WO1990008584A1

WO1990008584A1 - Novel aminoglycoside affinity media for separation of macromolecules

Info

Publication number: WO1990008584A1
Application number: PCT/US1990/000454
Authority: WO
Inventors: Padmanabhan P. Nair; George Kessie
Original assignee: The United States Of America, Represented By The Secretary, United States Department Of Commerce
Priority date: 1989-01-30
Filing date: 1990-01-30
Publication date: 1990-08-09
Also published as: IL93205A0; AU5036790A

Abstract

Matrix columns bearing aminoglycosides as ligands can be used for the separation and isolation of classes of macromolecules such as nucleic acids and proteins. The composition of loading and eluting buffers can be adjusted to change the relative affinity of the ligands for the macromolecules, thereby influencing their adsorption and desorption.

Description

NOVEL AMINOGLYCOSIDE AFFINITY MEDIA FOR

SEPARATION OF MACROMOLECULES

Background of the Invention

Field of the Invention

This invention relates to the separation of classes of nucleic acids and proteins.

Description of the Prior Art

Following the isolation and identification of streptomycin by Waksman and his colleagues [Proc. Soc. Exptl. Biol. Med. 55: 66 (1944)], a series of structurally related antibiotics, known as the aminoglycosides, were isolated from various species of Streptomyces. The aminoglycosides are characterized by the presence of multiple aminosugar residues linked together via glycosidic linkages. The mode of action of these broad spectrum antibiotics has been attributed to their ability to interfere with protein synthesis by virtue of their affinity for ribosomal RNA.

Cohen et al. [J. Biol. Chem. 168: 511-526 (1947); 235: PC55-56 (1960)] demonstrated the affinity of nucleic acids and proteins for streptomycin, and utilized this property for the differential precipitation of DNA, RNA, and proteins in biological extracts. Although affinity chromatography has gained widespread usage in the separation, purification, and isolation of nucleic acids and related proteins [Schott, "Affinity Chromatography: Template Chromatography of Nucleic Acids and Proteins," Dekker, New York (1984)], there have been no reports in which aminoglycosides were utilized as the ligand that was immobilized on a stationary phase.

Summary of the Invention

We have now discovered that matrices bearing aminoglycosides as ligands can be used for the separation and isolation of classes of macromolecules such as nucleic acids and proteins. The conditions of elution through the matrix column can be adjusted to change the relative affinity of the ligands for the macromolecules, thereby influencing their adsorption and desorption. In accordance with this discovery, it is an object of the invention to use aminoglycosides for the first time as ligands attached to matrices in columns for the separation of macromolecules.

It is also an object of the invention to

"tailor make" the column matrix according to the kind of separation desired by varying the ligand attached to the matrix.

A further object of the invention is to expedite various separations and fractionations by utilizing a matrix column for the separation of macromolecules.

Other objects and advantages of this invention will become readily apparent from the ensuing description.

Detailed Description of the Invention This invention utilizes the interaction between macromolecules (such as the nucleic acids) and aminoglycosides (such as streptomycin), and applies that interaction for the first time to the separation of nucleic acids on columns containing aminoglycosides as immobilized ligands attached to a matrix. Separations or fractionations performed by this invention employ a column that is packed with a matrix bearing aminoglycoside ligands, and the sample to be fractionated is eluted through the column with our solvent. The different macromolecules, because of differing strengths of ligand interactions, are eluted through the column at different rates.

The matrices contemplated by this invention must be appropriate for use as a support for immobilized ligands. Sepharose is suitable for this purpose, but it is anticipated that other matrices, such as agarose, glass beads, acrylamide, latex, polystyrene, and other synthetic and natural polymers, can also be used. A preferred derivatized sepharose is prepared by coupling dihydrostreptomycin to epoxy-activated sepharose and then deactivating unreacted sites with ethanolamine.

The use of dihydrostreptomycin as a ligand exemplifies the unique characteristics of the family of aminoglycosides, the aminocyclitols, and their derivatives and structural analogues in full or in part. A partial list of compounds that fall into this category are amikasin, gentamycin, puromycin, kanamycin, neomycin, streptobiosamine, streptidine, tobramycin, paromomycin, sisomycin, spectinomycin (aminocyclitol), and netilmicin.

The resulting affinity medium is packed into columns for the separation of classes of DNA. Retention of macromolecules on the column is determined by the choice of loading buffer and its pH and ionic strength. The profile of the eluting species is determined by the choice of eluting buffer, its pH and ionic strength, and the concentration of any chaotropic agents that might be added.

