JPH0334919B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION Thymosin α ₁ has _the formula H ₃ C−CO−
Ser−Asp−Ala−Ala−Val−Asp−Thr−Ser−
Ser−Glu−Ile−Thr−Thr−Lys−Asp−Leu−
Lys−Glu−Lys−Lys−Glu−Val−Val−Glu−
Glu-Ala-Glu-Asn-OH is a known biologically active peptide hormone. Thymosin alpha ₁ and its desacetyl congeners are
Woug and Merrifield (Biochemistry 19 , 3233−
3238 [1980]), the protected intermediates [Lys(Tfa) ^14,17,19,20 ] thymosin α ₁ and [Lys(Tfa) ^14,17,19,20 ] desacetyl- Thymosin alpha ₁
(Tfa=trifluoroacetyl). In addition, desacetyl-thymosin α ₁ is a recombinant
It can be produced by bacteria using recombinant DNA techniques. The present invention provides biologically active ribosome preparations that exhibit transacetylase activity.
The present invention relates to a method for producing thymosin _{α 1 directly from desacetyl-thymosin α 1 by} selectively acylating the N-amino group of desacetyl-thymosin α 1 using preparations. For purposes of the present invention, any biologically active ribosome prepared by art-recognized methods from animal or plant sources can be used. Biologically active ribosome preparations contain transacetylase activity. In particular, sources of ribosome preparations include any animal tissue, such as thymus, liver, reticulocytes, pituitary gland, muscle, heart, kidney, brain,
Also included are plant cells such as wheat germ. Particularly preferred sources of ribosome preparations are thymus and wheat germ. cytoplasmic component of tissue from wheat germ or thymus
constituency) can be disrupted by any art-recognized method that yields an extract containing biologically active ribosomes. To avoid reduction or loss of transacetylase activity contained upon storage, it is preferred that the ribosome preparations for this method be prepared fresh each day the method is performed. In preparing S-30 fraction containing ribosomes from wheat germ, Roberts et al. (Proc. Natl.
Particularly useful is the method of Acad. Sci. 70 , 2330-2334 [1973]) and Shackelford et al. (J. Biol. Chem.) in preparing ribosome preparations from the thymus.
254, 4220-4226 [1979]) is particularly suitable. Generally, ribosome preparations are prepared using a suitable buffer to give a PH value of about 6.5 to 7.5, such as Tris-(hydroxymethyl)-aminomethane hydrochloride (Tris-HCl), phosphate or N-2-hydroxyethylpiperazine- Advantageously prepared by homogenizing the cytoplasmic material in N'-2-ethanesulfonic acid (HEPES), the preferred PH value is 7.5, the buffer is preferably MgCl ₂ about 2-5 mM, preferably 3mM
and dithiothreitol (DTT) approx. 0.5-10m
M, preferably 1mM. The homogenized material is centrifuged at low temperature and the supernatant is removed free of surface layers of fat. The indicated S-30 fraction obtained by centrifugation is passed through a gel permeation column such as Sephadex G-25 (coarse), collecting the turbid fraction. This fraction is collected and centrifuged at about 180,000 xg to obtain a precipitate containing ribosomes. This precipitate can be used as is or can be further purified by art-recognized methods for isolating transacetylase therefrom. In the method of the invention, this precipitate is used after being dissolved in an aqueous buffer of Tris-HCl at a pH value of about 7.5 containing overlays of DTT and KCl of about 0.5-3.0M, preferably 1.5M. The optimal pH value is slightly below 7.5; above this pH value,
The degree of _NH2 -terminal acetylation is greatly reduced,
PH values below 6.5 result in a mixture of undesirable by-products. The solvent can also contain the above amount of MgCl ₂ . Surprisingly, it was found that the presence of KCl enhanced the _NH2 -terminal acetylation of desacetylthymosin _α1 . Acetylating agents useful in this method include suitably activated acetate compounds that can be tolerated by the transacetylase activity of the ribosomal preparation when carrying out the method, such as acetyl coenzyme A (acetyl coenzyme A).
CoA) or N-acetylthioethanolamine. A preferred acetylating compound is acetyl-CoA. tritiated acetyl
CoA ( ^3H -acetyl-CoA) can be used both for acetylation and as a label in the purity and identification of the final products of the invention. Although there are many sites that can be acetylated on a single molecule of desacetylthymosin _α , unexpectedly, the present method selectively acetylates the terminal amino group to form thymosin _α1.
It was found that this occurs. More specifically, the invention typically consists of a combination of the following steps: (a) freezing an aqueous solution containing acetyl-CoA as the acetylating agent and desacetylthymosin alpha ₁ as the substrate in a microscopic polypropylene test tube; dry. A suitable concentration range for the substrate is approximately 1 μM to approx.
200 μM, preferably 80 μM. A suitable concentration range for the acetylating agent is about 1 μM to about
500 μM, preferably 200 μM. (b) Add a buffered solution of the ribosome preparation to start the reaction, the amount added is not critical;
A preferred amount is about 13 μg ribosomes per 10 μg of reaction mixture. (c) Incubating the reaction mixture at about 30° to about 37°C, most preferably at about 35°C. Under these conditions, the reaction is approximately
10-20% complete in 20 minutes. (d) Adding an alkaline solution, such as NaOH, to a final concentration sufficient to destroy excess acetyl-CoA, but not significantly interfering with the integrity of the final product. The preferred final concentration of NaOH is about 1N, followed by incubation at about 35°C for about 15 minutes. The reaction mixture is then neutralized using a concentrated inorganic acid such as HCl. A 10% trichloroacetic acid solution can also be used to precipitate proteins. (e) The final product is obtained from the reaction mixture by any art-recognized method of isolation. The preferred method of isolation of the final product is chromatography. A particularly preferred chromatographic method is Rubinstein (Anal.Biochem, 98 , 1-
7 [1979]). The following examples further illustrate the invention, but are not meant to limit it in scope or intent. Example 1 (a) Preparation of wheat germ extract: Roberts and Paterson, Proc. Natl. Acad. Sci.
