RU1770359C - Recombinant plasmid dna pj db (msil), encoding of human interleukin-2 synthesis in yeast cells saccharomyces cerevisiae, method of its preparation, and yeast strain saccharomyces cerevisiae - a producer of human interleukin-2 - Google Patents

Abstract

The invention relates to biotechnology. The aim of the invention is to increase the yield of interleukin-2 in yeast cells by constructing recombinant pjDB plasmid DNA (MSIL), developing a method for constructing it and creating a strain transformed with this plasmid. The plasmid contains the yeast LEU 2 gene and a fragment of yeast two-micron DNA. ensuring its stable maintenance in yeast in a high number of copies; the coding region of the human interleukin-2 gene, flanked by the yeast PH05 gene transcription promoter and terminator, as well as bacterial DNA fragments that ensure plasmid replication in E. coli cells and carry the ampicillin resistance gene. The yeast cells of strain GC-1-GRF18 transformed with pjDB plasmid (MSIL) produce Inerleukin-2 in the order of 30-50 mg per liter of yeast culture, which corresponds to 10 units / ml. 3 s.p. f-ly. (L C

Description

The invention relates to biotechnology, in particular to genetic engineering, and is a recombinant plasmid DNA that provides a high level of production of human interleukin-2 in yeast cells, a method of constructing this plasmid DNA and a strain of yeast Saccharomyces cerevisiae-producer of human interleukin 2.

An object of the invention is to increase the yield of interleukin-2 in yeast cells.

The goal is achieved by the creation of the plasmid pjDB (MSIL), which provides the synthesis of interleukin-2 in its transformed yeast cells. The plasmid consists of the following elements:

fragment of plasmid DNA of bifunctional bacterial yeast vector pjDB207. limited to Hindlll and BamHI restriction sites, 6.6 kbp. comprising a bacterial ampicillin resistance gene, a bacterial replication initiation region, a fragment of a two-micron plasmid yeast, and a LEU2 yeast gene;

-BamHI - EcoRI of the yeast PH05 gene fragment containing the promoter of this gene, 0.52 kb;

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-E.coRI - Sail of the fragment of the plasmid pAA 12.13-23, bearing part of the interleukin gene-by-2 person, including all of the coding and part of the 3 non-coding region of this gene, 0.56 kb;

-Smal — BamH fragment of the polylinker portion of plasmid pUC19 8 bp long;

-Sau ZA-Pstl of the yeast PH05 gene fragment containing the transcription terminator of this gene, 0.37 kbp;

-Pst-Hindlll fragment polylinker plasmids pUC19 size 20 BP

The total plasmid size is 8.0 kb (molecular weight 5.2 Md).

To achieve the goal, a method has been developed for constructing the pjDB plasmid (MSlL), wherein the plasmid pAA 12.13 - 23 is hydrolyzed with Sail restriction endonuclease, then treated with DNA polymerase 1 (Klenov fragment and EcoRI restriction enzyme. The resulting DNA fragment including all coding and a portion of the 3-untranslated region of the human interleukin-2 gene are ligated with the pMS46 vector previously hydrolyzed with restriction enzymes EcoRI and Smal. From the resulting plasmid pMSILc using Sail and Hindlll restriction enzymes a DNA fragment carrying the promoter and terminator of transcription of the yeast PH05 gene with the interleukin-2 gene located between them.After purification, this fragment is ligated with the vector part of the plasmid pjDB207, which is also hydrolyzed by Sail and Hindlll restriction enzymes.8 The result is the plasmid pjDB (MSIL), providing a high level of production of interleukin-2 in yeast cells.

The choice of plasmid pMS46 is due to the fact that this plasmid contains the promoter and terminator of transcription of the PH05 yeast gene. the same located in the same orientation, and has a conveniently cloned half-linker region between the promoter and the transcription terminator. The promoter of the PH05 gene in this plasmid is modified in such a way that the coding region of the PH05 gene is completely removed in it, and a fragment of the polylinker of the pUC19 plasmid is inserted into the region corresponding to the leader region of mRNA.

A strain of Saccharomyces yeast is used to achieve the goal. cerevisiae GC-1-GRF18-pjDB (MSIL) obtained by transforming the strain GC-1-GRF18 with plasmid pjDB (MSIL).

The invention is illustrated by the following examples.

