JPH06336498A - New prolactin - Google Patents

Abstract

PURPOSE:To obtain a new prolactin having industrial advantage such as application to growth promoter in culture of fishes, showing action related to growth promotion of marine fishes, from prolactin derived from hypophysis of Acipenser mikadoi. CONSTITUTION:Hypophysis is extracted from Acipenser mikadoi before egg laying, freeze-dried, de-fatted, ground by a homogenizer in 35 % hydrochloric acid-acetone solution at 4 deg.C under cooling with ice and extracted with acid- acetone for one hour on ice. The extracted solution is ultracentrifuged to separate precipitate. The precipitate is dissolved in 1M acetic acid, subjected to gel filtration, its active fraction is purified by reversed phase high performance liquid chromatography and eluted with acetonitrile-containing 0.1% trifluoroacetic acid solution. The prepared fifth fraction is lyophilized to give the objective new prolactin which has about 23kD molecular weight measured by SDS-polyacrylamide gel electrophoresis and specific amino acid constitution obtained by hydrolysis with 0.6% phenol-containing 6N-hydrochloric acid and determination.

Description

Detailed Description of the Invention

[0001]

The present invention relates to a novel peptide,
Specifically, it relates to a peptide obtained from prolactin derived from the pituitary gland of sturgeon and having physiological activity.

[0002]

2. Description of the Related Art Natural products such as plants and animals contain a large amount of substances having activity on the living body. One of them, growth hormone, is a peptide secreted from the pituitary gland of vertebrates, and its main physiological action is known to be a growth promoting effect.

In recent years, the presence of prolactin, which is secreted from the anterior pituitary gland like growth hormone, is known as a protein having an amino acid sequence similar to that of growth hormone, and has attracted attention. Prolactin is generally considered to be a hormone having a wide range of reproductive effects such as secretion of milk in mammals, luteinizing action, and formation of pigeon's capsule milk in birds.
In lower vertebrates, amphibians are known to control metamorphosis and growth, and in fish, it is considered to be involved in regulation of osmotic pressure during adaptation to freshwater of broad-salt fish and freshwater fish.

Up to now, prolactin has been isolated from many kinds of animals from tetrapods to fish. However, although the presence of prolactin in marine fish is known from immunohistochemical studies, its isolation and physiological effects have not been studied.

On the other hand, by the present inventors, a growth hormone of sturgeon was extracted and isolated, and a gene encoding the amino acid sequence thereof, a recombinant vector incorporating the gene, and a transformation transformed with the recombinant vector A body and a production method using the transformant have been clarified (Patent Application 1993 No. 9948), and it is expected to be applied in the field of aquaculture industry in the future. In other words, by using growth hormone that can be prepared in large quantities, it usually takes seven years to grow into an adult fish.
It is an application to the field of aquaculture industry to make a great reduction.

However, as described above, in natural products such as plants and animals, there are many substances having an activity on the living body, and growth of the living body is not limited to growth hormone, but on the balance of many physiologically active substances. Has been adjusted. In particular,
It is easily imagined that a substance having a physiologically active action other than growth hormone including prolactin is required for application in the field of aquaculture industry in the future. Therefore, it was expected that a substance having a physiological action on marine fish, especially a substance having an action relating to growth promotion would be found and applied to the field of the aquaculture industry in the future.

[0007]

Means for Solving the Problems The present inventor has conducted diligent research to develop a substance having a physiological action on marine fish, particularly a substance having an action relating to growth promotion. As a result, SEQ ID NO:
The inventors have found that the novel peptide represented by 1 is prolactin, and completed the present invention.

That is, the present invention is as follows.

(1) A novel peptide having the following physicochemical properties (hereinafter referred to as prolactin of the present invention). The molecular weights measured by SDS-polyacrylamide gel electrophoresis are both about 23 kD. After hydrolysis using 6N-hydrochloric acid containing 0.6% phenol, the quantified amino acid composition is shown in Table 1. Table 1

(2) The novel peptide according to (1), which contains a peptide having the amino acid sequence represented by SEQ ID NO: 1 as a main constituent.

The prolactin of the present invention can be separated and purified by the following various methods.

For example, a tissue or cell containing the prolactin of the present invention, for example, the pituitary gland of sturgeon can be used as a starting material and a method known as a method for separating and purifying various proteins can be applied.

As a method for separating and purifying proteins, solubilization treatment by a homogenizer, ultrasonic cell disruption, etc., extraction treatment with a buffer solution containing various salts, solubilization or precipitation treatment with acid or alkali, and further organic solvent Extraction or precipitation treatment with, further extraction or precipitation treatment with an organic solvent, salting out with ammonium sulfate, dialysis, ultrafiltration using a membrane filter, gel filtration chromatography, ion exchange chromatography, countercurrent partition chromatography, Examples include high performance liquid chromatography, isoelectric point and gel electrophoresis, and these can be used alone or in appropriate combination.

