ES2367478T3 - PROCEDURE FOR THE NON-SPECIFIC ENRICHMENT OF BACTERIAL CELLS. - Google Patents

Abstract

Method for the unspecific purification of bacteria comprising the following steps: a) Contact a sample containing bacterial cells with a cationic polymer at an acidic pH value, forming a network of bacteria and polymers b) Add magnetic particles, depositing the particles magnetic particles in the network of bacteria and polymers c) Separate the bacteria from the sample using the magnetic particles deposited in the network.

Description

The present invention relates to a process for the nonspecific enrichment of bacterial cells by cationic or anionic polymers and magnetic supports.

The starting point of almost any processing, analysis or isolation of cellular components is cell enrichment, the "cell harvest." This is usually done by centrifugation. This centrifugation stage raises the main problem in the complete automation of the procedure such as the purification of plasmids since, in addition to the high technical expense for the integration of a centrifuge in a corresponding processing robot, extremely high precision in the position of Start and stop the centrifugation process. Therefore, the automatic procedures for the processing, analysis or isolation of cellular components generally begin with enriched cells, separated by centrifugation or sedimented already outside the processing robot. However, for example, for a rapid analysis of complete genomes, proteomes, but also for the rapid resolution of structures and functions in high performance procedures, as complete as possible automation of the relevant procedures in this regard is essential. In this respect, the automation of partial stages, for example, in genome analysis, is already very advanced: both bacterial culture and plasmid isolation can be performed automatically. However, to date a simple, cost-effective and complete automation of the procedure, including cell harvesting, has not been possible.

The conventional procedure of cell harvesting is the centrifugal separation of bacterial cultures. This requires a microtiter plate centrifuge, especially in procedures that will be designed for better performance.

An alternative procedure makes use of enrichment of cultured cells by filtration membranes, see, for example, WO01 / 51206, 3M Innovative Properties Co (US) and US5,464,541, Diagen Inst. F. Molekularbiologie; However, in highly enriched cell suspensions and the high viscosity of the solutions associated with them, the procedure is very prone to fail in those related to obstructions. The same applies to ion-exchanger-like binding to a porous matrix, combined with filtration or vacuum techniques (WO92 / 07863, Qiagen). This can present major problems, especially for automatic procedures.

An enrichment of microorganisms by procedures based on magnetic particles is in principle suitable for automation. To date, three enrichment procedures for bacteria have been described, which nevertheless have clear disadvantages with respect to strain specificity and preparation costs.

EP 1 113676 A2, Chemagen AG, and WO 01/53525 A2, Genpoint AS, describe a method for the enrichment of microorganisms on a solid phase by a non-specific ligand that is covalently or non-covalently immobilized on this solid phase . Solid phases within the meaning of this procedure are polyvinyl alcohol beads that can be magnetic or non-magnetic. As ligands, molecules that can serve as nutrients for microorganisms are considered as ligands. The disadvantage of this procedure is that certain ligands must be immobilized on a support, which leads to a clear rise in preparation costs. Another disadvantage is that for different strains, different ligands must eventually be immobilized, therefore the magnetic beads and the process cannot be used universally.

In another application, specifically in WO98 / 51696, Genpoint, mixtures of salts, alcohols and polymers comparable to polyethylene glycol or polyethylene glycol are described for the enrichment of bacteria on magnetic particles. The procedure described in WO98 / 51693 requires 5-20 minutes and polymer additions of 2-50% (weight / volume). These high concentrations of salts or polymers present problems in the processing of bacteria since, for example, 30% polyethylene glycol is very pasty, or high concentrations of salts interfere with the SDS-polyacrylamide gel electrophoresis.

WO00 / 29562, Merck Patent GmbH, describes a method for isolating plasmid DNA from microorganisms by a) acidification of the microorganism culture, incubation and mixing with the solid phase materials (silica magnetic particles) and b) subsequent plasmid purification . However, this procedure cannot be performed for all strains of E. coli without partly drastic yield losses. Another disadvantage of this procedure is the amount of pearls needed, since the pearl costs constitute a key aspect of the bacteria enrichment costs and especially the silica beads are relatively expensive. Since silica beads can also be linked to DNA in principle, performance losses can also be observed in the isolation of plasmids with silica beads.