Example 1. Escherichia coli bacteria cells containing plasmid pMS46 were grown overnight in 500 ml of LB growth medium (1% peptone, 0.5%

yeast extract, 1% sodium chloride) to which ampicillin was added at a concentration of 50 mg / l. Cells are harvested by centrifugation (5000 rpm, 10 min, 4 ° C), resuspended in 10 ml of buffer for

0 lysis (25 ml Tris-hydrochloride pH 8.0 buffer containing 10 mM EDTA and 50 mM glucose), 20 ml mg lysozyme are added and incubated for 10 min at 4 ° C. Next, 10 ml of 0.2 M sodium hydroxide containing 5% sodium dodecyl sulfate was added. After stirring carefully for about 1 minute, the solution was neutralized with 10 ml of 3M sodium acetate, pH 5.3. Next, the drug is maintained at 4 ° C for 1 h and

0 centrifuged at 20,000 rpm 0.6 volumes of isopropyl alcohol are added to the supernatant and the precipitate is separated by centrifugation at 5000 rpm at 4 ° C. The precipitate was dried in vacuo and dissolved.

5 in 3.5 ml of water, 3.5 g of cesium chloride and 100 μl of ethidium bromide solution (10 mg / ml) are added and centrifuged at 50,000 rpm for 12-16 hours in a centrifuge. After centrifugation, a strip is taken.

0 plasmid DNA (the lower of the two fluorescent bands in the center of the tube), ethidium bromide is extracted twice with an equal volume of isoamyl alcohol, the solution of cesium chloride is diluted with water in

5 2 times and precipitate DNA with two volumes of ethyl alcohol. The precipitate, separated by centrifugation (10,000 rpm, 5 min), was washed with 70% ethanol, dried in vacuo and dissolved in water (500 µl). The concentration of plasmid DNA is determined by the optical density of the solution at 260 nm, the purity of the drug is controlled by agarose gel electrophoresis (0.7% agarose in TBE buffer - 0.1 M Tris-borate containing

5 1 mM EDTA and 1 mg / L ethidium bromide).

Hydrolysis of the plasmid pM S46 with the Smal restriction enzyme was carried out in 10 mM Tris-hydrochloride buffer containing 10 mM. sodium chloride. 20 mm potassium chloride,

0 1 mm dithiothreitol. 30 units are added to 20 μg of DNA in a volume of 50 μl. restriction enzymes and the reaction is carried out at 30 ° C for 2 hours. The completeness of hydrolysis is monitored by agarose gel electrophoresis. Next, the DNA solution was diluted to 200 µl with 5C mM Tris-hydrochloride buffer containing 10 mM magnesium chloride, 100 mM sodium chloride, 1 mM dithiothreitol, 10 µg / ml bovine serum albumin (BC buffer) and treated with 50 m units. restriction enzymes EcoRI for 2 hours at 37 ° C. The reaction mixture is added to a well (2 x 50 mm) of an agarose gel prepared as described above and electrophoresis is carried out at a voltage of 5 V / cm for 2-3 hours. A well is cut out directly in front of the strip of the linear form of the plasmid ( 3 x 60 mm), a suitable dialysis membrane is placed in this well and filled with TBE buffer. Electrophoresis was continued for another 10-15 minutes, after which the buffer was removed from the well, extracted once with phenol and once or twice with butanol, 1/10 part of 3M sodium acetate buffer, pH 5.3, and three volumes of ethanol were added. . DNA was separated by centrifugation at 10,000 rpm for 10 minutes, the precipitate was washed with ethanol, dried in vacuo and dissolved in 100 µl of water.

Hydrolysis of 10 μg of plasmid pAA 12.13 - 23, the isolation of which is similar to the isolation of plasmid pMS46, is carried out using 30 units. Sail restriction enzymes in 50 μl of BC buffer for 2 hours at 37 ° C. Then, 80 μl of 100 mM Tris-hydrochloride pH 7.4 buffer containing 50 mM magnesium chloride and 10 units were added to the DNA solution. Klensky DNA Polymerase Fragment 1. After an incubation of 4 hours at room temperature, DNA polymerase was inactivated by heating at 65 ° C for 10 min, after which 100 μl of BC buffer was added and plasmid DNA was hydrolyzed for 30 units. restriction enzymes EcoRI for 2 hours at 37 ° C.

560 bp fragment cleanup carried out as described above for the vector fragment of plasmid pMS46, in addition to that, l / nu (5 x 3 mm) were cut out next to the well of the DNA preparation to be purified to apply molecular weight standards (lambda phage DNA, Pstl hydrolyzed). The desired band before elution is identified by comparing its mobility with the mobility of standard DNA fragments.

To obtain a plasmid designated as pMSIL DNA fragments. containing the promoter and terminator of the PH05 gene. ligated with a DNA fragment carrying the interleukin-2 gene. For this, DNA fragments from plasmids pAA 12.13-23 and pMS46, the purification of which is described above, are mixed in an amount of 0.1 μg in 10 μl of 70 mM Tris-hydrochloride buffer, pH 7.6, containing 5 mM dithiothreitol, 5 mM magnesium chloride, 1 mM ATP. 10 units were added to carry out the ligation reaction. DNA ligases are T4 phase and incubated overnight at 4 ° C.

