Method of producing recombinant protein

FIELD: medicine.

SUBSTANCE: method involves cultivation of recombinant koji mycelial fungi in a fluid medium, collection of recombinant protein from the prepared product. The culture medium contains as a raw substance, at least one selected from a group consisting of barley and wheat surface of which is completely or partially coated at least in the concentration 1 to 20% (weight/volume) and as nutrients, at least one inorganic salt. Recombinant koji mycelial fungi is produced by a procedure consisting in the fact that a gene coding a target protein is ligatured below a promotor of a gene coding an enzyme exposed to catabolite repression due to concentration of the nutrients, such as saccharides and amino acids, for preparing thereby the ligation product, and the ligation product is introduced in koji mycelial fungi as a host.

EFFECT: invention allows higher yield protein.

4 cl, 3 tbl, 1 ex

 

Technical area

The present invention relates to a method for producing a recombinant protein, more particularly to a method for producing a recombinant protein by using koji molds as the host body.

Background of the invention

Koji since ancient times, used as sources of enzymes for the production of fermented foods and drinks. As CODI for the production of fermented food and beverage traditionally used solid Koide, which is obtained by growing koji molds on the surface of the cereals and the like. Solid Kodi receive the traditional way of production. However, the method is a special method of cultivation, namely cultivation in the form of a solid culture that is unsuitable for large-scale production.

On the other hand, liquid Koide, which is a product of the culturing koji molds obtained by culturing koji molds in a liquid can be easily controlled in the culture, and it is a suitable method of cultivation for efficient production.

However, it is well known that liquid mould koji does not give sufficient enzymatic activity required for production is VA fermented foods and drinks, therefore, there are few examples in which the liquid Kodi used in real production (see non-patent documents 1-4).

Mold fungi koji easily multiply, respectively, can be prepared environment with low cost, and for cultivation does not require special equipment, so the cost of cultivation is low. Mold fungi koji used for the production of fermented food and beverage since ancient times, therefore, recognized as a safe organism-owner. Traditionally, therefore, attempts were made to embed genes derived from koji molds or from other organisms, by use of koji molds as the host body for high expression of genes for the production through this products derived from genes, i.e. recombinant proteins (see non-patent document 5).

In addition, it was reported about successful examples of production of recombinant protein in high yields using solid culture with wheat bran (see non-patent document 6). However, the production was carried out by a special method of cultivation, namely, firm culture, which is unsuitable for large-scale production.

On the other hand, it is considered that the nature of the liquid culture is insignificant to the number of proteins outside the cells, as described above, and therefore, is not suitable for large-scale production of recombinant protein.

Non-patent document 1: Y. Hata et al.: J. Ferment. Bioeng., 84, 532-537 (1997).

Non-patent document 2: Y. Hata et al.: Gene, 207, 127-134 (1998).

Non-patent document 3: H. Ishida et al.: J. Ferment. Bioeng., 86, 301-307 (1998).

Non-patent document 4: H. Ishida et al.: Curr. Genet., 37, 373-379 (2000).

Non-patent document 5: R. J. Gouka et al., Appl. Environ. Biotechnol., 47, 1-11 (1997).

Non-patent document 6: K. Tsuchiya, et al., Biosci. Biotech. Biochem., 58, 895-899 (1994).

Description of the invention

Issues that are addressed through the invention of

The object of the present invention is the provision of a method of large-scale production of recombinant protein by the method of liquid culture with the help of koji molds as the host body, traditionally considered unsuitable for the production of recombinant protein for the foregoing reasons.

Means for solving problems

The authors of the present invention have already proposed methods for manufacturing liquid Kodi with sufficient enzymatic activity (see Japanese Patent Application Nos. 2004-350661, 2004-352320, 2004-352324, 2004-378453 and 2005-290648, and JP-A-2003-265165). In these methods are used in the environmental quality of the raw material, the surface of which is entirely or partly covered with at least a film that protects the release of PI is athelny substances from raw material to the system of cultivation, and this brings the activity of enzymes, namely, amylolytic enzymes, cellulolytic enzymes and proteolytic enzymes. The methods are achieved enzymatic activity higher than the activity obtained in liquid culture using as raw substances polished barley or polished rice, which is a raw material for Shochu. Examples of koji molds include white mold koji fungi, black mold fungi koji, yellow molds koji and red molds koji.

Assume that the above methods of obtaining liquid Kodi increase the levels of transcription of the genes that encode the enzymes, subjected to catabolite repression due to the concentration of nutrients such as sugars and amino acids, and thus, the products (namely, the target enzymes)derived from these genes are formed and are secreted from cells koji molds.

The gene encoding the target protein are ligated below the promoter of the gene encoding the enzyme is subjected to catabolite repression due to the concentration of nutrients such as sugars and amino acids, to generate thereby the recombinant koji molds having included therein the product of ligation of cultured recombinant molds koji above methods of manufacturing a liquid koji, which formed as a result of the target recombinant protein in high yields.

An additional essential factor in the production of the recombinant protein is to be translated recombinant protein is correctly transported from the cells of the host body. Even if the levels of transcription simply increased, translated recombinant proteins can often accumulate to the cell or decompose under the action of enzymes in normal secreted by an organism of the host. Moreover, proteins that are correctly folded and have activity equivalent to the activity of the natural protein is not formed until such time as the recombinant protein has not undergone the necessary modifications during its transportation from the cells of the host body.

Taking this into consideration, it is highly likely to receive and secrete a large number of recombinant proteins by using the technology of producing liquid koji, as described above, using the koji molds as the host body. The target recombinant protein can also be obtained with high yields due to the fact that it can be produced outside of the host body moulds koji, as a fused protein of the recombinant protein with an enzyme, which normally is produced and secreted by the body-master moulds, cog is, and that protein contained in the culture supernatant, is cleaved at its site of ligation site-specific protease.

Thus, on the basis of the above data, the present invention is completed.

That is, the present invention according to claim 1 provides a method of obtaining a recombinant protein using recombinant koji molds that are formed by the transformation of the koji molds as the host body, comprising: culturing the recombinant koji molds in a liquid medium containing as culture raw material substance, at least one selected from the group consisting of grains, the surface of which is entirely or partly covered with at least the shell of the bean and/or tuber, the surface of which is coated, and amaranth and/or seed quinoa without preprocessing, such as the milling or grinding; and collecting the recombinant protein from the culture product.

The present invention according to claim 2 provides a method of obtaining a recombinant protein according to claim 1, wherein the recombinant molds koji get the fact that the gene encoding the target protein are ligated below the promoter of the gene encoding the enzyme is subjected to catabolite repression due to the concentration of nutrients, such as sugar in the water and amino acids, to obtain thereby the product of ligation, the ligation product is injected into molds koji as the host body.

The present invention according to claim 3 provides the method of obtaining a recombinant protein according to claim 2, wherein the promoter is a promoter of the gene encoding any enzyme selected from the group consisting of amylolytic enzymes, cellulolytic enzymes and proteolytic enzymes.