WO91 / 12079, Amersham, describes a procedure for the precipitation of cells on magnetic beads that bind unspecifically to polymers (cells). In this procedure, the order of addition of the necessary agents (alcohol and salt) and of the magnetic beads must be maintained. It is not possible to pre-mix the agents and / or agents and beads and, therefore, pipetting steps cannot be saved, which may be necessary in an automation for the optimal use of a robot. In addition, the magnetic particles that can be used are limited to cellulose / iron oxide particles.

US 6,284,470 B1, Promega, describes the formation of intact cell complexes with magnetic beads. The process is limited to silica particles in regard to the magnetic particles that can be used. But as this procedure prescribes that a volume of alcohol should be added to a volume of cell suspension, this procedure can hardly be applied without clear yield losses for the harvest of cells in deep well plates. In addition, high concentrations of alcohol can lead to a precipitation of proteins.

A series of studies have shown that cationic polymers can interact with bacteria. Experiments have resulted, for example, that cationic polymers such as, for example, chitosan (chitosan is deacetylated chitin) are very suitable for encapsulating or "sealing" live microbial cells for individual experiments (see, for example, Methods Enzymol. 1987, vol. 135, 259-268, or US5,489,401). Chitosan is also used for encapsulation of active biomaterials in pearl polymers (US 4,647,536 and WO01 / 46266). Chitosan is also used, if necessary also in modified form, as a coating of articles or test kits for immunoassays (US5,208,166).

However, publications concerning the influence of cationic polymers on hydrophobia of microbial cell walls had shown that although adhesion on hydrophobic surfaces at pH values above 3 clearly increased, at pH values below 3 decreased to almost 0% (Ref .: Goldberg, S., Doile,

R. J. and Rosenberg, M., 1990, J. Bacteriol., Vol. 172, 5650-5654).

In addition, the use of a procedure supported on magnetic particles still offers more advantages, since a separate crop of individual wells is made possible, for example, of a plate of deep wells and the entire plate must not be completely harvested. Therefore, for example, in the context of expression studies, they can be harvested as a function of time.

In summary, it can be seen that a simple procedure is not available for the efficient, rapid and cost-effective enrichment of bacterial cells that dispenses with a centrifugation stage and at the same time can be used for a broad spectrum of bacteria. Therefore, the objective of the invention is to provide a method for the nonspecific enrichment of bacterial cells that dispenses with the centrifugation step.

The objective is achieved by the object defined in the claims.

The invention is explained in more detail by the following figures:

Figure 1 graphically shows the crop yield performance of LE392 cells from E. coli (light bars) and TG2 (dark bars) using different magnetic particles. The values specify the% yield determined from the cloud ratio of the supernatant and the optical density of the cell suspension used. The abbreviations mean: Silica: MagPrep silica beads (MERCK company, Darmstadt), NH2: amino-polystyrene beads (Estapor company, MERCK-Eurolab); COOH: carboxy-polystyrene beads (Estapor company, MERCK-Eurolab); PS: polystyrene beads (Spherotech, Illinois, USA); FVA: polyvinyl alcohol beads (Chemagen, Baesweiler)

Figure 2 shows the result of the crop yield of cells at an acid pH value with silica beads (dark bars) and chitosan / amino polystyrene beads (light bars). Figure 2 compares the crop yield of cells according to the invention (light bars) with the procedure of WO00 / 29562 (dark bars). The cellular yields of the process according to the invention are set in this respect to 100%, the yields according to WO00 / 29562 were determined in relation thereto. All values are average values of several experiments with standard deviations. The harvesting of cells according to the method according to the invention was carried out as in Application Example 5. Document WO00 / 29562 / MERCK followed the manufacturer's protocol.

Figure 3 graphically depicts the result of the crop yield of cells for E. coli strains DH5a and HB101 using chitosan which is free in solution (clear bars) and chitosan immobilized on beads (chitosan pearls, Chemicell company, Berlin ) (dark bars). The values specify the yield in% determined from the ratio of the optical density of the supernatant and the cell suspension used.

Figure 4 shows in a graphical representation the result of the yield efficiency of the cell harvest (performed as in Application Example 5) by means of a selection of 15 cloning strains of E. coli. The yield in% (average values of several experiments, n = 27) determined from the ratio of the optical density of the supernatant and the cell suspension used is represented.

