Recombinant interleukin 2 people


(57) Abstract:

Usage: medicine, research practice. The essence of the invention: "inclusion bodies", the resulting expression in bacterial cells recombinant vector containing the native gene IL2man, by solubilization in a healing environment with chaotropes agent and subsequent purification by precipitation, high performance liquid chromatography reverse phase (RP-HPLC) using acidic eluant, breeding IL2-containing fractions acidic and re RP-HPLC in the acidic environment of the obtained recombinant IL2the man with the amino acid sequence of the natural form, but containing provisions 58, 105 and 125 restored cysteine residues and has the specific activity of at least 0.5 to 107E/mg 1 C.p. f-crystals, 9 Il., 3 table.

This invention relates to biologically active replicationmanager recombinant human interlekin-2 (called R-chil2in the restored form.

Natural human IL2that is a lymphokine that stimulates the multiplication of activated T cells, has three CIS whom 5 are connected by a disulfide bridge, and the cysteine at position 125 has a free sulfhydryl group (Robb, R. J and al. Proc. Natl. Acad. Sci USA. (1984) 81-6486-6490).

Methods for obtaining human IL2or its derivatives by recombinant DNA technology already described. For example, Taniguchi, T al. Nature (1983) 302 305-310 and Devos, R and al. Nucleic Acids Research (1983) 11 4307-4323, describe the cloning of the gene for human IL2and its expression in microorganisms, and Ju, C and al. J. Brol. Chem (1987) 262 5723-5731 received the expression of recombinant derived IL2. On the other hand, it is known that when IL2accumulated in the microorganism in the form of insoluble granules, it is in restored form, containing 3 tylnej group, and is devoid of activity (application for Japanese patent A 1257931). Therefore, it was considered that in order to get activated SLUDGE2I had to resort to the oxidation of the recovered protein found in the granules. To do this, after dissolving the protein in denaturing environment, the formation of a disulfide bridge 58-105, which is required for renaturation was carried out in a controlled oxidizing atmosphere. Describes various methods of oxidation, such as oxidation only oxygen (auto-oxidation by air) or in the presence of ions dvuhvalenten. Biochemistry (1987) 26 3129-3134).

After oxidative renaturation should be cleaned chromatography to remove oxidation products, corresponding to the formation of isomeric intramolecular bridges 58-125 and 105-125, as well as intermolecular bridges, inactivity which was proved by experiment and which can be separated by chromatography reverse phase according to Wang. A and al. Science (1984) 224 1431-1433 or Browing J. L and al. Anal Biochem (1986) 155 123-128.

The production of oxidized homogeneous recombinant IL2having a suitable biological activity based on protein, accumulated in the form of granules, raises technical problems regardless of the used method, as it requires multiple stages of purification, which results in a lowering of the yield of the target product.

The applicant has developed a new interleukin-2, for which does not require a stage of re-oxidation.

Thus, the invention relates to obtaining replicationmanager recombinant human interleukin 2 having, on the one hand, the following amino acid sequence:

< / BR>
as well as alleles and derivatives of this sequence, where X is methionine proyavlyayuschego biological activity, comparable to the activity of the oxidized IL2with the same sequence and containing a disulfide bridge in position 58-105.

Alleles and derivatives include sequence modified by substitution, deletion or insertion of one or several amino acids other than cysteine 58, 105, 125, thus, these products retain biological activity characteristic of the recovered IL2. The implementation of such modifications are well known in recombinant DNA technology, for example, methods of site-directed mutagenesis, listed Lather, R. F and Lecog, J. P in the journal Genetic Engineering Acad Press (1983) 31-50 or Smith, M and G. Mam.'s Genetic Engineering Principles and Methods Plenum Press (1981) 31-32. Under the restored form imply that remains cysteines that belong IL2contain free sulfhydryl group, the presence of which is determined, for example, spectrophotometric with dithiodipyridine as Colnago reagent. Biological activity restored form, obtained according to the invention, is determined by comparison with the corresponding oxidized form containing a disulfide bridge 58-105, by measuring the propagation of the leukaemic cell lines CTLL-2 mice, dependent IL2that is sitsa to a method for deglycosylation recombinant IL2man, having, on the one hand, the following amino acid sequence:

< / BR>
as well as alleles or derivatives of this sequence, where X is methionine or a hydrogen atom and three cysteine which in position 58, 105 and 125 are located in restored form, and on the other hand, exhibiting the biological activity of at least 0.5 to 107E/mg Unit activity IL2is defined as the quantity that gives 50% of the maximum response in the experience. As a benchmark, use the sample Biological Response Modifier Program (BRMP) reference Reagent human IL2(Jurkat), supplied by the National Cancer. Institute (NCI).

