JPH07165789A - Method for separating amino-terminal peptide of protein - Google Patents

Abstract

PURPOSE:To provide a general-purpose method for separating an N-terminal peptide of an N-terminal blocked protein, in which the operation is simply performed. CONSTITUTION:This method for separating an amino-terminal peptide of an amino-terminal blocked protein comprises a step for fragmenting the amino- terminal blocked protein, a step for reacting the resultant fragmented protein with an adsorbent capable of specifically adsorbing amino group and adsorbing only the fragment containing the amino group on the adsorbent and a step for recovering the amino-terminal fragment unadsorbed on the adsorbent.

Description

Detailed Description of the Invention

[0001]

The present invention relates to an amino terminal (hereinafter,
A method for separating an amino-terminal peptide of a protein whose “N-terminal” is sometimes blocked. 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, 80% of eukaryotic cell-derived soluble proteins contain α at the N-terminus.
-The amino group is blocked by a formyl group, an acetyl group or other groups, and the Edman degradation method cannot analyze these N-terminal blocked proteins.

Groups that block the N-terminal of soluble proteins derived from eukaryotic cells include formyl group, acetyl group, myristyl group, glucuronyl group, glycosyl group, pyroglutamyl group, dimethyl group, trimethyl group, aminoacyl group, Pyrrolidone carboxyl group and the like have been reported. N-terminal analysis methods for these blocked proteins include the method of Tsunazawa et al.
A method using releasing enzyme) and a method using DITC glass are known.

Of these, the method of Tsunazawa et al. (Hirano,
H., Komatsu, S., Takakura, H., Sakiyama, F and Tun
asawa, S. (1992) J. Biochem. 111, 754-757), N
Immobilize the blocked protein on the membrane,
The block group is removed by sequentially acting an acid and an enzyme, and the amino acid sequence is analyzed from the N terminus by the Edman degradation method. The N-terminal blocking group removed is identified by thin layer chromatography. With this method, among the above N-terminal blocking groups, only three types of blocking groups, a formyl group, a pyroglutamyl group, and an acetyl group, can be removed, and there is a drawback that the method is not versatile. In addition, the enzyme used in this method has a problem that it may not act as a protein as it is.

Further, in the method using an amino acid-releasing enzyme, a protein whose N-terminal is blocked is denatured and then fragmented by enzymatic digestion. An amino acid-releasing enzyme is allowed to act on this, and the patterns before and after the enzyme treatment are compared by reverse phase HPLC. The N-terminal blocking peptide is identified by utilizing the fact that only the N-terminal fragment from which the N-terminal blocking group has been removed changes the elution position. The isolated N-terminal blocked peptide is deblocked and then subjected to amino acid sequence analysis by the Edman degradation method. Since this method uses an amino acid-releasing enzyme that acts specifically on an aminoacyl group, other N-terminal blocked proteins cannot be analyzed, and they have the drawback of lacking versatility.

[0006] In the method using DITC glass (Kenichi Mitsunaga et al. (1993), Summary of the 44th Annual Meeting of Protein Structure Discussion), N-terminal blocked protein is fragmented with lysyl endopeptidase and then carboxypeptidase is added thereto. B is allowed to act 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, and the fragments other than the N-terminal blocking peptide are adsorbed on DITC glass,
The N-terminal block peptide is recovered. However, N
Fragments other than end-blocking peptides are completely DI
Since it is not adsorbed on TC 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 avidinylated immobilization 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 terminal peptides.

[0009]

As a result of earnest research, the inventors of the present invention have found that after protecting the ε-amino group of lysine in a protein, the protein is fragmented and this is an adsorbent that reacts specifically with the amino group. The present invention has been completed by finding that the above-mentioned object can be achieved by causing the adsorbent to adsorb fragments other than the N-terminal peptide fragment to the adsorbent.

That is, in the present invention, the step of fragmenting a protein whose amino terminus is blocked, and the resulting fragmented product are allowed to act on an adsorbent that specifically adsorbs an amino group, so that only the amino group-containing fragment is present. Provided is a method for separating an amino-terminal peptide of a protein whose amino terminal is blocked, which comprises a step of adsorbing to the adsorbent and a step of recovering an amino-terminal fragment not adsorbed to the adsorbent.

