RU2670071C1 - Pet21a-prochym recombinate plasmid, providing synthesis of chemeric b bos taurus proximosin protein, as well as escherichia coli bl21 (de3) plyse pet21a-prochym - producer of chemeric b bos taurus proximosin protein - Google Patents

Abstract

FIELD: chemistry.SUBSTANCE: group of inventions relates to a recombinant plasmid providing the synthesis of a chimeric protein of B Bos Taurus prochymosin, and a recombinant strain of E. coli, a producer of chimeric protein of B Bos Taurus prochymosin in E. coli cells. Recombinant pET21a-ProChym plasmid of 6504 base pairs (bp) contains the following structural elements essential to its functioning and expression of the target protein: a bacteriophage T7 promoter that provides efficient transcription of the controlled mRNA; a recombinant gene of B Bos Taurus prochymosin encoding the target chimeric protein; T7 terminator, which is the untranslated termination region of the transcription of the bacterial operon providing an effective termination of transcription; the sequence of the T7 expression tag, added to the composition of the nucleotide sequence of the gene of B Bos Taurus prochymosin to the N-terminus, to increase the yield of the target protein; a bla sequence encoding a beta-lactamase protein, which is a selective marker for the transformation of E. coli strains; a ColE1 sequence that replicates the plasmid in E. coli strains. Recombinant gene of B Bos Taurus prochymosin has the nucleotide sequence of SEQ ID NO: 1 from the nucleotide at position 5205 bp. to the nucleotide at position 6347 bp, encoding the target chimeric protein of B Bos Taurus prochymosin with the complete amino acid sequence of SEQ ID NO: 2, embedded at the FauND I and Sfr274 I restriction sites into the region of the pET21a polylinker containing the T7 phage RNA polymerase promoter and terminator. Proposed recombinant strain of Escherichia coli BL21 (DE3) pLysE pET21a-ProChym is obtained by transformation with the recombinant plasmid pET21a-ProChym and deposited in the Collection of bacteria, bacteriophages and fungi of the State Research Center of Virology and Biotechnology VECTOR of the Federal Service for Supervision of Consumer Rights Protection and Human Well-Being under registration number B-1362.EFFECT: group of inventions provides high production of pro-chymosin.2 cl, 5 dwg, 6 ex, 2 tbl

Description

The invention relates to a genetic construct (plasmid) for the synthesis of the chimeric protein prochymosin B of Bos Taurus, intended for hydrolysis of the peptide bond Phe105-Met106 in the kappa-casein molecule in the manufacture of cheese of hard and semi-solid varieties and the recombinant E. coli strain - producer of the chimeric protein prochymosin B Bos Taurus and can be used in the field of biotechnology and food industry.

Cheese production is the most profitable sector in the dairy industry. A key stage in the production of cheese is the coagulation of milk using milk-clotting enzyme preparations (MFPs). Coagulation of milk in this process is conditionally divided into two partially overlapping phases - primary or enzymatic and secondary - aggregation. During the primary phase, under the action of a milk-clotting enzyme, a Phe105-Met106 bond is broken in a kappa-casein (k-casein) molecule. As a result, two fragments are formed - para-κ-CN (residues 1-105), an N-terminal hydrophobic region, and a hydrophilic C-terminal casein macropeptide (residues 106-169). The hydrophobic para-κ-casein remains in the micelle, and the hydrophilic anionic casein-macropeptide "leaves" the serum. Thus, during the first phase of coagulation, under the action of chymosin (or other milk-clotting enzymes), the stability of micelles is impaired due to the removal of the glycomacropeptide site of k-casein from their surface [1, 2, 3, 4].

As a result of elimination of the stabilizing “hair” layer, micelles get the opportunity to get closer so much that their aggregation begins - the secondary phase of coagulation begins. Ultimately, the aggregation of destabilized micelles leads to the formation of a milk clot. Thus, the specific hydrolysis of the Phe105-Met106 bond provides the formation of a milk clot with the properties necessary for the manufacture of cheeses [3, 4].

In nature, there are many enzymes with proteolytic activity. However, rennet (SF) is considered a classic MFP that provides maximum yield and high quality cheese. The main active agent of SF is chymosin, an enzyme belonging to the class of aspartic proteases (EC 3.4.23.4). Cheese production volumes increase annually due to increased demand for this product. The increase in cheese production is inextricably linked to an increase in the need for milk-clotting preparations. In this regard, starting from the second half of the 20th century, there is a global shortage of rennet.