More specifically the invention relates to the production of replicationmanager recombinant interleukin-2 having the following amino acid sequence:

< / BR>
where X is methionine or a hydrogen atom and three cysteine which in position 58, 105 and 125 are located in restored form, and displaying biological activity comparable to the activity of natural IL2man.

Under comparable activity mean the same specific activity as the natural IL2isolated from leukemic Jurkat cells, i.e., 1,3 107E/mg (link WRMR), or activity, various the SUB>2according to the invention may contain additional N-terminal methionine, depending on the transformed microorganism, such as E. coli, in which it is expressed. In a preferred embodiment of the method preferred by the sequence containing methionine, but you can also use a mixture of the product containing methionine, with a product that does not contain methionine, or a product that does not contain methionine.

The method of obtaining replicationmanager recombinant restored IL2person includes removing SILT2accumulated in the form of granules in a microorganism transformed by the dissolution in the recovery environment using chaotropes agent, and then purification by precipitation with subsequent high-performance liquid chromatography reverse phase using acidic eluent, and then by request main elyuirovaniya fraction chromatography is subjected to cooling to a temperature of about -20oC, and then separated from the aqueous phase, which is diluted in an acidic medium, and then chromatografic to another column HPLC with a reverse phase in the acidic environment and secrete IL2.

Recombinant IL2, polucen which can be dissolved by the known methods using a concentrated solution chaotropes agent, such as a salt solution of guanidine 6-8 M, and then purified high-performance liquid chromatography reverse phase (referred to below RP-HPLC) on media, commercially available, preferably on the grafted silica, such as C3, C4, C8 or C18, acidic eluent having a pH of 1-4.

IL2can be blueraven from the column using a transport system consisting of organic acids, such as acetic acid or triperoxonane acid (denoted below TFU), and an organic solvent, such as acetonitrile. The main fraction, the elution of which is determined spectrophotometrically at 280 nm, is the starting product for the possible stages of cooling. Under cooling, it is understood that this main fraction collected at room temperature, is introduced into the environment, where the temperature is about -20oC, which gradually formed a solid aqueous phase, from which you can delete emerged fraction by decantation.

Thus, the separated aqueous phase is after dilution of the original product for RP-HPLC in an acidic environment specified below. Dilution of the aqueous phase is carried out in an acidic environment with a pH of 1-4, preferably 2 to 3. Diluted aqueous phase chromatographie C3, C4, C8 or C18, having pores of a suitable size for use with proteins, for example, with a diameter of at least 150 . For elution IL2use a gradient of increasing concentration of the lower alcohol, miscible with water and containing an organic acid.

Possible stage of cooling is carried out, in particular, in an aqueous solution of acetonitrile containing 0.1% triperoxonane acid, dilution is carried out with the aid of an aqueous solution of organic acid such as citric acid, and IL2eluted in the second chromatography using a solution comprising isopropanol, water and organic acid, such as citric acid.

An aqueous solution of acetonitrile containing 0.1% TFU acid, which is a preferred mixture is directed to the possible stage cooling, corresponds to the main faction, elyuirovaniya on the first chromatography. Especially preferably the dilution of the aqueous phase to conduct immediately or after a possible separation or after the first chromatography. This dilution is performed with the introduction of preferably at least 2 volumes of water containing 0.5-2% organic acids, such as formic, acetic, propionic, triol acid.

Second chromatography is that the aqueous phase which can be separated after cooling stage, and then dilution, direct on RP-HPLC using a media commercially available, such as grafted silica C3, C4, C8 or C18 with pore diameters of not less than 150 . The preferred medium is inoculated dioxide 04 VYD 300 consisting of silica gel with butylene groups grafted by covalent way, the pores of which have a diameter of 300 particles which have an average size of 15 to 20 μm. Elution IL2is conducted by means of the transporting system, including alcohol, such as propanol or isopropanol, and organic acid such as formic, acetic, propionic, triperoxonane or citric acid.