Figure 5 shows the result of the harvest of different types of bacteria. Figure 5 shows that the cell harvesting system according to the invention can be applied to a broad spectrum of bacteria of various groups (both gram-negative and gram-positive). The yield of the cell crop is represented respectively. Cells were harvested as in Application Example 7. E. faecalis: Enterococcus faecalis, B. vallismortis: Bacillus vallismortis, P. mirabilis: Proteus mirabilis, M. luteus: Micrococcus luteus, S. haemolyticus: Staphylococcus haemolyticus, C. freundii : Citrobacter freundii.

Figure 6 graphically shows the result of the crop yield of cells for E. coli strain JM83 using polylysine in 0.5 M HCl or 0.5 M HCl without polylysine. The values specify the yield in% determined from the ratio of the optical density of the supernatant and the cell suspension used. PL / HCl: polylysine in 0.5 M HCl; HCl: only 0.5 M HCl (control without polylysine)

Figure 7 shows the formation of a network of E. coli cells, chitosan and magnetic beads. The formation of this network is caused by the addition of free chitosan dissolved in 0.5 M formic acid to a suspension of E. coli (strain HB101) (C and D). In the control lot without chitosan (only 0.5 M formic acid) no network is formed (A and B), bacteria are present as before as individual cells. A and C: Light-field photographs, in A individual cells and magnetic pigments can be observed, in C aggregates can be observed. B and D: fluorescence photographs after staining of E. coli cells with propidium iodide / Syto9 (BacLightTM Viability Kit, Molecular Probes, Eugene, Oregon, USA). In B individual fluorescent cells can be observed, D shows the fluorescence of the cells incorporated in the reticular structure.

Figure 8 graphically shows the enzymatic activity of E. coli gala-galactosidase itself after cell harvesting according to the present invention or after harvesting by centrifugation. Values for enzyme activity in relative units are plotted against the proportion used in the 300 µl test of harvested cells (in%). Open diamond symbols reproduce the values for centrifuged cells. The triangles and squares show the values for bacteria that were harvested during the process according to the invention. Triangles: the cells were incubated after the addition of 30 µl of 125 µg / ml chitosan in 0.5 M formic acid for 3 minutes and separated for another 3 minutes by magnetic separation. Squares: The bacteria were incubated for 5 minutes after the solution was added and separated for another 5 minutes in the magnetic field.

Figure 9 shows the result of the enrichment of the N-Strep-T4-p12 protein by Strep-Tactin beads (IBA GmbH, Göttingen) after harvesting the E. coli strain that is expressed heterologously BL21 (DE3) by the process according to the invention. A: non-induced cells. B: induced cells; 1: crude lysate, 2: Strep-Tactin beads after N-Strep-T4-p12 binding; 3: N-Strep-T4-p12 protein eluted in native trimer form;

4: N-Strep-T4-p12 protein eluted in monomeric form (after heating at 95 ° C); 5: Strep-Tactin pearls after elution; 6: standard (purified N-Strep-T4-p12 protein)

Figure 10 shows the result of E. coli XL1Blue plasmid DNA isolation harvested according to the method according to the invention (as specified below in Application Example 4). 1: Harvest of the bacteria by the method according to the invention; 2: Harvest of bacteria by centrifugation; a: plasmid pUC19 digested with PstI; b: undigested plasmid; M: DNA standard (lambda / EcoRI / HindIII DNA).

The term used herein "purification" or "enrichment" means the separation of bacterial cells or cellular components from the aqueous solution, for example, from the culture medium in which the bacterial cells or cellular components are found. Purification or enrichment is carried out in this respect with the help of magnetic particles.

The term used here "non-specific enrichment" means that the enrichment is carried out under the conditions chosen regardless of the type, strain, gender, etc., of the bacteria.

A procedure for the non-specific purification of bacterial cells is described, comprising the following steps:

a) Contact a sample containing bacterial cells with cationic polymers at an acidic pH value or anionic polymers at a basic pH value,

b) Add a magnetic support,

c) Separate the magnetic support from the sample with the bacterial cells attached to it.

