CS199124B1 - Ion exchanger for the isolation of nucleic acids and proteins and method of preparing the same - Google Patents

Description

The present invention relates to ion exchangers for the isolation of nucleic acids and proteins. In its preparation, the ability of a carboxymethyl group attached to a cellulose dextran or polyacrylamide carrier to form a polyethyleneimine anion exchange ion exchanger to which aa can bind a nucleic acid in the next step, thereby forming an ehromatographic medium for column affinity chromatography of the enzyme metabolism enzymes is utilized.

Until now, various ehromatographic media developed abroad have been used to isolate anzymes and nucleic acids. They exploit differences in the ionogenic swelling of proteins and nucleic acids. These are cation exchangers such as carboxymethylcellulose, carboxymethyl aephadex, phosphocellulose and 1., and anion exchange diethylaminoethyl cellulose, diethylaminoethylsephadex and others.

These ehromatographic media aa are used as columns for column chromatography of enzymes, nucleic acids, and their components.

A common disadvantage of these ion exchangers is their slightly low selectivity * because they cannot divide nucleic acids and proteins from each other, since they are distinctly different in tone properties. As a consequence, other separation methods (gel filtration, gradient centrifugation, preparative) must also be used for purification of enzymes and nucleic acids.

199 124

198124 alektrophoresis and the like}. These are products of foreign companies whose import to us represents a significant item in foreign exchange funds. Given the current development classes in molecular biology research, he gave and expected that the consumption of chromatographic media would continue to increase, especially those having specific separation capabilities. The prior art methods for the preparation of chromatographic media containing nucleic acids ad either considerably demanding in the preparation of the basic carrier (cellulose bromination, ligand binding, etc.), or based on pure empiricity - binding of nucleic acids as a high molecular weight β cellulose or agarose. The reproducibility of these methods is low because, in addition to large laboratory routine, they require considerable homogeneity of the medium used, which is not always achievable with cellulose and agarose. These drawbacks are eliminated by the chromatographic medium which results from the binding of the nucleic acid to a polyethyleneimine-carboxymethyl-cellulose ion exchanger. How much in this reaction! Free polyethyleneimine positive / -NH - / * 'groups bind and negative groups / PO ^ Nucleic acids remain nucleic acid bases available through specific interaction and enzymes. In this simple manner, a chromatographic medium can be prepared which is capable of binding the substance from living ayemsis having affinity for nucleic acids.

The method of preparation of the ion exchanger comprises the following operations:

1. carboxymethyl cellulose active

100 g of powdered carboxymethyl cellulose are mixed with 1.5 l of 0.5 N NaOH. After standing for 30 minutes at room temperature, decant to distilled water to pH 8.0. Thereafter, the carboxymethylcellulose was removed with 1.5 L of 0.5 N HCl and decanted at room temperature after 30 minutes at room temperature to pH 5.5.

2. Polyethyleneimine treatment.

The polyethyleneimine stream of BASF Ludwlgehaffen (NSR) was designated as Polymin P (number 34-1460) and prepared as follows:

ml of Polymin F was mixed with β 80 ml of a buffer containing 0.02 M TRIS (hydroxymethylmilnamethene) pH 7.4 m B magnesium chloride and 0.1 m Hehelone III. The insoluble aa was removed by centrifugation at 3000 x g. The sediment aa was discarded and the supernatant was palliated for 20 hours against 2 L of buffer containing 0.02 M TRIS (hydroxymatyl amlnomethane) pH 7.4 and 0.1 m H chalaton III.

3. Preparation of Polyethylanimine - Carboxymethylcellulose (hereinafter FBI - Cli - Cellulose). The carboxymethylcellulose and Polymin P treated as described above were admixed at a ratio of 1: 1 to 1: 100, and occasionally stirred at a temperature of from 25 to 45 ° C for two hours and finally mixed at an isolated temperature of 15 hours. Then, PSI-CM-cellulose was loaded onto an emX cm column and washed aa at room temperature with 1.5 L buffer containing: 0.01 V IRIS (hydroxymethyl amlnomethane) pH 8.0; 1 m H 2 mercaptoethanol; 0.1 m M oelatone III; 1 M potassium chloride and glycerin in an amount to give a 5% solution of glycerin in buffer. The thus prepared chromatographic medium has a capacity of 89124: at most 50 mg bovine serum albumin per ml of wet chromatographic medium, wherein at pH 8.0 bovine serum albumin binds to carboxymethylcellulose.

4. Preparation of deoxyribonucleic acid - polyethyleneimine - carboxymethylcellulose (3dna DNA - EEI - CM - cellulose).

To a suspension of 5 g of EEI-CM-cellulose in 20 ml of a buffer containing 0.01 M TRIS (hydroxymethyl methane) pH 8.0 and 0.1 m M chelatone III. 250 mg deoxyribonucleic acid isolated from calf thymus in 10 ml buffer of the above composition was added. The deoxyribonucleic acid was denatured by heating to 100 ° C for 5 minutes before mixing with the cellulose. After stirring the suspension of PEI - CM - cellulose with DNA for 10 minutes. was centrifuged at 3000 x g. After drying at 50 ° C for 6 hours and homogenizing in a mortar, 15 ml of a buffer of the above composition containing 2 M magnesium chloride are added to the sediment. After mixing, the suspension was applied to a 2 x 10 cm column and washed to zero absorbance at 260 nm. The column is then washed with 200 ml of a buffer of the above composition containing 0.1 M potassium chloride and is ready for isolation of nucleic acid metabolism enzymes.

The prepared ion exchangers can be used to isolate proteins and nucleic acids by affinity chromatography of biological materials.

Claims (2)

OBJECT OF THE INVENTION An ion exchanger for the isolation of nucleic acids and proteins, characterized in that it consists of a nucleic acid, polyethyleneimine and a carboxymethyl group bonded to an inert carrier, for example cellulose, polydextran, polyacrylamide. 2. A method according to claim 1, wherein the nucleic acid or polynucleotide binds to a chromatographic medium prepared by reacting a polyethyleneimine with a carboxymethyl group bound to an inert carrier, for example cellulose, polydextran, polyacrylamide in a weight ratio of 1: 10 to 1: 100 at pH 7 to 9.0 and 45 ° C.