Useful effect of the invention

According to the present invention provides a method for mass production of recombinant protein by way of a liquid culture of koji molds as the host body. Mold fungi koji easy to breed, so the environment can be prepared at low cost, and does not require any special equipment. Mold fungi koji used for the production of fermented food and beverage since ancient times, so they are a safe organism-owner.

Next, a liquid culture of koji molds can be controlled more strictly compared to solid culture, so it is suitable for efficient production.

In addition, you can select multiple schemas production by using different raw substances and strains of koji molds and the efficiency is about and steadily in scale to obtain the target recombinant protein.

The best implementation for carrying out the invention

Further, the present invention will be described in detail.

The liquid medium used in the present invention contains at least one raw material for cultivation, selected from the group consisting of grains, the surface of which is entirely or partly covered with at least the shell of the bean and/or tuber, the surface of which is coated, and amaranth and/or seed quinoa without preprocessing, such as milling or grinding.

According to the present invention, examples of crops used as raw materials for cultivation include barley, rice, wheat, buckwheat, millet, Japanese millet, Italian millet, sorghum, Japanese, corn and the like. Raw materials for cultivation must have a surface entirely or partially coated or covered, at least shell (husk). You can use unpolished substance or a substance having an equal or higher ratio polishing, in which it was polished so that the shell, at least, remains on the surface of the grain, and you can use raw rice, raw barley and the like. In the case of rice, you can use raw rice, the rice is completely coated, and rice, partially covered by the shell.

When raw material for cultivation is barley, you can use unpolished material with a ratio of polishing 100%. An alternative, based on the fact that the ratio of the polishing unpolished material is taken as 100%, it is possible to use a material with a ratio of polishing is not less than the value determined by subtracting the ratio of the share of shell in barley (usually from 7 to 8%) from the ratio of the polishing unpolished material (100%), i.e. from about 92% to 93%.

The term "ratio polishing" refers to the residual value of the grain after polishing grain. For example, the term "percentage of polishing 90%means that 10% of the shell, or the like, on the part of the surface layer of the grain removed. According to the present invention, the term "unprocessed barley covers the grain in the range from unpolished polished barley to barley, with shells left on the surface of the grain, that is, a substance having a ratio of polishing of 90% or more. The term "shell" refers to the outer part, which covers the surface of the grain.

According to the present invention, examples of the bean and tuber used as raw materials for cultivation include soy bean, red beans, sweet potatoes and similar. These raw materials for cultivation of machining is shown only to those that wash off contamination on their skin (peel), but not subjected to processes such as cutting, grinding and the like, and used for preparation of liquid medium in the form of a fully coated.

The present invention beans and tubers as raw materials for cultivation can be subjected to heat or freezing remaining on them by the shell.

According to the present invention "amaranth" for use as raw materials for cultivation is a generic term for plants belonging to the genusAmaranthusfamilyAmaranthaceae. Among cereals amaranth has a high protein content and lysine content, which is one of the amino acids close to the soy bean. In addition, amaranth is a highly nutritious grain that contains large amounts of calcium, iron and fiber, compared to polished rice. Countries of origin are certain areas of South/Central America, India, the Himalayas and Nepal. On the other hand, quinoa is an annual plant of the familyAgathawhich is cultivated mainly in mountainous areas such as the Andes, located in southern Peru and Western Bolivia. Quinoa is rich in minerals, vitamins, proteins and food is diversified fibers.

Amaranth and quinoa, intended as raw materials for cultivation, can be used separately or in combination. These raw materials can be used directly for the preparation of liquid medium without prior processing, such as milling or grinding.

One of the above raw materials for cultivation, or a combination of two or more substances are used to prepare a liquid medium, as described above. The above raw materials for cultivation is mixed with water to prepare a liquid environment. The ratio of the components of the raw substances can be adapted within the target recombinant protein selectively formed and accumulated in the culture product mould koji.

For example, when using barley as raw materials for cultivation liquid medium is prepared by adding from 1 to 20% (weight/volume) of barley to water. When using unpolished barley liquid medium prepared by adding, more preferably, from 8 to 10% (weight/volume). When using the 95% - polished barley as a raw material substance, liquid medium is prepared by adding him, more preferably, from 1 to 4% (weight/volume).

Then, when used as raw materials for cultivation of unpolished rice is, who removed the shell, liquid medium is prepared by adding from 1 to 20%, preferably from 5 to 13%, or, more preferably, from 8 to 10% (each value represents the ratio mass/volume) unpolished rice to the water.

When using beans as raw materials for cultivation liquid medium is prepared by adding from 1 to 10% of the beans to the water, preferably by adding 8 to 10% of soybeans or from 1 to 2% (each value represents the ratio mass/volume) of red kidney beans to the water. When using tubers as raw materials for cultivation liquid medium is prepared by adding from 1 to 10% (weight/volume) of tubers to the water.

When using amaranth as raw materials for cultivation liquid medium is prepared by adding from 1.5 to 15%, preferably from 2 to 10%, or more preferably, from 2 to 8% (each value represents the ratio mass/volume) of amaranth to the water. When using quinoa as raw materials for cultivation liquid medium is prepared by adding from 1.5 to 7%, preferably from 2%to 6%, or more preferably, from 2 to 4% (each value represents the ratio mass/volume) quinoa to water.

The number used for mixing raw materials for cultivation can appropriately choose, PQS is LCU optimal number of vary degrees of polishing or varieties used raw materials for cultivation, used as the host body strain koji molds used promoter produced the recombinant protein and the like.

If the number of used raw materials for cultivation exceeds the upper limit, the viscosity of the culture fluid increases and the supply of oxygen or air required for the aerobic cultivation of recombinant koji molds, becomes insufficient. This reduces the oxygen content in the culture product, limits the development of culture and is not preferred. On the other hand, if the amount of used raw substances below the lower limit, the target recombinant enzyme in large quantities is not formed.

The starches contained in the raw material for cultivation, before cultivation can be pre-relativelayout. Gelatinisation starches can be obtained using, but without specific limitation, any of the traditional methods, including the method of steaming, the method of roasting and the like. Starches can be relativelayout on stage sterilization of a liquid medium, as described below, when they are heated to a temperature of gelatinization or higher, by sterilization at high temperatures and high pressures.

In addition to the above raw material is the material for cultivation in liquid medium, used according to the present invention, it is desirable to add organic matter, inorganic salts and the like as sources of nutrients.

For example, when using the white koji molds, such asAspergillus kawachiior black koji molds, such asAspergillus awamoriandAspergillus nigeras the host body is applied nitrate salt and phosphate salt, preferably, in combination, or more preferably, is applied sulfate salt in combination in addition to him. Examples of nitrate salts include sodium nitrate and potassium nitrate, and potassium nitrate are particularly preferred. Examples of phosphate salts include potassium phosphate and ammonium phosphate, and potassium phosphate are particularly preferred. Examples of the sulfate salt include sulfated heptahydrate magnesium sulfated heptahydrate iron and ammonium sulfate, and sulfated heptahydrate magnesium and sulfated heptahydrate iron are particularly preferred. Two or more of them can be used in combination.

