Method of preparative recovery of basic proteins from supramolecular structures of escherichia coli growing population

FIELD: medicine.

SUBSTANCE: what is presented is a method of preparative recovery of basic proteins from supramolecular structures of Escherichia coli growing population. The Escherichia coli cells are preserved in buffered 80-90% glycerol at -25°C. Then the sediment cells are washed in 3% triton X-100. It is followed by sediment extraction in salts of increasing concentrations: 0.14 M, 0.35 M; 2 M NaCl, 6 M in guanidine hydrochloride with 0.1% β-mercaptoethanol. Basic proteins are recovered from the prepared fractions by means of ion-exchange chromatography with amberlite resin IRC-50 of discontinuous gradient of guanidine hydrochloride: 6%, 8.9%, 10.6%, 13% on 0.1 M potassium phosphate buffer pH 6.8.

EFFECT: method enables producing fractions enriched by basic proteins with the use of a microamount of protein of Escherichia coli cell suprastructures.

7 dwg, 1 ex

 

The invention relates to biochemistry and molecular biology of prokaryotic cells and can be applied to the analysis of molecular-genetic mechanisms of formation of structure of prokaryotic cells and the role of protein components in their organization and remodeling of the genome that is necessary to reveal the ways regulation mechanisms of the impact of macro - and microorganisms, as well as the search for new targets for drugs and the development of environmentally friendly medicinal drugs.

The known method preparative highlight the major proteins of cell nuclei from calf thymus [1], which describes a method of fractionation of histones using ion-exchange chromatography on a column of amberlite IDC-50. The drawback of this method is that columns were used size 7.5×55 cm and 4.5×49 cm, to boot which should be sufficiently large amount of protein (2-3 g and 0.5-1 g, respectively), while when working with microorganisms selection of protein in this volume is extremely time-consuming.

There is a method of fractionation of plant histones on columns with amberlite IDC-50 [2], which describes the separation of basic proteins in the cell nuclei of maize seedlings. The drawback of this method is that it is impossible of refractional the th trace amounts of protein from cell suprastructures microorganisms (2-5 μg).

The above method of selection of plant histones on columns with amberlite IDC-50 [2] was adopted as a basis, in which initially the seedlings of maize isolate the cell nucleus, receive nuclear extract using 2 M sodium chloride and add 1 N. hydrochloric acid, and then applying the obtained protein on the column with amberlite IDC-50 and elution of histones in a linear gradient of guanidine hydrochloride, the determination of protein in the eluates turbidimetric. The drawback of this method is that the homogenization unacceptable for prokaryotes, because culture is composed of individual cells, located in the fabric, using 2 M sodium chloride was allocated only one fraction of proteins, protein content, which was determined by the turbidimetric method with low sensitivity threshold (3-5 µg protein in 0.1 ml of solution).

The purpose of the invention a method for preparative highlight the major proteins of the supramolecular structures of the growing populations of Escherichia coli using ion-exchange chromatography on a column of amberlite IDC-50.

This goal is achieved by the fact that in the preparative method of separating the main protein supramolecular structures growing populations of Escherichia coli from canned in the presence of buffered 80-90% glycerol cell off cell membrane 3% Tr the tone X-100, then the extraction is carried out by increasing salt concentrations: 0,14 M, 0.35 M; 2 M NaCl, 6 M guanidine hydrochloride with 0.1% β-mercaptoethanol, followed by separation of these fractions basic proteins using ion-exchange chromatography with amberlite IDC-50 in discontinuous gradient guanidine hydrochloride: 6%, 8,9%, 10,6%, 13% 0.1 M potassium-phosphate buffer pH 6.8.

The invention is illustrated by the following example.

Example. Experiments were performed throughout the life cycle of the cells of the strain E. coli JC-158 (Hfr PO1, thil, serA6, lacI22, relA1) [3], provided Stupak IV and Stupak EA, growing culture was carried out Tropinina T.S. (Institute of biology, Ufa branch of RAS, laboratory of mathematical and molecular genetics), and strain of E. coli 5A, provided Markusevi T.V. (Institute of biology, USC RAS, group genetics of microorganisms). In the work to grow used rich medium LB (Luria-Bertani). In 1 liter of distilled water was dissolved with stirring (magnetic stirrer type MM 2A): bacto-tripton (Difco, USA), 10 g; yeast extract (Difco, USA), 5 g; NaCl 10 g, pH to 7.5. The medium was sterilized at 120-123°C. the vapor pressure of 1 ATM in a standard autoclave.

