Editing Taq377 Purification (section)

== Expression and Purification of Polypeptides with DNA Polymerase Activity ==

A total volume of 41 of dYT medium, split into 800 ml aliquots in 21 flasks are inoculated with E. coli strain XL-1 Blue transformed with the pQE80L expression vector (Qiagen catalogue no. 32923) in which the reading frame encoding TaqΔ288, TaqΔ279 (Klentaq), the Stoffel fragment (TaqΔ290), and TaqWT has been inserted. Cells are grown in a shaking culture at 37° C. (shaking incubator, 250 rounds per minute). At an optical density of about 0.8 (measured at 600 nm) IPTG (Isopropylthiogalactoside) is added to a final concentration of 500 μM. Cells are further grown until an optical density of between 2 and 3 is reached. The cells are then sedimented by centrifugation and the pellet is resuspended in B50 buffer (25 mM Tris HCl pH 8.5, 0.1 mM EDTA, 5% glycerol, 1 mM DTT (dithiothreitol), 50 mM NaCl) at room temperature.

Purification Protocol 1

The cells are lysed using a French Press twice at 1,000 bar. NaCl is added to 1.5 M to dissolve polymerases from the DNA. Subsequently, the mixture is heated to 75° C. for 15 min on a water bath. Subsequently, the mixture is left in the water bath without further heating for another 15 min. The precipitate is separated from the supernatant by centrifugation. The supernatant is dialyzed against B100 buffer (25 mM Tris HCl pH 8.5, 0.1 mM EDTA, 5% glycerol, 1 mM DTT, 100 mM NaCl). Subsequently, chromatographic steps are performed. The first step uses a heparin sepharose column and elution of the TaqΔ288 polypeptide by means of gradient elution with B100 and B600 (25 mM Tris HCl pH 8.5, 0.1 mM EDTA, 5% glycerol, 1 mM DTT, 600 mM NaCl). Following dialysis of the TaqΔ288-containing fraction against B50 the enzyme is further purified using a Q sepharose column and for elution a gradient with B50 and B250 (25 mM Tris HCl pH 8.5, 0.1 mM EDTA, 5% glycerol, 1 mM DTT, 250 mM NaCl). Finally the fraction with the eluted TaqΔ288 is dialyzed against storage buffer (50% [v/v] glycerol, 100 mM KCl, 20 mM Tris HCl pH 8.5, 0.1 mM EDTA, 1 mM DTT, 0.25% Thesit).

Purification Protocol 2

Purification is facilitated if the His-tag is used during purification. 5 g sedimented cells are resuspended in 25 ml lysis buffer (50 mM NaH2PO4, 300 mM NaCl, 10 mM imidazole, 0.1 mM PMSF (phenylmethylsulfonyl fluoride), 1 mM DTT, pH 8.0) and lysed using ultrasound. DNase I (Roche Diagnostics GmbH, Mannheim) is added to a final concentration of 20 mg/ml and MgCl2 to a final concentration of 4 mM. The mixture is incubated at 25° C. for 30 min. Following heat incubation for 30 min at 72° C. the precipitate is sedimented by centrifugation (22,000×g). The cleared supernatant is loaded onto a 10 ml Ni-NTA superflow column (Qiagen). The column is washed with two volumes of buffer A (50 mM NaH2PO4, 300 mM NaCl, 10 mM imidazole, pH 8.0). The column is eluted with a volume of 100 ml of a linear gradient, starting with buffer A and ending with buffer B (50 mM NaH2PO4, 300 mM NaCl, 250 mM imidazole, pH 8.0). The His-tagged TaqΔ288 polypeptide is further purified by anion exchange chromatography with Q sepharose. Finally, the fraction containing the purified His-tagged TaqΔ288 polypeptide is dialyzed against storage buffer.

In order to remove the His tag, the Ni-NTA purified His-tagged TaqΔ288 polypeptide is dialyzed against Xa reaction buffer (20 mM Tris HCl pH 6.5, 50 mM NaCl, 1 mM CaCl2). Factor Xa protease (1 U per mg TaqΔ288 polypeptide) is added together with 1/10 of the total reaction volume of 10× concentrated reaction buffer. The mixture is incubated for 3 to 6 days at 4° C. Subsequently, the pH is raised to 8.0 by adding 1 M Tris HCl pH 8.5 (about 1/100 volume). Further, equilibrated Ni-NTA superflow material is added and the mixture is incubated for 30 min at room temperature under continuous agitation (rotation). The supernatant is separated from the Ni-NTA superflow material with uncleaved His-tagged TaqΔ288 polypeptide and heat-inactivated at 72° C. at 30 min. The following steps are anion exchange chromatography with Q sepharose, followed by dialysis against storage buffer.<br>

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[[Category:Protein_Purification]] [[Category:Protocols]]