More specifically, the pituitary gland obtained from the sturgeon by the usual method is immediately frozen with liquid nitrogen or dry ice and stored at about -80 ° C until use. The pituitary gland is crushed in a 35% hydrochloric acid-acetone solution (1:28) under cooling and then centrifuged. The precipitate obtained is extracted in 80% acetone. The extract is cooled,
Acetone is added, and the resulting precipitate is centrifuged and separated.
This precipitate is freeze-dried and dissolved in an appropriate buffer solution, and then subjected to gel filtration chromatography, such as Sephadex G-2.
Chromatograph 5. Elution fraction is 210n
measured by monitoring the absorbance at m,
Can be separated. Next, of the fractions thus obtained, the fraction containing the target prolactin is freeze-dried.

Next, the purified product is dissolved in an appropriate buffer solution and then subjected to high performance liquid chromatography, for example, ODS12.
It is subjected to high performance liquid chromatography using a column using a chemically modified silica gel such as 0T as a packing material. Prolactin can be obtained by collecting the fractions containing the target prolactin by monitoring the eluate with absorbance at 210 nm.

The gene encoding the amino acid of prolactin of the present invention can be produced by various methods shown below.

For example, poly (A) RNA is prepared from the pituitary gland of sturgeon, which is a cell producing prolactin of the present invention, and cDNA is synthesized using this as a template, which is then ligated to an appropriate vector to form a host cell. From the findings of the amino acid sequence analysis of prolactin obtained by the isolation / purification method of the present invention, a suitable DNA probe was synthesized, and a gene library was searched using the probe to obtain the desired DNA probe. A method of identifying a clone containing a cDNA encoding the amino acid sequence of prolactin and isolating it with a vector possessed by the clone, based on the DNA of the gene encoding the amino acid sequence of prolactin of the present invention, for example, the phosphate triester method , A method of chemically synthesizing a nucleic acid according to a conventional method such as the phosphoamidite method, a combination of these methods Method, and the like.

Next, the cDNA obtained as described above
Is inserted into an appropriate vector, then the vector thus obtained is introduced into an appropriate host and amplified by cloning, and then screened to obtain a cDNA encoding the amino acid sequence of the target prolactin.
A recombinant host containing A can be selected, and then the DNA encoding the amino acid sequence of prolactin of the present invention can be isolated from the plaque or colony of the recombinant host.

The gene expression vector encoding the amino acid sequence of prolactin of the present invention is introduced into a suitable host, particularly a host cell, according to a commonly known method to transform the host cell. By amplifying the host cells transformed in this way by a method such as culturing, it is possible to obtain a large amount of transformants, which are capable of producing a peptide having the amino acid sequence of prolactin of the present invention.

In growing or culturing the above transformant, conditions suitable for growing the transformant such as pH, temperature, aeration, agitation, and frequency of medium exchange can be appropriately determined by experiments or the like. The protein based on the gene expression of the present invention produced by growing or culturing the above transformant or by inducing the expression of the gene can be separated and collected by a usual operation.

Next, the present invention will be described in more detail with reference to examples.

Example 1 A pituitary gland was excised from a sturgeon having a body weight of 11 to 16 kg before spawning and freeze-dried. Defatted pituitary gland 0.5g
Under ice-cooling at 4 ° C, 35% hydrochloric acid-acetone (1:28).
After crushing with a glass homogenizer in 100 ml of solution 1
Acid-acetone extraction was performed on ice for hours. After extraction, it was centrifuged at 22000 G for 30 minutes at 0 ° C.
The obtained precipitate was further homogenized in 100 ml of 80% acetone and then extracted in ice-cooling at 4 ° C. for 1 hour, and 22000 in a centrifuge together with the centrifugation supernatant obtained above.
G was centrifuged for 30 minutes at 0 ° C. Acetone was precipitated by adding 3 l of acetone to the obtained extract in ice-cooling at 4 ° C. After removing the supernatant, the precipitate was freeze-dried. The obtained extract was about 54 mg.

The obtained precipitate was dissolved in 1M acetic acid and equilibrated with the same solution on a Sephadex G-25 column (2.7).
X 70 cm). Elution was carried out using 1 M acetic acid at 20 ° C., 70 ml of front flow, and 30 ml / hour of flow rate, and the eluate was measured for protein content by absorbance at a wavelength of 280 nm, and the amount collected per test tube was 3 ml, Three
Two gel filtration fractions were obtained. The obtained first fraction was freeze-dried to obtain 36.8 mg of white powder. Next, this was applied to a column using TSK-gel (ODS 120T,
0.46 × 25 cm) and purified by reverse phase high performance liquid chromatography. Elution was performed with a 0.1% trifluoroacetic acid solution containing acetonitrile at 40 ° C and 1 ml.
The protein content was measured by the absorbance at 220 nm at a flow rate of / min, and 7 fractions were obtained (Fig. 1). Further, the obtained fifth fraction was freeze-dried to collect the prolactin of the present invention. The resulting fifth fraction had the following physicochemical properties.

Molecular Weight The above fraction A was developed by SDS polyacrylamide gel electrophoresis, and it was found that the prolactin of the present invention has a molecular weight of about 23 kD. The 5th fraction obtained at the same time gave a single band. The standard molecular weight marker is a colored molecular weight marker LM manufactured by Pharmacia.
Wkit E, molecular weight 94,000; 67,000; 4300
0; 30000; 20000; 14000 were used.