The problem of SF deficiency is associated with an acute shortage of raw materials for its production - the abomasum of milk calves. This is due to the fact that in conditions of a decrease in the number of livestock, due to a sharp increase in its productivity, it is economically inexpedient to slaughter young animals that feed on milk. The scrapie epidemic - a prion disease of farm animals - has led to an additional reduction in the raw material base of natural MSF. The global deficiency of SF has initiated the search for its substitutes among the natural milk-clotting proteinases of animal, plant and microbial origin, which continue to this day.

The coagulants closest to chymosin, such as beef pepsin, some microbial, plant, and microfungal proteases, are currently used as substitutes for certain types of cheese. However, unlike natural chymosin, almost all of its known analogues have an increased level of nonspecific proteolytic activity, and some often outperform chymosin in thermal stability, which leads to deeper hydrolysis of caseins, a decrease in cheese yield and a decrease in its organoleptic characteristics [6, 7, 8, 9].

A modern solution to the problem of SF deficiency is not only the search for new raw material sources, but also the creation of technologies for the production of recombinant chymosin. The widespread use of recombinant chymosin as an MSF is associated with a number of advantages of this enzyme compared to other proteases. These include: the possibility of large-scale production of the enzyme; low non-specific proteolytic activity; predictability of coagulation reaction; lack of tissue components of farm animals; availability of kosher certificates.

Recombinant chymosin is one of the first recombinant enzymes to be widely used in industry. Currently, several approaches to the preparation of recombinant chymosin are described.

Known international application for the invention (WO 2009/027572, A1, IPC C12N 9/64; C12N 15/69; C12N 15/81, published 05.03.2009) [14] which describes a method for producing active buffalo chymosin, without the process of autocatalytic intermediate activation, mediated by a low pH, for coagulation of milk. For this, the buffalo chymosin gene was integrated into the DNA of Pichia pastoris yeast. During cultivation, chymosin is secreted into the extracellular medium in active form. Thus, the authors declare that the use of this recombinant buffalo chymosin, which is not subjected to the activation process using pH, is an additional advantage compared with the prior art, avoiding the intermediate activation process to obtain cheese masses.

The disadvantage of the genetic design of this invention may be that the authors use the natural gene Bubalus arnee bubalus. When a gene is transferred to another organism, the recipient due to differences in the mechanisms of transcription, translation, and post-translational modifications (higher and lower eukaryotes) can significantly decrease the yield of the product and its stability [10, 11]. To evaluate this in this invention is not possible, because the yield of the target product is not described. Also, in the genetic construct there are no amino acid tags (for example, 6 × His) that make it possible to obtain a highly purified product. In this invention, the authors, to determine the milk-clotting activity, use the culture supernatant, noting that when fermented into the culture medium, yeast proteases and other proteins can be secreted along with the target protein. This fact does not allow to accurately assess the specificity of the obtained chymosin. The invention does not disclose the use of this product under standardized cheese production conditions. In our invention obtained chymosin with a degree of purity of 98% (Figure 5, B), which is used to determine the enzymatic properties of the specified final product.

The active recombinant chymosin used for cheese making is obtained by activation of prochymosin. Therefore, for insertion into the expression vector, the prochymosin sequence is used [12, 13].

The closest analogue (prototype) is the preparation of recombinant chymosin and a method for its preparation (US application No. 20070166785, A1, IPC C12P 21/06, C07H 21/04, publ. 19.07.2007) [15]. The present invention describes the preparation of recombinant Bos taurus prochymosin in the Escherichia coli system, which includes the step of amplifying the natural Bos taurus prochymosin gene, its incorporation into the pET21b expression vector, transformation of the E. coli recombinant cell plasmid obtained, fermentation of the indicated E. coli to obtain prochymosin, methods purification, renaturation and activation of prochymosin to determine its milk-clotting activity.

The disadvantage of the genetic design in the prototype and, as a consequence, the producer, recombinant chymosin, as well as in the analogue described above (WO, 2009/027572), is the prochymosin gene that is not optimized for the selected expression system, which is especially reflected when the gene is transferred from a eukaryotic cell to prokaryotic due to which the yield of the product and its stability are reduced [10, 11]. In the claimed technical solution, the prochymosin gene was optimized (Codon Optimization OnLine) for expression in prokaryotic cells. In addition, the prototype does not provide data on the final purity of the product, as a result of which bacterial proteins may remain in the final product, which may affect the characteristics of the product.

