DD254029A1 - METHOD AND MEANS FOR DETERMINING THE AMINOGLUCOSID PHOSPHORTRANSFERASE ACTIVITY - Google Patents

Abstract

The invention relates to a method and means for determining the activity of aminoglucoside phosphotransferases. Areas of application are genetic engineering, biotechnology and medicine. Cell lysates or cell extracts are contacted with a cellulosic surface carrier in the presence of a mixture of ATP and 32P-g ATP. The phosphorylated kanamycin is visualized on the solid phase by autoradiography.

Description

Object of the invention

The object of the invention is to provide an improved method using an improved means for the qualitative and quantitative determination of the activity of aminoglucoside phosphotransferase.

Explanation of the essence of the invention

The object of the invention is to develop a method and suitable solid phases with bound antibiotics for the determination of the activity of aminoglucoside phosphotransferase. According to the invention the object is achieved in that the lysate of cells or the extract of cells with a cellulosic surface carrier following principal construction on:

HG-GH ₂ OI

wherein η is between 2500 and 15000, χ between η and 0.01 η and Kan is a Kanamycinrest, in the presence of a mixture of adenosine triphosphate (ATP) and ³² Py adenosine triphosphate in known media is brought into contact and the phosphorylated kanamycin the solid phase is visualized by autoradiography or scintillation counting.

A number of variants are possible for carrying out the method. In a first embodiment of the method according to the invention is based on cell lysates. A certain amount of the cell lysate is treated with a normalized area or mass of the loaded kanamycin solid phase in the presence of a fixed amount of ³² Py-ATP in a conventional buffer for this enzyme reactions for one hour at 37 ⁰ C incubated, then washed at Zimmtertemperatur and either autoradiographed or measured in a scintillation counter. This procedure can be carried out on plates with a large number of samples ^. In parallel approaches or kinetics of colony growth.

In a further embodiment of this method, drops of the lysate or cell extract are spotted onto a surface support according to the invention, e.g. B. in a transfer arrangement with a loaded with ³² Ρ-γ-ΑΤΡ filter paper in contact and incubated under similar conditions as above. Subsequently, as above, a large number of cell lysates or extracts can be evaluated simultaneously. In a third embodiment of this method is an electrophoresis gel with separated cell lysates or cell extracts

a) brought into contact with the surface support according to the invention under phosphotransferase reaction conditions, incubated with ³² Py-ATP and after washing steps the phosphorylated kanamycin determined by autoradiography, with a known surface support, for. As nitrocellulose, nylon membrane or activated cellulose, brought in the proteins under known conditions transferred (electrotransfer) and this loaded with the protein surface ^carrier with a surface ^{carrier according to the} invention in the presence of ^3z P - y-ATP brought into contact and then washed and evaluated by autoradiography, or

brought directly into contact with a surface support according to the invention in contact, transferred the proteins and the loaded, surface according to the invention then brought into contact with ³² Ρ-γ-ΑΤΡ, washed and then evaluated. In another variant of the method of the invention, bacterial colonies are grown on agar plates and lysed after a certain time. The carrier according to the invention is placed on these lysed bacterial colonies and reacted either directly or in a second experimental setup with ³² Ρ-γ-ΑΤΡ in a corresponding customary buffer. Then it is washed and evaluated. This variant can be carried out in a second form by first transferring or cultivating the bacterial colonies onto another surface support, eg nitrocellulose, nylon membrane or activated cellulose, and after lysis with the support according to the invention. B. under transfer conditions in the presence of ATP and ³² Ρ-γ-ΑΤΡ is reacted in a conventional buffer. It is then washed and evaluated, z. By autoradiography within one hour.

A preferred embodiment of the method according to the invention consists in the use of cell lysates which have been separated electrophoretically and the detection of one or more bands as phosphorylating enzyme. It is surprising in this connection that the proteins can be separated under denaturing conditions in sodium dodecylsulfate-polyacrylamide gels (SDS-PAA gels) and the proteins can be detected without renaturation in this form. Genetically engineered recombinations can express fusion proteins (resistance gene plus additional information). By comparing the lysates of various recombinants, their size can be determined by electrophoretic separation and reaction with a support according to the invention in the presence of ³² Ρ-γ-,, in which the SDS-PAA gels are separated strictly according to the molecular weight of the proteins.