The preferred mixture comprises isopropanol, water and citric acid, preferably 0.5 to 2%, particularly preferably of 0.5%, while the gradient with increasing concentration of isopropanol elution smaller fraction containing approximately 48% isopropanol, and then a large fraction containing about 59% isopropanol, and this last fractions contains active non-recombinant IL2man in the WMD IL2person in restored form, obtained by the method described above.

Referred to the resulting fraction can be stored at a temperature of from about 0oC to -20oC. It is stable in these conditions. You can also remove the isopropanol isotropes by distillation in vacuum. The resulting solution can be stored at a temperature of 4oC or immediately secrete IL2lyophilisation.

If the method is carried out without cooling the main fraction and separating the aqueous phase, the resulting diluted the main fraction stored at 4oC. It is stable and serves as the raw material of the second chromatography.

When dosed tirinya group, IL2obtained according to the invention has a 3 free sulfhydryl groups and exhibits a specific activity of 0.7 to 1.3 107E/mg in the experience of breeding lines CTLL-2, i.e., comparable with that which is manifested native IL2. This activity justifies the use of replicationmanager recombinant IL2person in restored form described above, as a medicine in the same way as native IL2when the number of indications where its immunomodulating activity, as well as its protivoopujolevy is mnozenie lymphocytes T, induction of cytotoxicity of NK cells (natural cell-killer) cells and LAK (lymphokine that activates the cell-killer), recovery of cellular immunity, protective effect against infection in the case of immune deficiency or secondary effect in relation to vaccines. The introduction may be direct or in Association with the corresponding method of immunotherapy, which is described Rosenberg S. A., and al. N. Engl.J.Med. (1985) 313, 1485-1492.

As well as native IL2IL2obtained according to the invention can be used alone or in Association with other means of immunomodulatory, such as interferon alpha, interferon gamma or with other therapeutic means.

Non-recombinant IL2person in restored form can be used to prepare pharmaceutical compositions containing as active principle.

The composition may be solid or liquid. Interleukin-2 of the invention may include in the recipe mix, prepared with known methods for the preparation of a pharmaceutical composition, where IL2mixed with a pharmaceutically acceptable carrier. This composition contains an effective amount of the IL2with suitable the thief IL2according to the invention after removal of the isopropanol azeotrope by distillation in vacuum, to which is added a filler soluble in water, and which lyophilizer. The word filler understand the substance soluble in water and which does not change the initial pH when the mixture is composed. Examples of fillers that can be injected, are given in the following order: glucose, ribose, sucrose, maltose, trehalose or restored sugar, such as mannitol. Prefer mannitol. Mannitol is added in concentrations from 10 to 50 mg/ml, preferably about 50 mg/ml when the concentration of IL2is between 0.05 mg/ml depending on the dose. The solution was sterile filtered in the absence of oxygen, distributed in ampoules-dosing and then lyophilizer. Lyophilized mixture may be reestablished by the introduction of a vial of distilled water, suitable for parenteral injection.

The composition according to the invention may be administered intravenously through a bolus or by continuous perfusion, by intramuscular, intraperitoneally by intrapleural or by subcutaneous injection. Effective dose IL2that depends on route of administration and CA6E/M2/24 h for an adult or child. The daily dose depends on the duration of the introduction, and should not be limited to the above doses.

For the introduction of a method of perfusion of the pharmaceutical composition, in particular, contains non-recombinant IL2person in restored form, water and organic acid, such as citric acid. Such a composition can be obtained from the lyophilized pharmaceutical composition of the invention, which is dissolved in the presence of a suitable media, which contributes to the stability of the active principle during perfusion, for example, glucose.

Under the preferred conditions of the lyophilized mixture is diluted with distilled water, entering her in a bubble for perfusion containing glucose at a concentration of 50 mg/ml, in the amount determined in accordance with the input dose.

The following figures illustrate some aspects of the invention.

Fig. 1A is a chromatogram analysis RP-HPLC of the crude extract of 8M guanidine of example 1.

Fig. 1B is a chromatogram analysis RP-HPLC standard IL2, reduced and oxidized.

Fig. 2 is a chromatogram-Analipsi cooling stage of example 1.

Fig. 4 is a chromatogram analysis OF HPLC fractions "59" example 1.

Fig. 5 is a gel electrophoresis SDS-PAGE sample 2.

Fig. 6A and 6b are peptide maps of example 2.

Fig. 7a and 7b is curves KD of example 2.

