Method of 26s- and 20s- proteasome pool division from cytoplasmic cell fraction

FIELD: medicine.

SUBSTANCE: tissue is homogenised in a buffer and centrifuged at 105000 g for 60-90 min at 0-4°C to produce a cytoplasmic fraction which is then incubated with 10 mM of phosphocreatine and 10 mcg/ml of phosphocreatine kinase for 25-45 minutes at 35°C. Cytoplasmic fraction proteins are divided by ammonium sulphate at three stages, at the first stage ammonium sulphate is added to 38% of saturation and centrifuged to isolate a precipitate containing a 26S-proteasome pool, at the second stage, a supernatant is added with ammonium sulphate to 42 % of saturation and centrifuged to isolate a precipitate containing ballast proteins, at the third stage to the supernatant is added with ammonium sulphate to 70 % of saturation and centrifuged to isolate a precipitate containing a 20S-npoteasome pool. Ammonium sulphate is added in portions during 20 min on a magnetic stirrer and further mixed for 20 minutes.

EFFECT: invention allows dividing native 26S- and 20S-proteasomes and isolating them in those amounts they exist in living cells, with preserving at most an undamaged 26S-proteasome structure.

3 cl, 1 ex

 

The invention relates to the field of biochemistry and can be used in research when developing drugs of new generation for the treatment of cancer, neurodegenerative , viral and other diseases.

An important role in maintaining the functional activity of cells play proteasome. The proteasome is multiobjective multiprotein protein complexes. The diversity of these forms can be reduced to two main pools - 26S-

and 20S proteasomes. 26S-proteasome consists of a proteolytic subunit 20S - and one or two 19S regulatory-subparticles. Proteasome carried out strictly controlled proteolysis and processing of regulatory proteins and play a key role in fundamental cellular processes: promoting cells in phases of the cell cycle, differentiation, apoptosis, signal, immune response. All organs and tissues of mammals contain multiple forms of proteasomes, differing in structure, hydrolysis of proteins and, consequently, the physiological role. The proteasome is also present in cells of malignant tumors.

Now for cancer treatment using anticancer drug bortezomib (bortezomib, Velcade®), acting on the proteasome. Bortezomib is administered into the bloodstream of patients and is used in conjunction with other against the tumor agents. However, acting on the total pool of proteasomes all organs, bortezomib causes side effects: fatigue, weakness, gastrointestinal disturbances, peripheral neuropathy, and a substantial deterioration of the General condition of the patients.

Known to increase the level 198-activator 26S-proteasome in hepatocellular carcinoma. Malignant tumors, including tumors of the liver, characterized by increased protein metabolism, which requires, in turn, increased the content of proteolytic enzymes, in particular 26S-proteasome [Astakhov, T.M., Delaunay, GV, lupine J.V., Abramova E.B., Uryvaev IV, Sharova N.P. Changing pool of proteasomes in the process of malignant transformation of liver cells of the mouse. // Acta naturae, 2010, No. 3, p.91-96//]. The results point to the prospective use of the 19S-activator 26S-proteasome, redundantly expressed in malignant tumors as targets for development of new anticancer drugs.

To reduce side effects in drug development of new generation it is necessary to study the action of drugs differentiation in the 26S-proteasome and 20S proteasome.

A method of obtaining proteasomes in crystalline form [patent WO 9842829, IPC C12N 9/60], which uses the following stages of purification: chromatography on a column of the hydroxyapatite, gelfiltration, FPLS system. Then the proteasome crystallized. When this partially destroys the structure of the 26S-proteasome.

Known purification method of the proteasome [patent US 6294363, IPC C07K 14/39]. This method is based on the immobilization of peptides with amino acid sequence that is homologous ubiquitin, column media (sorbent) and the binding of the 26S-proteasome with such columns. However, this method selects only the 26S-proteasome and purification of 20S proteasome is not provided.

A known method of preparation of the drug proteasome [JP 1309686, IPC C07K 14/39], which describes the allocation of subunits of the proteasome only in an inactive form.

A known method of purification of proteasomes [JP 4077498, IPC C12NK 15/09], which used a gel-filtration column chromatography on hydroxyapatite and the sorbent with immobilized heparin. All these procedures lead to the partial destruction by the 26S-proteasome and therefore unsuitable for analytical studies.

There is a method of allocating only the 26S-proteasome [JP 5292964, IPC A61K 38/46], which used the gel-filtration. This method leads to the partial destruction by the 26S-proteasome and allows you to get a fraction of the 26S-proteasome free from impurities 20S proteasome.