In the embodiment described above, which originate directly from bacterial colonies and is carried out either as a spot test or Abklatschtechnik or after the electrophoretic separation of the lysates, can be distinguished in this way between kanamycin resistant and non-resistant microorganisms, since only the former produce a signal. The essential agent of the process according to the invention is a surface carrier which contains an antibiotic bound via a spacer group of more than 2 atoms. It has been found that these agents according to the invention can be made particularly favorable from cellulose, when activated with 2,4,6-trihalo-s-triazines. Such an activation method is z. B. in DD-WP 154622 described. The essential steps of this process are:

a) reaction of the cellulose with caustic soda, e.g. 3N NaOH;

b) squeezing off unneeded sodium hydroxide solution;

c) activation with 2,4,6-trichloro-s-triazine, z. In a mixed solvent of xylene and acetone at a concentration of 5 to 6 wt .-% at room temperature within 10 to 120 minutes;

d) washing the thus activated support either with water or with acetone and / or with acetone: acetic acid mixtures

e) drying of the carrier or further processing.

In this way, activated cellulose is produced by 4,6-dichloro-s-triazines, which can be further reacted with amino-containing compounds. Such reactions are z. B. in DD-WP 237844 described. Preference is given to working with aqueous solutions of organic compounds having at least one amino group and at least one carboxylic acid or sulfonic acid group, which was adjusted to a certain pH, preferably in the range of 7-8. The reaction with sulfanilic acid has proved to be particularly favorable. The "hard" binding sulfonic acid group at the end of the side chain results in a firm binding of the antibiotic to the carrier and thus no losses in the necessary washing steps and consequently very sharp autoradiograms with very little or no background out

a) cellulose as a basic matrix,

b) 4,6-trichloro-s-triazine units attached thereto,

c) Sulfanilic acid residues bound to the carbon atoms characterized by the remaining chlorine atoms by substitution of the chlorine atoms via the NH ₂ group,

d) kanamycin bound to sulfanilic acid sulfonic acid groups via ionic interactions with amino groups of kanamycin

following basic structure:

-so:

where η, χ and Kan have the meaning as already defined.

The method of the invention thus provides easily accessible routes to solid cellulose-based kanamycin, so that kanamycin retains its efficacy in enzyme reactions and even after prolonged storage. Thus, in particular for the detection of the activity of aminoglucoside phosphotransferase in expressed bacterial colonies, a significant advance for rapid and quantitative determination has been achieved. By the use of cellulosic surface carriers, an agent according to the invention is provided for the process according to the invention which has a high binding capacity. The bound kanamycin remains well phosphorylatable under these conditions. In direct contact with bacterial cultures a high sharpness of the signal is maintained. By washing with water, the signal is not or very little washable.

The process according to the invention thus represents a substantial improvement in the method for determining the aminoglucoside phosphotransferase activity from separating gels directly onto the carrier. In addition, it is possible with the composition according to the invention and using the method according to the invention, in row cultures, the already achieved enzyme activity or in growth processes to carry out the changes over time in large numbers and to evaluate them by machine. The special surface of the carrier according to the invention allows the staining of the proteins with Coomassie brilliant blue, without the carrier having a background coloration. In addition to these advantages, the surface support according to the invention has a high mechanical stability.

embodiments example 1 Preparation of the agent according to the invention

A macromolecular mass with chemically active fillers according to DD-WP 154622 is prepared as follows: Cellulose paper is reacted for 15 minutes in 3 η NaOH with gentle shaking. This product is activated after squeezing the largest amount of caustic soda into an activation bath of 5.5% by weight of cyanuric chloride, 50% by weight of xylene and 44.5% by weight of acetone for 30 minutes with gentle shaking, followed by washing with acetone or acetone / acetic acid and dried. The product thus obtained is added to a solution of 10% by weight of the sodium salt of sulphanilic acid, which has been adjusted to a pH of 7.5 by means of sodium hydroxide solution, and reacted for 5 hours with gentle shaking. This product is washed 5x with distilled water for 15 minutes each and finally added to a solution of 1.5% by weight of kanamycin sulfate adjusted to pH 7.2 with triethanolamine and incubated therein with gentle shaking for 5 hours , This gives a carrier material laden with kanamycin.

