Isolation of lactoferrin from milk
First Claim
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1. A method for substantially purifying human lactoferrin from transgenic milk, comprising the steps of:
- contacting transgenic bovine milk or milk fraction containing human lactoferrin and an endogenous lactoferrin with a strong cation exchange resin under elevated ionic strength conditions which are at least 10 mM higher in monovalent cation concentration than milk for a suitable binding period, forming human lactoferrin-resin complexes;
removing the unbound milk or milk fraction components from the human lactoferrin-resin complexes; and
eluting the human lactoferrin from the recovered human lactoferrin-resin complexes with a salt solution having an ionic strength sufficient to elute the human lactoferrin from the human lactoferrin-resin complexes whereupon human lactoferrin substantially free from the endogenous lactoferrin is obtained.
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Abstract
The invention provides methods for purification of human lactoferrin from milk, especially milk of nonhuman species, and for separation of human lactoferrin from undesired macromolecular species present in the milk, including separation from nonhuman lactoferrin species.
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Citations
27 Claims
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1. A method for substantially purifying human lactoferrin from transgenic milk, comprising the steps of:
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contacting transgenic bovine milk or milk fraction containing human lactoferrin and an endogenous lactoferrin with a strong cation exchange resin under elevated ionic strength conditions which are at least 10 mM higher in monovalent cation concentration than milk for a suitable binding period, forming human lactoferrin-resin complexes; removing the unbound milk or milk fraction components from the human lactoferrin-resin complexes; and eluting the human lactoferrin from the recovered human lactoferrin-resin complexes with a salt solution having an ionic strength sufficient to elute the human lactoferrin from the human lactoferrin-resin complexes whereupon human lactoferrin substantially free from the endogenous lactoferrin is obtained. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15)
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- 16. A method for purifying native human lactoferrin from hLF degradation products in a sample, comprising the step of contacting the sample with a strong cation exchange resin under suitable binding conditions and eluting the native human lactoferrin as a resolvable peak by applying a salt gradient or in a stepwise mode and recovering the peak consisting essentially of native human lactoferrin having an intact amino terminal sequence.
- 19. A method for separating human lactoferrin from bovine lactoferrin in a sample, comprising contacting the sample with a strong cation exchange resin and eluting the human lactoferrin from the resin with a salt solution which preferentially elutes human lactoferrin but does not substantially elute bovine lactoferrin concomitantly with the human lactoferrin.
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23. A method for isolating substantially pure recombinant human lactoferrin from the milk of a transgenic bovine harboring a transgene which encodes the expression and secretion of recombinant human lactoferrin in its milk, comprising the steps of:
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obtaining milk or a milk fraction of said transgenic bovine containing recombinant human lactoferrin; contacting said milk or milk fraction with a strong cation exchange resin under suitable binding conditions 10 for binding of the human lactoferrin to the resin; eluting the recombinant human lactoferrin from the strong cation exchange resin with a stepwise salt gradient or linear salt gradient. - View Dependent Claims (24, 25)
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- 26. A method for removing LPS from a solution, comprising the step of contacting a solution containing LPS with a resin comprising immobilized hLF and recovering the portion of the solution that is bound to the resin comprising immobilized hLF.
Specification