HU179770B - Process for linking dns polymerase enzyme to solid vehicle and the application of this composition for the synthesis of polydeoxynucleotides - Google Patents

Description

The present invention relates to a process for the synthesis of polydeoxynucleotides from 2'-deoxynucleoside-5'-triphosphates in the presence of a divalent cation and optionally a primer complex of pH 6.5 to 9.5 with a free mercapto-free DNA polymerase enzyme. in a buffered aqueous medium at temperatures between 15 ° C and 42.0 ° C.

The sample primer complex is the same sample molecule as the polydeoxynucleotide to be produced.

It is known that enzymatic synthesis of polydeoxynucleotides is carried out in homogeneous processes. For example, J. F. Burd and R. D. Wells, J. Mol. Biol. 53: 435-459 (1970); F. J. Bollum, Procedures in Nunleic Acids Research, page 591, Ed, by Cantoni, G. L. and Davies, D. R., New York, Harper and Row (1966); J. Sági, A. Szabolcs, A. Szemző and L. Ötvös, Nucleic Acids Slit. 4, 2-6-2777 (1977). In these methods, the buffered reaction mixture contains the substrate, the sample primer complex, the enzyme and cations for optimal enzyme function. The reaction mixture is incubated, the enzyme is denatured after completion of the reaction (for example, by heating the reaction mixture), and the precipitated enzyme protein is removed from the mixture. From the resulting polydeoxynucleotide mixture, high molecular weight polymers are recovered by dialysis.

The polydeoxynucleotides thus produced are used as DNA models for the physical, chemical, biochemical and biological study of nucleic acids.

A commonly used DNA model is a double helix copolymer of 2'-deoxyadenosine-5'-monophosphate (dAMP) and thymidine-5'-monophosphate (dTMP):

... -dAMP-dTMP-d AMP-dTMP ...

abbreviated as pofi d (A-T).

For DNA-dependent DNA polymerases, the nucleotide sequence of the new DNA strand is determined by the DNA nucleotide sequence of the sample introduced into the reaction mixture. Therefore, the same pofi d (A-T) sample primer complex is required for the thesis d (A-T). By introducing the appropriate primer complex, various polydeoxynucleotides such as poly (dA) can be prepared. poly (dT), pofi d (G-C), pofi d (I-C), pofi (dG) .poli (dC), pofi (dl) .pofi (dC), etc., where A = adenine, T = thymine, G = guanine, I = inosine, C = = cytosine.

However, some DNA polymerase enzymes are capable of synthesizing so-called de novo pofi d (A-T) without such an exogenously introduced primer complex. An example of such an enzyme is the E. coli DNA polymerase I enzyme.

The disadvantage of the known methods for the enzymatic synthesis of polynucleotides is that the enzyme can be used only once and that, when the reaction mixture is processed, removal of the enzyme involves denaturation.

It is an object of the present invention to provide a method for overcoming these drawbacks.

The present invention is based on the discovery that the above object can be achieved by the synthesis of polydeoxynucleotides using an enzyme fixed to a solid support. so it is

-1179770 can be removed from the reaction mixture by simple filtration and reused.

A further basis of the invention is the discovery that DNA polymerase enzymes having a free mercapto moiety which is not part of the active center of the enzyme can be used for attachment to a solid support. Namely, through these groups, the enzyme can be linked to a suitable carrier by substitution of the proton without attachment causing loss or loss of activity. Accordingly, the present invention provides a process for the synthesis of polydeoxynucleotides from 2'-deoxynucleoside-5'-triphosphates with a free mercapto-free DNA polymerase enzyme in the presence of a divalent cation and optionally a primer of 6.5 and 9.5, respectively. in a buffered aqueous medium at a temperature between 15 ° C and 42.0 ° C. According to the present invention, the DNA polymerase enzyme is covalently bonded to a solid support capable of forming a mercapto group bond.

In a preferred embodiment of the process of the invention, the immobilized reagent containing a mercapto group is used as a solid support.

Solid supports capable of forming a bond with a mercapto group are suitable for attachment of the enzyme. Such are modified agarose-based immobilized mercapto reagents such as Thiopropyl-Sepharose 6B (Pharmacia Information Booklet, 1977) or Activated Thiol-Sepharose 4B (Pharmacia Information Booklet, 1974).

The structure of Thyopropyl-Sepharose 6B is illustrated in Figure 1, where the matrix is modified agarose. Thiopropyl-Sepharose 6B 20 contains 2-thiopyridyl groups per 1 ml of swollen gel and 1 g of lyophilized gel swelled at ca. Volume 3 ml. The immobilized mercapto group protected and activated by the 2-thiopyridyl group can be liberated in various ways, but can also be reacted directly with the mercapto group to form a disulfide bond. The molar extinction coefficient of the downstream 2-thiopyridone at 343 nm was 8.08 x 10 ³ M -1 cm ^-1 .

The selected solid support is swollen in potassium phosphate buffer, pH 7.4, loaded on a column and loaded with the enzyme dissolved in the buffer.