An aspect of the present invention is to provide a method for the unspecific purification of bacterial cells comprising the following steps:

a) Contact a sample containing bacterial cells with cationic polymers at an acidic pH value, forming a network of bacteria and polymers,

b) Add magnetic particles, depositing magnetic particles in the network of bacteria and polymers,

c) Separate the bacteria from the sample using the magnetic particles deposited in the network.

In particular, the procedure is characterized in more detail by the following stages:

The culture of bacteria is mixed with cationic or anionic polymers, especially with unbranched cationic polymers, preferably with chitosan (e.g., FLUKA article No. 22741, 22742 or 22743, Sigma C-3646 or Roth 5375.1) or polylysine (for example, Sigma P2636). Preferred anionic polymers are dextran sulfate, chondroitin sulfate, polyglutamate, polymalate, polygalacturonic acid, polyphosphates and sulfated oligosaccharides.

The bacteria / polymer culture mixture must have a pH value corresponding to the polymers used together with the corresponding buffers. The pH value is in the case of cationic polymers preferably less than or equal to 3 or 4, in the case of anionic polymers preferably greater than or equal to 8. The pH value can be brought to the desired pH value or before the addition. of the polymers by adjusting the culture of bacteria by adding buffer solutions or directly from acid or base. The pH value can also be adjusted so that the polymers are dissolved in a corresponding buffer solution or directly in acid or base. The pH value can also be adjusted so that after the addition of the polymers they are brought to the desired pH value by adjusting the bacteria / polymer culture mixture by adding buffer solutions or directly from acid or base.

The cationic polymers can be dissolved in acidic buffer or directly in acidic. The solution is adjusted so that the culture mixture of cationic bacteria and polymers gives the desired pH value for the process. For the process according to the invention, an acid pH value of the bacteria / polymer culture mixture is preferred. The pH value can be adjusted with buffers or discretionary acids, preferably with 0.5 M HCl / 25% acetic acid, 0.5 M HCl or 0.5 M formic acid. The buffer used depends especially on the type of processing. of the bacteria or the subsequent procedure. If the process according to the invention is used for the purification of plasmids, HCl / HAc at a pH value in the range of about 2 to about 3 is preferred as a buffer, since at a pH value less than 1.8 it is hydrolyzed the DNA. If the process according to the invention is used for protein purification, formic acid at a pH value in the range of about 4 is preferred as a buffer, since for the isolation of functional proteins the pH value will be in the range of about 4 to about 9.

Cationic polymers are present in a concentration range in which sufficient purification of the bacteria is guaranteed. The polylysine is used for precipitation with a concentration in the range of 12.5 µg / ml of culture at 150 µg / ml of culture, preferably with a concentration in the range of 25 µg / ml to 150 µg / ml, especially with a concentration in the range of 40 µg / ml to 75 µg / ml, especially with a concentration of 50 µg / ml. In addition, the optimum concentration also depends in this respect, together with the type of bacteria or the strain of bacteria, on the source of supply or on the molecular weight of the polylysine used.

Chitosan is used for precipitation with a concentration in the range of 0.05 µg / ml of culture to 100 µg / ml of culture, preferably with a concentration in the range of 0.25 µg / ml to 50 µg / ml, especially with a concentration in the range of 0.5 µg / ml to 50 µg / ml, in addition especially with a concentration in the range of 1 µg / ml to 50 µg / ml, especially with a concentration of 2.5- 50 µg / ml. Other preferred concentrations that may depend on the buffer used result from the following table. In addition, the optimum concentration also depends in this respect, together with the type of bacteria or the strain of bacteria, on the source of supply or the molecular weight of the chitosan used and the degree of deacetylation of the chitosan. An overview of the preferred chitosan concentrations is found in the following table.