The present invention will be described in detail below.

First, the ε-amino group of lysine in a protein whose amino terminus is blocked is protected. Lysine ε
The -amino group can be protected by, for example, a method of succinylating an ε-amino group, a method of converting to a maleyl group, a method of converting to a citraconyl group, or a protection with a Braunitour reagent. These methods are known per se, and specific methods for succinylation are described in detail in the Examples below. In addition, N whose structure should be determined by amino acid analysis, etc.
If it is known in advance that the terminal fragment does not contain lysine, this step need not be performed, and the step described below may be started.

Then, in the previous step, the protein in which the ε-amino group of lysine is protected is fragmented. When the previous step is not performed, this fragmentation step is the first step. The fragmentation method is not particularly limited, and it can be performed by a chemical or biological method. Among these, the method of treating with endopeptidase is preferable. Preferred examples of endopeptidases include, but are not limited to, α-chymotrypsin, trypsin, V8 enzyme, pepsin and thermolysin.

In the subsequent step, the fragmented product obtained in the previous step is allowed to act on an adsorbent that specifically adsorbs an amino group,
Only the amino group-containing fragment is adsorbed on the adsorbent. When protecting the ε-amino of lysine in the first step, the remaining amino group is the α at the N-terminus of each fragment.
-Only amino groups. Therefore, all fragments having an α-amino group at the N-terminus are adsorbed on the adsorbent,
Since only the N-terminal fragment of the protein originally has an N-terminal blocked, it does not have an amino group and is not adsorbed to the adsorbent. Also, as mentioned above, protection of the ε-amino group of lysine can be omitted if it is known that the N-terminal fragment to be analyzed does not have lysine, but in this case it is present. The amino groups are only the N-terminal α-amino group of each fragment and the ε-amino group of lysine present in fragments other than the N-terminal fragment. Therefore, even in this case, similarly to the above, only the N-terminal fragment is not adsorbed, and the other fragments are adsorbed to the adsorbent. Thus, in any case, all the fragments except the N-terminal fragment of the protein are adsorbed by the adsorbent, and only the N-terminal fragment is not adsorbed.

As the adsorbent for specifically adsorbing amino groups, CNBr-activated agarose gel (for example, Sepharose B4 (trade name, manufactured by Pharmacia), periodate-activated agarose, epoxide-activated agarose, carboxyalkyl derivatized) Examples thereof include agarose, 2-fluoro-1-methylpyridine-activated agarose, etc. The carrier of the adsorbent may be silica gel, polyacrylamide gel, methacrylate gel, dextran gel, or cellulose in addition to the carrier using the above agarose. Etc. can be used, etc. The method of allowing the fragmentation product to act on the adsorbent is a method of column chromatography, that is,
It can be carried out by packing the adsorbent in a column and applying the fragmented product to the column. This operation can be performed by, for example, plugging the tip of the syringe with a cotton plug or the like, inserting the adsorbent into the syringe, adding a buffer solution and fragmentation product, and reacting with the adsorbent. The adsorbent is C
Specific examples in the case of NBr-activated agarose gel are described in detail in the examples below.

[0016] In the previous step, fragments other than the N-terminal fragment of the protein are adsorbed to the adsorbent, so that the desired N-terminal fragment can be recovered by recovering the fragment not adsorbed on the adsorbent. it can. This can be performed by collecting the flow-through fraction when the adsorbent is used as the adsorbent for the column chromatography. In particular, in the case of filling the adsorbent in the syringe described above, it can be easily performed by pressing the plunger and collecting the liquid discharged from the syringe.

The N-terminal fragment thus recovered can be analyzed by a conventional method to identify the N-terminal blocking group and determine the amino acid sequence. These are preferably performed using MS, more preferably MSI-MS-MS. In addition, prior to these analyzes
Desalting by liquid chromatography is preferred.