Fig. 8 is a curve mitogenic effect on human lymphocytes in example 3.

Fig. 9 is a curve of toxicity obtained in relation to lines To 562 and DAUDI.

The following examples illustrate the invention but do not restrict it.

Example 1. Purification of the biologically active restored p-chil2from granules.

Pellets obtained by centrifugation of the cultures of E. coli strain transformed with a plasmid containing the coding sequence of natural IL2and can accumulate IL2in the form of granules inside the cells, as described, for example, SATO, T al., J. Biochem. (1987) 101 525-534. Cells obtained in the fermenter with a volume of 10 l, is subjected to the gap homogenizer (Manton Gaulin.

Based on these cellular residues, selected and washed, (90-170 g wet weight) IL2dissolve 2.5 volumes of buffer solution Tris, HCl 20 mm pH 8, containing guanidine hydrochloride (GU, HCl) 8M and Dimitrii the NECS VYDAC C4 (0,46 x 15 cm) 300 , 5 microns, at a speed of 2 ml/min with a linear gradient of acetonitrile (from 30 to 70% for 10 min) containing 0.1% TFU, spectrophotometric detection was performed at 280 nm or 210 nm, the peak area which was determined at 280 nm, after calibration with standard IL2(Fig. 1A and Fig. 1B). IL2then precipitated down to 2M concentration of Si, HCl, in the presence of DTT.

After washing the precipitate using a 0.1% aqueous solution of TFU to obtain pH surfaced part 5.0 IL2dissolved in an aqueous solution containing 20% acetonitrile and 0.1% TFU. The new solution, which contains analytical RP-HPLC (Fig. 2) more than 85% restored IL2shows biological activity below 0.01 107E/mg restored IL2and is the content of sulfhydryl groups in the number 2,85 SG/mol restored IL2. The fraction obtained solution, corresponding to about 200 mg IL2by RP-HPLC, diluted to bring the concentration of acetonitrile below 10% in 0.1% TFU, and then transferred into a column VYDAC C4 (5.7 x 30 cm). IL2elute with a flow rate of 100 ml/min using a linear gradient of acetonitrile (30-80% over 40 min) containing 0.1% TFU, at a concentration of about 60% of acetonitrile in a large peak is found, is it restored IL2, then subjected to a stage of slow cooling from room temperature to -20oC 1oC and removed by decantation of the upper organic phase rich in acetonitrile. The lower phase, containing a lot of water and also containing IL2can be stored in frozen form. After thawing the resulting solution after dilution with two volumes of a 0.5% aqueous solution of citric acid, transferred to a column VYDAC C4 (5.7 x 30 cm), which is being launched with a linear gradient of isopropanol (20-70% over 40 min) containing 0.5% citric acid, with a flow rate of 50 ml/min Effluent, monitored spectrophotometrically at 280 nm, shows a sequential elution of a small peak at a concentration of about 48% isopropanol (fraction "48") and a main peak at a concentration of about 59% isopropanol (fraction "59") (Fig. 3). Faction "59" collect. It is stable when stored at 0oC for at least 24 h in the absence of air.

After removal of the isopropanol azeotrope by distillation in vacuum faction "59" analyze analytical RP-HPLC and find homogeneous peak (Fig. 4), suirvey at 60% acetonitrile, whereas the oxidized IL2(standard) eluted at about 57% acetonitrile (Fig. 1B). Faction "59", which after removal of the absence of air, for at least one week, or immediately lyophilizer or mixed to obtain a pharmaceutical composition. Analyze the biological activity of the lyophilized fractions "59" test "in vitro" multiplication of cells CTLL-2. Its specific activity is equal to 1,30,5 107E/mg and is comparable with the activity of natural IL2.

The content of free sulfhydryl groups on the faction "59", lyophilized defined by the colorimetric method with dithiodipyridine is 2,94 SG/mol, compared with 0.76 to SG/mol for the reference of the oxidized IL2.

150 - 300 mg, titrated analytical RP-HPLC, restored R-IL2man, containing 3 groups SG, biologically active, homogeneous by RP-HPLC, obtained from the faction "59" in the fermenter tank capacity 10 litres

Example 2. Purification of the recovered biological active R-IL2.

Act as in example 1, except that it is not subjected to "main faction of the cooling phase, and then separating by decantation, and immediately diluted with 2 volumes of a 0.5% aqueous solution of citric acid. Faction "59" is collected and processed as in example 1.