Bacterial cells used in the experiment was originally in agar columns LB (1 liter environment 1.5 g agar-agar, Difco, USA) and kept at ambient temperature the re 4°C. At this temperature, and the almost complete absence of oxygen is slow all physiological processes in cells. In order to transfer cells in normal physiological condition, namely aerobic respiration, bacterial culture from the agar column under sterile conditions endured by using loops in liquid LB medium in an amount of 5 ml, in chemical beaker with a volume of 20 ml, closed with a cotton-gauze tube and incubated at 37°C, 160 rpm for laboratory thermostatted shaker (P-E) for 16 hours. Separately grown well-formed colony of bacteria from the agar LB medium was one case replanted using loops in liquid LB medium in a volume of 5 ml and were incubated at 37°C, 160 rpm for 7 hours. Then 100 µl of the grown cell culture was one case replanted in fresh LB liquid medium in an amount of 5 ml and were incubated at 37°C, 160 rpm for 16 hours. 2 ml of cell culture were added into a cuvette with a working length 5,075 mm and measured the optical density on the photoelectric colorimeter concentration (ck-2) at a wavelength of 590 nm. This value was 1.0. In fresh LB liquid medium in the amount of 120 ml 500 ml flask were inoculated 120 ál 16 hour culture and incubation was carried out at 37°C, 160 rpm for 7 hours and 10 minutes. The first sample was taken after 50 minutes p is after the beginning of incubation. The optical density of the first sample was 0.005. For further analysis were selected samples in a volume of 1.5 ml of Cells were precipitated by centrifugation at 12,000 rpm in Eppendorf centrifuge for 5 minutes the Supernatant was removed, precipitation dry a little. To precipitation was added 50 μl of medium of the following composition: 80-90% glycerol in 0.01 M Tris-HCl buffer pH 6.8 with the addition of 0.005 M MgCl2; 0.025 M KCl; 0.003 M CaCl2; 0.005 M NaCl for preservation of cells at minus 25°C. Subsequent samples were taken every 20 minutes for up to 7 hours 10 minutes. Further precipitation cells were washed with 3% Triton X-100 in the medium of the following composition: M triethanolamine (tea)-HCl pH 6.8; 0.005 M MgCl2; 0.025 M KCl; 0.003 M CaCl2; 0.005 M NaCl, pH 6.8; was shaken for 30 min on microsecure (Micro-shaker type 326 m, Poland), followed by centrifugation at 4000 rpm (K-23, East Germany) for 20 min to remove cell membrane, after which the precipitate washed twice in the medium of the following composition: 0.005 M MgCl2; 0.025 M KCl; 0.003 M CaCl2; 0.005 M NaCl; 0.01 M Tris-HCl pH 6.8, followed by centrifugation under the above conditions.

Bakteriostaticheskie proteins were extracted to 0.14 M NaCl, 0.01 M Tris-HCl pH 6.8 buffer. Faction reprocessing with the cellular residue was isolated by extraction of sediment 0.35 M NaCl, 0.01 M Tris-HCl pH 6.8 buffer. Next, the precipitate was fractionally suspendirovanie in Tris-HCl buffer 2 is NaCl, getting faction, strongly bound with cellular balance. In the sediment remained a fraction containing the cell balance with the cell membrane. Subsequent extraction was performed 6 M guanidine hydrochloride with 0.1% β-mercaptoethanol in Tris-HCl buffer. In the above buffer precipitate was completely dissolved. Cell fractions were stored at - 196°C in nitrogen.

