Purification and administration of DNA repair enzymes

• US 5,272,079 A
• Filed: 02/05/1993
• Issued: 12/21/1993
• Est. Priority Date: 07/06/1988
• Status: Expired due to Term

• Current Assignee: Applied Kinetics Inc.
• Original Assignee: Applied Kinetics Inc.
• Inventors: Yarosh, Daniel B.
• Primary Examiner(s): Patterson, Jr., Charles L.
• Application Number:
  US08/014,358
• Time in Patent Office: 319 Days
• Field of Search: 435/193, 435/196, 424/94.5, 424/94.6, 424/450
• US Class Current: 435/193
• CPC Class Codes: A61K 38/00 Medicinal preparations containing peptides peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00
• CPC Class Codes: A61K 9/127 Liposomes
• CPC Class Codes: C12N 9/1007 Methyltransferases (general) (2.1.1.)
• CPC Class Codes: C12N 9/22 Ribonucleases RNAses, DNAses catalytic nucleic acids C12N15/113

Abstract

Methods for purifying DNA repair enzymes are provided in which an aqueous solution of a DNA repair enzyme in an impure state is applied to a molecular sieve column having an exclusion limit which will retard the DNA repair enzyme but will not retard contaminants larger than the DNA repair enzyme. The DNA repair enzyme in an enhanced state of purity is eluted isocratically from the molecular sieve column in an elution buffer and applied directly to a DNA affinity column in the same buffer without intermediate dialysis, ultrafiltration, or other procedures. The DNA repair enzyme is eluted from the DNA affinity column using, for example, a salt gradient. The method is rapid, inexpensive, simple to perform, and has been found to produce a homogeneous final product. In accordance with other aspects of the invention, the purified DNA repair enzymes are encapsulated in liposomes and administered to living cells in situ. This form of administration has been found to be non-toxic to the cells and to result in increased incision of damaged DNA, enhanced DNA repair synthesis, and increased cell survival after exposure to ultraviolet light. In certain preferred embodiments, DNA repair enzymes are administered in pH sensitive liposomes.

Claims

1. A process for purifying a DNA repair enzyme comprising:

(a) contacting an aqueous solution of the DNA repair enzyme in an impure state with a molecular sieve having an exclusion limit which is (i) larger than the molecular weight of the DNA repair enzyme and (ii) smaller than the molecular weight of at least some of the impurities in the extract such that the DNA repair enzyme will pass through the molecular sieve at a slower rate than at least some of the impurities in the aqueous solution;

(b) isocratically eluting the DNA repair enzyme from the molecular sieve with an aqueous elution solution so as to obtain the DNA repair enzyme in one or more selected fractions of the eluate in a state of enhanced purity, said aqueous elution solution being chosen so that complexes can form between the DNA repair enzyme and selected nucleic acids;

(c) without further purification, contacting the one or more selected fractions of step (b) in said aqueous elution solution with one or more selected nucleic acids which have been immobilized on a solid support so as to form immobilized nucleic-acid/DNA-repair-enzyme complexes between the DNA repair enzyme and the one or more selected, immobilized nucleic acids;

(d) washing the immobilized complexes to remove at least some of the remaining impurities; and

(e) eluting the DNA repair enzyme from the one or more selected, immobilized nucleic acids so as to obtain the DNA repair enzyme in one or more selected fractions of the eluate in a state of further enhanced purity.

2. The process of claim 1 wherein the exclusion limit of the molecular sieve is (i) larger than the Stokes'"'"' radius of the DNA repair enzyme and (ii) smaller than the Stokes'"'"' radius of at least some of the impurities and wherein the molecular sieve comprises a gel filtration column.

3. The process of claim 2 wherein the exclusion limit of the molecular sieve is larger than about twice the Stokes'"'"' radius of the DNA repair enzyme and smaller than about 35 Angstroms.

4. The process of claim 1 wherein the molecular sieve comprises a gel filtration column.

5. The process of claim 4 wherein the exclusion limit of the molecular sieve is larger than about twice the molecular weight of the DNA repair enzyme and smaller than about 60,000 daltons.

6. The process of claim 1 wherein the one or more selected nucleic acids are single-stranded deoxyribonucleic acids.

7. The process of claim 1 wherein the DNA repair enzyme is eluted from the one or more selected, immobilized nucleic acids using an aqueous elution solution which contains a gradient of a material capable of disassociating the DNA repair enzyme from the one or more selected, immobilized nucleic acids.