The technical result of the claimed invention is the creation of a genetic construct and on its basis a recombinant strain of Escherichia coli - producer of prochymosin B Bos taurus, which provides higher production of prochymosin (up to 40% of the total protein of the cell) with a purity of up to 98% (example 4, figure 5, A, B).

The indicated technical result is achieved by creating a recombinant plasmid pET21-ProChym with a size of 6504 base pairs (bp), characterized by the physical and genetic map shown in FIG. 4, providing synthesis of the 41 kDa prochymosin B chimeric protein B Bos taurus in Escherichia coli BL21 (DE3) pLysE cells containing the recombinant Bos taurus prochymosin B protein gene having the nucleotide sequence of SEQ ID NO: 1 (see FIG. 1 and Appendix) from nucleotide at position 5205 bp to the nucleotide at 6347 bp, encoding the target chimeric protein prochymosin B of Bos taurus with the full amino acid sequence of SEQ ID NO: 2 (see Fig. 3 and Appendix), inserted at the restriction sites FauND I and Sfr274 I in the region of the vector polylinker pET21a containing the promoter and terminator of RNA polymerase of phage T7.

The indicated technical result is also achieved by the creation of a recombinant strain of Escherichia coli BL21 (DE3) pLysE pET21-ProChym, a producer of the chimeric protein prochymosin B of Bos taurus E. coli obtained by transformation of the recombinant plasmid pET21-ProChym according to claim 1 and deposited in the Collection of bacteria, bacteriophages and fungi and fungi FBUN SSC WB "Vector" Rospotrebnadzor under registration number V-1362.

The inventive strain Escherichia coli BL21 (DE3) pLysE / pET21-ProChym is a producer of recombinant prochymosin B Bos taurus, which provides specific hydrolysis of the Phe105-Met106 peptide bond in the casein molecule and is intended for use in the manufacture of hard and semi-hard cheeses, as follows.

When developing a recombinant prochymosin producer strain, the sequence Bos taurus B (Swiss-ProtID: P00794) was selected. The choice of the sequence of prochymosin B rather than preprochymosin or chymosin is explained as follows. Chymosin is synthesized in the body of animals in the form of preprochymosin. Preprochymosin is a protein consisting of 365 amino acid residues, 16 of which are a hydrophobic leader sequence, which plays an important role in the secretion of chymosin through the cell membrane; it is not required for expression in the prokaryotic system [16]. In previous studies, it was found that the expression of mature chymosin leads to the production of a product that does not have milk clotting activity (MA) [17]. There are two alternative forms of chymosin - chymosin A and B. These forms differ from each other by one amino acid residue, aspartate or glycine at position 244, respectively, while chymosin B is more common and has a more stable structure (Fig 1) [18].

To ensure a high and stable yield of the target product, the codon composition of the selected sequence of prochymosin B Bos taurus for expression in E. coli was optimized. In the course of theoretical design, codons used in eukaryotic cells were replaced by codons used by prokaryotes. The sequence of the T7 expression tag was added to the nucleotide sequence of the gene at the N-terminus. The optimized sequence has been synthesized.

Plasmid pET21a was selected as the expression vector. After cloning the synthesized sequence in the vector obtained by the recombinant plasmid, the transformation of TOP10 bacterial cells was carried out to produce plasmid DNA.

The E. coli strain BL21 (DE3) pLysE was selected as the producer. The choice of the recipient strain is due to the fact that it carries the T7 phage RNA polymerase gene, which is necessary to ensure efficient transcription of the target genes in the vector construct under the control of the T7 phage promoter, as well as the fact that this strain is defective in protease synthesis, which is essential increases the yield of synthesized heterologous proteins.

When cultured, the E. coli strain BL21 (DE3) pLysE / pET21a-ProChym provides the synthesis of recombinant, enzymatically active prochymosin B of Bos taurus at a level of 40% of the total bacterial protein (see Figure 5, A).

By optimizing the codon composition and adding the T7 tag sequence, the yield of the target protein increases, since studies have shown that prochymosin is produced in small quantities without adding this region [17]. At the C-terminus of the gene, a fragment encoding six histidines (6 × His) is laid, in order to be able to purify and concentrate the target protein using metal chelate chromatography.