Chitosan concentrations

Chitosan LMW (FLUKA, article number 22741) Chitosan MMW (FLUKA, article number 22742) Chitosan HMW (FLUKA, article number 22743)

HCl / HAc Concentration range for precipitation

0.05 µg / ml to 1 µg / ml 0.05 µg / ml to 5 µg / ml 1 µg / ml to 20 µg / ml

Optimal range

0.5 µg / ml to 5 µg / ml 0.5 µg / ml to 5 µg / ml 1 µg / ml to 5 µg / ml

Optimum

2.5 µg / ml 2.5 µg / ml

Formic acid

Concentration range for precipitation

0.5 µg / ml to 50 µg / ml 0.5 µg / ml to 50 µg / ml 1 µg / ml to 100 µg / ml

Optimal range

0.5 µg / ml to 50 µg / ml 0.5 µg / ml to 50 µg / ml 1 µg / ml to 50 µg / ml

Optimum

12.5 µg / ml to 50 µg / ml 12.5 µg / ml to 50 µg / ml 12.5 µg / ml to 50 µg / ml

5

10

fifteen

twenty

25

30

After the addition of the polymers, the bacteria / polymer culture solution will preferably be mixed by pipetting up and down or stirring. By mixing, all the components involved are quickly and immediately contacted.

The acidified or basic polymer / bacteria mixture can be processed immediately. However, the mixture can also be incubated for a short period of time, preferably 3-10 minutes, especially preferably for 5 minutes. The incubation temperature may rise from room temperature to 40 ° C, 37 ° C is preferred, especially preferably room temperature.

The acidified or basic polymer / bacteria mixture is mixed with a magnetic support. Preferably, the magnetic supports are magnetic particles (hereinafter also called magnetic pearl), for example, polystyrene beads or magnetic pigments. Within the process according to the invention, superficially functionalized polystyrene beads can be used, especially with NH2, COOH or OH surfaces, with the NH2 surface being preferred. As functionalized particles, pearls that can be obtained commercially are used, for example, amino beads (EM2 100/40, diameter 1.43 µm) or carboxy beads (EM1 100/40, (23710) diameter 1.3 µm) of the company Estapor. As magnetic pigments can be used pearls that can be obtained commercially, for example, Bayferrox 318M, Bayoxid E 8706 or Bayoxid E8713H (Bayer AG). The preferred diameter of the beads is in the range of 0.5 µm to 2 µm, a diameter in the range of 1 µm to 5 µm is preferred, especially preferably about 1 µm.

The process according to the invention contains the formation of a network of bacteria and polymers in which magnetic particles of any type are deposited, for example, polystyrene beads or pigments. By this, bacteria can be separated by particles found in the network by the application of a magnetic field.

The process according to the invention is not limited in this respect to a certain type of superficially functionalized magnetic particles, but any type of magnetic particle, that is, for example, pearl or pigment, of a different surface can be used. An overview of common types of magnets with different surfaces is summarized in Tables A and B. If magnetic pigments are used in the process according to the invention, then they can be dissolved in both alcohols and aqueous buffers.

Table A represents in table form all magnetic particles so far tested and which are functional in the process according to the invention. Table A: magnetic pigments, sorted according to manufacturers. Table B: magnetic beads of different base matrix with different functionalized groups, ordered according to manufacturers. PS: polystyrene; Silica: silica matrix; PVA: polyvinyl alcohol.