Example 3. Physico-Henichesk is th R-IL2located in the faction "59" example 1, is studied by the following properties.

1) Uniformity

Electrophoresis SDS-PAGE is carried out on the gel with two phases /gel concentration and gel migration, respectively, with 5% and 15% acrylamide/ containing 10% SDS.

The sample is preheated for 2 min at 100oC in a buffer solution containing 3% SDS and 5% mercaptoethanol.

Migration with a buffer solution containing 1% SDS, gives colour silver, which shows a single band corresponding to a purity higher than 99% on the residue in 2 μg (Fig. 6).

2) Molecular weight determined by electrophoresis

In a reducing environment determine the apparent MW of about 15 kDa, which corresponds to the calculated MV 15420 (Fig. 5).

3) Amino acid composition

The sample containing 25 μg of the recovered p-IL2man according to the invention in 0.5 ml of water, is introduced into a glass tube for hydrolysis, to which was added 0.5 ml of concentrated hydrochloric acid containing 31.7% of TFU and 4.8% thioglycolic. The tube is sealed under vacuum, and then conducting hydrolysis at 155oC for 40 minutes

The hydrolysis product is then pariveda to dryness under reduced pressure. ction AA 511 (0,46 x 15 cm) using a gradient of pH (3 to 5) and sodium chloride (0 to 70 g/l) in a buffer solution of citrate, at 60oC, with a flow rate of 0.5 ml/min and detection by fluorescence after drainage orthophtalaldehyde at the outlet of the column. The results are given in table. 1.

Values are average numbers obtained for 2 gidrolizom (repeated) and 2 chromatograms, respectively repeated. The composition corresponds to the composition of the native IL2containing an additional N-terminal methionine.

4) N-terminal amino acid sequence

N-terminal sequence identified by the method of micropaleontology using an automatic way of degradation of Edman. Analysis of 15 µg restored biologically active p-IL2man according to the invention on micropaleontol gas phase Applied Biosystems A, coupled with HPLC 120A, allowed us to identify the following amino acids FGF (phenylthiohydantoin), are given in table. 2.

Consistently 20 N-terminal residues is consistent with theoretical order of sequences of native IL2. Found about 10% SILT2without methionine.

5) Peptide map obtained with methyl cyanide

700 mg of the recovered IL2according to the invention (about 53 nmol) dissolved in 4 ml of Oh temperatures, dilute with water, and then subjected to lyophilization. The reaction mixture was analyzed using RP-HPLC on a column of Bondapack C18 PF (0,46 x 20 cm) using a gradient of acetonitrile varying from 0 to 70%, containing 0.1% TFU, at a speed of 1 ml/min at room temperature and with spectrophotometric detection at 220 nm. The peptide map of the restored p-IL2man (Fig. 6A) shows the fragmentation that is different from the fragmentation of the oxidized IL2pattern (Fig. 6b).

6) Circular dichroism

The spectra of circular dichroism (CD) is determined at room temperature on spectrograde Jobin Yvon Mark V. a Sample of the recovered p-IL2human, lyophilized after RP-HPLC performed with a linear gradient of isopropanol, which was replaced with 0.5% citric acid in example 1 0.1% formic acid, and subsequent removal by distillation of isopropanol and drying, absorb the acetic acid with a concentration of 1 mg/ml Used capacity be 0.01 cm and 0.5 cm respectively for peptide region (185-250 nm) and for the aromatic region (260-320 nm). Range of solvent is calculated from the spectrum IL2for each sample. The results are given in the ellipticity of 0 (the average weight for the remainder IL2man according to the invention has a spectrum, which indicates the presence of an ordered secondary structure. Definition % alpha helix shows an insignificant difference with oxidized ethanol IL2(% alpha helix 50%).

Fig. 7b shows the range of KD in the near UV: oxidized IL2the standard is indicative of KD, which indicates asymmetric environment for aromatic residues, whereas the restored p-IL2man according to the invention has a KD little revealing, but different.

Example 4. Biological activity

The biological activity of the recovered p-IL2man according to the invention is evaluated by experiments in vitro or ex vivo".