The invention is illustrated by the following graphic figures. In FIG. 1 shows the nucleotide sequence of the gene of the chimeric protein prochymosin ProChym. In FIG. 2 shows the amino acid sequence structure of the preprochymosin B of Bos taurus (Swiss-ProtID: P00794). In FIG. Figure 3 shows the amino acid sequence of the chimeric protein prochymosin B of Bos taurus encoded by the ProChym gene. In FIG. 4 depicts the physical and genetic map of the recombinant plasmid pET21a-ProChym containing the sequence of the chimeric bkel of prochymosin Bos taurus. In FIG. 5A, B are photographs of electrophoretic separation: (Fig. 5, A) - E. coli BL21 (DE3) pLysE / pET21a-ProChym cell lysate in 12% PAG (1 - negative control (culture of E. coli BL21 (DE3) pLysE without plasmids); 2 - E. coli BL21 (DE3) pLysE pET21-ProChym cell lysate; 3 - E. coli BL21 (DE3) pLysE pETys-ProChym debris lysate after homogenization and separation from the soluble fraction; 4 - molecular weight marker); (Fig. 5, B) - PAGE electrophoresis of the preparation of the chimeric protein prochymosin B Bos taurus (1 - sample of recombinant chymosin B Bos taurus translated into BL21 (DE3) pLysE without propeptide, 2 - sample of the recombinant chimeric protein prochymosin B taurus (obtained from using the inventive plasmid and producer strain), 3 - molecular weight marker).

For a better understanding of the essence of the invention, the following are examples of its implementation. All standard genetic engineering and microbiological manipulations, as well as amplification and DNA sequencing, were carried out according to known methods (Maniatis T., Fritsch E, Sambrook J. Molecular cloning, M .: Mir, 1984 [19]; DNA cloning. Methods. Ed. D. Glover, Translated from English, Moscow, Mir, 1988 [20]; Saiki RK, Gelfand DH, Stoffel S., Sharf SJ, Higuehi R., Horn GT, Mullis KB, Eriich HA Science, 1988. V 239, No. 4839, p. 487-491; Sanger F., Nicklen S., Coulson AR Proc. Nat. Acad. Sci. USA, 1977, V. 74, p. 5463-5467) [21].

Example 1. Synthesis, insertion and analysis of a DNA fragment comprising the complete coding sequence of the prochymosin B Bos taurus gene.

The inventive plasmid contains the following structural elements essential for its functioning and expression of the target protein:

- promoter of the bacteriophage T7, providing efficient transcription of controlled mRNA; recombinant gene encoding the target chimeric protein (prochymosin B Bos taurus gene); the untranslated region of the transcription termination of the bacterial operon, which ensures the efficient termination of transcription;

- bla bacterial operon encoding beta-lactamase protein, which is a selective marker for the transformation of E. coli strains;

- a bacterial site for the initiation of replication of the ColEl type, which ensures replication of the plasmid in E. coli strains.

The selected nucleotide sequence of the prochymosin B Bosta gene (Swiss-ProtID: P00794) was synthesized on an ABI 3400 DNA / RNA Synthesizer. The synthesized gene was cloned into the pGH cloning vector (DNA Synthesis, Moscow). To obtain the Bos taurus prochymosin gene, PNR was performed using oligonucleotide primers corresponding to the initial part of pET21a + proChym- (S) (contains the propeptide and tag T7 providing an increased level of expression of the target protein) and terminal pET21a + Chym (B) using high-precision DNA polymerase AmpliTaq Gold from Thermo Fisher Scientific (USA) (Table 1).

PCR was performed using a BIS PCR amplifier BIS-N LLC (Russia). A 50 μl reaction mixture containing 2 μg DNA (pGH plasmid with the chymosin gene), 10 pM of each primer (pET21a + proChym- (S) and pET21a + Chym (B), 67 mM Tris-HCl (pH 8.8), 15 mM ammonium sulfate, 2.5 mM magnesium chloride, 0.01% Tween-20, a mixture of deoxynucleotide triphosphates (dATP, dTTP, dTTP, dTTP 2.5 mM each) and 1 unit of AmpliTaq Gold DNA polymerase from Thermo Fisher Scientific ( USA); the reaction was carried out at the following parameters: 50 s - 96 ° C, 30 s - 60 ° C, 1 min - 72 ° C (30 cycles).

