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  Methods and kits for purifying his-tagged proteinsUSPTO Application #: 20050209129Title: Methods and kits for purifying his-tagged proteins Abstract: Disclosed are methods of separating a target molecule from a non-target molecule using zinc- or cobalt-charged solid supports. (end of abstract)
Agent: Michael Best & Friedrich, LLP - Madison, WI, US Inventors: Tonny Johnson, Rebecca Godat, Laurie Engel USPTO Applicaton #: 20050209129 - Class: 514006000 (USPTO) Related Patent Categories: Drug, Bio-affecting And Body Treating Compositions, Designated Organic Active Ingredient Containing (doai), Peptide Containing (e.g., Protein, Peptones, Fibrinogen, Etc.) Doai, Heavy Metal Containing (e.g., Hemoglobin, Etc.) The Patent Description & Claims data below is from USPTO Patent Application 20050209129. Brief Patent Description - Full Patent Description - Patent Application Claims
CROSS-REFERENCE TO RELATED APPLICATIONS [0001] This application claims priority to U.S. Provisional Patent Application Ser. No. 60/502,544, filed Sep. 12, 2003, that application being incorporated herein by reference, in its entirety. STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT [0002] Not applicable. INTRODUCTION [0003] 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. [0004] 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. [0005] Rabbit reticulocyte lysate is a particularly useful expression system for obtaining expression of eukaryotic sequences. Rabbit reticulocyte lysate-based systems have been found to support co-translational and post-translational modifications of expressed proteins. However, because reticulocyte lysate includes large concentrations of hemoglobin, and because of the difficulties associated with separating hemoglobin from his-tagged proteins, reticulocyte lysate expression systems have not been fully exploited for expressing his-tagged proteins. [0006] Lytton et al. (WO 02/37100 A2) discloses that removal of hemoglobin from his-tagged proteins produced in a rabbit reticulocyte lysate may be effected by first binding the hemoglobin and his-tagged proteins to a nickel nitrilotriacetic acid (Ni-NTA) resin in the presence of imidazole, followed by step-wise elution of hemoglobin and his-tagged proteins using an imidazole gradient. [0007] There is a need in the art for simplified methods of separating heme proteins from his-tagged proteins that are amenable for use in high throughput systems. SUMMARY OF THE INVENTION [0008] In one aspect, the present invention provides methods for separating heme proteins from his-tagged protein in a starting material. The starting material is contacted with a zinc- or cobalt-charged solid support under conditions in which the support preferentially binds to his-tagged protein relative to its binding to hemoglobin. The conditions include no imidazole or imidazole in a concentration of from about 0 mM to about 60 mM. Suitably, imidazole may be present in a concentration of about 10 to about 40 mM, or from about 10 mM to about 20 mM. [0009] In another aspect, the present invention provides kits for separating heme proteins from his-tagged proteins in a starting material. The kits include a zinc- or cobalt-charged solid support and a binding buffer that comprises no imidazole or imidazole in a concentration of from about 10 mM to about 60 mM. Optionally, the kits may further comprise an elution buffer comprising imidazole in a concentration from about 100 mM to about 3 M. In an alternative embodiment, the kits comprise an elution buffer comprising EDTA in a concentration of from about 10 mM to about 0.5 M, most suitably about 50 mM, or an elution buffer having a pH of less than about 6. The kits may further comprise instructions for performing the method according to the present invention. BRIEF DESCRIPTION OF THE FIGURES [0010] FIG. 1 shows a fluoroimage of electrophoretically separated proteins from rabbit reticulocyte lysate. [0011] FIG. 2 shows a fluoroimage of electrophoretically separated proteins from rabbit reticulocyte lysate. [0012] FIG. 3 shows proteins from rabbit reticulocyte lysate separated by SDS-PAGE and stained with GelCode.TM.. [0013] FIG. 4 shows Western blot of electrophoretically separated proteins from rabbit reticulocyte lysate probed with anti-renilla luciferase antibody. [0014] FIG. 5 shows a Western blot of proteins from rabbit reticulocyte lysate electrophoretically separated by SDS-PAGE and probed with anti-hemoglobin antibody. [0015] FIG. 6 shows a fluorescent scan of electrophoretically separated proteins. [0016] FIG. 7 is an image of electrophoretically separated proteins. [0017] FIG. 8 is a Western blot of electrophoretically separated proteins. [0018] FIG. 9 is an image of electrophoretically separated proteins. [0019] FIG. 10 is an image of electrophoretically separated proteins. Continue reading... Full patent description for Methods and kits for purifying his-tagged proteins Brief Patent Description - Full Patent Description - Patent Application Claims Click on the above for other options relating to this Methods and kits for purifying his-tagged proteins patent application. ###  How KEYWORD MONITOR works... a FREE service from FreshPatents1. Sign up (takes 30 seconds). 2. Fill in the keywords to be monitored. 3. Each week you receive an email with patent applications related to your keywords. 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