According to the invention, an enzyme is used which contains a free mercapto group that can be chemically reacted. Such is the DNA polymerase type I enzyme (Cornberg polymerase), which can be isolated from Escherichia coli. It is a subunit spherical molecule with a molecular weight of 109,000 Daltons and 975 amino acids. Its active center contains, inter alia, a lysine and an arginine molecule. The molecule has a single disulfide bond and a mercapto group. The latter can be reacted with iodine acetate and mercury salts without loss of activity.

Like E. coli, DNA polymerase I enzymes isolated from other bacteria, such as Bacillus subtilis, Micrococcus lisodeicticus and Mycobacterium tuberculosis, are not inhibited by mercapto reagents and are therefore likely to be immobilized in a similar manner to E. coli. However, DNA polymerase type II and III enzymes isolated from the above bacteria are inhibited by mercapto reagents and therefore cannot be fixed on a solid support as described above. Mammalian ^two organization staff from so far isolated DNA polymerases (EC. 2.7.7.7.) From the polymerase α and γ polymerase also inhibited, and the polymerase is not inhibited by β-mercaptoethanol reagents.

The enzymes on the column, dissolved in buffer, are washed in the column with additional buffer and the mixture is circulated 10 times with a peristaltic pump for maximum fixation. The reaction proceeds according to Scheme 1, where E = DNA polymerase I and K = Sepharose-O-CH ₂ -CH-CH ₂ -.

I

OH

The amount of fixed enzyme can be determined directly by measuring the light absorption of the downstream 2-thiopyridone group or indirectly by measuring the amount of DNA produced by enzyme-catalyzed synthesis.

After the fixation reaction is completed, the buffer is lowered and the gel is washed with additional fresh buffer, first until the flow buffer has the same spectrum as the pure buffer, which is about 10%. Wash with 10 ml and then with an additional 40 ml at 1 ml / min. For spectrophotometry, a Zeiss Specord UV VIS recording spectrophotometer was used.

The enzyme fixed as described above is then used to synthesize the polydeoxynucleotides. The reaction mixture used for the synthesis contains the deoxynucleotides used as the substrate and the nino primer complex in an aqueous medium.

The reaction mixture is buffered with the amount and quality of the enzyme used for optimal function

Buffer to pH 6.5 to 9.5. In addition, a metal salt, preferably a niagnose salt, is added to the reaction mixture in an appropriate concentration to ensure optimal function of the enzyme.

The column was tempered to 37 ° C and the reaction mixture also at 37 ° C was applied, which was then circulated with a peristaltic pump.

The progress of the reaction can be monitored in various ways, such as by measuring the radioactivity of the insoluble material, by paper chromatography, by analysis of the final product or by control. reaction.

In the case of radioactivity measurement, the reaction mixture must also contain a labeled substrate. For example, [ ³ HJdATP] may be used. The measurement is performed by taking a 25 μΐ sample of the column mixture at various intervals using a Hamilton micropipette and measuring the aliquots on a GF / C glass filter disk (2.5 cm diameter, Whatman). It is dropped into a cold (0-5 ° C) mixture of 5% trichloroacetic acid (TCA) and 1% sodium pyrophosphate solution (10 ml solution / pc glass filter, 0-5 ^° C, 10 min), washed twice 5% TCA solution, twice with 96% ethyl alcohol, twice with diethyl ether and air dried. The radioactivity of the acid-insoluble polymer molecules (polymer number greater than 10) was measured on a Packard spectrometer in a toluene cocktail. This method can also be used to monitor the control reaction.

Paper chromatography can be used for quantitative monitoring of the reaction with radioactive substrates, whereas for inactive substrates it is used for the qualitative monitoring of the reaction. On a 1 cm x 15-18 cm DE 81 (DEAE-Cellulose, Whatman) paper strip, 5 to 25 μΐ of reaction mixture was run and run with 0.15 M NH ₄ HCO ₃ : the R 1 of the nucleotides is between 0.35 and 0.55 , the polynucleotides remain at baseline.

-2179770

The gel was flushed and washed (checked at 260 nm) with a 0.65-fold volume of 2.5 M NaCl - 0.038 M EDTA and kept at 70 ° C for 10 minutes. After cooling, shake three times with an equal volume of chloroform-isoamyl alcohol (24: 1). The two phases are separated (by centrifugation or on phase separation paper, Whatman), and the aqueous phase is dialysed twice with water (Bio-Fiber 50 Minibeaker, agitation, 4 ° C, continuous operation). The conductivity of the dialysis solution is measured. After the conductivity at 0.8-1 liters of double-distilled water has reached the value of double-distilled water (0.2-0.6 additional 0.4-

Dialyze with 0.5 L of water. It is then lyophilized to dryness, taken up in 10 ^-3 M TRIS.HCl (pH 7.4), 10 ^-4 M EDTA (500 μΐ), and aliquots taken from the resulting solution are determined for concentration, UV spectra, thermal hyperchromicity, template activity. The polymer solutions were stored at -25 ° C.

Control reaction: Add 165 volumes of the reaction mixture (equivalent to that used in the immobilized enzyme reaction) and incubate at 37 ° C. Sampling (25 μl) at the same time as the reaction to be controlled on a GF / C glass filter; processing, measuring as previously described.