70, 2330-2334 (1973),
A 30,000×g supernatant (S-30) of wheat germ was prepared. Wheat germ (6g) with equal weight of sand and PH
Tris-HCl 50mM, MgCl ₂ 3mM and
It was ground for 1 minute in a chilled mortar with 20 ml of Tris-HCl buffer solution containing 1 mM DTT. The homogenate was centrifuged at 30,000 xg for 20 minutes at 0-2°C and the supernatant was separated without a surface layer of fat.
Pass the S-30 fraction through a Sephadex G25 (coarse) column (60 x 2 cm) and add 1mM DTT.
14 by 50mM Tris-HCl with pH value 7.5 containing
Equilibration was performed at a flow rate of ml/min. Preserves cloudy fraction. Centrifugation was performed at 180,000 xg for 2 hours at 2°C. Precipitate the Tris-HCl buffer solution 0.75
ml to obtain a wheat germ ribosome preparation. (b) Acetylation of desacetylthymosin α ₁ by wheat germ ribosome preparation: A solution containing [ ³ H]acetyl CoA (2.1 nM) and desacetylthymosin α ₁ 0.4 nM was lyophilized in a micro polypropylene test tube. did. Acetylation was initiated by addition of 10μ of wheat germ ribosome preparation. After culturing at 35° C. for 20 minutes, 40 μg of an aqueous solution containing 80 μg (24 nM) of thymosin α ₁ was added as a carrier. This is followed by
NaOH was added to a final concentration of 1N and incubated at 35°C for 15 min to degrade excess acetyl-CoA.
This solution was neutralized with concentrated hydrochloric acid. Conversion of desacetylthymosin α ₁ to thymosin α ₁ was 15%. (c) Reversed phase high performance liquid chromatography (HPLC) Rubinstein (Anal.Bio chem. 98 , 1-7
[1979]) using a reverse phase column (Lichrosorb RP-8, 10 μm; 4.6×
250 mm) and subjected to high performance liquid chromatography (HPLC). This column was mixed with 1M formic acid/
0-40% (V/
Elute with a linear gradient of V) at a flow rate of 0.33 ml/min for 2 hours, collecting fractions every 2.5 minutes.
At 20 second intervals, 5μ of the sample was placed into the fluorescence detection system.
The samples were also counted for radioactivity. HPLC analysis showed the presence of a radioactive peak co-eluted with the thymosin α ₁ carrier, verifying the absence of acylated ε-NH ₂ -lysyl groups.
Partial acetylation of ribosomal proteins was observed, but was small compared to acetylation of desacetylthymosin alpha ₁ . The labeled product of the thymosin alpha ₁ peak is the _NH2 -terminal tryptic peptide _H3C -CO-Ser-
Asp−Ala−Ala−Val−Asp−Thr−Ser−Ser
-Glu-Ile-Thr-Thr-Lys was identified as thymosin alpha ₁ by tryptic digestion and amino acid analysis. Example 2 (a) Preparation of thymus extract Thymus (12 g) obtained from two freshly killed calves was treated with 3mM _MgCl2 , 25mM NaCl,
Homogenized in a Dcunce homogenizer with 2 volumes of 20mM Tris-HCl with a pH value of 7.5 containing 1mM DTT and 250mM sucrose.
This homogenate was centrifuged at 12,000 x g for 10 min.
The supernatant fraction was centrifuged at 30,000 xg for 10 minutes. The S-30 fraction was transferred to a Sephadex G-25 (rough) column (60 x 2 cm) pre-equilibrated with 50mM Tris-HCl with a pH value of 7.5 containing 1mM DTT and _3mM MgCl2 at a flow rate of 1.4ml/min. ) passed through. The cloudy fraction indicating the rejected volume of the column was the source of the transacetylase. All operations are 0-2
I did it at ℃. (b) Acetylation of desacetylthymosin α ₁ [ ³ H]acetyl-CoA (1.05 nM) with and without desacetylthymosin α ₁ (204 pM)
The solution containing was freeze-dried in a micro polypropylene test tube. The reaction was initiated by addition of 5 μg of thymus S-30 fraction (4.1 μg). After incubating for 20 min at 35°C, reduce the reaction to 10%.
Stopped by addition of 2 ml CCl ₃ COOH. Filter the precipitate using Millipore filters
(0.45 μm) and the liquid was diluted with 5% CCl ₃ COOH.
The cells were washed with water, dried and analyzed for radioactivity in 10 ml of Aquasol. Acetylation was 0.3%.

Claims (1)

[Claims] 1. N-desacetylthymosin α ₁ and acetyl-CoA or N-acetylthioethanol in an aqueous medium in the presence of a biologically active ribosome preparation exhibiting transacetylase activity. 1. A method for producing thymosin α ₁ , which comprises selectively acetylating the N-amino group of N-desacetylthymosin α ₁ by reacting with an amine. 2. The method according to claim 1, wherein the ribosome preparation is prepared from plant cells. 3. The method according to claim 1, wherein the ribosome preparation is prepared from malt. 4. The method according to claim 1, wherein the ribosome preparation is prepared from animal tissue. 5. The method according to claim 1, wherein the ribosome preparation is prepared from the thymus of an animal. 6. The method according to any one of claims 1 to 5, wherein the PH value of the reaction medium is slightly lower than 7.5. 7. The method of claim 6, wherein the reaction mixture contains dithiothreitol and KCl. 8. The method of claim 7, wherein the reaction mixture further comprises _MgCl2 .