Anthony R. Torres

Preparative high-performance liquid chromatography of proteins on an anion exchanger using unfractionated carboxymethydextran displacers

It has been previously demonstrated that carboxymethyldextran (CMD) displacers fractionated on an ion-exchange column can be used for the resolution and high capacity displacement chromatography of proteins on high-performance ion-exchange columns. It is shown, in this manuscript, that high resolution and high capacity are obtainable using unfractionated CMD displacers. Up to 25 mg of a mixture of ovalbumin, alpha-lactalbumin, and soy-bean trypsin inhibitor along with many impurities were separated on a 3.3-ml Altex DEAE-5PW column using three unfractionated CMD displacers and a final CMD displacer. A method for removing the displacers from proteins, on a hydrophobic interaction column, is also described.

Purification of complex protein mixtures by ion-exchange displacement chromatography using spacer displacers

The anion-exchange separation of complex protein mixtures by displacement chromatography using spacer displacers driven by high-affinity final displacers is demonstrated. Guinea pig serum was separated on a medium-resolution adsorbent using a single heterogeneous mixture of carboxymethyldextran displacers to space the protein components. Mouse liver cytosol was separated on a low-resolution adsorbent using six carboxymethyldextran spacer displacers of increasing column affinity. The demonstration of the purification of alkaline phosphatase from E. coli periplasm by displacement chromatography on a high-performance liquid chromatography column is reviewed. The benefits of spacer displacers for separating minor components from complex biological protein mixtures is discussed. A simplified method for preparing carboxymethyldextran displacers is presented.

Purification of Gc-2 globulin from human serum by displacement chromatography: A model for the isolation of marker proteins identified by two-dimensional electrophoresis

A protein that was initially known only as a minor spot in two-dimensional electrophoresis patterns of serum obtained from certain psoriasis patients, particularly those with a pustular component to their disease, has been purified by two stages of ion-exchange displacement chromatography on DEAE-Sephacel at different pH levels, followed by elution chromatography on hydroxylapatite. The purification was followed by examining the column fractions directly by two-dimensional gel electrophoresis. The capacity of the displacement system, which utilized carboxymethyldextrans as displacers, was very high; 6 ml of dialyzed serum applied to a 7-ml column in the initial stage resulted in a very substantial enrichment of the target protein. The second displacement stage yielded a highly purified product, contaminated only by A-1 lipoprotein. The latter was removed by hydroxylapatite chromatography. The purified protein was subsequently identified as Gc-2 globulin, a vitamin D-binding protein, by immunological procedures. The results demonstrate the effectiveness of ion-exchange displacement chromatography in focusing resolving power on the relatively narrow range of affinities represented by the target protein and its immediate neighbors in a chromatogram, as well as the applicability of the system to the isolation of a protein known only by its position in a two-dimensional electrophoretic pattern.

Parative high-performance liquid chromatography of proteins on ion-exchange columns with carboxymethyl dextrans as displacers

Abstract It is shown that carboxymethyldextrans can be used to displace proteins from high-performance ion-exchange columns. The high resolving power of the method is demonstrated by the total separation of the A and B genetic variants of the β-lactoglobulins, which have only 0.1 pH difference in their isoelectric points. The separation of up to 12.8 mg of the β-lactoglobulins on a 150-μl column demonstrates the potential for high capacity in displacement chromatography.

Purification of Escherichia coli alkaline phosphatase on an ion-exchange high-performance liquid chromatographic column using carboxymethyl dextrans

Carboxymethyl dextrans (CM-Ds) were used on an HPLC ion-exchange column to obtain significantly enriched alkaline phosphatase (EC 3.1.3.1) from a sample of Escherichia coli periplasmic space proteins without significant loss of enzymatic activity. The ability of CM-Ds to separate alkaline phosphatase even when the column was 80-85% saturated with protein demonstrates the potential for high column capacity using CM-Ds. In addition, the fractions containing alkaline phosphatase and CM-Ds were reapplied to the same ion-exchange column under different buffer conditions and purified to homogeneity by salt gradient elution chromatography, thus demonstrating the compatibility of CM-Ds with the latter chromatographic method. The two-step chromatographic procedure yielded enzyme of purity comparable to that of electrophoretically purified E. coli alkaline phosphatase obtained commercially. These studies demonstrate that HPLC displacement chromatography is a mild procedure which allows rapid, quantitative purification of an enzyme. Scaling up with larger columns should allow purification of enzymes of a commercial basis.

Purification of monoclonal antibodies by complex-displacement chromatography on CM-cellulose☆

A single high-capacity chromatographic procedure was used to purify a monoclonal antibody from ascites fluid. A new displacement procedure involving a displacer protein complex is described. The scale-up from 1 ml to 450 ml of ascites sample was easily accomplished with this complex-displacement system. A monoclonal antibody recovery of 79% was measured for the largest-scale purification.