RU2510821C1 - Strain of bacteria bacillus pumilus mk-10 with low proteolytic activity and its application - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: invention relates to field of biotechnology, namely to microorganisms and ferments, and represents strain of bacteria Bacillus pumilus MK-10 All-Russian collection of industrial microorganisms B-10742. Claimed strain is obtained from initial strain of bacteria Bacillus pumilus 3-19 by inactivation of gene of major serine protease on chromosome.

EFFECT: invention makes it possible to extend assortment of bacteria stains, applied as recipient with low proteolytic activity in production of heterologous proteins.

3 cl, 3 dwg, 1 tbl, 1 ex

Description

The claimed technical solution (the proposed strain) relates to the field of biotechnology, namely to microorganisms and enzymes. It can be used in the microbiological industry and genetic engineering.

The presence of a defect in the genes of dominant proteases or a deficiency of such genes in bacteria makes them (bacteria) universal producers of recombinant proteins, for example, used for the production of drugs with stable properties.

A known bacterial strain Bacillus brevis 31-OK [1] with low proteolytic activity, obtained by spontaneous mutation after transformation of the Bacillus brevis HPD31 strain with plasmid pNU2OOhGH carrying the human growth hormone gene. The disadvantage [1] is the dependence of the strain productivity on the synthesis of exoenzymes on the composition of the nutrient medium. A specific nutrient medium is required with the obligatory addition of, for example, polypeptone P1 at a concentration of up to 2%, the absence of which significantly reduces the yield of enzyme production. This dependence narrows the list of culture media used for the cultivation of the strain (Bacillus brevis 31-OK), which in turn significantly limits the scope of this strain as a producer of recombinant proteins, for example, in the manufacture of drugs.

Known obtained by repeated UV (ultraviolet) irradiation, the protease-defective bacterial strain Bacillus amyloliquefaciens A50-32 [2], with 0.3% protease activity. The disadvantage of the bacterial strain Bacillus amyloliquefaciens A50-32 [2] is the presence of uncontrolled and unpredictable mutations, such as regulatory mutations. These mutations have a pleiotropic (multiple) effect on the synthesis of exoenzymes. Due to this drawback (uncontrolled and unpredictable mutations), the vital products of the strain (exoenzymes), for example, those used for the production of drugs, are unstable in properties and action, which significantly limits the scope of the analogue.

The closest essentially proposed strain (prototype) is a protease-deficient strain of bacteria Bacillus subtilis GX4924 [3] with directed inactivation of the subtilisin gene (abbreviated name of the gene is apr). The disadvantage of the prototype [3] is the presence of “interference” in the genome of the indicated bacterial strain — the gene of another major neutral protease (the abbreviated name of the gene is npr), whose share is up to 54% in the total proteolytic pool of enzymes (prototype vital products). The presence of a protease that acts as an obstacle causes additional degradation of recombinant polypeptides that quantitatively limit the synthesis of useful exoenzymes, reduces the quantity and quality of the resulting product, for example, used in pharmaceuticals for the manufacture of drugs in the form of enzymatic additives, for example, for the treatment of pancreatic exocrine insufficiency.

The claimed technical solution is illustrated in Fig.1 - 3 and the table on which are presented:

Figure 1 presents the scheme for the phased creation of a triple recombinant construct consisting of the left (L), right (R) flanking regions of the aprBp gene of Bacillus pumilus 3-19 and the antibiotic resistance gene erythromycin erm (E) using the plasmid pGEM-T.

Figure 2. The scheme of integration of a triple recombinant construct inactivating the aprBp gene in the Bacillus pumilus 3-19 genome is presented. Designations: aprBp is the subtilisin-like protease gene, erm is the antibiotic resistance gene erythromycin, the wavy line indicates the chromosome Bacillus pumilus 3-19 and the chromosome Bacillus pumilus MK-10, the dashed line indicates the integration path. In figure 3. Colonies of the original Bacillus pumilus 3-19 strain (control) and the knockout / protease-negative Bacillus pumilus MK-10 strain (experiment) on 2% milk agar are presented.

The aim of the invention is to obtain a strain of a bacterial recipient of the species Bacillus pumilus with low proteolytic activity for the production of pure homologous and recombinant proteins, increasing the purity of bacteria used for useful purposes.

The goals are achieved in that the bacterial strain Bacillus pumilus MK-10 VKPM B-10742 obtained from the original bacterial strain Bacillus pumilus 3-19 by inactivation of the major serine protease gene on the chromosome is used as a recipient with low proteolytic activity. The bacterial strain Bacillus pumilus MK-10 VKPM B-10742 is used as a recipient with low proteolytic activity. A bacterial strain is used to produce homologous proteins of Bacillus pumilus. The bacterial strain is used to produce heterologous proteins of the genus Bacillus.