There is also known a method of selection by the 26S-proteasome [JP 6022759, IPC A61B 10/00], which used ultracentrifugation. But analytically for the research, this method cannot be applied, as a significant part of the 26S-proteasome remains mixed with 20S proteasomes.

It is known selection of proteasomes from the liver (non-lymphoid organ) and spleen (lymphoid organs) albino rats of Wistar breed different ages of postnatal development, liver, spleen and lungs of healthy mice of Balb/c and developed a seven-day ascitic carcinoma Krebs-II, vaccinated mice of Balb/c [Astakhov T.M. Changing pool of proteasomes in postnatal development and in malignant transformed cells of rodents. // Abstract of thesis Cand. Biol. of Sciences, Moscow, 2007//]. Pools 26S and 20S proteasomes were separated by the method of two-stage fractionation clarified homogenates of organs with different amounts of ammonium sulfate. Adding ammonium sulfate taken up 0-40% of saturation, was deposited mainly by the 26S-proteasome, and at 40-70% saturation was deposited 20S proteasome. However, get the pools with a large number of ballast proteins, which complicates further research.

At the stage of purification of proteasomes in the process of separation is destroyed

26S-proteasome on 20S and 19S-subunit, thus distorting the results.

When developing the method of separation of pools 26S and 20S proteasomes proceeded from the known fact that the forms of 26S and 20S are deposited with different amounts of ammonium sulfate. When 0-40% from saturation osadetz is, mainly, the 26S-proteasome, with 40-70% - 20S-proteasome [Ganoth et al., 1988; Eytan et al., 1989; Ben-Shahar et al., 1999]. After fractionation with ammonium sulfate previously carried out further purification as pool 26S and a pool of 20S proteasomes by various methods. In this process of purification by the 26S-proteasome in more or less collapsed.

The task to be solved by the invention is the development of the method of separation of 26S and 20S proteasomes in the native form and in quantities in which they exist in living cells, in which the structure of the 26S-proteasome remained intact as much as possible.

To solve this problem the method of separation of pools 26S and 20S proteasomes from the cytoplasmic fraction of cells includes a homogenization of the tissue in a buffer containing 30 mm Tris-HCl, pH 7.5, 100 mm NaCl, 1 mm EDTA, 1 mm dithiothreitol, 10%glycerol, 5 mm MgCl2, 2 mm ATP, 10 mm Na2S2O5, 0.5 μg/ml leupeptin, 1 µg/ml pepstatin and 1 µg/ml of Aprotinin in the ratio of 1:3 at 0-4°C, centrifugation of the homogenate at 105000 g for 60-90 min at 0-4°C with getting the cytoplasmic fraction, incubation cytoplasmic fraction with 10 mm phosphocreatine and 10 μg/ml phosphocreatine for 25-45 min at 35°C and separation of proteins in the cytoplasmic fraction of ammonium sulfate in three stages, the first stage of adding ammonium sulfate up to 38% of trasimene and centrifuged to separate the precipitate, containing a pool of the 26S-proteasome, in the second stage to the supernatant add ammonium sulfate to 42% from saturation and centrifuged to separate the precipitate, containing blastnye proteins, in the third stage to the supernatant add ammonium sulfate to 70% saturation and centrifuged to separate the precipitate, containing a pool of 20S proteasomes, and centrifugation to separate the precipitation is carried out at 10000 g for 30 min at 0-4°C, with a residue containing a pool of the 26S-proteasome, is dissolved in a buffer containing 20 mm Tris-HCl, pH 7.5, 1 mm EDTA, 1 mm dithiothreitol, 50%glycerol, 5 mm MgCl2, 2 mm ATP, 10 mm Na2S2O5, 0.5 μg/ml leupeptin, 1 µg/ml pepstatin, 1 μg/ml Aprotinin, and the residue, containing a pool of 20S proteasome, is dissolved in a buffer containing 20 mm Tris-HCl, pH 7.5, 1 mm EDTA, 1 mm dithiothreitol, 50%glycerol, 10 mm Na2S2O5, 0.5 μg/ml leupeptin, 1 µg/ml pepstatin, 1 μg/ml Aprotinin.

In the private version of the incubation solution containing pool 268-proteasome, is carried out with 10 mm phosphocreatine and 10 μg/ml phosphocreatine for 25-45 min at 35°C to restore the structure of the 26S-proteasome.

In another private embodiment, the ammonium sulfate added in portions over 20 min on a magnetic stirrer, and then stirred for 20 minutes

To solve subsequent problems separation pools 26S and 20S proteasomes are what ispolzovaniem ammonium sulphate as the sole admission division of proteasome pools.