TO

BASF

HQ carbonyl iron powder

Pigment 345

Bayer

Bayferrox 318M

Bayoxide E8706

Bayoxide E8713 H

Bayoxide E8710 H

Bayoxide E8707 H

Bayoxide E8706 H

EPCOS

N27 Ferrite Powder

MERCK

Iriodin 600

OMIKRON

Iron whisk powder, black

Iron oxide black, strongly colored

Vogt

Fi324 Ferrite Powder

B

is standing

NH2  $

COOH

$

hydrophobe

$

OH

$

Spherotech

NH2  $

COOH

$

hydrophobe

$

Polysciences

BioMagNH2  $

BioMagCOOH

$

PS, paramagnetic

$

NH2, paramagnetic

$

MERCK

Silica MagPrep Silica

Micromod

Sicastar M-NH2  Silica

Sicastar M-plain

Silica

Nanomag C, plain

Ferrofluid

Chemagen

M-PVA 011, OH PVA

B

M-PVA 012, OH

PVA

M-PVA 013, OH

PVA

5

10

fifteen

twenty

25

30

35

40

Four. Five

Magnetic particles are present in a concentration range in which sufficient purification of the bacteria is guaranteed; however, the costs of purification are within a justifiable framework. Preferably polystyrene beads are added with a solution of 1% polystyrene beads at a concentration of approximately 1/10 to 1/70 of the bacterial culture volume. A concentration of about 1/20 to 1/50, especially 1/20 or 1/50, is especially preferred. The concentration in this respect depends on the size of the bacterial culture volume. If the volume of the culture of bacteria amounts, for example, to 0.2 or 1 or 1.5 ml, then a concentration of the 1% polystyrene beads solution of 1/20 is preferred, to a volume of the culture of bacteria of, for example, 10 ml a concentration of the 1% polystyrene beads solution of 1/50 is preferred. The higher the volume of the bacteria culture, the lower the concentration of polystyrene beads. Magnetic pigments are preferably used as a 5% solution with a 1/10 volume concentration of the bacteria culture.

After the addition of the magnetic particles, the bacteria / polymer / magnetic particles culture solution will preferably be mixed by pipetting up and down, stirring or by the magnet. By mixing, all the components involved are quickly and immediately contacted.

The mixture can be processed immediately. However, the mixture can also be incubated for a short period of time, preferably 3-10 minutes, especially preferably for 5 minutes. The incubation temperature may rise from room temperature to 40 ° C, 37 ° C is preferred, especially preferably room temperature.

Next, the bacteria attached to the magnetic support are separated from the sample. A The mixture of bacteria / polymer / magnetic particles is brought to a magnetic field and the complex precipitates.

The magnetic particles bound to the bacteria by the preceding process step can be separated from the bacteria depending on the type of the next processing stage, for example, plasmid preparation, RNA or genomic DNA preparation, for example, by shaking and discarding or be used later in the process steps together with the bacteria. If the bacteria have to be separated from the magnetic particles, the bacteria / polymer / magnetic particles can be resuspended again in the corresponding buffer after separation of the sample and removed and, for example, transferred to a new reaction vessel therewith. retention time of the magnetic particles by means of the magnet. A Tris buffer (200 mM, pH 8-9.5) is preferably used to dissolve the bacteria in the bacteria / polymer / magnetic network. For the solution, a stirring stage is preferably carried out at high revolutions at, for example, 1200 rpm (Eppendorf stirrer).

The process according to the invention can be carried out in all the usual reaction vessels, preferably in Eppendorf reaction vessels of any type or microtiter plates.

Bacteria enriched with the process according to the invention can be all types of bacteria, preferably eubacteria and archaebacteria, especially E. coli, especially BL21, JM109, E. coli B, DH1, LE392, E. coli K12, DH5α, NM522, E. coli C, DH10b, Sure, GM2163, TG2, HB101, Top10, InvaF ', XL1Blue, JM83, but also Micrococcus spec. especially Micrococcus luteus, Proteus spec. especially Proteus mirabilis, Enterococcus spec. especially Enterococcus faecalis, Citrobacter spec. especially Citrobacter freundii, Bacillus spec. especially Bacillus vallismortis, Staphylococcus spec. especially Staphylococcus haemolyticus.

The process according to the invention can be used as a substitute for centrifugation, and thus automatic purification of bacterial cells is made possible. With the described procedure, bacteria from a culture can be nonspecifically enriched in the course of approximately 5-10 minutes. Since the concentration of the cationic polymers used with clearly less than 1% is very low, enrichments can be carried out essentially more profitably than in the process according to the state of the art. In addition, essentially lower amounts of beads of 1-15 beads per bacterium are used in the process according to the invention instead of 30-50 beads per bacterium, for example, in WO00 / 29562.

The following examples serve only to explain and in no way limit the scope of the invention.

one.

 Preparation of chitosan solution

Chitosan was weighed, resuspended in 0.5 N HCl / 25% HAc and the solution was heated until (approximately 8090 ° C) that the chitosan dissolved completely. The solution was then diluted and adjusted to 25 µg / ml in 0.5 N HCl / 25% Hac (= "working solution" H working solution) or to 125 µg / ml in 0.5 N formic acid (= working solution of "working solution" A). The working solution (25 µg / ml) remained stable in solution at room temperature and was stored.

2.

 Preparation of the polylysine solution

Chitosan was weighed, resuspended in 0.5 N HCl, adjusted to 0.5 mg / ml in 0.5 N HCl (= "working solution" working solution). The working solution was stored at -20 ° C until use.

3.