The proposed strain of Bacillus pumilus MK-10 receive, for example, as described below. Using known computer programs, for example, Oligo Calc software [4], oligonucleotide primers are designed in a known manner [5] using the NCBI (National Center for Biotechnology Information) public database [6]. The sequences of the subtilisin-like protease gene (aprBp) and the antibiotic resistance gene erythromycin (erm) are used, for example, from the NCBI public database. The primer sequences are shown in the table.

Table Primers used to obtain the strain of Bacillus pumilus MK-10 Primer Name Primer sequence Sub left up 5 'GAAGTCAAACAAACGGTGTCC 3' Sub left low 5 'GAATACCAACATGACGAATCCCCCACGTTGTAATCAAGGACC 3' Sub right up 5 'GGGCACTCTAATAACTACCAAAATTGGTGTTCTTGGGGGTCG 3' Sub right low 5 'AGAGAGGGATCCGAGAGGCAGGGGGTGACGTCTTTTC 3' Em up 5 'GGGATTCGTCATGTTGGTATTC 3' Em low 5 'ATTGGTAGTTATTAGAGTGCCC 3' SP6 5 'GATTTAGGTGACACTATAG 3' T7 5 'TAATACGACTCACTATAGGG 3'

Further actions are shown in FIG. Using the polymerase chain reaction (hereinafter referred to as PCR), the left (L) and right (R) flanking regions of the aprBp gene are amplified with the genomic DNA of the bacterial strain Bacillus pumilus 3-19 using oligonulotide pairs Sub left up / Sub left low and Sub right up / Sub right low respectively. PCR to obtain the erm gene is carried out from the plasmid pCS9 using primers Em up / Em low. As a result, amplitudes of single structures are generated (with a length of, for example, ~ 1 kb or one kilobase).

Then the amplitudes of the single structures are used as raw materials and double structures are obtained by PCR: the left flanking region aprBp + erm (LE) and the right flanking region aprBp + erm (ER). Moreover, oligonucleotide pairs Sub left up / Em low (LE) and Em up / Sub right low (ER), respectively, are used in PCR. Amplification products (double structures), electrophoretically corresponding to the expected length (amplification products) of ~ 2 kb, are isolated from agarose gel, for example, using a set of reagents from Fermentas (Lithuania).

The resulting double constructs (LE and ER) are cloned into the pGEM-T Easy plasmid (Promega, USA) by ligation at the sticky T-ends of the plasmid and the A-ends of the amplifications. The known bacterial strain Escherichia coli XL-1 Blue is transformed with a ligase mixture. Transformation is carried out, for example, by a known method [7]. A suspension of transformed cells is plated on agar plates containing 5-bromo-4-chloro-3-indoyl-beta-D-galactopyranoside, for example, at a concentration of 40 mg / ml, isopropyl-β-D-1-thiogalactopyranoside, for example at a concentration of 32 mg / ml and ampicillin, for example, at a concentration of 20 mg / ml. Use, for example, reagents of the company Fermentas (Lithuania). After incubation of the transformants for 24 hours at plus 37 ° C, white (containing LE and ER cloned constructs) and blue (containing no constructs) colonies of bacterial cells are formed on the plates.

White bacteria colonies were selected from which PCR screening was performed from flanking primers (T7, SP6) for the presence of an insert in plasmid pGEM-T Easy (amplification product) of ~ 2 kb. From the positive (containing the amplification product) bacterial colonies, the plasmids pGEMTE-LE and pGEMTE-ER with inserts LE and ER, respectively, are isolated by alkaline lysis.

The amplification reaction is carried out and a triple recombinant construct (LER) is obtained. To do this, use the plasmids pGEMTE-LE and pGEMTE-ER as the template and primers Sub left up / Sub right low. The amplification reaction product corresponding to the expected electrophoretic length of ~ 3 kb is purified from an agarose gel, for example, using a Fermentas kit (Lithuania).

The final step in obtaining the proposed protease-deficient strain of bacteria Bacillus pumilus is the transformation of bacterial cells of Bacillus pumilus 3-19 obtained triple recombinant design. The transformation is carried out according to the method [8], while using synthetic minimal medium Specisen 1/11. The salt base of the Specisen medium (%): K ₂ NRA ₄ × 3H ₂ O - 18.34; KH ₂ PO ₄ (anhydrous) - 6.0; (NH ₄ ) ₂ SO ₄ - 2.0; citrate - Na - 1.2. Solutions are prepared separately: glucose - 40%; casamic acids - 200 mg / ml; Mg ₂ SO ₄ - 0.2 g / ml. Specialized Medium I: per 100 ml H ₂ O: glucose - 2 ml; casamic acids - 2 ml; yeast extract - 2 ml; Mg ₂ SO ₄ - 0.1 ml; salt base - 10 ml. Specisen II medium: per 100 ml H ₂ O: glucose - 2 ml; casamic acids - 1 ml; Mg ₂ SO ₄ - 8 µ1; salt base - 10 ml).