Separation of proteins cytoplasmic fraction ammonium sulfate is carried out in three stages. In the first stage, add ammonium sulfate to 38% from saturation, which allows the selection pool 26S-proteasome without loss to get rid of impurity protein, preventing further studies. At the second stage separation add ammonium sulfate to 42% from saturation. Adding ammonium sulfate from 38% to 42% from saturation allows you to separate the interfering research ballast impurity proteins having a molecular weight in the range of molecular masses of the subunits of the proteasome. At the third stage, add ammonium sulfate to 70% saturation. Adding ammonium sulfate from 42% to 70% of saturation allows to obtain a pool of 20S proteasomes without loss, free from the proteins that interfere with future research.

Zentrifugenbau homogenate with getting cytoplasmic fractions are within 60-90 min, because during this time of the homogenate deposited kernel and microsomal fraction. The process of zentrifugenbau carried out at 0-4°C, as at these temperatures reduced activity of the present enzyme.

Incubation cytoplasmic fraction and a solution containing a pool of the 26S-proteasome, are within 25-45 minutes This time is optimal for reconstructing the structure of the 26S-proteasome at 35°C.

The invention illustriou is conducted by the following example.

Example.

Tissue rat liver was washed in a standard phosphate buffered saline, obsessively on filter paper, weighed, separated (100 mg) and homogenized in the homogenizer glass-glass (Braun Melsungen) in 300 μl of buffer containing 30 mm Tris-HCl (pH 7.5), 100 mm NaCl, 1 mm EDTA, 1 mm dithiothreitol, 10%glycerol, 5 mm MgCl2, 2 mm ATP, 10 mm Na2S2O3, 0.5 μg/ml leupeptin, 1 µg/ml pepstatin and 1 µg/m Aprotinin at 4°C.

The obtained homogenate was centrifuged at 105000 g for 60 min at 4°C and collected 250 µl of the supernatant (cytoplasmic fraction). Under these conditions centrifugation DNA and microsomal structures remain in the sediment.

The cytoplasmic fraction was incubated with 10 mm phosphocreatine and 10 μg/ml phosphocreatine for 45 min at 35°C to maintain the structure of the 26S-proteasome.

Spent separation fractionation of proteins from the cytoplasmic fraction with ammonium sulfate in three stages. When this fraction (pool) 26S-proteasome received when using ammonium sulfate in the range of 0-38% of saturation, fraction (pool) 20S proteasome - when using ammonium sulfate in the range of 42 to 70% of saturation.

Step 1. Received a fraction of the 26S-proteasome using ammonium sulfate taken up 38% from saturation: 250 ál of the clarified homogenate was added to 57.1 mg of dry ammonium sulfate stands on the s for 20 min on a magnetic stirrer in an ice bath. After complete dissolution of the ammonium sulfate, the drug was left on a magnetic stirrer for 20 min for the formation of insoluble under these conditions, the fraction of proteins and then centrifuged at 10,000 g for 30 min at 4°C. the Precipitate containing a pool of the 26S-proteasome, was dissolved in 300 μl of buffer (20 mm Tris-HCl, pH 7.5, 1 mm EDTA, 1 mm dithiothreitol, 50%glycerol, 5 mm MgCl2, 2 mm ATP, 10 mm Na2S2O5, 0.5 μg/ml leupeptin, 1 μg/ml of pepstatin, 1 μg/ml Aprotinin), were incubated with 10 mm phosphocreatine and 10 μg/ml phosphocreatine for 45 min at 35°C to restore the structure of the 26S-proteasome and kept at -20°C for 4-5 months.

Step 2. To adosados fluid was added ammonium sulfate to 42% from saturation: 230 μl adosados fluid was added to 9.9 mg of dry ammonium sulfate and formed precipitate as in step 1, and then centrifuged at 10,000 g for 30 min at 4°C. the Obtained precipitate containing ballast proteins, and adosado fluid (220 μl). The precipitate contains the ballast protein, which complicates further research, for example, when carrying out electrophoresis of the protein forms a "he". Therefore, it was excluded from further work. So this step is important for the subsequent analytical studies of the proteasome.

Step 3. Received a fraction of the 20S proteasome using sulfate as mania in the range 42-70% of saturation: to 220 µl of the supernatant liquid, obtained in the second step, was added 41 mg of dry ammonium sulfate and formed precipitate as in step 1, and then centrifuged at 10,000 g for 30 min at 4°C. the Obtained precipitate and adosado liquid. The precipitate, containing a pool of 20S proteasome, was dissolved in 300 μl of buffer (20 mm Tris-HCl (pH 7.5), 1 mm EDTA, 1 mm dithiothreitol, 50%glycerol, 10 mm Na2S2O5, 0.5 μg/ml leupeptin, 1 μg/ml of pepstatin, 1 μg/ml Aprotinin) and kept at -20°C for 6-7 months. Adosado liquid of the third stage was excluded from the work, as it did not contain proteasomes.

