Method of recovering microbially produced recombinant ricin toxin a chain

• US 4,689,401 A
• Filed: 09/09/1986
• Issued: 08/25/1987
• Est. Priority Date: 03/06/1986
• Status: Expired due to Term

• Current Assignee: Chiron Corporation (Bayer AG)
• Original Assignee: Cetus Corporation (Bayer AG)
• Inventors: Ferris, Robert
• Primary Examiner(s): SCHAIN, HOWARD
• Application Number:
  US06/905,283
• Time in Patent Office: 350 Days
• Field of Search: 530/377, 530/396, 530/397, 530/403, 530/416, 530/808, 530/809, 424/85, 424/88, 435/68, 435/70, 435/172.3, 435/240, 435/241, 435/172.3
• US Class Current: 530/396
• CPC Class Codes: C07K 14/415 from plants
• CPC Class Codes: Y10S 530/808 Materials and products related to genetic engineering or hybrid or fused cell technology, e.g. hybridoma, monoclonal products
• CPC Class Codes: Y10S 530/809 Fused cells, e.g. hybridoma

Abstract

Substantially pure, intracellularly produced, soluble recombinant ricin toxin A (RTA) is recovered from transformed cells by disrupting the cell membrane, removing insoluble cell membrane materials from the disruptate, adjusting the pH of the cell membrane material-free solution to 6 to 6.5 and the conductivity to 1.25 to 1.75 millisiemens, passing the adjusted solution through a bed of SP-cellulose cation exchanger, and eluting the substantially pure RTA from the bed.

Claims

1. Method of recovering recombinant intracellularly produced, soluble ricin toxin A chain (RTA) from an aqueous suspension of transformed microorganisms containing the RTA comprising:

(a) disrupting the cell membrane of the microorganisms at a pH that keeps the RTA in solution;

(b) removing insoluble cell membrane materials from the disruptate at a pH that keeps the RTA in solution;

(c) adjusting, if necessary, the pH of the solution resulting from step (b) to about 6 to about 6.5 and the conductivity of said solution to about 1.25 to about 1.75 millisiemens;

(d) then passing said solution through a bed of an SP-cellulose cation exchanger, whereby RTA in the solution is retained by the exchanger; and

(e) eluting RTA from the bed to produce a solution whose protein content is at least about 90% by weight RTA as determined by SDS-PAGE.

2. The method of claim 1 wherein step (a) is effected by high pressure cycling and is carried out under reducing conditions.

3. The method of claim 1 wherein step (b) is effected by diafiltration.

4. The method of claim 1 wherein said conductivity is about 1.45 to about 1.55.

5. The method of claim 1 wherein the cation exchanger is composed of cellulosic backbones crosslinked with vinyl polymer carrying pendant sulfopropyl functional groups and the bed is configured such that the solution is passed radially through the bed.

6. The method of claim 1 wherein said protein content is at least about 95% by weight RTA as determined by SDS-PAGE.

7. The method of claim 5 wherein step (a) is effected by high pressure cycling, step (b) is effected by diafiltration, the conductivity is about 1.45 to about 1.55 millisiemens, and the protein content is at least about 95% by weight RTA as determined by SDS-PAGE.

8. The method of claim 1 including (f) chromatographing the eluted RTA using a phenyl-sepharose column, whereby the purity of the RTA is increased to above about 99%.

9. The method of claim 1 wherein steps a-f are singly or in combination run at a temperature that reduces formation of precipitates comprising RTA.

10. The method of claim 9 wherein said temperature is in a range from one sufficient to keep solutions from freezing to about 10°

C.

11. The method of claim 9 wherein said temperature is about 4°

C.

12. Ricin toxin A chain (RTA) recovered from an aqueous suspension of transformed microorganisms containing the RTA by the steps comprising:

(a) disrupting the cell membrane of the microorganisms at a pH that keeps the RTA in solution;

(b) removing insoluble cell membrane materials from the disruptate at a pH that keeps the RTA in solution;

(c) adjusting, if necessary, the pH of the solution resulting from step (b) to about 6 to about 6.5 and the conductivity of said solution to about 1.25 to about 1.75 millisiemens;

(d) then passing said solution through a bed of an SP-cellulose cation exchanger, whereby RTA in the solution is retained by the exchanger; and

(e) eluting RTA from the bed to produce a solution whose protein content is at least about 90% by weight RTA as determined by SDS-PAGE.

13. Ricin toxin A chain of claim 12 wherein step (a) is effected by high pressure cycling and is carried out under reducing conditions.

14. Ricin toxin A chain of claim 13 wherein step (b) is effected by diafiltration.

15. Ricin toxin A chain of claim 12 wherein said conductivity is about 1.45 to about 1.55.

16. Ricin toxin A chain of claim 12 wherein the cation exchanger is composed of cellulosic backbones cross-linked with vinyl polymer carrying pendant sulfopropyl functional groups and the bed is configured such that the solution is passed radially through the bed.

17. Ricin toxin A chain of claim 12 wherein said protein content is at least about 95% by weight RTA as determined by SDS-PAGE.

18. Ricin toxin A chain of claim 16 wherein step (a) is effected by high pressure cycling, step (b) is effected by diafiltration, the conductivity is about 1.45 to about 1.55 millisiemens, and the protein content is at least about 95% by weight RTA as determined by SDS-PAGE.

19. Ricin toxin A chain of claim 12 including (f) chromatographing the eluted RTA using a phenylsepharose column, whereby the purity of the RTA is increased to above about 99%.

20. Ricin toxin A chain of claim 12 wherein steps a-f are singly or in combination run at a temperature that reduces formation of precipitates comprising RTA.

21. The method of claim 20 wherein said temperature is in a range from one sufficient to keep solutions from freezing to about 10°

C.

22. The method of claim 20 wherein said temperature is about 4°

C.