CS199123B1 - Method for removal of nucleic acids from proteins solution - Google Patents

Description

The invention relates to a method of removing nucleic acids by column iontomenide ehromatography from proteins using an anion exchanger prepared by attaching a polyethyleneimine to a carbaxymethyl group of cellulose, dextran, or polyacxyl amide, and using the abilities of this chromatographic handle to be more acidic than nucleic acids.

Nucleic acids are an astonishing wonder of the high-molecular cell material. Because they are involved in basic biological processes and have a significant negative charge, they willow and and β the other component of the cell, in particular but the proteins.

Several methods have been developed to isolate nucleic acids from proteins (e.g., phenol extraction, inactivation of proteins with sodium eol dodecyl sulfate, etc.). All these methods cause protein denaturation. Until now, we have no simple way to eliminate nucleic acids from a protein solution. Most commonly, nucleic acids are removed from the protein solution by precipitation of etreptomycin sulfate. This method is disadvantageous in particular when enzymes for the proteosynthetic system are isolated in vitro. Another method of removing nucleic acids is precipitated by polyethylene glycol. The disadvantage of this method is the non-reproducibility of fumes. Nucleic acid elimination method

199 123 acids from a protein solution by precipitating a polyethyleniline solution has the disadvantage that large volumes of buffer solutions are used in the solubilized proteins from preelpitate, as a result of which the protein concentration is low in the obtained solution, which compromises the further procedure of enzyme isolation.

These disadvantages are overcome by the nucleic acid elimination method of the column chromatograph and, using lontomycin prepared by a carboxymethylcellulose reaction and polyethylene amine, which utilizes the capability of this thromatographic medium, it has been cured with nucleic acids as solidly as bleachamines. The leached bleaches are released from the ionic wash by washing with a buffer solution of pH 7 to pH9 with an ionic strength of 0.25M and a nucleic acid buffer of pH 7 to pH 9 and with a ionic strength greater than 1M.

Example 1

To do this, 5g of carboxymethylcellulose is decanted with distilled water and then with 200 ml of 2M NaOH. Carboxymethylcellulose aa is washed with water until the pH drops to 8. Then 200 ml of HCl are added and after standing for 1 hour it is washed with distilled water to pH 5.0. The activated cellulose is then treated with 20 ml of 5% polyethylene amine in a buffer containing 0.1% Tris-hydroxymethyl-aminomethane pH 7.2 and 1 mM 2-mercaptoethanol.

After vigorous stirring for 24 hours, the suspension is applied to a 2 x 15 cm column and washed with 1000 ml of a buffer containing 0.01 M Tris-hydroxymethyl-amine-ethanoate pH 8.0, ships ehelat III and 2 M potassium chloride. Then wash with 500 ml of the above-mentioned buffer containing 0.1 M potassium chloride instead of 2M potassium chloride. A 1.5 x 5 cm column was loaded at a rate of 1 ml / mln. loaded with 100 mg of bovine ssrumalbumin in 5 ml of buffer of the above composition, through which the column was washed with a volume of 50 ml. The release of lontoms albumin was monitored with a UV analyzer. The noisy speed was 1ml / mln. Under these conditions, no blot was released from the column. It was further eluted with a buffered solution of the above composition still containing 0.3M potassium chloride. Under these conditions, virtually all of the flash-loaded flask was released from the chromatographic medium. Next, the column was washed with 50 ml 0.5M KCl. Finally, the column was washed with 50 ml of 1M KCl in the indicated buffer. 1 ml of this prepared chromatographic grip is made from a buffer containing 0.01 M Tris-hydroxymethyl-amino-ethanoate pH 8, 1 mM Ehelat III and Ο, ΙπΜ 2-mercaptoethanol, at least 100 mg bovine serum albumin.

15 mg of quanic ribonucleic acid was loaded onto this column under conditions as for protein loading. The results are shown in Tab. 1. The washed column is capable of opalizing the RNK, indicating that the elution of this chromatographic medium with high ionic strength (211 KOI) at pH 8.0 n releases the bond between the carboxymethyl group and polyethylene ml.

Table ¢ .1

Binding of yeast RNA to an IEI-CM-cellulose column. (15 mg RNA applied).

Concentrated potassium chloride in elution buffer Quantity released RNA in mg % RNA released relative to no amount applied 0.1M 0 0 0.2M 0,005 0.03 0.5M 0.08 0.5 1M 14.9 99.47 2M 0 0

Example 2

Using the chromatographic medium prepared according to Example 1, nucleic acids are removed from the crude S. aureofaclene cell extract by adding 30 ml of crude S. aureofaclene cell extract in a buffer containing 0.3 M KCl, obtained by mechanical treatment, to 10 ml of HSI-CM cellulose. disintegrate 50 g wet cells. This crude extract has a considerable nucleic acid content, a 280/260 abeorbance ratio = 0.53.

After stirring for 2 hours at 4 [deg.] C. of the crude β FEI-CM cellulose extract, the mixture was centrifuged and the aupernatant had a significant decrease in nucleic acid content (280/260-1.15 abeorbance ratio). no. Second

Table no. 2

Removal of nucleic acids from crude cell extract.

* Absorbance (nm) 280 260 280/260 Pdvodná vzarka 0.63 1.17 0.53 After treatment with EI-CM-cellulose 0,575 0.45 1.16

Claims (1)

A method for removing nucleic acids from a protein solution, characterized in that the mixture of protein and nucleic acids obtained from biological materials in a 0.25M ionic pH buffer and pH 7.0 - 9.0 is applied to the column using an ion exchange resin prepared by reaction of polyethylene and carboxymethyl group bound to an inert carrier such as cellulose, polydextran, polyacrylamide and washed with an O.25M ionic strength buffer at ρΗ7 · 0 - 9.0.