The concentration of inorganic salts in a liquid medium using white koji molds or black koji molds pick up within, to the target recombinant protein was selectively formed and accumulated in the culture product ples is of evich koji fungi. To be specific, the concentration of the nitrate salt is from 0.1 to 2.0%, preferably from 0.2 to 1.5%, the concentration of the phosphate salt is from 0.05 to 1.0%, preferably 0.1 to 0.5%, and the concentration of sulfate salt is from 0.01 to 0.5%, preferably 0.02 to 0.1% (each value represents the ratio mass/volume).

When using yellow koji molds, such asAspergillus oryzaeandAspergillus sojae, nitrate salt, a phosphate salt and sulfate salt is used, preferably together in a liquid medium. Examples of nitrate salts include sodium nitrate and potassium nitrate, and sodium nitrate are particularly preferred. Examples of phosphate salts include potassium phosphate and ammonium phosphate, and potassium phosphate are particularly preferred. Examples of sulfate salts include sulfated heptahydrate magnesium sulfated heptahydrate iron and ammonium sulfate, and sulfated heptahydrate magnesium and sulfated heptahydrate iron is particularly preferred. Two or more of these inorganic salts can be used in combination.

The concentration of inorganic salts in a liquid medium using yellow koji molds pick up within, to the target recombinant protein was selectively formed and accumulated in the culture product of the koji molds. To the set of specific, the concentration of nitrate salt is from 0.1 to 2.0%, preferably from 0.2 to 1.5%, the concentration of the phosphate salt is from 0.05 to 1.0%, preferably 0.1 to 0.5%, and the concentration of sulfate salt is from 0.01 to 0.5%, preferably 0.02 to 0.1% (each value represents the ratio mass/volume).

Organic substances and inorganic salts, with the exception noted above inorganic salts, respectively, can be added as sources of nutrients to the liquid medium of the present invention. These additives are not particularly limited, as they are commonly used for culturing koji molds. Examples of the organic substance include rice bran, wheat gluten, corn syrup, soybean meal and low fat soy beans. Examples of inorganic salts include water-soluble compounds such as ammonium salt, potassium salt, calcium salt and magnesium salt. Two or more organic and/or inorganic salts can be applied simultaneously. Additional quantities of them are not particularly limited as to cause propagation of recombinant fungal mould koji. Additional quantity of organic material is, preferably, from about 0.1 to 5% (weight/volume) and the additional amount of inorganic salt the leaves, preferably, from about 0.1 to 1% (weight/volume).

Adding these nutrient sources in excess of the upper limit is not preferred because it inhibits the growth of the recombinant koji molds. Quantity is less than the lower limit is not preferable because the target recombinant protein is not produced in large quantities.

Thus obtained liquid medium can optionally be subjected to processing, sterilization, and handling procedures are not particularly limited. For example, this can be a method of sterilization with an increased temperature and pressure held at a temperature of 121°C for 15 minutes.

Sterilized liquid medium is cooled to a temperature of cultivation, and then seeded with recombinant molds koji in a liquid environment.

Recombinant molds koji used in the present invention, are a product obtained by the transformation of the koji molds as the host body, and can be a product cultivated in the above liquid medium by means described below cultural way. Used as the host body molds koji can be a product that produces enzymes, subjected catabolite the repression due to the concentration of nutrients, such as sugars and amino acids. Examples thereof include white mold fungi koji, such asAspergillus kawachiiblack mold fungi koji, such asAspergillus awamori and Aspergillus nigerand yellow molds koji, such asAspergillus oryzae and Aspergillus sojae.

Recombinant molds koji according to the present invention obtained by ligating the gene encoding the target protein following promoter, and introducing the product of ligation in molds koji as the host body. Any promoter can be used in the present invention, since it expresses the underlying gene in mycelial fungi koji as the host body, and is preferred promoter of the enzyme, which high output is produced outside the cells koji molds. More preferably the promoter of the gene encoding the enzyme is subjected to catabolite repression due to the concentration of nutrients such as sugars and amino acids. Specific examples thereof include promoters of genes encoding amylolytic enzymes, such as glucoamylase (GlaA or GlaB) and α-amylase (AmyB), cellulolytic enzymes such as glucanase (EglA), and proteolytic enzymes, such as protease (PepA).

According to the present invention the recombinant molds koji to multiwire the t using the above raw materials for cultivation, therefore, the splitting of nutrients such as sugars and amino acids in raw substance takes a long time, and the rate of release of nutrients into the system of cultivation is limited. Accordingly, activates the promoter of the gene encoding the enzyme is subjected to catabolite repression, due to the concentration of these nutrients, and increases the level of transcription of the gene encoding the target protein, which is located below the promoter, resulting in mass quantity, the formation of the target recombinant protein.

According to the present invention, the gene encoding the target protein may be a material that can be Express in mycelial fungi koji as the host body, and may be a cDNA or chromosomal DNA. In the present invention, the term "protein" includes glycoproteins. The gene encoding the target protein is not limited to genes derived from koji molds. You can also use genes derived from organisms of other species, because the genes are suitable for the production of recombinant proteins using koji molds as the host body.

In addition to the promoter and gene, codereuse target protein, the product of ligation with the terminator, a selective marker and the like, Legerova the data in it, may be optionally introduced into recombinant molds koji according to the present invention. The terminator can be a product that operates in mycelial fungi koji as the host body, and preferably the use of terminator enzyme, which is formed with high yield outside the cells koji molds.

According to the present invention the transformation of the koji molds as the host body can be performed in a standard way, such as the method of introducing the plasmid vector in protoplast of the host body in the presence of PEG (Unkles et al., Mol. Gen. Genet., 218, 99-104 (1989)).

As a vector, you can use any plasmid, provided that it is suitable for the host body moulds koji. For example, the plasmid can be created using pPTRI, pPTRII (TAKARA BIO INC.) and the like, depending on the task. However, the choice of these plasmids is not limited.

To introduce the gene encoding the above-noted target protein in the above-mentioned vector, it is possible to apply well-known method. One way involves splitting the selected gene encoding the target protein, the appropriate restriction enzyme, the embedding of the cut gene in the area of recognition by the enzyme restriction or land for multiple cloning the corresponding vector DNA is La ligating the thus cut out of the gene in the vector.

Mold fungi koji, transformed, as described above, were cultured using the appropriate selective medium. After that, the formed colony allocate for the preparation of recombinant koji molds, which are built in the gene encoding the target protein.

Thus obtained recombinant molds koji can be used for cultivation of one strain or for the cultivation of a mixture of two or more homologous or heterologous strains. This allows you to use any form of spores or mycelium obtained in the previous cultivation. However, preferably used mycelium, because it requires a shorter period of time for the logarithmic phase of growth. The number of recombinant koji molds for cultivation in liquid medium is not particularly limited, but the number of spores may be in the range from approximately 1×104up to 1×106in ml liquid medium. In the case of mycelium preferably seeded with from about 0.1 to 10% pre-grown liquid culture.

The temperature of the cultivation of the recombinant koji molds may preferably be set from 25 to 45°C, or more preferably, from 30 to 40°C, but not particularly limited because it does not effect the overwhelmingly on growth. If the temperature of cultiv the simulation below, there is a tendency contamination by infectious microorganisms, because the growth of the recombinant koji molds slowed.

