Taq-Citraconylation

Patents:  5,677,152,  6,183,998 and 6,479,264.

Citraconylation of TaqΔ279

3 mg of purified His-tagged TaqΔ279 polypeptide in 3 ml reaction buffer (50 mM HEPES, 300 mM KCl, 1 mM EDTA, pH8.5) are reacted with 2 ÎŒl of citraconic acid anhydride for 1 h at room temperature. The mixture is subsequently dialyzed against storage buffer (63% [w/v] glycerol, 100 mM KCl, 20 mM Tris HCl pH 8.5, 0.1 mM EDTA, 0.5% Tween 20, 1 mM DTT).

Citraconylation of Taq DNA Polymerase

Dialyze Taq DNA polymerase at 1.3 mg/ml  against 1000× sodium borate at pH 8.63. (This step is not critical). Taq DNA polymerase  in a Tris buffer (50 mM Tris-HCl, 1 mM EDTA, 65 mM KCl, pH 7.5) can be directly without dialysis against sodium borate.

Purchase citraconic anhydride (11.06M) from Sigma. Dilute  citraconic anhydride (11.06M )  100-fold in DMF (N,N dimethyl formamide).

Make a dilution series of the citraconic anhydride solution  by repeated 2-fold dilutions in DMF.

Add 4 ÎŒl of diluted citraconic anhydride solution to 400 ÎŒl Taq DNA polymerase solution (with sodium borate dialysis), resulting in solutions containing molar ratios of citraconic anhydride to Taq DNA polymerase of approximately 80/1, 40/1, 20/1, and 10/1. Incubate the solutions overnight at 40° C. to inactivate the Taq DNA polymerase. The resulting citraconylated Taq DNA polymerases are referred to herein as 80×, 40×, 20×, and 10× Taq DNA polymerases.

Inactivation and Heat Recovery of DNA Polymerase Activity using Citraconic Anhydride

This example describes activity measurements of the citraconylated Taq DNA polymerases of Example 2 both before and after re-activation of citraconylated Taq DNA polymerase by heat incubation. The effect of pH on the amount of activity recovered following heat reactivation of the citraconylated Taq DNA polymerases was measured.

Dilute samples of citraconylated Taq DNA polymerase 1/200 in a buffer consisting of 10 mM Tris-HCl, 100 mM KCl, 2 mM MgCl 2 , 0.5% Tween 20, 0.5% NP-40, 16% glycerol. (pH 8.25 at room temperature). Diluted samples of citraconylated Taq DNA polymerase were incubated at 90° C. for 20 minutes or maintained at room temperature as a control. Following treatment, samples were diluted 1/5 in enzyme dilution buffer (25 mM Tris-HCl, 50 mM KCl, 1 mM β-mercaptoethanol, 0.5% Tween 20, 0.5% NP-40, 0.1% gelatin) and the activity was assayed as described in Example 1. The DNA polymerase activities following treatment am shown below. The molar ratio refers to the molar ratio of citraconic anhydride to Taq DNA polymerase used in the modification reaction. Each of the activities is the average two activities measured from duplicate samples.
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Activity (% of control) molar ratio unheated 90° C. incubation
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Control 100
80X 0 16
40X 0 28
20X 3.7 38
10X 38 63
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80× (or higher) citraconylated Taq DNA polymerase in a commercial kit to allow greater manufacturing tolerances.

Similar experiments were carried out using a buffer adjusted to pH 7.75 at room temperature. The results are shown below.
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Activity (% of control) molar ratio unheated 90° C. incubation
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Control 100
80X 0 61
40X 0 67
20X 3.570
10X 35 77
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The amount of DNA polymerase activity recovered was greater at lower pH.

The activities of the 80× and 160× Taq DNA polymerases (without sodium borate dialysis) were measured before and after reactivation by heat incubation essentially as described above. The buffer pH used for the incubations was 8.0 at room temperature. The results am shown below. Each of the activities is the average two activities measured from duplicate samples.
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Activity (% of control) molar ratio unheated 90° C. incubation
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Control 100
160X 0 19
80X 0 29
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Amplifications were carried out using 1/20, 1/40, and 1/80 dilutions of the 240× modified Taq DNA polymerase 

Dilutions were made in a buffer consisting of 20 mM Tris-HCl, pH 8.0 (at room temperature), 100 mM KCl, 0.1 mM EDTA (ethylenediaminetetraacetic acid), 1 mM DTT (dithiothreitol), 50% glycerol, 0.5% Tween 20, 0.5% Nonidet P40 (AmpliTaq®storage buffer, Perkin Elmer, Norwalk, Conn.). For comparison, amplifications also were carried out using 1/10, 1/20, 1/40, and 1/80 dilutions of unmodified Taq DNA polymerase.

EXAMPLE 4

Activity of TaqΔ288

DNA polymerase activity is determined using standard procedures by first hybridizing an M13 primer to M13 mp9ss DNA at 65° C. and then incorporate radioactively labeled α32dCTP. Incorporation is measured by liquid scintillation counting. The activity is compared to a master lot of TaqWT preparation which serves as a reference. Table 2

TABLE 2
Specific activities of polypeptides with DNA polymerase activity
TaqWT ~100 kU/mg
TaqΔ288  ~77 kU/mg
TaqΔ279 ~370 kU/mg
TaqΔ290 −111 kU/mg

Regarding the presence or absence of the His tag in the TaqΔ288 polypeptide no differences regarding DNA polymerase activity have been detected.

Citraconylation of TaqΔ288

3 mg of purified His-tagged TaqΔ288 polypeptide in 3 ml reaction buffer (50 mM HEPES, 300 mM KCl, 1 mM EDTA, pH8.5) are reacted with 2 μl of citraconic acid anhydride for 1 h at room temperature. The mixture is subsequently dialyzed against storage buffer (63% [w/v] glycerol, 100 mM KCl, 20 mM Tris HCl pH 8.5, 0.1 mM EDTA, 0.5% Tween 20, 1 mM DTT).

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Chemical modification of Taq DNA polymerase:

This example describes the chemical modification of Taq DNA polymerase using cis-aconitic anhydride. Recombinant Taq DNA polymerase (Taq) purified from E. coli was used at a concentration of 1.22 mg/mL in 50 mM Tris, 300 mM KCl, 1 mM EDTA, 0.2% Tween 20, pH 8.0 at 25° C.

For the modification reaction, the cis-aconitic anhydride was solved in methanol in a final concentration of 22.0 mg/mL. 1061 μL of cis-aconitic anhydride solution were added to 106.1 mL Taq DNA polymerase solution. The solution was incubated overnight at 4° C. to inactive the Taq. After the chemical modification the enzyme was dialyzed against a storage buffer (20 mM Tris, 100 mM KCl, 0.1 mM EDTA, 1 mM DTT, 0.2% Tween 20, 50% glycerol, pH 9.0 at 25° C.). After dialysis the sample was diluted 1:29 with the storage buffer. For the chemically modified enzyme the term FastStart Taq DNA Polymerase (FS Taq) was used.

The activity of the FS Taq was measured as described in example IV. The enzyme was incubated at 80° C. for three hours to activate the polymerase activity. The enzyme was diluted (1:300-1:700) in storage buffer and the activity was determined as described below. Enzyme samples that were not incubated at 80° C. for three hours showed no activity (<1%).