Amino Acid Composition The novel peptide of the present invention was added to 6N-containing 6% phenol.
After hydrolyzing at 110 ° C for 24 hours using hydrochloric acid, it was reacted with phenylisothiocyanate to give a phenylthiocarbamyl amino acid derivative, and a column using TSK gel (ODS-80TM, 0.46 x 25 cm) was used. Purified by reverse phase high performance liquid chromatography. For elution, constituent amino acids were quantified by a proportional gradient (isopropanol: acetonitrile). Also, Cys was confirmed by the formic acid oxidation method. The results are shown in Table 1.

Table 1

Amino Acid Sequencing and Identification of N-Terminal Amino Acids To reduce 200 μg of the prolactin of the invention, it was reacted with dithiothreitol for 4 hours and further alkylated with 4-vinyl pyridine. Next, the alkylated prolactin of the present invention was treated with Lys endopeptidase [conditions: E / S = 1/60, 0.05 M ammonium bicarbonate (pH 8.0), 37 ° C., 18 hours], or Asp-N. Endopeptidase [Condition: E /
S = 1/200, 0.05 M ammonium bicarbonate (pH 8.0), 37 ° C., 18 hours]. The large fragment obtained with Asp-N endopeptidase was further digested with trypsin [conditions: 0.2 M ammonium acetate (pH 8.0), 37 ° C, 6 hours]. The obtained fragment was purified by reverse phase high performance liquid chromatography using a column (ODS 120T) using TSK-gel. For elution, a 0.1% trifluoroacetic acid solution containing isopropanol in a proportional gradient was used. The obtained peptide is an automatic sequencer (Shimadzu, PSQ-
It was analyzed and determined in 1).

From the above results, it was determined that the prolactin of the present invention is a peptide having the sequence shown in SEQ ID NO: 1.

Further, since such a peptide has an essential part for exhibiting activity and a part not directly related to activity expression, those having the same peptide sequence as the novel peptide of the present invention are included in the present invention. Be done.

Next, in order to confirm the physiological activity of the prolactin of the present invention, plaque (0.01 μm) of the present invention obtained in Example was added to juvenile sturgeon from which the pituitary was removed.
g / mouse) or physiological saline was intraperitoneally administered to increase body weight and measure sodium concentration in blood. In addition, the measurement of blood sodium concentration,
GENERAL AND COMPARATIV
E ENDOCRINOLOGY) 70, 407-41
7 (1988).

As a result, it was confirmed that the sturgeon juveniles administered with the prolactin of the present invention showed a significant increase in both body weight and blood sodium concentration in comparison with those administered with physiological saline.

[Brief description of drawings]

FIG. 1 is a high performance liquid chromatography chart of an extract obtained from the pituitary gland of sturgeon.

[Sequence list]

SEQ ID NO: 1 Sequence length: 204 Sequence type: Amino acid Sequence type: Peptide Sequence characteristics method: E sequence 5 10 15 Ser Pro Leu Cys Gly Gly Leu Gly Cys Pro Pro Pro Ile Leu Leu 20 25 30 Ser Asp Leu Leu Glu Arg Ala Thr Gln Leu S er Ser Arg Leu His 35 40 45 Ser Leu Ser Arg Thr Val Thr Ala Gly Leu A sp Pro His Phe Ser 50 55 60 Pro Leu Leu Lys Pro Arg Pro Ser Ser Leu Cys His Thr Ser Ser 65 70 75 Leu Ala Thr Asp Glu Asn Lys Glu Gln Ala L eu hr Leu Gln Gln 80 85 90 Glu Gln Leu Leu Ser Leu Ile Met Ser Leu L eu Arg Ser Trp Thr 95 100 105 Pro Pro Leu Met Phe Leu Val Arg Glu Ala G ln Ser Leu Pro Pro 110 115 120 Asn His Ser Leu Ser Gly Ser Leu Ser Trp G ln Thr Ala Glu Leu 125 130 135 Ser Gln Ser Gln Lys Leu Ala Lys Gly Leu G lu Thr Ile Leu Asn 140 145 150 Arg Phe Asp Pro Ser Ala Ala His Lys Ala S er Phe Gly Asn Ala 155 160 165 Asp Asp Leu Trp Lys Gly Gly Ala Ser Asp P he Pro Gly Ser Asp 170 175 180 Arg Lys Ser Arg Leu Leu Asn Phe Tyr Phe L eu Leu Ser Cys Phe 185 190 195 Arg Arg Asp Ser His Lys Ile Asp Ser Phe L eu Lys Leu Leu Arg 200 Cys Arg Ala Gln Glu Asn Gly Gly Cys

Claims (2)

[Claims] 1. A novel peptide having the following physicochemical properties. The molecular weight measured by SDS-polyacrylamide gel electrophoresis is about 23 kD. After hydrolysis using 6N-hydrochloric acid containing 0.6% phenol, the quantified amino acid composition is shown in Table 1. Table 1 2. A peptide containing the amino acid sequence represented by SEQ ID NO: 1 as a main constituent.
The novel peptides described.