JPH0413698A - Bioactive peptide - Google Patents

Abstract

NEW MATERIAL:A bioactive peptide having following physicochemical properties. molecular weight: about 7900 measured by SDS electrophoresis and about 8500 measured by HPLC, isoelectric point: 7.00, bioactivity: having chymotrypsin- inhibiting activity. USE:Used as a chymotrypsin inhibitor having pH-stability and heat-stability. PREPARATION:For instance, blood plasma is isolated from blood in larva of domesticated silkworm Bombyx mori k03 by centrifuge and ammonium sulfate is added to the blood plasma, then precipitate precipitated at 30-70% saturation is centrifuged, thus resultant precipitate is dissolved in 0.05M tris-hydrochloric acid buffer solution (pH6.8) and resultant buffer solution is dialyzed, then subjected to anion-exchange column chromatography, thus non-adsorbed fraction is collected and purified by subjecting to affinity column chromatography using chymotrypsin-combining column to afford the aimed bioactive peptide having chymotrypsin-inhibiting activity.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Field of Application] The present invention relates to a physiologically active peptide that exhibits chymotrybusin inhibitory activity.

[Prior art] When separating and purifying various substances having peptide bonds from microorganisms, various organs, culture media, etc., or when culturing microorganisms to produce proteins, etc., and when producing various physiological activities for living organisms. When working with peptides, it is often experienced that the presence of protease reduces the yield of the protein, or that the target protein is not obtained at all. Therefore, in such cases, protease activity may be suppressed by raising or lowering the temperature or adding salt, etc.
Or, something like deactivating it is done. However, with these methods, it is inevitable that the target substance, which is a protein with peptide bonds like protease, will be affected in some way, and for example, when microorganisms are made to make proteins, the growth of microorganisms is often inhibited. .

[Problem to be solved by the invention] To solve the above problem, it is possible to use an inhibitor that acts only on protease, that is, a protease inhibitor [Means for solving the fJ problem] Bioactive peptide of the present invention The gist is that it has the following physical and chemical properties.

Molecular weight: Approximately 7.900H as measured by SDS electrophoresis
Approximately 8,500 as measured by PLC Isoelectric point: 7. o.

Physiological activity: Has chymotrybusin inhibitory activity [Effects and Examples] The physiologically active substance of the present invention is derived from Bombyx morik0, a strain of Japanese silkworm Bombyx morik0, which is maintained by the Oaza Japanese Silkworm Genetic Resource Center.
It was able to be purified from the plasma of No. 3, and can be identified by the following physical and chemical properties.

1) Molecular weight: approximately 7,900° as measured by SDS polyacrylamide gel electrophoresis; approximately a, soo as measured by HPLC.

2) Isoelectric point: 7.00 3) Predicted primary structure of 20 amino acids from the N-terminus: ^5p
-Glu-Pro-Thr-Thr-Lys-Pro-
Phe-Cys-Glu-Gln-Ala-Phe-G
ly-^5p-Cys-Gly-Thr-Pro-Ty
r-Physiological activity: Indicates chymotrybusin inhibitory activity.

(K i =0.17MM using casein as a substrate) Furthermore, as shown in the examples, it also has excellent thermal stability and pH stability. It is also expected to have physiological activities other than chymotrybusin inhibitory activity.

The physiologically active peptide of the present invention (hereinafter abbreviated as Cl-2) can be obtained by using domestic silkworm plasma as a raw material and purifying it by a conventional method. For example, after salting out ammonium sulfate, it can be purified to a high degree of purity by combining DEAE or CM-ion exchange chromatography and affinity chromatography using chymotrivcin. Furthermore, since the primary structure of amino acids from the N-terminus has been determined, it is now possible to extract (isolate) the gene, and it is also possible to mass-produce it by applying genetic recombination technology.

Hereinafter, a detailed description will be given based on examples.

Example 1 Purification Method Plasma was collected on the 3rd day of the 5th instar of larvae of ``patch'' υ-μxri k03, and blood cells were removed by centrifugation.

Ammonium sulfate was added to the above domestic silkworm plasma 20a + 1 to 30
The precipitate, which settled at ~70% saturation, was centrifuged. A small amount of 0.05M )-lis-hydrochloric acid solution (pH a, a) was added to the obtained precipitate to dissolve it, and then dialyzed against the same solution.

Ion exchange column chromatography DEAE-5e equilibrated with the above Tris-HCl buffer
The active fraction obtained by ammonium sulfate fractionation was passed through a phadex A50 column (manufactured by Pharmacia) (2 x 40 cm), washed with the same Il street solution, and the non-adsorbed (or passed through) fraction was injected with the active fraction of Cl-2. I got my share. The elution pattern is shown in Figure 1. After combining and concentrating the active fractions, 0.05M sodium phosphate 1! Dialysis was performed against a buffer solution (p) 17.4), and a supernatant was obtained by centrifugation.