 Harvest of bacteria at the 0.2 ml scale of culture: Network formation

The culture of HB101 of E. coli was carried out overnight in a 0.5-well 96 ml polypropylene microtiter plate with 0.2 ml of culture per well. For the culture, 20 µl of working solution (“working solution”) of chitosan A or 20 µl of 0.5 M formic acid without chitosan were added to the control lot and 20 µl of a 5% solution of the Bayoxid magnetic pigment E8706 in PBS and mixed by pipetting up and down. After incubation at room temperature for 5 minutes, the samples were examined under a microscope to check the formation of a network (Figure 7).

Four.

 Harvest of bacteria at the 1.5 ml scale of culture: DNA isolation

The XL1Blue culture of E. coli transformed with plasmid pUC19 was carried out in a 96-well deep well plate overnight with each 1.25 ml of culture volume per well in LB medium with 100 µg / ml ampicillin . Then, 125 µl of working solution ("working solution") of chitosan H and 125 µl of a 5% pigment pearl solution (Bayferrox 318M in isopropanol) were added to the culture overnight and mixed well by pipetting up and below. After an incubation at room temperature for 5 minutes, the bacteria / pearl complexes were separated in a magnetic separator (Bilatec AG, Mannheim). The supernatant was completely removed and plasmid DNA was isolated from the cell pellet by the Wizard MagneSil ™ plasmid purification system from the DNA isolation kit (Promega, Madison, USA) according to the manufacturer's protocol (Fig. 10)

5.

 Harvest of bacteria at the 10 ml scale of culture

The JM83 culture of E. coli was carried out in a polypropylene tube overnight with 10 ml of culture volume. After overnight cultivation, 1 ml (1/10 volume) of working solution of chitosan H was added and mixed by pipetting up and down twice. Then 0.2 ml of a solution of 1% amino-polystyrene beads was added. Then, the solution was mixed by pipetting up and down (2 times). An incubation followed at room temperature for 5 minutes. The tube was inserted 10 minutes into the magnetic field of a permanent magnet (Bilatec AG, Mannheim) for sedimentation of bacteria / pearl complexes. The supernatant was discarded and the bacterial cells were processed directly.

6.

 Cultivation of E. coli crops

E. coli cultures were grown in standard media such as LB, 2xYT, SOB or SOC (Maniatis, 1987) overnight. To each 200 µl of the cultures overnight, 20 µl of working solution ("working solution") of chitosan H was added to each and to each 20 µl of amino-polystyrene beads and mixed well. After 5 minutes of incubation, the bacteria / pearl complexes were separated in a magnetic separator. In the use of the mentioned means, it may be necessary, if necessary, to adapt the concentrations of the work solution ("working solution") and beads.

7.

 Harvest of bacteria at the 0.2 ml scale of culture

The culture of 0.2 ml of each of the cultures of Enterococcus faecalis, Proteus mirabilis, Staphylococcus haemolyticus was carried out in a 96 microwell plate overnight at 37 ° C. The culture of 0.2 ml of each of the cultures of Bacillus vallismortis, Micrococcus luteus, Citrobacter freundii was carried out in a 96 microwell plate overnight at 30 ° C. To the overnight culture was added 20 µl (1/10 volume) of working solution ("working solution") of chitosan H. Then 10 µl of a solution of 1% aminopolystyrene beads were added. Then, the solution in the well was mixed by pipetting up and down (1 or 2 times). An incubation followed at room temperature for 5 minutes. The microtiter plate was incubated 5 minutes in a magnetic separator (Bilatec AG, Mannheim) for sedimentation of the bacteria / pearl complexes. The supernatant was discarded and the bacterial cells were processed directly.

8.