A suspension of competent triple recombinant construct Bacillus pumilus 3-19 cells is plated on agar plate with antibiotic erythromycin, for example, at a concentration of 20 Hg / ml. After incubation for 24 to 48 hours at + 37 ° C, for example, three independent clones growing on erythromycin medium are selected. Additional verification of the presence (or absence) of the protease gene is performed using PCR. The presence of the erm gene insert (inside the aprBp protease gene) revealed by PCR confirms the destruction of the functional gene, i.e. The achievement of the purpose of the invention is the production of a modified bacterial strain having the aprBp ::. erm genotype, referred to as Bacillus pumilus MK-10.

Description of the proposed strain

The bacterial strain Bacillus pumilus MK-10 was deposited in the All-Russian collection of industrial microorganisms of the Federal State Unitary Enterprise GosNII Genetika under the registration number VKPM V-10742. The proposed strain is derived from a strain of Bacillus pumilus 3-19 and contains genes for minor proteases, for example glutamyl endopeptidase, adamalysin-like metalloprotease.

Genetic features of the strain

The proposed bacterial strain Bacillus pumilus MK-10 carries the antibiotic resistance gene erythromycin, flanked by portions of the subtilisin-like protease gene of Bacillus pumilus 3-19. The aprBp gene is inactivated by homologous recombination (Figure 2).

Cultural and morphological features of the strain

The proposed strain of bacteria Bacillus pumilus MK10 - aerobic, gram-positive bacteria. The cells are rod-shaped, straight or curved, located in clusters or separately. The size of the cells of a one-day culture grown on an agar medium is 14-15 microns. Free spores appear on the fortieth growth hours (no more than 5%), then their number increases and reaches its maximum value (up to 80%) in the late stationary growth phase. When growing on an agar medium Luria-Bertani (hereinafter referred to as LB), the colonies have a rougher surface and uneven edges compared to smoother and more even colonies of the original Bacillus pumilus 3-19 strain.

Physiological and biochemical properties

Growth medium, for example LB: tryptone - 10 g / l, yeast extract - 5 g / l, NaCl - 10 g / l, pH 8.5 with the antibiotic erythromycin, for example, in a final concentration of 20 µg / ml. To create a solid nutrient medium, an additional 2% agar is added. The optimum growth temperature is + 37 ° C.

Proteolytic activity of the proposed strain

The activity of the strain is determined, for example, in the following way.

Determination of the proteolytic activity of the protease-deficient strain of Bacillus pumilus MK-10 is carried out on a medium containing 2% milk agar. The presence of proteolytic activity in this case is determined by the appearance of the enlightenment region on milk agar (zone of splitting of milk proteins). As a result of selection (selection), for example, of three colonies that do not have enlightenment zones on the dairy medium, the proposed strain is obtained.

Marked proteolysis zones on milk agar (2% agar and 1% milk) are observed only in the initial bacterial strain Bacillus pumilus 3-19. The proposed strain of Bacillus pumilus MK-10 enlightenment is less pronounced compared to the original strain of Bacillus pumilus 3-19 (Figure 3), which indicates the inactivation of the gene of secreted subtilisin-like protease. The presence of residual lysis zones is explained by the presence of activity of other secreted proteases, for example metalloproteases. Thus, the proposed bacterial strain Bacillus pumilus MK-10 has a low proteolytic activity.

An example of the application of the proposed bacterial strain Bacillus pumilus MK-10 is given below.

EXAMPLE. The use of the proposed strain for the production and synthesis of recombinant homo- and heterologous proteins of bacillus, used to create drugs.

