SU1187835A1 - Method of affine chromatography of biologically active substances - Google Patents

Abstract

1. METHOD OF AFFINE CHROMATOGRAPHY viologuSkI ACTIVE vizvchstv characterized in that, in order to accelerate the method, the complex ligand-affinant is chromatographed on a sorbent at a salt concentration gradient of 0.02-1.5 M. that, in order to increase the purity, the target product is chromatographed on a sorbent, in which the exclusion molecular weight is equal to the molecular weight of the ligand, and the lower resolution limit is equal to the molecular weight of the affinant.

Description

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The invention relates to biochemistry, in particular, to methods for the production and EFFICIENCY of macromolecules from microbiological and other types of raw materials.

The aim of the invention is to accelerate the process by increasing the purity of the target product.

PRI me R. 1. A column of 2.5 I 95 cm with Spheroi 1000 (resolution mol. M. For proteins 800000-5000000) was equilibrated with 1.5 l of 20 mM Tris-HCl buffer, pH 7.5, containing 1.5 M KCl. Then in the column was injected with a linear concentration gradient KC1 from 1.5 to 0.1 M (160 ml) at a rate of 70 ml / h. 3 ml of albumin (20 mg) c. 0 mM Tris-HC1 buffer pH 7.5, containing O, 1 M KCl and 60 mg of blue dextran (mol.m. 2,000,000) were applied onto the column following the salt gradient and washed with 10 mM Tris-HC1 buffer, pH 7.5 - 0.1 M KS1. The albumin elution profile is shown in FIG. For comparison, the elution profile of albumin obtained by chromatography without blue dextran is shown. Figure 2 shows a densitogram obtained after electrophoresis of albumin before and after purification by this method.

Example 2. A column of 1.6 jf 40 cm with Toyopearl-75G (resolution limits in mol.m. for proteins 500000 50000000) was equilibrated with 0.3 l of 0.05 M sodium acetate buffer (pH 4.0) containing 1.5 M KS1, with a speed of 60 ml / h. Then, a concentration gradient of KCl was introduced into the column from 1.5 to 0.05 M (30 ml). 0.8 ml of a mixture containing 3 mg of calf thymus DNA and 3.0 mg DNase 1 in 0.05 M sodium acetate buffer (pH 4.0) was applied onto the column after a salt gradient and washed with 0.05 M sodium acetate buffer (pH 4.0) containing 0.05 M KCl. DNA was first eluted from the column, then DNase 1 (2.48 mg) and a trace of impurity contaminating the enzyme preparation.

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Claims (2)

1. METHOD FOR AFFINITY CHROMATOGRAPHY OF BIOLOGICALLY ACTIVE SUBSTANCES; characterized in that, in order to accelerate the method, the ligand-affinity complex is chromatographed on a sorbent at a salt concentration gradient of 0.02-1.5 M. 2. The method according to claim 1, characterized in that, in order to increase the degree of purity, the target product is chromatographed on a sorbent in which the molecular weight of exclusion is equal to the molecular weight of the ligand, and the lower limit of resolution is equal to the molecular weight of the affinity. F0 SL I