If the host body use yellow molds koji, the enzymatic activity is enhanced by controlling the temperature of cultivation in accordance with the growth phase yellow koji molds. To be specific, the culture temperature can be maintained between 25 and 35°C, preferably from 28 to 33°C during the phase of cell proliferation, which begins from the start of the cultivation and ends after 12 to 36 hours from the time of launch and during the subsequent phase of production of the enzyme from 35 to 45°C, preferably from 37 to 42°C.

The equipment for cultivation may be any of those designed to work with liquid culture. Mold fungi koji need to cultivate aerobic, therefore, the cultivation should be carried out under aerobic conditions, in which oxygen or air is fed to the environment. In addition, preferably the mixing of the medium during cultivation for raw material, oxygen and recombinant molds koji evenly distributed in the plant under cultivation. The conditions of mixing and the degree of aeration can be arbitrary, since the culture conditions support Givaudan aerobic, and can be appropriately selected depending on the equipment for cultivation, viscosity and the like.

Recombinant molds koji grown using the above method of cultivation to obtain in the culture product of the target recombinant protein with a high output.

According to the present invention the recombinant protein is then collected from the educated cultural product of the koji molds. You can use any well-known method as the method for collecting the recombinant protein. For example, you can choose the way that cultural product is filtered, centrifuged or the like to obtain the culture supernatant, and that the culture supernatant optional concentrate, purify or similar through the adsorption resin, electrophoresis or the like.

Examples

Hereinafter the present invention will be described in more detail by means of examples and the like. However, the present invention is not limited.

Example 1 (Method of production of recombinant protein by using yellow koji molds as the host body)

(Cooking environment)

The composition of the liquid medium for yellow koji molds consisted of the following: 2,0% 98%-polished barley (Starling, produced by the led in Australia), 1.2% of sodium nitrate, 0.8% potassium chloride, 0.4% of potassium phosphate, 0.2% of sulfated heptahydrate magnesium and 0.08% sulfated heptahydrate iron (each value represents the ratio mass/volume).

As control was used environment DPY (containing 2% dextrin, 1% polypeptide, 0,5% yeast extract, 0.5% of potassium phosphate and 0.05% of magnesium sulfate (each value represents the ratio mass/volume)).

In a 100-ml conical flask for mixing were placed in 20 ml of medium, respectively, and were sterilized by autoclaving at 121°C for 15 minutes.

(Recombinant mould koji)

As the recombinant koji molds was used niaD 300-Der fI, which was deposited with the access number FERM BP-10667 in International Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology, Tsukuba Central 6, 1-1-1 Higashi, Tsukuba, Ibaraki, Japan (the Old name and address: Fermentation Research Institute, Agency of Industrial Science and Technology, Ministry of International Trade and Industry, 1-1-3 Higashi, Tsukuba, Ibaraki, Japan) on July 30, 1997, and which was transferred to the international Depository of the initial securities Depository on August 28, 2006. Recombinant mould koji niaD 300-Der fI was obtained by the method described in JP-A-11-75840, using as the host body mutant on the assimilation of nitrate strain niaD 300Aspergillus oryzae,by introducing Der fI E(-1)K, then e is th DNA fragment, obtained by the replacement of the codon of glutamic acid at the 3'-end on the lysine codon in the cDNA sequence of the precursor glycoprotein Der fI, which is the primary allergen present in theDermatophagoides farinae(see H. Shoji, et al., Biosci. Biotechnol. Biochem., 61(10), 1668-1673, 1997).

In DNA niaD 300-Der fI glaA promoter derived fromAspergillus oryzae, Legerova in the position of the upstream DNA Der fI E(-1)K and amyB terminator derived fromAspergillus oryzae, Legerova in a position downstream in DNA Der fI E(-1)K.

(Cultivation of recombinant koji molds)

Approximately 106conidia obtained recombinant fungal mould koji niaD 300-Der fI was inoculable in 20 ml of medium prepared as described above, respectively, and increased with shaking at 30°C and 100 rpm for 24 hours.

(Purification of recombinant protein Der fI E(-1)K)

After cultivation in liquid medium of each culture fluid was centrifuged at 3000×g and 4°C for 10 minutes. Endoglycosidase Hf (Biolabs Company) was added directly at a concentration of 10 units/ml to the culture supernatant and left for reaction at 37°C for 3 hours to effect the removal of sharidny circuits. Formed after the reaction liquid was passed through a column of strong unionamerica (trade name: QMA, Waters Corporation)equilibrated with a solution of 20 mm FOS is Atego buffer (pH 6,0), for adsorption of α-amylase, a large number of which were present in the reaction liquid. For cleavage sequence predecessor Der fI E(-1)K lysyl-specific endopeptidase (Wako Pure Chemical Industries Inc.) was added to the fraction that passed without adsorption is carried on the QMA column, so that the final concentration was 10 μg/ml thereafter, the reaction product were dialyzed against 50 mm Tris-HCl (pH 9,0) over night at 4°C.

Thus obtained concentrate was loaded with a column of DEAE-Sephacel (Amersham Bio-Sciences K.K.), equilibrated with 50 mm Tris-HCl (pH 8.0)and washed 20 mm Tris-HCl (pH 8.0) in an amount of 3 times the column volume. Then the Mature recombinant protein Der fI E(-1)K, which was adsorbed on the column was blueraven using a concentration gradient of NaCl. The fraction containing the Mature recombinant protein Der fI E(-1)K revealed using Western analysis using antibodies against Der fI collection with the greatest purity of the fractions and used as a purified sample. Electrophoresis in nitrilotriacetate-polyacrylamide gel (SDS-PAGE), it was confirmed that the purity of the purified samples is 90% or more.

The purified sample was subjected to quantitative analysis for protein content using the BCA Protein Assay Reagent Kit (Pierce Biotechnology, Inc.).

(Results) the Yield of recombinant protein Der fI E(-1)K

When you use among the s DPY received approximately 8 mg of the Mature recombinant protein Der fI E(-1)K per liter of medium. When using a liquid medium for yellow koji molds were obtained approximately 24 mg of the Mature recombinant protein Der fI E(-1)K per liter of medium.

In this method found that the present invention recombinant protein was formed in an amount three times the amount that is formed using the method using traditional environment DPY.

Recombinant protein Der fI E(-1)K has sacharides circuit different from sacharides chain of natural Der fI. However, recombinant protein Der fI E(-1)K showed the ability to bind to IgE and the ability to irritate the skin, which were equivalent to those of natural Der fI. Thus, the recombinant protein used as an alternative natural Der fI for preparation of antibodies, Allergy treatment and the like.

Experimental Example 1 Determination of the promoter activities of genes different fermento in white mycelial fungi koji for Shochu)

To confirm that the promoters of genes different fermento white koji molds are not applicable to the present invention, the levels of expression of these promoters was determined by the following method.

<Used strain>Aspergillus kawachiiNBRC4308

<Conditions of cultivation> the medium Composition was as follows: 2,0% 98%-polished barley (Starling, made in the den in Australia), 0.2% of sodium nitrate and 0.3% potassium phosphate (each value represents the ratio mass/volume). 100 ml of medium were placed in 500-ml conical flask for mixing and sterilized by autoclaving at 121°C for 15 minutes. Approximately 106conidiaAspergillus kawachiiNBRC4308 was inoculable in 100 ml of medium obtained as described above, and cultured with shaking at 37°C and 100 rpm for 18 hours.