Example 2 Determination of aminoglucoside phosphotransferase activity of polyacrylamide gels enzymes

Lysates of bacteria with and without kanamycin resistance gene are added to an SDS-PAA gel and the proteins are separated by gel electrophoresis. Thereafter, the gel is placed in a solution of 20 mM Tris-HCl buffer, pH 7.2 for 15 minutes; 125mMol ammonium chloride, 1 mM dithiothreitol, 1OmMoI magnesium chloride, 2 mmol ³² Py-ATP (diluted to a specific activity of 0,3mCi / mmol) were incubated.

The carrier prepared according to Example 1 and 3 layers thick filter paper are soaked with the same solution. The following structure is used: At the bottom a glass plate, then the three layers of soaked filter paper, then the gel, then the impregnated carrier according to the invention with kanamycin, a layer not soaked thick filter paper, some layers of pulp, finally again a glass plate and a weight of 100 g. This assembly is incubated in a closed plastic tube for 30 minutes at 37 ° C. Thereafter, the experimental arrangement is disassembled and separated the carrier according to the invention from the gel. Initially, the gel is stained with Coomassie Brilliant Blue. The gel detects a complete residual band pattern, i. nothing was transferred from the gel to the carrier

 The support according to the invention is washed with water for 4 × 15 minutes and dried. Subsequently autoradiographed (2 hours at room temperature). Autoradiography shows, after development in the position where the phosphotransferase was present in the gel among the many lysate proteins, very sharp and proportional to the amount of enzyme present, blackening (bands); These can be precisely assigned to the enzyme and its quantity or activity. Using stained marker proteins, the molecular weights of the enzyme and fusion proteins are accurately determined.

Example 3 Test to distinguish bacterial colonies with and without kanamycin resistance gene

E. coli bacteria of strain DH1 with and without plasmid containing the neogen of TN-5 were grown on agar plates under usual conditions. After 12 hours, the agar plate with the colonies was covered with a nitrocellulose membrane and further cultured. After another six hours, this membrane was carefully removed and used in the following experimental setup: On a glass plate are three layers thick filter paper, which are impregnated with the same buffer as in Example 2, placed on the agent of the invention according to Example 1 is placed on this the nitrocellulose membrane with the lysed colonies (lysis by incubation in a lysis buffer of 20 mM Tris-HCl, pH = 8.0, 0.1% TritonX-100 and 5 mg / ml lysozyme, 10 minutes at teat temperature), then several layers of thick filter paper again , a glass plate and a weight of 250g. This arrangement is kept in a plastic box for one hour at 37 ° C. Finally, the structure is dismantled, the loaded agent according to the invention washed several times and autoradiographed. The positions on the agar plate on which the colonies were grown with the Neogen showed very strong, sharp signals after 2 hours, while no signals (blackening) occurred in the colonies without the Neogen.

Claims (8)