At the end, the reaction mixture was separated by electrophoresis on a 1% agarose gel, and the reaction product, a single fragment of 1.1 kb, was detected. The fragment was isolated from the gel using a set of "Gel Extraction Kit" company Qiagen (Germany). Then, the isolated PCR product and previously prepared and purified plasmid pET21a were treated with restriction endonucleases FauND I and Sfr274I (SibEnzyme, Novosibirsk).

The hydrolysis reaction was carried out under conditions recommended by the manufacturer. To purify the restriction vector, plasmid DNA was applied to a 1% agarose gel and isolated from the gel using the Gel Extraction Kit from Qiagen (Germany). The ligation reaction was carried out using T4 bacteriophage DNA ligase (SibEnzyme, Novosibirsk). The reaction was carried out at + 4 ° C during the night. The obtained ligase mixture was transformed (as described in example 2) chemically competent cells of E. coli strain TOP 10.

After transformation, clones were selected on LB agar medium containing the antibiotic ampicillin (50 mg / ml). Plasmid DNA was isolated from the colonies by alkaline lysis. To analyze the presence of the prochymosin gene insert in the recombinant plasmid, PCR with specific primers was used (table 1, primers 1 and 2). The amplification products were separated on a 1% agarose gel, followed by staining with ethidium bromide (0.5 μg / ml). Positive colonies were added to 5 ml of LB medium with ampicillin (50 μg / ml) and grown overnight at 37 ° C at 170 rpm. Then plasmid DNA was isolated from bacterial cells using commercial Qiagen DNA mini kit kits according to the manufacturer's recommendations. The primary structure of the obtained plasmids was confirmed by sequencing. Sequencing was carried out according to the Sanger method in the Genomics Center of the Siberian Branch of the Russian Academy of Sciences (Novosibirsk).

Sequencing of plasmid DNA of positive clones in the region of the insertion of the prochymosin B gene of a cow allowed selection of clones with no defects in the inserted genes (insertion, deletion, substitution), after which plasmid DNA was generated and isolated from the selected clones.

Example 2. Obtaining a recombinant strain of Escherichia coli BL21 (DE3) _P LysE / pET21a- ProChym

The obtained recombinant plasmid pET21a-ProChym was used to transform E. coli BL21 (DE3) pLysE cells using calcium chloride. The cell culture was _grown to OD _λ600 = 0.5. father 1.5 ml of the suspension was centrifuged at 6000 rpm for 2 min in an Eppendorf 5415 R centrifuge. The cell pellet was suspended in 700 μl of 0.2 M CaCl ₂ , incubated at 4 ° C for 15 min, then the cells were precipitated by centrifugation at 4000 rpm for 3 min. The cell pellet was suspended in 100 μl of 0.2 M CaCl ₂ . 50-70 ng of plasmid DNA was added to the cell suspension, incubated for 30 min at 4 ° C, then for 5 min at 37 ° C. 1 ml of LB medium was added to the cell suspension and incubated at 37 ° C for 1 hour. 100 μl of culture was scattered on a Petri dish with agar medium supplemented with ampicillin to a concentration of 100 μg / ml. The remainder of the culture was precipitated by centrifugation at 4000 rpm for 3 min, the supernatant was taken, suspended in 100 μl of LB and plated on a plate with agarized LB medium containing 100 μg / ml of ampicillin. The dishes were placed in a 37 ° C thermostat and incubated overnight. The next day, individual BL21 (DE3) pLysE / pET21a- ProChym colonies were identified.

The strain E. coli BL21 (DE3) pLysE carrying the recombinant plasmid pET21a-ProChym is a producer of recombinant prochymosin. Bos taurus is characterized by the following features: high physiological stability (the decrease in the activity of the enzyme produced after 50 generations does not exceed 20%) and a high yield of the active product.

Cells grow well on commonly used culture media. When grown on liquid LB and YT media, intense even turbidity forms.

Physiological and biochemical characteristics

The optimum cultivation temperature is from 30 to 37 ° C, the optimum pH is 7.6. The source of nitrogen is organic compounds (in the form of tryptone, yeast extract).

Significant when using these strains is their sensitivity to kanamycin (up to 25 μg / ml). Resistant to ampicillin (up to 100 μg / ml) due to the presence of pET21a-Chym plasmids.