[0018]

EFFECTS OF THE INVENTION According to the present invention, selective separation of the 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 is made efficient. In addition, the method of the present invention is capable of separating all blocked N-terminals, and has the advantage of versatility.

[0019]

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 to 3 As the N-terminal block protein, horse cytochrome C
(Manufactured by Sigma), bovine brain calmodulin (provided by Dr. Isobe of Tokyo Metropolitan University), or ovalbumin (manufactured by Sigma) was used. The following treatment was performed for about 50 μg of protein. That is, the protein was dissolved in 0.5 ml of 0.5 M sodium carbonate buffer (pH 8.3, containing guanidine hydrochloride). Then, succinic anhydride 10 mashed with this
0 μg was added 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-
The solution was desalted using 25 columns (diameter 0.78 cm, length 30 cm, manufactured by Pharmacia) and 0.1 M ammonium hydrogen carbonate as a mobile phase. The obtained sample was subjected to the following chymotrypsin treatment.

200 succinylated proteins (cytochrome C: 183 μg, calmodulin: 246 μg, ovalbumin: 519 μg) obtained as described above were used.
It was dissolved in μl of 0.1 M sodium carbonate solution. Then TLK-α-chymotrypsin was added to the 2% concentration and 3
The reaction was carried out at 7 ° C for 16 hours.

About 200 μg of CNBr-activated agarose gel (Sepharose B4, manufactured by Pharmacia) was applied to 1c.
A mx5 cm disposable syringe was filled. The tip of the syringe was covered with a cotton plug. This syringe was used as a reaction container. The gel filled in the syringe was washed with 40 ml of 1 mM hydrochloric acid, and about 200 μg of the sample dissolved in 200 μl of 0.1 M sodium hydrogen carbonate buffer (pH 8.3) was applied to the syringe. While rotating the syringe at 5 rpm,
The reaction was carried out at room temperature for about 2 hours. The plunger of the syringe was pressed to recover about 200 μl of solution. 1 more gel
Rinse with ml of the same buffer. Reverse phase chromatography (TSK gel ODS 80TM (0.46 cm x 25 c
m, manufactured by Tosoh Corporation). The mobile phase was 0.1% trifluoroacetic acid and the mobile phase modifier was acetonitrile.

As an example, the chromatographic results for ovalbumin are shown in FIG. The lower part of Fig. 1 is CN
It is a chromatogram about the chymotrypsin digestion material before a Br activation agarose process, and the upper part is a result about the liquid after a CNBr activation agarose process. As is apparent from this, CNBr-activated agarose treatment separated a single peptide. Similar results were obtained for the other two types of proteins.

1 part of the obtained single-peak peptide
It was hydrolyzed at 10 ° C. for 6 hours and subjected to amino acid analysis. As a result, it was in agreement with the already known amino acid composition of the N-terminal portion of each protein. Furthermore, ESI-MS-MS
When sequence analysis was performed from the N-terminal blocking group and the N-terminal, each peptide was in agreement with the already known data.

[Brief description of drawings]

FIG. 1 shows a reverse-phase chromatogram of ovalbumin separated by the method of the present invention and a reverse-phase chromatogram of a chymotrypsin digestion product before treatment with CNBr-activated agarose gel.

Claims (4)

[Claims] 1. A step of fragmenting a protein whose amino terminal is blocked, and the obtained fragmentation product is allowed to act on an adsorbent that specifically adsorbs an amino group, and only the amino group-containing fragment is adsorbed on the adsorbent. A method for separating an amino-terminal peptide of a protein whose amino terminal is blocked, which comprises a step of adsorbing and a step of recovering an amino-terminal fragment not adsorbed by the adsorbent. 2. The method according to claim 1, wherein the adsorbent that specifically adsorbs the amino group is a CNBr-activated agarose gel. 3. The method according to claim 1 or 2, further comprising a step of protecting the ε-amino group of lysine in the protein prior to the step of fragmenting the protein whose amino terminus is blocked. 4. The method according to claim 3, wherein the step of protecting the ε-amino group of lysine is performed by succinylation of the ε-amino group.