8. The process of claim 1 wherein the DNA repair enzyme is a homogeneous protein at the end of step (e) as determined by SDS polyacrylamide gel electrophoresis.

9. The process of claim 1 wherein the DNA repair enzyme is T4 endonuclease V.

10. The process of claim 1 wherein the aqueous solution of the DNA enzyme in an impure state is obtained by (i) disrupting cells containing the DNA repair enzyme, (ii) removing cell debris, and (iii) collecting the resulting solution, said solution comprising the aqueous solution of the DNA enzyme in an impure state.

11. The process of claim 1 wherein the aqueous solution of the DNA enzyme in an impure state is obtained by (i) disrupting cells containing the DNA repair enzyme, (ii) centrifuging the disrupted cells, (iii) collecting the supernatant, (iv) precipitating the proteins in the supernatant, and (v) resuspending the precipitate in the aqueous elution solution used to elute the DNA repair enzyme from the molecular sieve, said resuspended precipitate comprising the aqueous solution of the DNA enzyme in an impure state.

12. The process of claim 11 wherein prior to being contacted with the molecular sieve, the resuspended precipitate of step (v) is dialyzed against the aqueous elution solution used to elute the DNA repair enzyme from the molecular sieve.

13. The process of claim 11 wherein the nucleic acids from the disrupted cells are precipitated prior to centrifugation step (ii).

14. The process of claim 7 wherein the material capable of disassociating the DNA repair enzyme from the one or more selected, immobilized nucleic acids is selected from the group consisting of salts, acids, detergents, and competing ligands.

15. A process for producing a liposome-encapsulated DNA repair enzyme comprising:

(a) growing cells which synthesize the DNA repair enzyme;

(b) preparing an extract of the cells which contains the DNA repair enzyme in an impure state;

(c) contacting the extract with a molecular sieve having an exclusion limit which is (i) larger than the molecular weight of the DNA repair enzyme and (ii) smaller than the molecular weight of at least some of the impurities in the extract such that the DNA repair enzyme will pass through the molecular sieve at a slower rate than at least some of said impurities;

(d) isocratically eluting the DNA repair enzyme from the molecular sieve with an aqueous elution solution so as to obtain the DNA repair enzyme in one or more selected fractions of the eluate in a state of enhanced purity, said aqueous elution solution being chosen so that complexes can form between the DNA repair enzyme and selected nucleic acids;

(e) without further purification, contacting the one or more selected fractions from step (d) in said aqueous elution solution with one or more selected nucleic acids which have been immobilized on a solid support so as to form immobilized nucleic-acid/DNA-repair-enzyme complexes between the DNA repair enzyme and the one or more selected, immobilized nucleic acids;

(f) washing the immobilized complexes to remove at least some of the remaining impurities;

(g) eluting the DNA repair enzyme from the one or more selected, immobilized nucleic acids so as to obtain the DNA repair enzyme in one or more selected fractions of the eluate in a state of further enhanced purity; and

(h) encapsulating at least a portion of the purified DNA repair enzyme contained in the one or more selected fractions of step (g) in liposomes.

16. The process of claim 15 wherein the exclusion limit of the molecular sieve is (i) larger than the Stokes'"'"' radius of the DNA repair enzyme and (ii) smaller than the Stokes'"'"' radius of at least some of the impurities and wherein the molecular sieve comprises a gel filtration column.

17. The process of claim 16 wherein the exclusion limit of the molecular sieve is larger than about twice the Stokes'"'"' radius of the DNA repair enzyme and smaller than about 35 Angstroms.

18. The process of claim 15 wherein the molecular sieve comprises a gel filtration column.

19. The process of claim 18 wherein the exclusion limit of the molecular sieve is larger than about twice the molecular weight of the DNA repair enzyme and smaller than about 60, 000 daltons.

20. The process of claim 15 wherein the one or more selected nucleic acids are single-stranded deoxyribonucleic acids.

21. The process of claim 15 wherein the DNA repair enzyme is eluted from the one or more selected, immobilized nucleic acids using an aqueous elution solution which contains a gradient of a material capable of disassociating the DNA repair enzyme from the one or more selected, immobilized nucleic acids.

22. The process of claim 15 wherein the DNA repair enzyme is a homogeneous protein at the end of step (g) as determined by SDS polyacrylamide gel electrophoresis.

23. The process of claim 15 wherein the DNA repair enzyme is T4 endonuclease V.

25. A composition comprising liposomes having a DNA repair enzyme encapsulated therein prepared by the process of claim 15.

24. A composition comprising liposomes having a DNA repair enzyme encapsulated therein.