One of the selected clones was designated as Escherichia coli BL21 (DE3) pLysE / pET21a- ProChym, grown in 5 ml of LB liquid medium containing 100 mg / l ampinicillin to OD _λ600 = 5.0, an equal volume of sterile 30% glycerol was added to the culture , poured into 500 μl into sterile 1.5 ml tubes, frozen and stored at - 70 ° C.

Example 3. The accumulation of biomass BL21 (DE3) pLysE / pET21-ProChym and the selection of recombinant prochymosin In Bos taurus

The obtained E. coli clones of strain BL21 (DE3) pLysE after transformation with recombinant plasmid pET21a- ProChym were expanded in 5 ml of YT × 2 medium overnight. The resulting overnight culture was added 1.5 ml to 750 ml flasks containing 150 ml of YT × 2 medium and grown to a density OD _λ600 = 0.8. After that, IPTG was added to the cultures to a final concentration of 1 μM to induce the T7 phage promoter and cultivation was continued for 5 hours at a temperature of 37 ° C, 170 rpm. The biomass of bacterial cells was besieged by centrifugation (7000 rpm), then the biomass was homogenized in a lysis buffer using a Soniprep 150 Plus ultrasonic homogenizer. Debris was separated by centrifugation at 16,000 rpm for 15 min, 4 ° C.

The biomass of bacterial cells was besieged by centrifugation (7000 rpm, 9000 g). The pellet was suspended in lysis buffer (20 mM Tris, 300 mM NaCl, 0.1% Twin 20, pH = 8.0) using a Soniprep 150 Plus ultrasonic homogenizer. Debris (a precipitate formed by centrifuging a suspension of homogenized tissue and containing dilapidated cells) was separated by centrifugation at 16,000 rpm (31,000 g) for 15 min at 4 ° C.

After separation, both fractions were analyzed in 12% PAG for the presence of the target protein. The results showed that the entire target protein is in the insoluble fraction (Figure 5, A).

Example 4. Purification of Recombinant Prochymosin In Bos taurus

Since in Example 3 it was shown that the target protein is in inclusion bodies, for subsequent purification, debris was dissolved in a base buffer (20 mM Tris, 300 mM NaCl, pH = 8) containing 8 M urea.

Since a 6 × His fragment was inserted into the nucleotide sequence of the C-terminal regions of the recombinant protein, it was purified using metal-chelate chromatography on Ni-NTA agarose.

A column containing Ni-NTA agarose was equilibrated with basic buffer: 20 mM Tris, 300 mM NaCl, 8M urea, pH = 8.0. After disintegration, the debris was dissolved in a base buffer with 8 M urea and applied to the column. The column was washed with 10 volumes of wash buffer (base buffer with 8 M urea + 20 mM imidazole). The desorption of proteins associated with the sorbent was carried out with a base buffer with a stepwise (step 50 mM) increasing (20-300 mm) concentration of imidazole. It was found that recombinant prochymosin elutes at an imidazole concentration of 250 mM. Quantitative analysis showed that 1.5 g of prochymosin is produced per 1 liter of culture medium (Figure 5). In this case, the mass of purified prochymosin is from 30 to 40% of the total mass of BL21 (DE3) pLysE / pET21-ProChym cells. Protein purity was monitored by electrophoresis on a 12% polyacrylamide gel (Fig. 5A).

Example 5 Resuscitation and Activation of Recombinant Prochymosin In Bos tauru

Refolding of the target protein eluted from the metal-chelate sorbent and its purification from urea and imidazole were performed by dialysis. Dialysis was carried out in two stages: once against a buffer containing 50 mM Tris, 300 mM NaCl, pH = 10.25, not more than 5 hours. Then four times against a solution containing 50 mm Tris, pH = 7.5.

After dialysis, the concentration of samples used to determine milk coagulation activity was determined. Also, for comparison, the previously obtained recombinant chymosin B Bos taurus, translated into BL21 (DE3) pLysE without a propeptide, i.e., without activation, was taken.

No. 1. Recombinant prochymosin c = 0.2 mg / ml

No. 2. Recombinant chymosin c = 0.8 mg / ml

To confirm the presence of specific enzymatic activity, an analysis of milk coagulation activity was carried out. For this, the enzyme had to be converted to the active form.

The activation of prochymosin was carried out by stepwise lowering and increasing the pH. All operations were performed in plastic dishes at room temperature. To control the initial milk clotting activity (MA) of recombinant prochymosin, an aliquot of the inactive enzyme was selected.