claims: 1. A method for the qualitative and quantitative determination of aminoglucoside-phosphotransferase activity in cell lysates or cell extracts, optionally after electrophoretic separation of the proteins contained therein by the detection of enzymatic activity by phosphorylation of kanamycin, characterized in that the lysate or the extract with a cellulosic surface carrier following basic structure j HO ₅ -C. SCÜ H ^ N-Kan Ö Hj where η is between 2500 and 15000, χ between η and 0.01 η and Kan is a kanamycin residue, in the presence of a mixture of adenosine triphosphate and ³² Py adenosine triphosphate is contacted in known media and the phosphorylated kanamycin on the solid phase visualized by autoradiography. 2. The method according to claim 1, characterized in that electrophoretically separated cell lysates are used, wherein one or more bands are detected as a phosphorylating enzyme. 3. The method according to claim 1 and 2, characterized in that the electrophoretic separation of the proteins on sodium dodecyl sulfate polyacrylamide gels (SDS-PAA gel) takes place and in the thus separated proteins, the phosphotransferase is detected. 4. The method according to claim 1 and 2, characterized in that cell lysates of different recombinants are located in the plasmid genetically engineered kanamycin resistance genes are electrophoretically separated and the size of fusion proteins is determined. 5. The method according to claim 4, characterized in that the separation of the cell lysate is carried out on SDS-PAA gels. 6. The method according to claim 1, 2 and 4, characterized in that a distinction is made between Kanamycin-resistant and kanamycin-resistant microorganisms on spot tests or after electrophoretic separation of lysates containing the phosphotransferase. 7. The method of claim 1, 2, 4 and 6, characterized in that a distinction is made between bacterial colonies after their lysis between kanamycin-resistant colonies and kanamycin-non-resistant colonies. 8. A means for determining the aminoglucoside phosphotransferase activity characterized in that it is a surface carrier consisting of a) cellulose, b) 2,4,6-trichloro-s-triazine units attached thereto, to the carbon atoms characterized by the remaining chlorine atoms by substitution of chlorine atoms via the NH ₂ group bound Sulfanilsäurereste, to the sulfanilic acid sulfonic acid groups via ionic interactions with amino groups of kanamycin bound Kanamycin the following basic structure ^ - I ι ' HC-CHp-C- ά / ί-Τ where η, χ and Kan have the meaning as already defined exists. Field of application of the invention This invention relates to a method of determining the activity of aminoglucoside phosphotransferases, particularly neomycin phosphotransferase (NPT II), in cell lysates or colonies. Field of application of the invention are genetic engineering, biotechnology, molecular biology and medicine. Characteristic of the known state of the art A number of methods for detecting and analyzing the expression of cloned genes have already been developed. Colony hybridization allows clones to be rapidly screened for specific DNA sequences. In immunoassays, in turn, the clones that produce specific antigens are detectable. By means of the recombinant technique, gene segments or genes for specific proteins can be introduced into cells (eg bacteria). For the selection of recombinants u.a. Resistance genes used against antibiotics. The resistance to antibiotics is usually based on the effect of the corresponding enzymes, which are encoded by the resistance genes, on the antibiotics. Colony tests can then distinguish resistant from non-resistant cells. To determine the resistance enzyme activity z. B. bound to solid phases antibiotics can be used, which grow in the presence of a substrate only the resistance genes containing cells. These are bound to solid phases antibiotics necessary. The binding of antibiotics and other drugs to solid phases is known. This bond is preferably carried out via covalent bonds. By the hydrolysis of these bonds in vivo then a controlled release of active ingredient becomes possible. The preparation of such products and their application is z. See, for example, S. Dumitriu et al., Cellulose Chemistry and Technology (1985) 19, p. 601 and p. 609. As solid phases serve here cellulose derivatives to the z. For example, be bound to 6-aminopenicillanic acid, 7-aminocephalosporanic acid, tetracycline, ampicillin, kanamycin, neomycin, chloramphenicol or oxytetracycline. Generally, it is found that this type of coupling gives low reactivity. Another possibility for the coupling of antibiotics to solid phases is the use of quaternizable nitrogen atoms of the antibiotics for binding to acidic ion exchangers. The products produced by this process are z. B. for the determination of neomycin phosphotransferase from cell extracts, as described by B. Reiss, R. Sprengel, H. Will and H. Schaller in Gene (1984) 30, pp 211-218, is described. As a solid phase hierein commercially available phosphate cellulose paper is used in which the phosphate groups are linked directly via the OH groups of the cellulose. In this relatively complex process, the resistance enzyme is first electrophoretically separated in a polyacrylamide gel, then offered in an additionally applied agarose ³² Ρ-γ-ΑΤΡ and kanamycin (not carrier fixed) as substrates under enzyme reaction conditions and then tried, the phosphorylated Kanamycin on phosphate cellulose paper by capillary blotting technique. .Nachteile of the method are the step of radioactivity containing agarose, the plurality of steps which required washing with hot water (80 ⁰ C) (losses) and long washing times (3 to 16 hours) in order to reduce the background of the support material. As a result diffuse autoradiographies are obtained, which make evaluation difficult or even impossible for complex tasks. From VIKiselev, AFGorkun and VO Rechinski is reported in Anal. Biochem. (1985) 146, p.434, describe a method for determining enzyme activity in bacterial colonies using kanamycin linked to carboxymethylcellulose (CM-cellulose). Only colony detection is possible with this method. Diffuse autoradiography may indicate that part of the kanamycin can be washed off the support by its very tight binding to cellulose or partial hydrolysis. To this process as a whole it can be said that derivatized cellulose supports of this type in which cellulose derivatives with direct coupling of the ionically active group to the cellulose chain are mechanically relatively unstable and hydrolytically susceptible, the former resulting in insufficiently strong binding of the antibiotic to the support and the latter leads to increased background in the detection techniques (primarily autoradiography).