

[0022]

EXAMPLES The present invention will be described more specifically below based on examples. However, the present invention is not limited to the following examples.

Examples 1 and 2 Horse cytochrome c was used as the N-terminal block protein.
(Manufactured by Sigma) and bovine brain calmodulin (provided by Dr. Isobe of Tokyo Metropolitan University) were used. The following treatment was performed for about 100 μg of protein. That is, the protein was dissolved in 1 ml of 0.5 M sodium carbonate buffer (pH 8.3, containing guanidine hydrochloride). Next, 100 μg of ground succinic anhydride was added to this in 3 portions. The solution was stirred at 50 ° C. for 3 hours. At this time, the pH was kept constant by adding sodium carbonate. Then Sephadex G-25 column (diameter 0.78 cm, length 3
0 cm, manufactured by Pharmacia) and used as a mobile phase.
The sample was desalted with 1M ammonium bicarbonate. The obtained sample was subjected to the following chymotrypsin treatment.

The succinylated protein (cytochrome c: 200 μg, calmodulin: 180 μg) obtained as described above was dissolved in 200 μl of 0.1 M ammonium carbonate solution. Next, TLCK-α-chymotrypsin was added to a concentration of 2% and reacted at 37 ° C. for 16 hours.

The chymotrypsin digestion product was fractionated by the following reverse phase chromatography, and each peptide was acetylated. That is, after adding acetic anhydride: pyridine: water = 30: 30: 5, the mixture was reacted at room temperature for 2 hours, then water was added at a ratio of 5 and the mixture was allowed to stand for 30 minutes. Each peptide after acetylation was subjected to reverse phase chromatography (TSKgelODS 80TM 0.46).
cm x 25 cm, Tosoh Corporation) and the elution positions before and after the acetylation treatment were compared. The mobile phase was a 0.1% trifluoroacetic acid aqueous solution and the mobile phase modifier was acetonitrile.

As an example, the results of chromatography on horse cytochrome c are shown in FIG. The lower part of Fig. 1 shows
It is a chromatogram about the chymotrypsin digestion product of the cytochrome c before acetylation processing, and 1 to 5 are the patterns obtained by acetylating and fractionating each fraction obtained by digesting chymotrypsin. That is, Fraction 4 contained an N-terminal fragment whose elution position did not change before and after the acetylation treatment. The other peptides in Fraction 4 are narrow peptides. As is clear from this, the N-terminal fragment was isolated by acetylating the digest of the protein with the N-terminal blocked. Similar results were obtained with calmodulin.

One part of the obtained single-peak peptide was
It was hydrolyzed at 55 ° C. for 20 minutes and subjected to amino acid analysis. As a result, the amino acid compositions of the already known N-terminal portions of the two proteins were in agreement. Sequence analysis from the N-terminal blocking group and the N-terminal was carried out by ESI / MS / MS, and it was in agreement with the already known data of each peptide.

[Brief description of drawings]

FIG. 1 N of horse cytochrome c identified by the present invention
It is a figure which shows the reverse fragment chromatogram of the terminal fragment (fraction 4) and the chymotrypsin digestion product before acetylation processing.

Claims (4)

[Claims] 1. A step of fragmenting a protein, a step of acetylating the obtained fragment, and a fragmentation product before and after the acetylation treatment are subjected to reverse phase chromatography to change the elution position in the reverse phase chromatography. A method for separating an amino-terminal peptide of an amino-terminal blocked protein, which comprises a step of recovering a fragment that does not exist. 2. The method according to claim 1, further comprising the step of protecting the ε-amino group of lysine in the protein prior to the step of fragmenting the protein. 3. The method according to claim 1, wherein the step of protecting the ε-amino group of lysine is performed by succinylation of the ε-amino group. 4. The method according to any one of claims 1 to 3, wherein the step of fragmenting the protein is carried out by reacting the protein with an endopeptidase.