To activate recombinant prochymosin in the sample, with constant stirring, 2.0 M Hcl was added to pH 3.0. Stirring was stopped and the mixture was incubated at pH 3.0 for 2 hours. After the incubation time, the pH of the sample was adjusted to 6.2 using 0.5 M NaOH. In a sample activated in this way, MA was determined.

Example 6 Determination of the milk-clotting activity of recombinant activated chymosin (after activation, prochymosin passes into chymosin)

To determine the MA used a standardized dry milk substrate ("Substrate ST.3"), manufactured by VNIIMS (Uglich). To prepare for work 25.0 gr. the substrate was dissolved in 230 ml of 0.01N CaCl ₂ for 1 h at room temperature with constant stirring. The pH of the resulting mixture was adjusted to 6.5 using 1.0 M NaOH. The final volume of the solution was adjusted to 250 ml of 0.01 N CaCl ₂ .

The activity of the test samples was determined relative to the industry control sample of rennet (OKO SF), certified by MA, produced by OJSC "Moscow rennet plant". To prepare for work, 80 ml of distilled water was added to 1.0 g of OKO SF, stirred for 30 minutes at 20-25 ° С, the mixture volume was brought up to 100 ml and kept in a water bath at 35 ° С for 15 min. Before determining the MA, the test samples were kept for 15 minutes at 35 ° C.

The determination of MA was carried out at 35 ° C. 2.5 ml of a standardized substrate solution was added to a dry glass tube and heated at 35 ° C for 5 minutes. 0.25 ml of the test sample was added to the test tube with the substrate, the stopwatch was turned on, the contents of the tube were immediately thoroughly mixed. The duration of coagulation of the substrate was noted by a stopwatch from the moment the sample was introduced until the first flakes of coagulation of the substrate appeared. The appearance of flakes was checked by periodically applying a drop of the reaction mixture with a glass rod to the wall of the tube.

The milk clotting activity of the test samples was expressed in arbitrary units per 1 ml of the drug (conventional units / ml) and was calculated by the formula:

MA = 0.01 * A * T1 / T2, where:

A - certified milk-clotting activity of OKO SF in arbitrary units;

T1 - the duration of coagulation of the substrate with OKO SF;

T2 - the duration of coagulation of the substrate with the test sample.

To convert the conventional units of MA into international milk-clotting units (IMCU), a reduction factor of 129.1 was used. The results of the study are presented in table 2.

Comparative enzymatic activity showed that recombinant prochymosin after activation exhibits high milk-clotting activity (examples 3 and 4).

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Claims (2)

1. Recombinant plasmid pET21a-ProChym with a size of 6504 base pairs (bp), providing synthesis of the chimeric protein prochymosin In Bos taurus of 41 kDa in Escherichia coli cells BL2 \ (DE3) pLysE and containing the following essential for its functioning and expression of the target protein structural elements: promoter of the bacteriophage T7, which provides efficient transcription of controlled mRNA; recombinant prochymosin B Bos taurus gene encoding the target chimeric protein; T7 terminator, which is an untranslated region of the termination of transcription of a bacterial operon, which provides an effective termination of transcription; the sequence of the expression tag T7 added to the nucleotide sequence of the prochymosin B Bos taurus gene at the N-terminus to increase the yield of the target protein; a bla sequence encoding a beta-lactamase protein, which is a selective marker for transformation of E. coli strains; the ColE1 sequence for plasmid replication in E. coli strains, the recombinant prochymosin B protein gene of Bos taurus has the nucleotide sequence of SEQ ID NO: 1 from the nucleotide at 5205 bp. to the nucleotide at position 6347 bp, encoding the target chimeric protein prochymosin B of Bos taurus with the complete amino acid sequence SEQ ID NO: 2, inserted at the restriction sites FauND I and Sfr274 I in the polylinker region of the pET21a vector containing the promoter and RNA polymerase terminator phage T7. 2. The recombinant strain Escherichia coli BL21 (DE3) pLysE pET21a-ProChym is a producer of the chimeric protein prochymosin B of E. coli ba taurus obtained by transformation of the recombinant plasmid pET21a-ProChym according to claim 1 and deposited in the collection of bacteria, bacteriophages and fungi Vector "Rospotrebnadzor under registration number B-1362.

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