Thus obtained ligase mixture transform cells of E. coli HB101. For this, E. coli cells are grown in 100 ml of LB medium at 37 ° C and intensive

stirring until the optical density at 600 nm is 0.4-0.5. The culture is cooled on ice, centrifuged at 3000 rpm for 10 min at 0 ° C, resuspended in 50 ml of 0.1 M calcium chloride,

incubated in an ice bath for 40 minutes, centrifuged again under the same conditions, suspended in 5 ml of calcium chloride solution, glycerol added to 20%. The competent cells thus obtained are packaged in 200 µl aliquots and stored frozen at -70 ° C until use. In order to transform, the competent E. coli cells are thawed in an ice bath, a mixture of ligase reaction products is added to the cell suspension and incubated in an ice bath for 40 minutes. Then the cells are subjected to heat shock for 2 min at 42 ° С, after which they are incubated in 1.5 ml of LB medium at 37 ° С

for 1 h. The cell suspension is concentrated by centrifugation at 30,000 rpm for 10 min and rubbed on the surface of the plate with the same nutrient medium containing 2% agar and 50 mg / l

-ampicillin.

Plasmid DNA was isolated from the individual transformant clones obtained by the described method in a manner analogous to the plasmid isolation method pMS46;

the difference is that the cultivation of E. coli cells is in 10 ml of culture medium and all volumes of solutions are correspondingly reduced by 50 times in comparison with the indicated ones. In addition, instead of centrifuging in solution

cesium chloride use treatment with pancreatic RNase. For this, the nucleic acid precipitate obtained by precipitation with isopropanol was dissolved in 100 µl of restriction buffer and treated with 10 µl of an RNase solution (1 mg / ml) for 30 min at 37 ° C. Such a preparation is used for restriction analysis of the structure of plasmids in individual clones. In the case of plasmid pMSIL, the assay is carried out as follows. To portions of 3 μl of the plasmid preparation, 7 μl of water are added and then individual portions are treated with the following combinations of restriction endonucleases (10 units): Sal I

+ Hindlll (the desired plasmid gives a fragment of 1760 bp), EcoRI + Hindlll (fragment 960 bp), BamHI + Sail (fragments 275, 520 and 560 bp). From a transformant clone containing the plasmid selected in this way

pMSIL, plasmid DNA is preparatively isolated as described for plasmid pVS46.

In order to obtain the shuttle plasmid pjDB (MSIL), the Sall-Hindlll fragment of the pMSIL plasmid 1.76 kbp was preparatively purified by the same methods. and a vector fragment of plasmid pjDB207 hydrolyzed by the same restriction enzymes. Ligating these two fragments, carrying out the transformation of E. coli cells and restriction analysis of the transfermant restriction enzyme clones EcoRI, Sal + Hindlll, BamHI + Sail, select a clone containing the plasmid pjDB (MSIL). Plasmid DNA was preliminarily isolated from the cells of this clone by the above-described method and used to transform yeast cells, as described in Example 2.

Example 2 In order to obtain the human strain Interleukin-2 synthesizing yeast strain Saccharomyces serevisiae, the yeast cells of strain GC-1-GRF18 were transformed with the plasmid pjDB (MSIL) as follows. The yeast strain of the recipient is grown on UEPO medium until the cultural optical density is reached at 600 nanometers 2-4. The cells are washed twice with water and once with 0.1 M sodium citrate buffer containing 1 M sorbitol, suspended in the same buffer and treated first with 150 μl of mercaptoethanol for 15 min at 30 ° C and then with 150 μl of glucuronidase for 30- 60 min at the same temperature. The spheroplasts thus obtained are washed three times with one-molecular sorbitol, suspended in 0.01 M Tris-HC buffer containing 0.01 M calcium chloride and 1 M sorbitol, and after adding 10 μg of pjDB plasmid DNA (MSIL), they are incubated for 30 min at room temperature. Then, a 44% solution of polyethylene glycol 4000, sup-, was added to the cell suspension. keep it for 30 min at 30 ° C, then 2 min at 42 ° C and seeded by deep seeding on SC medium. containing 1% agar and histidine at a concentration of 50 mg / L.

The yeast strain Saccharomyces cerevisiae GC-1-GRF18-pjDB (MSIL) is characterized by the following features.

Morphological signs. The cells are round, slightly oval in shape, about 5-10 microns in size, in some of the cells daughter cells or kidneys attached to their surface are observed.

Cultural signs. Cells grow well on complete organic media: PEP - 2% peptone, 2% glucose; PEPFO - 2% peptone, 2% glucose, 0.15% monosubstituted potassium phosphate. YEPD - 2% peptone, 1% yeast extract, 2% glucose.