1) Activity in vitro on human cells

A. Experience lymphoblastic transformation

In addition to the proliferative activity at the cellular generations of mice, such as CTLL-2, which allows the biological dose IL2restored p-chil2according to the invention exhibits mitogenic effect in a dose-dependent comparable with the effect expressed oxidized IL2standard normal circulating human lymphocytes, which is yadernykh cells

Experience is incubated on circulating mononuclear human cells in the presence of IL2and the cytotoxic effect was determined in relation to the tumor target cells, respectively erythroleukemias generation To 562 (sensitive to NK cells ) and generation DAUDI produced from lymphoma B (resistant to NK cells) by measuring vysalivaniya 51 CR for 4 h

The results, expressed in lytic units 106cells (LE/n), show that the restored p-IL2man according to the invention is capable, depending on the dose, respectively, to increase the activity of NK or inducing cytotoxicity of T lymphocytes against tumor targets, which can be compared with the known activity of natural IL2(Fig. 9).

2) Activity ex vivo in mice (stimulation of peritoneal macrophages)

In normal mouse Balb/c and MRL -+/+ serial intraperitoneal injection of a first recombinant gamma interferon in rats during suboptimal doses (100-300 F), and then 24 hours later restored p-IL2man according to the invention initiates oxidative mechanisms of phagocytic cells is determined by measuring chemiluminescent the 4 h after injection of IL2. The effect is dose-dependent, comparable to the effect observed with the oxidized IL2standard.

The results are shown in table. 3.

Example 5. Pharmaceutical composition for injection

An aqueous solution of the restored p-IL2person, the relevant fraction "59" according to the invention, from which the isopropanol was removed by azeotropic distillation in vacuum, diluted as necessary with the help of degassed and saturated with nitrogen aqueous solution of mannitol at the rate of 100 mg restored IL2/ml and 50 mg/ml mannitol. After filtration on a membrane thickness of 0.22 μm, sterile distribution 1 ml vials and lyophilization flask-dose sealed under nitrogen atmosphere and stored at a temperature of 4oC before use.

Example 6. Pharmaceutical composition for continuous perfusion.

An aqueous solution of the restored p-IL2person, the relevant fraction "59" inventions, removed isopropanol azeotrope by distillation in vacuum, diluted as necessary with the help of degassed and saturated with nitrogen aqueous solution of mannitol ratio of 500 µg restored IL2/ml and 50 mg/ml mannitol. After filtration, the membrane thickness of the TA and stored at a temperature of 4oC before use. Then the content of each flask was dissolved by the introduction of 1 ml of sterile distilled water. The solutions corresponding to 7 bottles-doses (about 35106units), is introduced into the container Viaflex R containing 500 ml of solution for perfusion of 5% of TravenolROf glucose.

1. Recombinant interleukin 2 (IL 2) the person obtained pellets of the product of expression of the bacterial cells after their transformation vector containing the native gene IL 2 person, by solubilization in a healing environment with chaotropes agent and subsequent purification by precipitation, high performance liquid chromatography reverse phase (RP-HPLC) using acidic eluant, breeding IL 2-containing fractions acidic and re RP-HPLC in an acidic environment, including cysteine in positions 58, 103 and 125 in restored form, with mol. m about 15 KD and an amino acid sequence H-Ala-Pro-Tre-Ser-Ser Ser-Tre-Liz-Liz-Tre-GLn-LEU-Gli-LEU-Glu-GIS-LEU-LEU-LEU-ASP-LEU-GLn-Met-Ile-Met-LEU-Tre-hair-Liz-Hairdryer-Tyr-Met-Pro-Lys-Lys-Ala-Tre-Glu-LEU-Lys-GIS-LEU-GLn-GIS-LEU-Glu-Glu-Glu-LEU-Lys-Pro-LEU-Glu-Glu-Val-LEU-ASN-LEU-Ala-GLn-Ser-Lys-ASN-Hairdryer-GIS-LEU Arg-Pro-Arg-ASP-LEU-Ile-Ser-ASN-Ile is her-ASN-Arg-TRP-GLn-Ser-Ile-Ile-Ser-Tre-Lei-Tre or a similar sequence, which contains provisions that differ from 58, 105 and 125, amino acid substitutions deletions or insertions that do not affect biological activity, where X is methionine or hydrogen, and has mitogenic, proliferative, macrohistory and inducing cytotoxicity managernew cells the activities of the native IL-2.

2. Interleukin 2 on p. 1, characterized in that it exhibits a specific activity of at least 0.5 to 107U/mg.


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