Next were proof that the fraction obtained from the sludge in the first stage, contains a pool of 26S-proteasome, and the fraction obtained from the precipitate in the third stage, contains a pool of 20S proteasomes. Proof were: 1) Western blotting using antibodies to the α1 subunits, 2, 3, 5, 6, 7, members of both proteasome pools, and using antibodies to subunit Rpt6, part of the 19S-subunit 26S-proteasome; 2) inhibitor analysis chymotrypsinogen activity pools 26S and 20S proteasomes.

The proposed invention allows to obtain pools of 26S and 20S proteasomes in the native form and in quantities in which they exist in the cells that maintain the structure of the 26S-proteasome stable for 4-5 months, and the structure of the 20S proteasome within 6-7 m the months, and also will allow to carry out analytical studies on the action of various compounds, including drugs, cytoplasmic 26S and 20S proteasome in various cells and organs in vivo and in vitro, which is valuable for fundamental science and may eventually find application in practical medicine.

1. Method of separating pools 26S and 20S proteasomes from the cytoplasmic fraction of cells, including tissue homogenization in a buffer containing 30 mm Tris-HCl, pH 7.5, 100 mm NaCl, 1 mm EDTA, 1 mm dithiothreitol, 10%glycerol, 5 mm MgCl2, 2 mm ATP, 10 mm Na2S2O5, 0.5 μg/ml leupeptin, 1 µg/ml pepstatin and 1 µg/ml of Aprotinin in the ratio of 1:3 at 0-4°C, centrifugation of the homogenate at 105000 g for 60-90 min at 0-4°C To produce the cytoplasmic fraction, incubation cytoplasmic fraction with 10 mm phosphocreatine and 10 μg/ml phosphocreatine for 25-45 min at 35°C and separation of proteins in the cytoplasmic fraction of ammonium sulfate in three stages, the first stage add ammonium sulfate to 38% saturation and centrifuged to separate the precipitate, containing a pool of the 26S-proteasome at the second stage to the supernatant add ammonium sulfate to 42% from saturation and centrifuged to separate the precipitate, containing ballast proteins, in the third stage to the supernatant add ammonium sulfate to 70% of the t saturation and centrifuged to separate the precipitate, containing a pool of 20S proteasomes, and centrifugation to separate the precipitation is carried out at 10000 g for 30 min at 0-4°C, while the precipitate, containing a pool of the 26S-proteasome, is dissolved in a buffer containing 20 mm Tris-HCl, pH 7.5, 1 mm EDTA, 1 mm dithiothreitol, 50%glycerol, 5 mm MgCl2, 2 mm ATP, 10 mm Na2S2O5, 0.5 μg/ml leupeptin, 1 µg/ml pepstatin, 1 μg/ml Aprotinin, and the residue, containing a pool of 20S proteasome, is dissolved in a buffer containing 20 mm Tris-HCl, pH 7.5, 1 mm EDTA, 1 mm dithiothreitol, 50%glycerol, 10 mm Na2S2O5, 0.5 μg/ml leupeptin, 1 µg/ml pepstatin, 1 μg/ml Aprotinin.

2. The method according to claim 1, characterized in that the incubation solution containing a pool of the 26S-proteasome, is carried out with 10 mm phosphocreatine and 10 μg/ml phosphocreatine for 25-45 min at 35°C to restore the structure of the 26S-proteasome.

3. The method according to claim 1, characterized in that the ammonium sulfate added in portions over 20 min on a magnetic stirrer, and then stirred for 20 minutes



 

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Glypican-3 antibody // 2427588

FIELD: medicine.

SUBSTANCE: versions of antibodies bound with glypican-3 in a site with amino acid residues 1-563 are offered. Each version is characterised by the fact that it contains three CDRs of a light chain and three CDRs of a heavy chain. There are described: coding polynucleotide, and also a based expression and a host cell on the basis of the vector. There are disclosed: a method of producing the antibody with using a host cell, a cell growth inhibitor on the basis of the antibody, versions of application of the antibody for treating cancer or hepatoma. There is described peptide for producing glypican-3 antibodies containing residues 546-551 of glypican-3. The offered new antibodies exhibit higher cytotoxicity as compared with known glypican-3 antibodies and are specific to a certain site of glypican-3.

EFFECT: invention use can find further application in cancer therapy.

16 cl, 20 dwg, 2 tbl, 27 ex

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