In addition to complete organic media, the cells grow well on a mineral composition composition: 0.67% Yeast nitrogen base (Difco), 2% glucose (SC medium), with the addition of 50 mg / l histidine, as well as other synthetic yeast media containing the additive 50 mg / l histidine.

When growing on solid media, they form smooth, round, cloudy colonies with a matose surface, light cream in color, the edge is even.

When growing in liquid media, they form an intense smooth haze. The culture has a characteristic smell of yeast.

Physiological and biochemical characteristics. Cells grow in the range of 4 - 37 ° C, with an optimum of 30 ° C.

When grown under aerobic conditions, the cells significantly acidify the culture medium. The optimum pH for growth is 3.5-5.5. .

Cells can use many simple organic compounds as a carbon source, in particular glucose, sucrose, glycerin.

As a source of nitrogen, subject to the addition of histidine, cells can use both mineral salts in the ammonium form and simple organic compounds - urea, amino acids.

Cells are capable of both aerobic and anaerobic growth.

Essential features of the strain are the need for histidine and leucine prototrophy.

The content of interleukin-2 in strain GC-1-GRF18-pjDB (MSIL) was 1 million units / ml of cell extract. In terms of weight, the production of interleukin-2 reaches 30-50 mg per liter of yeast culture.

The yeast strain Saccharomyces cerevisiae GC-1-GRF18-pjDB (MSIL) -npofly-cent human interleukin-2 was deposited in the All-Union Collection of Industrial Microorganisms under the number VKMP-U791.

Thus, the invention allows to achieve a level of synthesis of human interleukin-2 30-50 mg per liter of yeast culture, which corresponds to 106 units. per 1 ml of cell extract. An advantage of the invention is also that, in comparison with the known producers of interleukin-2 based on E. coli, the yeast has no toxic properties and, therefore, it is possible to more effectively purify interleukin-2 from harmful impurities.

Claims (3)

SUMMARY OF THE INVENTION 1. Recombinant plasmid DNA pjDB (MSIL), which synthesizes human interleukin-2 in Saccharomyces cerevisiae yeast cells with a mol. 5.2 Md and a size of 8.0 kb containing - Hindlll - BamHI - a fragment of the plasmid DNA of a bifunctional bacterial-yeast vector pjDB207 of 6.6 kb, including the bacterial gene for ampicillin resistance, bacterial replication initiation region, a fragment of a yeast two-micron plasmid and the yeast gene LEU2; BamHI - EcoRI - a fragment of the yeast pH05 gene, containing the promoter of this gene 0.52 kb in size .; EcoRI-Sal is a fragment of plasmid pAA 12, 13-23, carrying part of the human interleukin-2 gene, including all of the coding and part of the 3-non-coding region of this gene, 0.56 kb in size: Smal - BamHI - polylinker fragment plot plasmid pUC19 size 8 bp .; SauSA - Pstl — yeast PH05 gene fragment containing the transcription terminator of this gene, 0.37 kbp in size. Pst - Hindlll - fragment of the polylinker of plasmid pUC19 20 bp .; the bacterial gene Ba, which ensures the synthesis of betalactamase in bacteria and resistance to ampicillin; a bacterial ori locus that provides plasmid replication in E. coli cells; a fragment of two-micron DNA of yeast, providing autonomous replication of the plasmid in yeast; LEU2 yeast gene, which ensures the biosynthesis of isopropyl malate dehydrogenase in yeast and the possibility of growth of Ieu2 mutants on selective media without leucine; yeast PH05 gene promoter providing initiation transcriptions of the interleukin-2 gene; PH05 gene transcription terminator, providing the correct termination of transcription of the interleukin-2 gene in yeast; unique restriction sites: 2EcoRI, Hindlll, Sail. 2. A method for producing recombinant plasmid DNA pjDB (NSIL), which provides the synthesis of human interleukin-2 in yeast cells Saccharomyces cerevisiae, consisting in the fact that the fragment of the interleukin-2 gene from plasmid pAA 12, 13-23, limited by the restriction sites EcoRI and Sail, is ligated with the vector part of the plasmid pMS46 hydrolyzed by restriction enzymes EcoRI and Smal, from the obtained plasmid PMSIL by restriction enzyme hydrolysis by Sail and Hindll fragment DNA comprising the promoter of the yeast gene PH05 docked in the correct orientation, the coding part of the interleukin-2 gene and the transcription terminator of the PH05 gene, then this fragment is ligated with the vector part of the plasmid pjDB207 hydrolyzed by Sail and Hindlll restriction enzymes, clones containing the necessary constructs are selected according to restriction analysis data. 3. The strain of yeast Saceharomyces cerevisiae VKPM U-791 - producer of human interleukin-2.