Protein purification on immobilized metal affinity resins effected by elution using a weak ligand
AssigneeBiogen, Inc.
InventorsMark A. Staples, Christopher A. Pargellis
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Abstract
A process for the purification of proteins from solutions containing contaminants of similar net charge and molecular weight is provided, comprising contacting a solution containing the desired protein with an immobilized metal affinity chromatography resin in a buffer containing a low concentration of a weak ligand for the chelant of the resin. The adsorbed protein is then eluted using a buffer having a high concentration of the same weak ligand, e.g., Tris. Particularly preferred features employ agarose-iminodiacetic acid resins having copper cations and are especially useful in obtaining preparations of homogeneous, stable rsT4 proteins.
Claims32
A process for separating, in a partially purified protein sample, a protein having surface metal-binding amino acid residues from a proteinaceous impurity having similar molecular weight and molecular charge to said protein which remains associated with said protein after the partial purification of said protein sample, said process comprising the steps:
- (a) contacting the partially purified protein sample with an immobilized metal affinity chromatography resin, said resin comprising a matrix resin linked to a bidendate chelator bound to divalent metal ions, in a binding buffer consisting essentially of salt and a weak ligand, the affinity of the weak ligand for the divalent metal ions being less than that of said protein and said impurity;
- and
- (b) selectively eluting the protein or the impurity using an elution buffer consisting essentially of salt and a higher concentration of the weak ligand than in the binding buffer.
In a process for purifying a protein having surface metal-binding amino acid residues which includes steps effecting the elimination of impurities on the basis of molecular weight and net charge, the improvement wherein impurities not removed in previous steps are removed by the steps:
- (a) contacting the protein and remaining impurities with an immobilized metal affinity chromatography resin, said resin comprising a matrix resin linked to a bidendate chelator bound to divalent metal ions, in a binding buffer consisting essentially of salt and a weak ligand, the affinity of the weak ligand for the divalent metal ions being less than that of said protein and said impurities;
- and
- (b) selectively eluting the protein or the impurities using an elution buffer consisting essentially of salt and a higher concentration of the weak ligand than in the binding buffer.
A process for purifying a recombinant soluble T4 protein, comprising the steps of:
- (1) contacting culture medium containing recombinant soluble T4, filtered free of the source cells which produced the recombinant soluble T4, with a cationic exchange resin;
- (2) eluting the adsorbed proteins from the resin on the basis of their net charge;
- (3) applying the fraction or fractions of eluate from the cationic exchange resin containing the recombinant soluble T4 to an anionic exchange resin;
- (4) applying the wash fraction or fractions from step (3) containing recombinant soluble T4 to an immobilized metal affinity chromatography resin, said immobilized metal affinity chromatography resin comprising a matrix linked to a bidentate chelator bound to divalent metal ions, using a binding buffer consisting essentially of a low concentration of a weak ligand for the metal ion of the immobilized metal affinity chromatography resin;
- and (5) eluting the recombinant soluble T4 using as an eluant a buffer consisting essentially of a higher concentration of the weak ligand than in binding buffer.
Dependencies are inferred from the claim text.