As a control for comparison, the cultivation was performed with the medium of the same composition and under the culture conditions described above, except that instead of 98%-polished barley in the environment used 65%-polished barley or product crushed 98%-polished barley (each of which is from Stirling, made in Australia).

<Obtaining total RNA> After completion of the cultivation, the cells were rapidly collected and thoroughly destroyed in the presence of liquid nitrogen. From the destroyed cells of mold koji with the set for the allocation of total RNA (i.e., RNeasy Plant mini kit, production QIAGEN) according to the attached Protocol was isolated total RNA.

<cDNA Selection> From the obtained total RNA using High-capacity cDNA Archive Kit (manufactured by Applied Biosystems) according to the attached Protocol was synthesized cDNA.

<Quantitative real-time PCR> Quantitative real-time PCR Khujand is used along by using the obtained cDNA as template and using primers designed on the basis of the main sequences of the target genes of enzymes, as described below, to thereby determine levels of gene expression of enzymes. Each primer used for quantitative real-time PCR was designed using Primer Express software (manufactured by Applied Biosystems). Sequences of the primers are presented exactly as shown below. Gene H2A, encoding histone was used as internal standard for quantitative way to compare.

PCR and detection of the signal was performed using SYBR Green PCR Master Mix (manufactured by Applied Biosystems) as a reagent for quantitative real-time PCR according to the attached Protocol. PCR and detection of the signal was performed by using ABI PRISM 7700 (manufactured by Applied Biosystems).

<Sequence genes and primers that were used>

(1) Gene glucoamylase gla-1 (derived fromAspergillus kawachii, GenBank No. D00427)

Direct primer: 1589-ccagctcgacctatagcagcat (SEQ ID NO: 1 in the List of sequences)

Reverse primer: 1761-aagtctgatggcgacgagct (SEQ ID NO: 2)

Pairs of primers were designed to amplify a DNA fragment from 1589 to 1780-th nucleotide pairs above gla-1 (GenBank No. D00427).

(2) Gene acid α-amylase asaA (derived fromAspergillus kawachii, GenBank No. AB008370)

Direct primer: 994-cggcacggcagatgatc (SE ID NO: 3)

Reverse primer: 1044-gaatgtacctcatggtcgacgtc (SEQ ID NO: 4)

Pairs of primers were designed to amplify a DNA fragment from 994 to 1066-th nucleotide pairs above asaA (GenBank No. AB008370).

(3) the Gene for α-amylase amyA (derived fromAspergillus kawachii, GenBank No. AB109452)

Direct primer: 1874-acactcctgggcacattcg (SEQ ID NO: 5)

Reverse primer: 1989-ttacaccaacgacatagccct (SEQ ID NO: 6)

Pairs of primers were designed to amplify a DNA fragment from 1874 to 2009 nucleotide pairs above amyA (GenBank No. AB109452).

(4) Gene histone H2A (derived fromAspergillus niger, GenBank No. Y15320)

Direct primer: 289-actgaacaagctcctgggtca (SEQ ID NO: 7)

Reverse primer: 322-ccagggtggtgtcctcccc (SEQ ID NO: 8)

Pairs of primers were designed to amplify a DNA fragment from the 289th to 340-th nucleotide pairs above H2A (GenBank No. Y15320).

<Results> Levels of gene expression of relevant enzymes quantify relative expression level of histone H2A. The results are shown in table 1. In the experimental graphs (the present invention) when using 98%-polished barley levels of expression of corresponding genes increased compared to levels of expression in the control charts. Thus, it was found that the promoters of the genes of these enzymes have been used effectively in the method received what I recombinant protein of the present invention.

Table 1
Used raw substanceThe level of expression
gla-1The present invention (98%-polished barley)8,49
Control 1 (product of grinding of 98%-polished barley)3,95
Control 2 (65%-polished barley)2,35
asaAThe present invention (98%-polished barley)2,89
Control 1 (product of grinding of 98%-polished barley)1,99
Control 2 (65%-polished barley)1,05
amyAThe present invention (98%-polished barley)23,86
Control 1 (product of grinding of 98%-polished barley)17,65
Control 2 (65%-polished barley) 14,59

Experimental Example 2 (determination of the promoter activity of genes of enzymes in black koji molds for Shochu)

To confirm that the promoters of genes of various enzymes in black koji molds suitable for the present invention, the levels of expression of these promoters were defined as follows.

In particular,Aspergillus awamoriNBRC4388 were grown in the same way as in Experimental Example 1. Then total RNA was extracted from cells obtained after cultivation in the same way as in Experimental Example 1, and the synthesized cDNA. Then the levels of gene expression of target enzymes, as described below, were quantified using the obtained cDNA as a matrix in the same way as in Experimental Example 1. Sequences of primers used for quantitative real-time PCR, represented as described below.

<Sequence genes and primers that were used>

(1) the Gene for α-amylase amyA (same amyA, as described in Experimental Example 1)

Used the same pair of primers (SEQ ID nos: 5 and 6), as in Experimental Example 1.

(2) Gene acidic protease pepA (derived fromAspergillus awamori, GenBank No. M34454)

Direct primer: 793-ttttgggactggcctttagct (SEQ ID NO: 9 in the List is sledovatelnot)

Reverse primer: 900-ttcttcgacaccgtcaagtcc (SEQ ID NO: 10)

A pair of primers was designed to amplify a DNA fragment from 793 to 920-th base pair above pepA (GenBank No. M34454).

(3) Gene histone H2A (H2A, as described in Experimental Example 1)

Used the same pair of primers (SEQ ID nos: 7 and 8), as in Experimental Example 1.

<Results> Levels of gene expression of relevant enzymes quantitatively evaluated as values relative to the expression level of histone H2A. The results are shown in Table 2. In the Experimental graphs (the present invention) when using 98%-polished barley levels of expression of corresponding genes increased in comparison with the levels of expression in the control charts. Thus, it was found that the promoters of the genes of these enzymes have been used effectively in the method of obtaining a recombinant protein of the present invention.

Table 2
Used raw substanceThe level of expression
amyAThe present invention (98%-polished barley)19,1
Control 1 (product of grinding of 98%-polished barley)0,1
Control 2 (65%-polished barley)0,3
pepAThe present invention (98%-polished barley)2,4
Control 1 (product of grinding of 98%-polished barley)0,7
Control 2 (65%-polished barley)1,5

Experimental Example 3 (determination of the promoter activity of genes of enzymes in yellow mold fungi koji for sake)

To confirm that the promoters of genes of various enzymes in the yellow mold fungi koji applicable to the present invention, the levels of expression of these promoters were defined as follows.

