Method of recovering microbially produced recombinant ricin toxin a chain
First Claim
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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.
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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.
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Citations
22 Claims
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1. Method of recovering recombinant intracellularly produced, soluble ricin toxin A chain (RTA) from an aqueous suspension of transformed microorganisms containing the RTA comprising:
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(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. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11)
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12. Ricin toxin A chain (RTA) recovered from an aqueous suspension of transformed microorganisms containing the RTA by the steps comprising:
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(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. - View Dependent Claims (13, 14, 15, 16, 17, 18, 19, 20, 21, 22)
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Specification