A distinctive property of the original Bacillus pumilus 3-19 strain from which the proposed strain is derived is the presence of one major serine subtilisin-like protease in the chromosome of the gene. That is, the proposed strain, unlike the prototype, lacks a second major secreted protease, the presence of which would lead to the degradation of recombinant proteins and reduce the efficiency (level of synthesis and quality of the target protein) of the resulting product. Inactivation of the gene of the only major enzyme (subtilisin-like protease) allows you to get a strain of Bacillus pumilus MK-10 with low, compared with the prototype, proteolytic activity. As a result, the total proteolytic activity of the strain is reduced by 2.5 times compared with the activity of the original strain. Thus, the proposed strain of Bacillus pumilus MK-10 provides its use as a recipient for the expression of homologous genes from bacteria of the genus Bacillus pumilus. This property is associated with the absence of Shine Dalgarno sequence variations in the promoters of genes isolated from Bacillus pumilus strains. Therefore, the proposed strain is most effective in the production of industrially important enzymes, for example, subtilisin-like protease Bacillus pumilus, which has the potential to create thrombolytic drugs [9]. That is, the proposed strain allows you to get rid of the possible prototype changes in the efficiency of protein translation inherent in the prototype and thereby achieve the stability of the properties of the products obtained as a result of the vital activity of these microorganisms and realized in biotechnology for the production of drugs, for example, used in medicine, veterinary medicine, agricultural production and food production.

The proposed strain as a host is used, for example, by the method described below for the expression of homologous Bacillus pumilus genes.

1. The bacterial cells of the proposed Bacillus pumilus MK-10 strain are transformed with a plasmid containing a recombinant protein gene (for example, the subtilisin-like protease gene aprBp of Bacillus pumilus strain 3-19) under the control of an inducible or constitutive promoter. The obtained transformed cells are grown on a nutrient medium, for example, LB, at an incubation temperature of + 37 ° C. The induction of protein synthesis is carried out in accordance with the selected promoter.

2. Samples for protein analysis are taken from QOL by separating the supernatant from the cell mass, for example, 1 hour and 16 hours after induction.

3. Extracellular proteins in the sample (culture fluid or QOL) are characterized by Laemmli polyacrylamide gel electrophoresis [10].

4. Recombinant proteins are further purified by various methods, for example using affinity chromatography, gel filtration, ion exchange chromatography, salting out.

Thus, the example shows that the proposed bacterial strain Bacillus pumilus MK-10 by reducing the degradation of the protein product allows to obtain recombinant proteins, for example, for use in medicine, veterinary medicine, in agriculture for crop production.

In addition, the proposed strain is used to express heterologous bacillus genes according to the above method.

An example of the application of the proposed bacterial strain shows its usefulness in biotechnology in the industrial production of recombinant proteins. The use of the proposed strain also helps to identify the regulatory properties of the inactivated AprBp enzyme, which contributes to fundamental molecular biology. The proposed strain has an inventive step, since the published literature has not identified protease-deficient strains of the species Bacillus pumilus.

USED SOURCES

1. Kajino T., Kato K., Miyazaki S., Asami O., Hirai M., Yamada Y., Udaka S. Isolation of a protease-deficient mutant of Bacillus brevis and efficient secretion of a fungal protein disulfide isomerase by the mutant / J Biosci Bioeng. 1999.87 (1): 37-42.

2. Plotnikova T.O., Selivanova G.N., Iomantas Iu.V., Kozlov IuI. Isolation and analysis of protease-deficient mutants of Bacillus amyloliquefaciens I Genetika. - 1992.28 (5): 66-72.

3. Fahnestock S.R., Fisher K.E. Protease-deficient Bacillus subtilis host strains for production of Staphylococcal protein A / Appl. Environ. Microbiol. 1987. 53 (2): 379-84.

4.http: //www.basic.northwestern.edu/biotools/oligocalc.html

5. Maniatis T., Fritsch E., Sambrook J. Methods of genetic engineering. Molecular Cloning // - M .: Mir, 1984.

6.http: //www.ncbi.nlm.nih.gov/

7. Sambrook J. Molecular cloning: a laboratory manual / J. Sambrook, EFFritsch, T. Maniatis. - 2 ^nd ed. NY: Cold Spring Harbor Laboratory Press, 1989.

8. Harwood, C. R., S. M. Cutting. 1990. Molecular biological methods for Bacillus. John Wiley & Sons, Chichester, England.

9. Itskovich E.L., Lyutova L.I., Balaban N.P., Mardanova A.M., Shakirov E.V., Sharipova M.R., Leshchinskaya I.B., Rudenskaya G.N. Thrombolytic and anticoagulant properties of the thiol-dependent serine proteinase Bacillus intermedms 3-19 / Questions of medical chemistry, 1999, No. 5.

10. Laemmli, U.K. Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4 [Text] / U.K. Laemmli // Nature. - 1970. - V.227. - P.680-685.

Claims (3)

1. The bacterial strain Bacillus pumilus MK-10 VKPM B-10742 is a recipient with low proteolytic activity. 2. The bacterial strain according to claim 1 is used to produce homologous Bacillus pumilus proteins. 3. The bacterial strain according to claim 1 is used for the production of heterologous proteins of the genus Bacillus.

Images