<Used strain>Aspergillus oryzaeNRIB40

<Conditions of cultivation> the medium Composition was as follows: 2,0% 98%-polished barley (Starling, made in Australia), 1.2% sodium nitrate, 0.8% potassium chloride, 0.4% of potassium phosphate, 0.2% of sulfated heptahydrate magnesium and 0.08% sulfated heptahydrate iron (each value represents the ratio mass/volume). 100 ml cf the water was placed in 500-ml conical flask for mixing and sterilized by autoclaving at 121°C for 15 minutes. Approximately 106conidiaAspergillus oryzaeRIB40 was inoculable in 100 ml of medium obtained as described above, and cultured with shaking at 30°C and 100 rpm for 42 hours.

As a control for comparison was prepared culture with the same medium composition and cultivation conditions as described above, except that instead of 98%-polished barley used 65%-polished barley or product grinding 98%-polished barley (each of which were from Stirling, made in Australia).

Then from cells obtained after culturing in the same manner as in Experimental Example 1, provided the total RNA and synthesized cDNA. Then quantitatively determined the levels of gene expression of target enzymes, described below, using the obtained cDNA as a matrix in the same way as in Experimental Example 1. Sequences of primers used for quantitative real-time PCR, represented as described below. The gene encoding histone H4 was used as an internal standard for the method of quantitative comparison.

<Sequence genes and primers that were used>

(1) Gene glucoamylase glaA (isolated fromAspergillus oryzae, GenBank No. D01035)

Direct primer: 1247-cgtgcagatcgtccaaacct (SEQ ID NO: 11 in the List of the placenta is of valnontey)

Reverse primer: 1357-acttctcacggccaacaacc (SEQ ID NO: 12)

A pair of primers was designed to amplify a DNA fragment from 1247 to 1376-th base pair above glaA (GenBank No. D01035).

(2) the Gene for α-amylase amyA (isolated fromAspergillus oryzae, GenBank No. AB021876)

Direct primer: 21762-cactcctgggcacattcgt (SEQ ID NO: 13)

Reverse primer: 21875-gttacaccaacgacatagccctc (SEQ ID NO: 14)

Pairs of primers were designed to amplify a DNA fragment from 21762 to 21897-th nucleotide pairs above amyA (GenBank No. AB021876).

(3) Gene β-glucanase celB (isolated fromAspergillus oryzae, GenBank No. D83732)

Direct primer: 1137-caaactgggaatgccacaaa (SEQ ID NO: 15)

Reverse primer: 1187-tgaagacggagagaactattccatg (SEQ ID NO: 16)

Pairs of primers were designed to amplify a DNA fragment from 1137 to 1211-th nucleotide pairs above celB (GenBank No. D83732).

(4) Gene acidic protease pepA (isolated fromAspergillus oryzae, GenBank No. D13894)

Direct primer: 897-cgctagcaagattagcgatcagt (SEQ ID NO: 17)

Reverse primer: 958-gctttcagctcgatcaacactg (SEQ ID NO: 18)

Pairs of primers were designed to amplify a DNA fragment from 897 to 979-th nucleotide pairs above pepA (GenBank No. D13894).

(5) Gene histone H4 (isolated fromAspergillus oryzae, GenBank No. AB033943)

Direct primer: 110-cgtgacaacatccagggtatca (SEQ ID NO: 19)

Reverse primer: 171-tcaagcgtatctctgccatga (SEQ ID NO: 20)

Pairs of primers were sconst frowny thus, in order to amplify a DNA fragment from 110th to 191-th nucleotide pairs above H4 (GenBank No. AB033943).

<Results> Levels of gene expression of relevant enzymes quantitatively evaluated as values relative to the expression level of histone H4. The results are shown in table 3. In the Experimental graphs (the present invention) when using the product grinding 98%-polished barley levels of expression of corresponding genes increased in comparison with the levels of expression in the control charts. Thus, it was found that the promoters of the genes of these enzymes have been used effectively in the method of obtaining a recombinant protein of the present invention.

Table 3
Used raw substanceThe level of expression
glaAThe present invention (98%-polished barley)0,31
Control 1 (product of grinding of 98%-polished barley)0,04
Control 2 (65%-polished barley)0,10
amyAThe present invention (98%-polished barley)21,0
Control 1 (product of grinding of 98%-polished barley)3,3
Control 2 (65%-polished barley)9,3
celBThe present invention (98%-polished barley)0,018
Control 1 (product of grinding of 98%-polished barley)0,003
Control 2 (65%-polished barley)0,006
pepAThe present invention (product of grinding of 98%-polished barley)0,15
Control 1 (product of grinding of 98%-polished barley)0,07
Control 2 (65%-polished barley)0,05

Industrial applicability

The present invention provides a method of mass production of recombinant protein by using koji molds as the host body. Mold fungi koji cultivar the Ute in a low cost environment, special equipment for cultivation is not required, therefore, necessary protein is obtained with low cost. In addition, molds koji used for the production of fermented food and beverage since ancient times, and they are largely safe body-master, and generated recombinant proteins can be used for various purposes.

1. A method of obtaining a recombinant protein using recombinant koji molds, which are obtained by transformation of the koji molds as the host body, including:
cultivation of the recombinant koji molds in a liquid medium, which contains as raw materials for culturing at least one selected from the group consisting of barley and wheat, which surface is entirely or partly covered with at least a shell, in a concentration of from 1 to 20%(wt./about.) and as nutrients, at least one inorganic salt; and
collecting the recombinant protein from the culture product, where
recombinant molds koji get the fact that the gene encoding the target protein are ligated below the promoter of the gene encoding the enzyme is subjected to catabolite repression due to the concentration of nutrients, such as charity and amino acids, to obtain thereby the product of ligation, the ligation product is injected into molds koji as the host body.

2. The method according to claim 1, characterized in that the promoter is the promoter of the gene encoding any enzyme selected from the group consisting of amylolytic enzymes, cellulolytic enzymes and proteolytic enzymes.

3. The method according to claim 1 or 2, characterized in that use inorganic salt selected from the group consisting of nitrates, phosphates and sulfates.

4. The method according to claim 1 or 2, characterized in that use inorganic salt containing a combination of nitrate, phosphate and sulfate.



 

Same patents:

FIELD: medicine.

SUBSTANCE: invention refers to a method of producing a mutant lactobacillus Streptococcus thermophilus, to a milk ferment, a method of producing a fermented milk product and to the fermented milk product. The offered invention can be used for producing the fermented milk products with improved storage characteristics. A method of producing the mutant lactobacillus Streptococcus thermophilus characterised by weaker postoxidation, than a parent strain is implemented by introducing in DNA a genome of said parent strain of mutation codon 552 coding histidine, of teh domain HA of lactose permease. Said mutation induces replacement of said histidine by amino acid which is distinct from serine, tyrosine, histidine and threonine.

EFFECT: invention allows producing the mutant lactobacillus Streptococcus thermophilus characterised by weaker postoxidation and suitable particularly for producing the fermented milk products.

10 cl, 5 dwg, 3 tbl, 2 ex

FIELD: medicine.