Affinity column chromatography Chymotrivucin binding 5e equilibrated with sodium phosphate buffer
The supernatant was passed through a Pharose 4B (Pharmacia) column (1xlOcm) and washed with the same II street solution. 0.1M sodium acetate buffer ()adjusted to pH 0 with ICI, O,SM NaCl and 4MUre
(containing a) to obtain purified Cl-2. The elution pattern is shown in Figure 2.

Table 1 shows the volume, total protein amount, specific activity, total chymotrybusin inhibitory activity, and recovery rate at each purification step. Activity staining was performed using the following method.

Experimental method> Measurement of chymotrybusin inhibitory activity using the method of Kunitz et al. (J, Gen, Physiol,
30, 291-310° 1947).

Add 0.1 ml of the sample solution to 0.22 ml of chymotrivcin solution (200 pool) and incubate at 37°C for 10 minutes, then add H dissolved in the buffer solution in advance.
aw+marsten casein at a final concentration of 30 mg
/ml. The amount of reaction liquid at this time was 0.3
It was set to 2m1. After reacting at 37°C for 10 minutes, trichloroacetic acid was added to a final concentration of 5% to stop the reaction. After centrifuging the reaction solution, the absorbance at 280 nm (A2♂0) of the supernatant was measured. . Similar measurements were made for those without the sample solution added, and when A2a without the sample solution was added and A266 with A1 added was B, the inhibitory activity was expressed as (A-B)/AX100. That is, the amount of activity that inhibits 40 pmole of chymotrivcin by 50% under the above reaction conditions was defined as 1 unit (u) of chymotrivcin inhibitor.

Acrylamide gel strain Davis, B. J., and Ornstein, L.
et al.'s method (^nn.

N, Y, ^cad, sc1.121. Art 2,32
1-349, 404-427.1964).

Activity staining Llriel and Berges method (Nature
218, 57B-580° 1968).

In this method, the areas that show chymotrybusin activity are stained, so the areas that are not stained, that is, the areas where chymotrybusin inhibitors are present, are not active and come out white, making it clear where chymotrivcin inhibitors are present. .

As shown in Table 1, the specific activity was purified to about 407 times with a recovery rate of about 5% from the raw material. The specific activity of the purified product was 11,070 u/a+g protein.

Example 2 Physicochemical Properties> The following analyzes and tests were conducted using the purified product obtained in Example 1.

1 Sat 1 1) Measurement by SDS-polyacrylamide gel electrophoresis Laeo+m1i method (Nature, 227; 68
Figure 3 shows the results of 12.5% gelsDs electrophoresis using 0.0,1970), which revealed that the molecular weight in the reduced state was approximately 7,900.

2) Measurement TSKgal by HPLC (non-reduced state)
l+3000 SW [Manufactured by Tosoh ■, column (8 x 30
0+m)] is shown in FIG. 4. The molecular weight in a non-reduced state (natural state) was found to be about 8.500.

From the above results, it is estimated that Cl-2 is a monomer.

Mpholine (manufactured by Pharmacia) (2%v
Isoelectric focusing gel electrophoresis with a pH gradient of 3.5-10 was performed using a 6.4% polyacrylamide gel (300 V, 5 hr) using 1 V), and the migration position was determined by activity staining. On the other hand, another gel that was electrophoresed in a 5 mm
Each sample was sliced, the pH was measured, and the isoelectric point of the chymotrybusin inhibitor was determined from the electrophoresis position. The results are shown in FIG. 5, and it was found that the first isoelectric point was 7.0.

The primary structure was determined by primary tank Edman degradation of 20 amino acids from the N-terminus. First, using an aminoacid sequencer 890 M/E (manufactured by Beckman), phenylthiohydantoin (pTH);
! Obtain a conductor and place the conductor in an ODS column (4,6X 25
0wl11) H using [Manufactured by Tosoh ■, 120T]
Identified by PLC, one of the 20 amino acid residues on the N-terminal side
The following structure was determined.

The estimated primary structure was as follows.

Asp-Glu-Pro-Thr-Thr-Lys-P
ro-Phe-Cys-Glu-Gln-^1a-Ph
e-Gly-Asp-Cys-Gly-Thr-Pro
-Tyr- The method of Moore and 5tain et al.
ds in Enzymology q;819.196
3). i.e. 0.0 in the purified sample.
Fe free 6 containing 5% mercaptoethanol
After adding N-HCI and hydrolyzing at 110° C. for 24 hours, the constituent amino acid ratio was determined using an automatic amino acid analyzer [Aminoacid Analyzer Model 655A, manufactured by Hitachi ■].

