Methods and kits for purifying his-tagged proteins
a technology for purifying kits and his-tagged proteins, which is applied in the direction of peptide/protein ingredients, extracellular fluid disorder, separation processes, etc., can solve the problems of reticulocyte lysate expression systems that have not been fully exploited for expressing his-tagged proteins, and it is difficult to separate his-tagged proteins from material containing hem
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example 1
Preparation of Zinc- or Cobalt-charged Nitrilotriacetic Acid-modified Magnetic Silica Particles
[0039] Siliceous oxide-coated magnetic particles were modified with nitrilotriacetic acid (NTA) to produce 3-[[[bis(carboxymethyl)amino]acetyl]amino]-propyl magnetic silica particles (NTA-modified magnetic silica particles), as described in U.S. application Ser. No. 10 / 689,176, filed Oct. 20, 2003, which is incorporated by reference in its entirety.
[0040] Zinc-charged particles were prepared as follows. Particles in a 4-ml aliquot of NTA-modified particles (10% w / v in water) were separated from the liquid using magnetization and the liquid discarded. The particles were contacted with 4 ml of ZnCl (100 mM) and rocked on a horizontal shaker (5-10 rpm) for 15 minutes. The particles were separated from the liquid using magnetization and the liquid discarded. The particles were contacted with a fresh 4-ml aliqout of ZnCl (100 mM), rocked on a horizontal shaker (5-10 rpm) for 15 minutes, and t...
example 2
Expression of His-tagged Proteins in Rabbit Reticulocyte Lysate Using Coupled Transcription / translation
[0042] An expression vector encoding his-tagged Renilla luciferase was expressed using TNT® T7 Quick Coupled Transcription / Translation System, Cat# L1170TNT (Promega Corp., Madison, Wis.) according to the manufacturer's instructions.
example 3
Purification of His-tagged Proteins from Rabbit Reticulocyte Lysate
[0043] His-tagged Renilla luciferase, prepared as described in Example 2, was purified from the coupled transcription / translation reaction mixture as follows. A 50-μl aliquot of the reaction mixture was combined with 150-μl binding buffer A (sodium phosphate buffer 100 mM, imidazole 20 mM, and NaCl 400 mM) buffered at a pH in the range of 6.0 to 8.0, and combined with 30 μl (3 mg) of zinc charged particles, prepared as described in Example, for 15-15 minutes at room temperature. The particles were separated from the solution (“flowthrough solution”) using magnetization, and the particles were washed with 200 μl binding buffer three to five times. Proteins were eluted by thoroughly mixing the particles with 100 μl elution buffer (HEPES 100 mM (pH 7.5) and imidazole 500 mM), separating the particles from the elution buffer by magnetization, and collecting the eluted proteins.
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