 Harvest of live bacteria

To 300 µl of each of an overnight culture of BL21 (DE3) of E. coli were added in a 0.5-well 0.5 microtiter plate of 0.5 ml 30 µl of working solution ("working solution") of chitosan A and 30 µl of a 5% pigment pearl solution (Bayferrox 318M in PBS) and mixed well by pipetting up and down. Then followed an incubation step at room temperature for 3 or 5 minutes. Next, the bacteria / pearl complexes were sedimented by incubation for 3 or 5 minutes in a suitable magnetic separator (Bilatee AG, Mannheim). The supernatant was discarded and the bacteria / pearl sediments were each resuspended in 200 µl of 200 mM Tris at pH 8.0. As a control, 300 µl of culture was centrifuged overnight and the sediment was also resuspended in 200 µl of 200 mM Tris at pH 8.0. The viability of the cells was checked by plating the dilution series on agar plates and the colonies were counted after overnight incubation. As another test for the activity and viability of the cells, the enzymatic activity of the propia-galactosidase of the cells was measured (Apte et al., 1995, Wat. Res. 29, 1803-1806) (Fig. 8).

9.

 Protein Isolation:

The Strep-Tag system was used to isolate proteins from bacteria harvested with the process according to the invention. For this, p12 (Selivanov, NA, et al., 1988 Nucleotide and deduced amino acid sequence of bacteriophage T4 gene 12, Nucleic acids Res.; 16 (5): 2334) was fused according to standard molecular biology procedures with a Strep- Tag (Strep brand) of the N end (US 5506121) and expressed as described by Burda, MR & Miller S. (Europ. J. Biochem, 1999, 265: 771-778). The culture of E. coli strain expressing NStrep-T4-p12 BL21 (DE3) pNS-T4-p12p57 was carried out in a 96-well deep plate with 1.25 ml of culture per well. At a DO600 cell density of approximately 0.5 it was induced with 1 mM IPTG for 3 h. Then, per well, 125 µl of working solution ("working solution") of chitosan A and 125 µl of a solution of 5% pigment beads (Bayoxid E8706 in PBS) were added, mixed well by pipetting up and down and incubated for 5 minutes at room temperature. Then, the bacteria / pearl complexes were pelleted by incubation of 5 minutes in a suitable magnetic separator (Bilatec AG, Mannheim). The supernatant was completely removed and the pellet was resuspended in 150 µl of buffer A (200 mM Tris at pH 8.0, 200 mM NaCl, 10 mM EDTA, 0.05% Tween 20). The bacteria were then removed from the bacterial / pearl network by means of a stirring step (5 minutes at 1200 rpm, shaker from the company Eppendorf). The beads were separated from the cells that were now in the supernatant by incubating several times in the magnetic separator and the supernatant was transferred to a new well of a 0.5 well 96 ml PP microtiter plate. The cells were then lysed by adding lysozyme (1.6 mg / ml), stirring for 2 minutes at 750 rpm and 15 minutes incubation at room temperature and the DNA was digested by adding 5 µg / ml DNase. The heterologously expressed N-Strep-T4-p12 was attached to the StrepTactin beads by adding 10 µl of 5% StrepTactin beads (IBA company, Göttingen) and 20 minute incubation under gentle agitation and enriched with the lysate. The N-Strep-T4-p12 was eluted from the beads by the addition of 5 mM biotin (Fig. 9).

Claims (3)

1.- Procedure for the non-specific purification of bacteria comprising the following steps: a) Contact a sample containing bacterial cells with a cationic polymer at an acidic pH value, forming a network of bacteria and polymers 5 b) Add particles magnetic, depositing the magnetic particles in the network of bacteria and polymers c) Separate the bacteria from the sample by the magnetic particles deposited in the network. 2. Method according to claim 1, wherein after step a) and / or stage b) an incubation stage is introduced. 3. Process according to one of the preceding claims, wherein the cationic polymer is chitosan or 10 polylysine 4. Method according to one of the preceding claims, wherein chitosan is added with a concentration in the range of 0.05 to 100 µg / ml of culture and polylysine with a concentration in the range of 12.5 to 150 µg / ml of culture. 5. Method according to one of the preceding claims, wherein the pH value is adjusted with HCl / HAc to 15 less than or equal to 3 or with formic acid less than or equal to 4. 6. Method according to one of the preceding claims, wherein the magnetic particles have a diameter of 0.5 to 5 µm. 7. Method according to one of the preceding claims, wherein the magnetic particles are a Magnetic pigment or a magnetic polystyrene pearl, especially with a surface of NH2, COOH or OH. Method according to one of the preceding claims, in which the purified bacteria are E. coli, Bacillus spec, Proteus spec, Micrococcus spec, Staphylococcus spec or Citrobacter spec. Silica