Methods and kits for purifying his-tagged proteins

#Not applicable.

#Histidine-tagged proteins are recombinant proteins designed to include a polyhistidine tail (his-tag) that facilitates purification of the proteins from in vitro expression systems. The preferential binding of the his-tag to metal chelating resins has been exploited in purifying his-tagged proteins from undesired contaminating proteins using immobilized metal affinity chromatography (IMAC). Metal chelating resins typically include a transition metal such as Ni or Co.

#Like his-tagged proteins, heme proteins (e.g., hemoglobin or myoglobin) bind to metal chelating resins. When both his-tagged proteins and heme proteins are applied to a metal chelating resin, the heme proteins co-purify with his-tagged proteins. It is therefore difficult to separate his-tagged proteins from material containing heme proteins to obtain a preparation of his-tagged proteins of acceptable purity without a significant amount of contaminating heme proteins.

#FIG. 1 shows a fluoroimage of electrophoretically separated proteins from rabbit reticulocyte lysate.

#FIG. 2 shows a fluoroimage of electrophoretically separated proteins from rabbit reticulocyte lysate.

#FIG. 3 shows proteins from rabbit reticulocyte lysate separated by SDS-PAGE and stained with GELCODE™ Blue Stain Reagent, product No. 24592 (Pierce Chemical, Rockford, Ill.).

#The present invention provides methods and kits for separating a heme protein from a target polypeptide in a sample comprising heme protein and the target polypeptide material using zinc- or cobalt-charged solid support. Suitably, the target polypeptide includes a polyhistidine tag of from five to six histidine residues.

#The ability to separate heme proteins from a target protein of interest is particularly important in applications in which there are contaminating heme proteins, for example, when isolating proteins from reticulocyte lysate, from whole blood, or other bodily fluids or samples containing hemoglobin. Although purification schemes can be developed to separate virtually any proteins, such methods frequently require many steps and numerous reagents, are time- and labor-intensive, and are, therefore, not amenable to use in high throughput screening or assays.

#As described in the Examples below, zinc- or cobalt-charged nitrilotriacetic acid (NTA) modified siliceous-oxide coated magnetic particles contacted with a sample comprising hemoglobin and his-tagged proteins in the absence of imidazole or in the presence of low levels of imidazole (10 to 60 mM) were found to preferentially bind to the his-tagged proteins, relative to binding of heme protein to the particles, such that an increase in purity of his-tagged protein from a hemoglobin-containing starting material was achieved. The term magnetic refers to paramagnetic particles, magnetic particles as well as particles capable of being magnetized. As described in the Examples, under conditions that allow preferential binding to his-tagged protein, hemoglobin binding to zinc- or cobalt-charged solid supports is minimal, whereas his-tagged proteins are bound with sufficiently high efficiency relative to similar supports charged with nickel. The methods of the invention afford an increase in purity of his-tagged proteins, relative to hemoglobin, of 1-fold or greater. Suitably, the increase in purity is least 2-fold, 2.5-fold, 5-fold, 10-fold, 20-fold, 50-fold, or 100-fold.