SUBSTANCE: Streptomyces caespitosus 3810/OE strain is a mitomycin C producer prepared from a stock strain recovered from soil by processing with chemical mutagens followed by stage selection, and deposited in the Collection of Cultures of State Institution G.F.Gauze Research Institute for Investigation of New Antibiotics of the Russian Academy of Medical Science, No. INA 01082. For producing mitomycin C, the strain is grown on a sowing medium of the following compoisiton (%): glucose - 0.6-1.4, starch - 0.6-1.4, soya flour - 2-5, corn steep - 0.6-1.0, KH2PO4 - 0.01-0.03, chalk - 0.05-0.12, (NH4)2SO4 - 0.06-0.14, main water, pH before sterilisation 6.8-7.2 and then is transferred to an enzyme medium of the following composition (%): starch - 0.6-1.4, sucrose - 1-3, soya flour - 2-4, corn steep - 0.6-1.0, CoCl2•6H2O - 0.007-0.012, KH2PO4 - 0.02, chalk - 0.05-0.12, (NH4)2SO4 - 0/06-0.04, FeSO4•7H2O - 0.01-0.02, MgSO4•7H2O - 0.01-0.03, NaCl - 0.2-0.6, main water, pH before sterilisation 6.8-7.2. The strain is grown in an aeration environment for 100-120 h, then mycelium is removed by centrifugation or filtration, and a supernatant containing an end product is extracted by mixed acetonitrile and methyl or ethyl alcohol. A level of strain produced mitomycin C is 85 to 100 mcg/ml.

EFFECT: improved method of producing antibiotic mitomycin.

2 cl, 1 ex

FIELD: medicine.

SUBSTANCE: method provides introducing a preparation of active dry yeast Saccharomyces cerevisiae in water in the ratio 1:10. Then the mixture is exposed for 4-6 h at temperature 23-27°C. The produced suspension is layered to reactivated yeast and a supernatant. The supernatant is removed.

EFFECT: method allows producing reactivated yeast with more complete living function recovery.

2 tbl, 3 ex

FIELD: food industry.

SUBSTANCE: nutrient medium contains soya flour and distilled water at preset quantitative ratios.

EFFECT: Deinococcus radiodurans yield increase.

2 tbl, 8 ex

FIELD: food industry.

SUBSTANCE: lysine-producing gram-positive bacterial strain for biologically active compounds delivery to ruminant animals is grown by way of at least single passage through the growth medium containing a quantity of lysozyme which is effective for bacteria cell walls growth induction; these bacteria are resistant to protozoal intake. The bacterial strain is extracted from the medium containing lysozyme and used for preparation of a feed supplement for ruminant animals passing through the ruminant animal paunch. The ruminant animals feeding method involves addition of an effective quantity of the feed supplements (passing through the paunch) to the feed ration.

EFFECT: increase in resistance of the lysine-producing gram-positive bacterial strain to inactivation in the paunch which strain is used for biologically active compounds delivery to ruminant animals.

17 cl, 5 dwg, 2 tbl, 13 ex

FIELD: chemistry.

SUBSTANCE: culture medium contains dry nutrient agar, glucose, 5- aminosalicylic acid, an extract of nutrient yeast, paraaminobenzoic acid, bromthymol blue, tris-buffer, sodium carbonate, brilliant green and microbiological agar.

EFFECT: invention shortens duration of identifying klebsiell.

3 ex

FIELD: medicine.

SUBSTANCE: method provides collection of an investigated material from a surface with a tampon made of an elastic finely porous material wetted in a solution inhibiting the development of other microorganisms. The collected investigated material with tampons is kept in the same solution for 18-24 hours. The investigated material is separated from a tampon and centrifuged. The precipitation is neutralised with 1% citric acid (in the ratio 1:1). No more than one-third of the investigated material is separate from the precipitation. The separated precipitation portion is placed on a phase-contrast slide to conduct phase-contrast microscopy. The residual precipitation is placed on a nutrient medium to be cultivated thereon if more exact quantitative assessment of mycobacterial pollution of the investigated surface is required.

EFFECT: invention allows reducing mycobacteria detection time.

2 tbl, 1 ex

FIELD: medicine.

SUBSTANCE: strain is obtained by insertion into genome of Escherichia coli, isolated from clinically healthy representative of canine family, of genes, determining synthesis of microcin C51, B-subunit of toxin, as well as vaccine version of elt-operon and A-subunit, enhancing immune response. Strain possesses expressed adhesion to mucous membrane of intestine of animals of canine family. Frozen dried culture of Escherichia coli EB387 represents probiotic medication for protection of animals of canine family against toxicoses, induced by cytotonic toxins of A1B5 type.

EFFECT: normalisation and stabilisation of qualitative and quantitative composition of gastrointestinal tract microflora in representatives of canine family.

2 cl, 3 ex

FIELD: medicine.

SUBSTANCE: nutritional medium contains liquid phase, content of liquid phase is determined by biological peculiarities of particular microorganism and solid phase. Solid phase contains coagulated serum and agarose in specified ratio of components.

EFFECT: invention allows to increase reliability of estimation of results of microorganism culture growth and development.

1 ex

FIELD: medicine.

SUBSTANCE: claimed is powder composition, possessing lipase activity. Composition contains filtering auxiliary material(s) and product, obtained by fine grinding of lipase, originating from Thermomyces sp., immobilised on silicon carrier(s), to average particle size 1 mcm or larger to smaller than 300 mcm. Also claimed are methods for re-etherification of fats and oils and for etherification with application of obtained lipase powder composition.

EFFECT: powder composition by the invention possesses improved lipase activity.

10 cl, 1 dwg, 2 tbl, 8 ex

FIELD: medicine.

SUBSTANCE: hybridoma techniques involving the fusion of myeloma of Sp-2/0 mice and lymphocytes and splenocytes of BALB/c mice immunised by a conjugate of benzo[a]pyrene and a bovine serum albumin, with using polyethylene glycol of molecular weight 1500 for cell fusion, followed by limit dilution cloning are used to produced a Mus. Musculus BpBal animal hybrid cultured cell strain deposited in RKKK(P) of the Institute of Cytology of the Russian Academy of Sciences, Registration No. 723 D producing the benzo[a]pyrene and benz[a]anthracene specific monoclonal antibodies. The hybridoma strain produces the monoclonal antibodies mAB showing high Bp and Ba specificity and low cross-responsiveness with non-cancerogenic polycyclic aromatic hydrocarbons (PAH) and with endogenous compounds (aromatic amino acids and steroid hormones).

EFFECT: using the BpBal mAB allows producing sensitive and specific test systems for definition cancerogenic PAHs, the related antibodies in human and animal biological fluids, and searching immune mimetic peptides and producing anticancerogenic PAH vaccines.

1 dwg, 3 tbl, 3 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention concerns area of molecular biology and biochemistry, and can be used in medicine. There is offered mutein conjugate of the blood coagulation factor VIII (FVIII) wherein a residue not being cysteine in position 41, 129, 377, 388, 468, 491, 556, 1804, 1808, 1810, 1812, 1813, 1815 and/or 2118 is substituted by a cysteine residue with polyethylene glycol (PEG) where a PEG molecule is bound with a polypeptide in a mutant cysteine residue.

EFFECT: improved pharmacokinetic properties of the FVIII as an ingredient of the conjugate under the invention with preserved a procoagulant factor activity allows presenting new FVIII PEG-muteins for producing of a pharmaceutical compositions for treating hemophilia.