The amount of protein was determined by the binding protein with Kumasi bright blue G (Loba, Austria) [4]. The method was used in the case micronanoelectronice of protein determination. The obtained fraction was passed through a column amberlite IDC-50 (polymethacrylate synthetic resin with free carboxyl groups). The resin was used in the form of a powder obtained by grinding in a ball mill. Pulverized resin sifted through a sieve of 200 mesh, and then repeatedly rinsed with water to remove the smallest particles were dried and rinsed with acetone. To achieve higher chromatographic efficiency were cyclization. To 100 g of resin was added 500 ml of 4 N. NaOH was stirred for 3 h followed by laundering on the filter with water until neutral and translated in an acidic form, passing through it a 500 ml 4 M HCl, the excess HCl was removed by washing with water. This resin-filled column size of 0.4 x 4.5, see column inflicted protein in the amount of 20-50 mcg, restorany is 6% of the guanidine hydrochloride in 0.1 M potassium phosphate buffer pH 6.8. The rate of elution was 6 ml/h Preparative separation of basic proteins in the proteome of cells of E. coli were carried out in discontinuous gradient guanidine hydrochloride: 6%, 8,9%, 10,6%, 13% 0.1 M potassium-phosphate buffer pH 6.8. The content of protein in the eluates was determined by the method of Bradford in our modification [4]. We used domestic product guanidine hydrochloride ("Rahim"), which was previously cleared. Supercriticality drug has a high absorption in the ultraviolet. Recrystallization was carried out according to the method described Onions [1]. The concentration of guanidine-hydrochloride was determined refractometrically at room temperature. Calculation of concentrations was carried out on the basis of the refraction index [5], using the following relationship:

where n25Gu HCl is the refractive index guanidine hydrochloride (amount dependent on the concentration of the drug); n250.1 M sodium phosphate buffer is the refractive index of this buffer (constant for a given concentration); figure 25 indicates the temperature at which was held refractometric studies.

The above-described method was able to separate basic proteins from "sour" obtained as described above, supramolecular structures (BP, HC-I HC-II KO's) at 4 faction: 0-fraction (nesoderzhaschii "sour" proteins) is 3 faction, obtained by elution of 8.9%, 10.6% and 13% concentrations of guanidine hydrochloride (polietilenoksidnye main gestonorone proteins).

Figure 1 shows the growth curve of the strain E. coli JC-158. On the ordinate axis shows the optical density of the periodic culture. On the x-axis shows the age of periodical culture in min, measured over 7 hours and 10 minutes in a growing culture of cells on the background of the growth phases of the growing population of E.coli.

Figure 2 shows the chromatographic elution profile of proteins on a column with IDC-50 using a discontinuous gradient of guanidine hydrochloride, buervenich of supramolecular structures growing populations of E. coli. On the axis of ordinates indicate the protein content in the sample, on the x-axis is the number of samples. The following notation is used: BP - bacteriology, HC-I - supramolecular structure, narodnozabavni with cell balance (KO), HC-II - supramolecular structure, tightly bound with a cluck, CLUCK - cell balance. The figure shows that the fraction gestonorone proteins are highlighted when the following concentrations of guanidine hydrochloride: 8,9%, 10,6%, 13%, 0.1 M potassium-phosphate buffer pH 6.8; peak obtained by elution of 6% guanidinopropionic is nesoderzhaschii ("sour") supramolecular protein structures.

Figure 3 shows the dynamics of the content of total protein in step-gradient fractions guanidine hydrochloride, e is of new supramolecular structures growing populations of E. coli. On the x-axis shows the growth of a population of E. coli with the designation of phases of growth, minutes On the y - axis the protein content of 1 cell, vermiform. The following notation is used: 1 - the dynamics of content "sour" protein lirovannomu 6% guanidine hydrochloride of supramolecular structures (nesoderzhaschii on a column with IDC-50); 2 - dynamics of total protein, lirovannomu 8,9%, 10.6% and 13% guanidine hydrochloride (sum of delayed positively charged basic proteins on the column with IDC-50). The analysis shows relatively the same dynamics of the content of basic and acidic proteins".

Figure 4 shows the protein content of 6% fractions of guanidine hydrochloride, buervenich of supramolecular structures growing populations of E. coli. On the x-axis shows the growth of a population of E. coli with the designation of phases of growth. On the y - axis the protein content of 1 cell, vermiform. The following notation is used: BP - bacteriology, HC-I - supramolecular structure, narodnozabavni with cell balance (KO), HC-II - supramolecular structure, tightly bound with a cluck, CLUCK - cell balance. The figure shows the dynamics of the content of "sour" unbound with amberlite IDC-50 proteins.

Figure 5 shows the protein content of 8.9% fraction of guanidine hydrochloride, lirovannomu of supramolecular structures growing populations of E. coli. who and the x-axis shows the growth of a population of E. coli with the designation of phases of growth. On the y - axis the protein content of 1 cell, vermiform. The following notation is used: BP - bacteriology, HC-I - supramolecular structure, narodnozabavni with cell balance (KO), HC-II - supramolecular structure, tightly bound with a cluck, CLUCK - cell balance. The figure shows the dynamics of the content of the main positively charged proteins, contacting amberlite IDC-50, buervenich solution of 8.9% of guanidine hydrochloride.