The immobilized enzyme is stored and reused as follows:

After the reaction mixture was drained and washed, the gel was washed with additional 100 mL of 60 mM potassium phosphate buffer followed by 20 mL of 0.02% sodium azide solution and stored therein or removed from the column at 3-6 ° C. After washing out the sodium azide solution, the enzyme can be re-used and hardly decreases in its original activity (0-5%). The most frequently re-tested and most stable immobilized enzyme was the E. coli DNA polymerase T grade II enzyme, with no change in activity over six months.

The main advantages of the process according to the invention are as follows:

(a) The DNA polymerase enzyme covalently bonded to the solid support undergoes a heterogeneous phase reaction upon synthesis of the deoxypolinucleotides and may be removed from the reaction mixture without denaturation upon completion of the reaction.

(b) The fixed enzyme may be re-used.

(c) The fixed enzyme may also be used in continuous mode.

The following examples illustrate the process of the invention.

Example 1

a) Preparation of Thiopropyl-Sepharose g lyophilized, commercially available Thiopropyl-Sepharose 6B mercaptive reagent, which is the reagent of Figure 1, was swollen for 15 minutes at room temperature in 60 mM potassium phosphate buffer (pH 7.4). . It is filled into a 1.5 x 15 cm glass column with a sheath and a glass filter at the bottom and washed with 200 ml of the above buffer to wash out the factory-added stabilizing agents.

b) Immobilization of the DNA polymerase enzyme

Approx. In 0.5 ml buffer, 100 units (20 μΐ) of E. coli MRE 600 DNA polymerase I, grade I (product of Boehringer-Mannheim GmbH) with a specific activity of 3850 cg / mg of protein were added with a Hamilton micropipette. (1 unit DNA polymerase enzyme incorporates 10 mM total nucleotide at 37 ° C into the acid-insoluble polymer for 30 minutes in the presence of an appropriate sample primer complex in defined buffer.) Slowly (0.1-0.2 ml / min) gel and washed with additional 10 ml of buffer at room temperature and circulated 10 times with a peristaltic pump.

c) Synthesis of polydide (A-T)

The column was tempered to 37 ° C with a thermostat and then introduced into the gel with 10 ml of the following reaction mixture: 60 mmol potassium phosphate pH 7.4, 6 mmol magnesium chloride, 1 mmol [ ^{1 *} 3H] dATP (specific) activity of 10.9 degradation / min.pmol), 1 mM dTTP, 100 μτηόΐ (P) -activated poly d (A-T) primer complex dissolved in 10 ml of double-distilled water. Discard 2-3 ml of buffer corresponding to dead volume (checked by spectrophotometer) and circulate the reaction mixture at 0.5 ml / min using a peristaltic pump. The progress of the reaction is monitored by radioactivity measurement.

hours after the reaction, the amount of polymer formed was 3.04 mg based on the incorporation of [ ³ H] dAMP into the acid insoluble material. The substrate utilization is 45%.

The resulting polyd (A-T) is dialyzed to separate co-molecular weight materials. The amount of pure high molecular weight native polyd (A-T) is 2.2 mg.

Example 2

150 units (100 μapt) of E. coli MRE 600 DNA polymerase I, grade II (product of Boehringer-Mannheim GmbH) was immobilized on the immobilized mercaptive reagent.

Example 1. Synthesis was also carried out according to the method described in Example 1. The substrate used is 2 mM [ ³ HJdATP and 2 mM dTTP. The total amount of polymer synthesized in this way over 4 hours was 2.37 mg based on radioactivity and 18% substrate utilization. The isolated and purified polymer has a low molecular weight (5-200,000 Dalton) in an amount of 1.78 mg.

Example 3

45 units (50 μΐ) of 6940 units / mg of protein specific activity E. coli MRE 600 DNA polymerase I large fragment enzyme (product of Boehringer-Mannheim GmbH) were immobilized on the immobilized reagent as described in Example 1. Synthesis was also carried out according to the method described in Example 1. The substrates used are 0.5 mM [ ³ H] dATP and 0.5 mM dTTP. The total amount of polymer synthesized in this way over 24 hours was 2.74 mg based on radioactivity,

-3179770 utilization 82%. The isolated purified fluff d (A-T) is high molecular weight (> 200,000 Dalton) in an amount of 2.12 mg.

Claims (2)

Patent claims 1. A process for the synthesis of polydeoxynucleotides from 2'-deoxynucleoside-5'-triphosphates in the presence of a free mercapto group DNA polymerase enzyme, a divalent cation and optionally the same primer complex as the desired polydeoxynucleotide in the presence of 6,5 9.5 in a buffered aqueous medium at a temperature between 15 ° C and 42.0 ° C, characterized in that the DNA polymerase enzyme A fixed enzyme covalently bonded via a disulfide bridge is used between the mercapto group of 5 enzymes and the solid support containing the mercapto group. 2. The process of claim 1 wherein the solid support is an immobilized reagent containing a 2-thiopyridone protected mercapto group. ------- «4 Figure 1