The number of each amino acid was estimated based on the results of molecular weight measurement by HPLC. The results are shown in Table 2.

As is clear from Table 2, it has the characteristic of not containing Net and old S.

Table 1) Number of amino acids estimated from the molecular weight (8,500) measured by HPLC [inhibitory activity against chymotrivcin] Various amounts of Cl were added to chymotrivcin (200 pmole).
After adding -2 and incubation at 37°C for 10 minutes, chymotrybuscin activity was measured using casein as a substrate. The measurement results are plotted as newer-Burk's reciprocal plot, and Cl-2 is obtained from the diagram shown in Figure 6.
The Ki value was 0.77 μM.

[100% Inhibition Ratio to Chymotrybusin] After adding various amounts of Cl-2 to chymotrybcin (200 pmole) and incubating at 37°C for 10 minutes, chymotrybcin activity was measured using casein as a substrate. Figure 7 shows the relationship between the amount of chymotrybusin inhibited and the amount of Cl-2.
Since the molecular weight of C34,21,,1) is 25.200, Cl with a molecular weight of 8,500 estimated by HPLC
100% inhibition ratio of -2 to chymotrivcin [I/E
The molar ratio of l is 2.24:1.

1. The residual activity of protease after reacting various proteases with Cl-2 was measured. The amount of each protease used and the method for measuring the activity are shown in Table 3 below.

Table 3 Fruit table The results are shown in Table 4.

Cl-2 showed inhibitory activity against chymotrivcin, and almost no inhibitory activity against other proteases.

Figure 8 shows the results of investigating how the chymotrivcin inhibitor activity changed after the samples were left at room temperature for 24 hours at a pH of 2 to 12. In addition, the m-force used in this reaction) at night was
Use glycine-hydrochloric acid buffer for pH 2-3, acetic acid for pH 4-5! ! For pH 6-7, use phosphate buffer, for pH 6-7
Tris-hydrochloric acid Mmtlt was used for pH 9, glycine-sodium hydroxide buffer was used for pH 10 to 11, and glycol-sodium hydroxide buffer was used for pH 12.

The figure shows that even at around pH 2, where stability is considered to be the worst, 75% of the inhibitory activity is still maintained, indicating that this bioactive peptide exhibits excellent pH stability.

The bioactive peptides of Tadasujidai Mamemoto were dissolved in phosphate buffer (pH 7, 4) and the residual activity was examined after heat treatment at various temperatures for 10 minutes. From the results shown in Figure 8, 10 at 90°C.
Even after treatment for 1 minute, 75% of the chymotrybusin inhibitory activity was maintained, indicating that this bioactive peptide has excellent heat resistance.

5hiff reaction (Zaccharias, R.J., M.
orisson, J.H.

and Woodlock, J. J., Anal, Bi.
ochem, 31, 148.1969) was negative, and after Cl-2 electrophoresis, Western blo
Transfer to a membrane at
nin Lectin Kit I (manufactured by Vector)]
There was no fluorescent band even when reacted with (negative result)
Therefore, it is considered that there is no binding of 1181 in this physiologically active peptide.

[Effects of the Invention] The physiologically active peptide having chymotrybusin inhibitory activity of the present invention has excellent pH stability and thermal stability as described above, and its activity can be applied to various fields. For example, when added during the production and purification of a substance having a peptide bond, the yield can be increased, and by coexisting with a physiologically active substance having a peptide bond, the physiologically active substance may be administered orally. It can be applied to various fields.

[Brief explanation of the drawing]

Figure 1 shows the elution pattern of DEAE column chromatography, Figure 2 shows the elution pattern of affinity column chromatography, Figure 3 shows the results of molecular weight measurement by SDS electrophoresis, and Figure 4 shows the results of molecular weight measurement by HPLC. Figure 5 is a graph showing the results of isoelectric focusing, Figure 6 is a graph showing the inhibitory activity of chymotrivcin when Cl-2 casein is used as a substrate, and Figure 7 is a graph showing the results of isoelectric focusing.
Inhibition molar ratio of Cl-2 to chymotrivucin IM [1
/E], FIG. 8 is a graph showing the pH stability of Cl-2, and FIG. 9 is a graph showing the thermal stability of Cl-2.

Claims (1)

[Scope of Claims] A physiologically active peptide characterized by having the following physicochemical properties. Molecular weight: Approximately 7,900 HP as measured by SDS electrophoresis
Approximately 8,500 as measured by LC Isoelectric point: 7.00 Physiological activity: Has chymotrybusin inhibitory activity