Figure 6 shows the protein content of 10.6% fraction of guanidine hydrochloride, lirovannomu of supramolecular structures growing populations of E. coli. On the x-axis shows the growth of a population of E. coli with the designation of phases of growth. On the y - axis the protein content of 1 cell, vermiform. The following notation is used: BP - bacteriology, HC-I - supramolecular structure, narodnozabavni with cell balance (KO), HC-II - supramolecular structure, tightly bound with a cluck, CLUCK - cell balance. The figure shows the dynamics of the content of the main positively charged proteins, contacting amberlite IDC-50 and buervenich step-gradient of 10.6% guanidine hydrochloride.

Figure 7 presents the protein content of 13% fraction of guanidine hydrochloride, lirovannomu of supramolecular structures growing populations of E. coli. On the x-axis shows the growth of a population of E. coli with the hereafter is rising phases of growth. On the y - axis the protein content of 1 cell, vermiform. The following notation is used: BP - bacteriology, HC-I - supramolecular structure, narodnozabavni with cell balance (KO), HC-II - supramolecular structure, tightly bound with a cluck, CLUCK - cell balance. The figure shows the dynamics of the content of the main positively charged contacting amberlite IDC-50 proteins, buervenich supinate-gradient 13% guanidine hydrochloride.

Analysis Fig.5-7 shows the frequency of occurrence of major gestonorone proteins extracted from supramolecular structures growing population of E.coli on the background of the phases of the periodic growth of the culture. At work, adopted for the prototype [2], using this method it was possible to separate basic proteins from the "sour" of proteins in the fractions obtained with 2 M NaCl in cell nucleus of eukaryotes. This method allowed us to obtain fractions enriched in histone H1, histone H2A and H2B as well as, histones H3 and H4. We believe that the approach will make it possible to identify gestonorone proteins in prokaryotic cells, to determine their role in cell metabolism, involved in the styling and structuring nucleoid in the growth of the bacterial population and to determine the amino acid composition of proteins obtained. Knowledge of the biochemical processes that occur at different phases of growth of a population of bacteria, allows the t to reveal the regulatory mechanisms of the impact of macro - and microorganisms, and promotes the search for new targets for drugs that allows controlling the bacterial communities inhabiting the human body.

Sources of information

1. Luck J.M., Rasmussen P.S., K. Satake, Tsvetikov A.N. Further studies on the fractionation of calf thymus histone. // The J. of Biological Chemistry. 1958, V.233, N 6, P.1407-1414.

2. Ivanova E.A. Fractionation plant histones on columns with amberlite IDC-50. // Proceedings of the third scientific conference of young scientists. Ufa: Bashkir branch of the USSR Academy of Sciences, 1972, P.54-55.

3. Myrphy D.B., Pembroke J.T. Transfer of the IncJ plasmid R391 to recombination deficient E. coli K12; evidence that R391 behaves as a conjugal transposon. // FEMS Microbiology Letters, 1995, V.134, P.153-158.

4. Ivanova E.A., vagina GH Way to obtain nuclear fractions with proteinases and inhibitory activity. Copyright certificate 1733471 // B. I. 1992. Vol.18, Pp.96.

5. Bonner J., Chalkley G.R., Dahmus, M., D. Fambrough, F. Fujimura, R.C. Huang, J. Huberman, R. Jensen, Marushige, K., Ohlenbusch H., Olivera Century, Widholm J. Isolation and characterization of chromosomal nucleoproteins. // Methods Enzymology. 1968, Acad. Press. New York. V.XII, part B, sec. V, ch. VII, P.25-31.

The preparative method of separating the main proteins of the supramolecular structures of the growing population of Escherichia coli, including the preservation of cells in buffered 80-90% glycerol at minus 25°C, followed by removal of the cellular membrane with 3% Triton X-100 extraction increasing salt concentrations: 0,14 M, 0.35 M; 2 M NaCl, 6 M guanidine hydrochloride with 0.1% β-mercaptoethanol and separation is observed from the above fractions of basic proteins using ion-exchange chromatography with amberlite IDC-50 in discontinuous gradient guanidine hydrochloride: 6%, of 8.9%, or 10.6%, 13% at 0.1 M potassium-phosphate buffer pH 6.8.



 

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