The following examples are intended only to further illustrate the invention and are not intended to limit the scope of the invention which is defined by the claims.

Example 1

Preparation of Dihydrostreptomycin-sepharose. Epoxyactivated sepharose 6B (Pharmacia/LKB, Inc.), 2.0 g, was swollen in 100 ml of distilled water for 1 hr. The gel was washed thoroughly on a sintered glass funnel with 300 ml of distilled water and then suspended in 50 ml of 0.01 M sodium borate (pH 11.0) containing 150 mg of dihydrostreptomycin sulfate. The mixture was agitated continuously for 18 hr at room temperature to allow the dihydrostreptomycin to couple to the epoxy-sepharose 6B. The resulting gel was washed sequentially with 100 ml of the borate buffer (0.01 M, pH 11.0), with distilled water until the washings were neutral, with 100 ml of sodium acetate buffer (0.1 M, pH 4.0), and then with 100 ml of sodium borate buffer (0.1 M, pH 8.0). The gel was then reacted with 50 ml of 2 M ethanolamine, pH 9.0, for 4-6 hr and washed with distilled water until the washings were neutral. Then followed washes with sodium borate buffer (0.1 M, pH 8.0, containing 0.5 M sodium chloride) and with sodium acetate buffer (0.1 M, pH 4.0, containing 0.5 M sodium chloride). The gel was then suspended in either borate buffer (0.01 M, pH 8.0) or in ammonium chloride (0.3 M, pH 7.6).

Example 2

Affinity medium prepared by the method of Example 1 was placed in a small column and wahsed with ammonium chloride (0.3 M, pH 7.6). Each DNA from the sources indicated in Table I was dissolved separately in 0.3 M ammonium chloride at pH 7.6, passed through the column, and then washed successively with 0.3 M ammonium sulfate at pH 7.6 and 0.5 M guanidine hydrochloride in 0.1 M sodium phosphate buffer at pH 7.6. Effluents were assayed for DNA by UV absorption at 260 rm using an extinction coefficient of E 1% = 200. The results in Table I show how the elution profile of a eukaryotic DNA differed from that of a prokaryotic DNA.

Example 3

The procedure of Example 2 was repeated with the proteins indicated in Table II. Effluents were assayed for protein by UV absorption at 280 rm. The analytical results were given in Table II.

Example 4

The column-loading procedure of Example 2 was repeated, except that the DNA's were dissolved separately in ammonium chloride buffers at the ionic strengths indicated in Table III and passed individually through the affinity column. The results from effluent assays (Table III) show that the retentivity of the DNA's was selectively altered by changes in ionic strength of the ammonium chloride loading buffer.

It is understood that the foregoing detailed description is given merely by way of utilization and that modification and variations may be made therein without departing from the spirit and scope of the invention.

Table I

Percent of DNA Eluted

Human Escherichia

Buffer Placenta coli

Ammonium chloride, 0.3 M, pH 7.6 4.3 63.8

Ammonium sulphate, 0.3 M, pH 7.6 71.8 26.2

Guanidine HCl, 0.5 M in sodium 2.8 7.5

phosphate, pH 7.6

Table II

Percent of Protein Eluted Calf Bovine Serum

Buffer Thymus Albumin Mucin

Ammonium chloride, 0.3 M, pΗ 7.6 100 98 55

Ammonium sulfate, 0.3 M, pH 7.6 0 0 0

Guanidine hydrochloride, 0.5 M in 0 0 0 sodium phosphate, pH 7.6

Table III

Percent of ENA Bound

Human Escherichia

Ionic Strength Placenta coli

0.1 100 67

0.2 100 50

0.3 96 36

0.4 17 15

Claims

WHAT IS CLAIMED IS:

1. A column for the separation and isolation of macromolecules selected from the group of nucleic acids and proteins comprising as an affinity medium a matrix bearing a covalently attached ligand selected from the group of aminoglycosides and aminocyclitols.

2. The column as described in claim 1 wherein said matrix is agarose.

3. The column as described in claim 1 wherein said ligand is dihydrostreptomycin.

4. The column as described in claim 1 wherein said macromolecules are nucleic acids.

5. The column as described in claim 1 wherein said macromolecules are proteins.

6. The column as described in claim 1 wherein said matrix is selected from the group consisting of agarose, glass beads, acrylamide, latex, and polystyrene.

7. The column as described in claim 1 wherein said ligand is selected from the group consisting of amikasin, gentamycin, puromycin, kanamycin, neomycin, streptobiosamine, streptidine, tobramycin, paromomycin, sisomycin, spectinomycin, and netilmicin.