12 cl, 38 dwg, 8 tbl, 1 ex

FIELD: medicine.

SUBSTANCE: neutral detergent Cevtalone is used for recovery and purification of a low-molecular ribonucleoproteid fraction from DNA and high-molecular RNA. Cevtalone forms from water-insoluble salts with DNA and RNA thereby enables consistent precipitation of DNA and high-molecular RNA from the solution. As a result, the solution contains the low-molecular ribonucleoproteid fraction which then precipitated with Cevtalone. The produced precipitate is dissolved in ethanol for pure ribonucleoproteid precipitation. Cevtalone salts of nucleic acids are resistant to ribonuclease enzymes thereby the given method can be staged with great time intervals (up to several days) without loss of biological properties of recovered ribonucleoproteids. The given method of recovery allows to producing high-purity low-molecular ribonucleoproteids (the ribonucleoproteid contents in the prepared samples is 90 %) in preparative amounts.

EFFECT: recovery of low-molecular nuclear ribonucleoproteid from tissues of mammals with using non-toxic and low-cost reagents.

1 ex

FIELD: chemistry.

SUBSTANCE: washed Tibetan milk mushroom is put into a glass vessel and 65.7 l (1:3) tap water at temperature (45±1)°C is added. The mixture is leached with Na2CO3 to pH 8.2-8.3 on phenolphthalein. Crushed pancreatic gland of a cow is added in amount of 0.101 kg. 2% chloroform is added to the overall volume and the vessel is tightly plugged with woolen-gauze sponge with permanganate and then put into a heat chamber at temperature (48±1)°C. The vessel is kept for 9-10 days, while shaking during the first days every 10-15 minutes for 4-5 minutes, and during the next days every 1.5-2 hours for 4-5 minutes. The flow of the enzymatic process is controlled based on increase in amine nitrogen. After 9-10 days, increase of the said indicator stops. The heat chamber is switched off and the hydrolysate is left for one day. The hydrolysate is then filtered with a filter cloth. 2% chloroform is then added to the filtrate, pluged with a rubber plug and stored at temperature ranging from 2 to 8°C. Amine nitrogen in the ready hydrolysate is in amount of 567.8 l/g. The colour is transparent brown.

EFFECT: cheap and simple method of obtaining enzymatic hydrolysate from Tibetan milk mushroom, high output of bacterial biomass with high dry substance index.

2 cl, 4 tbl, 4 ex

FIELD: medicine.

SUBSTANCE: invention concerns anti-PSMA antibodies which contain variable sites of heavy and light chains; the antibody does not contain fucosyl residues. Amino acid sequences of said chains are presented in the formula. Antibodies under the invention are a monoclonal antibody, a humanised or chimeric antibody, a human antibody. Also, there is described a method of inhibition of PSMA+ cell growth, such as tumour cells by contact of said cells with the anti-PSMA antibodies.

EFFECT: antibodies show higher, antibody-dependent prostate cancer cell cytotoxicity as compared with a fucosyl antibody form.

32 cl, 3 tbl, 6 ex

FIELD: medicine.

SUBSTANCE: there are described isolated mouse antibody selectively bound with antigen F1 of Yersinia pestis, and an antigen-binding fragment (Fab). There is presented yeast cell producing the described antigen-binding fragment (Fab). There is offered method for producing described antigen-binding fragment. Also, there is presented method for Yersinia pestis detection and a kit for detection of antigen F1 of Yersinia pestis.

EFFECT: invention presents a Yersinia pestis detection reagent.

9 cl, 12 dwg, 7 ex

FIELD: medicine.

SUBSTANCE: there are described humanized antibody selectively binding antigen F1 of Yersinia pestis and its antigen-binding fragment (Fab). There is presented yeast cell producing the described antigen-binding fragment. There is offered method for producing described antigen-binding fragment.

EFFECT: invention presents a Yersinia pestis detection and plague treatments reagent.

8 cl, 6 dwg, 3 ex

FIELD: medicine.

SUBSTANCE: in the method nutritional medium is used for cultivation of cells, transformed with the gene, encoding target product. One of peculiarities of nutritional medium is the following property: total concentration of amino acids is higher than approximately 70 mM; ratio of total molar quantity of glutamine to total quantity of asparagine is lower than approximately 2; ratio of total molar quantity of glutamine to total quantity of all amino acids is lower than approximately 0.2; ratio of total molar quantity of inorganic ions to total quantity of all amino acids is approximately from 0.4 to 1 or combined total quantity of glutamine and asparagine per unit of volume is in the range of approximately from 16 mM to 36 mM. Such system of cultivation ensures high levels of antibody production. Methods of cultivation can include change of temperature, usually reduction of temperature when culture reaches 20-80% of maximal cell density, pH value, osmolarity, level of chemical activator and their combinations. As alternative or supplement, described are methods-versions, in which levels of lactate and/or ammonium in the culture after reaching peak values decrease in the course of time.

EFFECT: invention ensures high output of antibodies with simultaneous reduction of undesirable products.

48 cl, 76 dwg, 27 tbl, 17 ex

FIELD: chemistry.

SUBSTANCE: method involves incubation of Klebsiella pneumoniae on a nutrient broth at temperature 37°C to obtain a broth culture, followed by an enzyme immunoassay reaction. Ballast proteins are separated in 2 M ammonium sulphate to obtain a solution. Iron-ammonia alums are then added to the obtained solution to saturate lactoferrin with iron. The iron-saturated solution is then desalinated and buffered with a phosphate buffer with subsequent application onto a column with cationic sephadex G-50/8.5x9 cm/ and elution with discontinuous gradient of sodium chloride concentration is carried out to obtain lactoferrin which is contained in fractions with salt concentration of 0.25 M and 0.3 M.

EFFECT: invention increases output of the desired product.

1 tbl, 3 ex

FIELD: medicine.

SUBSTANCE: invention can be used for hypertension prevention. A biologically active peptide is characterised by the amino acid sequence LLYQQPVLGPVRGPFPIIV. Said peptide is prepared of milk by enzymatic hydrolysis. It is followed with purification and ultrafiltration of the prepared hydrolyzates with using the membranes of pore diametre 10 and 15 kDa at pH 6.0-6.5.

EFFECT: invention allows producing a biologically active peptide which exhibits antihypertensive activity.

1 ex

FIELD: agriculture.

SUBSTANCE: ferments have improved characteristics compared to a ferment of wild type selected from high thermal stability, activity in wide pH range, high specific activity, wide substrate specificity and proteolytic stability. Besides, nucleic acids are proposed, which code these ferments, a vector containing these nucleic acids, a hostcell, which carries the vector, and a method to produce proposed phytase ferments from hostcells. This invention also relates to a food product or an animal fodder and to the method to make food product or fodder for animals, which includes addition of phytase ferment according to this invention to one or several ingredients of the prepared product or fodder.

EFFECT: using phytase ferments according to this invention as additives to animal fodders makes it possible to improve availability of organic phosphorus and to reduce contamination of environment with a phosphate.

28 cl, 3 dwg, 2 tbl, 11 ex

Up!