Paul.xu at 21:57, 16 March 2012

2012-03-16T21:57:07Z

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== '''Protocol 1''' ==

1. Grow Rosetta BL21(DE3) pLysS transformed with pET30b-sso7d-Taq_d289 (377) in 6L LB Kan, at 37 oC shaking 180 rpm. 

2. Induce with 1 mM IPTG for 3~ 4 hours at 37oC (add 1 ml 1 M IPTG to 1 L culture). 

3. Spin down cells, 6000 rpm for 5 min. 

4. Using DI water, transfer E coli cells to 50 ml VWR tubes, spin down cells, 6000 rpm for 5 min. 

5. Freeze at -20 oC (store cells at -20 oC if not use right away). 

6. Add 200 ml bacterial lysis '''Buffer A''' containing 10 mM imidazole. 

7. Lyse cells by thawing at room temperature. 

8. Transfer cells to a beaker, homogenize gel like stuff. 

9. Sonicate if it is still sticky. 

10. Boil at 75 oC for 30 min, cool to 4 oC on ice. 

11. Spin down pellet at 12000 rpm for 20 min at 4 oC. 

12. Collect supernatant, spin again at 25000 or 30000 rpm for 20 min at 4 oC. 

13. Collect cleared supernatant. Load supernatant to 10 ml Ni column. 

14. Wash with '''washing buffer''' until there is no detectable protein (10 ÎŒL eluate + 75 ÎŒL Bradford reagent). 

15. Wash with '''washing buffer II''' until there is no detectable protein (10 ÎŒL eluate + 75 ÎŒL Bradford reagent). 

16. Elute Taq with elution '''buffer B'''. Collect 5 ml fractions. Check each fraction by SDS-PAGE. 

17. Combine relatively pure fractions.  Concentrate protein using 10 K MWCO spin column. 

18. Load 5 ml Taq to HiLoad 16/60 Superdex 200 PreGrade column, equilibrated with '''GF Buffer'''. 

19. Elute with GF buffer. Collect 3 ml per fraction. 

20. Use an ELISA plate, check fractions containing Taq protein using 75ÎŒL Bradford reagent. 

21. Take 10 ~ 15 ul to run SDS-PAGE to locate fractions containing Taq. 

22. Combine fractions containing Taq, dialyse against '''Storage Buffer A''', dialyse 3 X 75 ml, 3 hour each, then 75 ml overnight. 

22. Measure protein concentration. Dilute to ~ mg/ml with '''Storage Buffer A + 1/100 Storage Buffer B (ratio of A:B is 100:1).''' Use Invitrogen Taq and Qiagen Taq  as "reference standard" to estimate specific activity. Dilute protein to 5 units/ÎŒL. 

'''Buffers:'''

Buffer A: 10 mM Imidozale, 20 mM Tris-HCl pH 8.0, 600 mM NaCl

Buffer B: 300 mM Imidozale, 20 mM Tris-HCl pH 8.0, 600 mM NaCl

Washing Buffer: 20 mM Imidozale, 20 mM Tris-HCl pH 8.0, 600 mM NaCl

Washing Buffer II: 50 mM Imidozale, 20 mM Tris-HCl pH 8.0, 600 mM NaCl 

GF buffer: 20 mM Tris-HCl (pH 8.0), 0.1 mM EDTA, 100 mM NaCl. 

'''Storage Buffer A:''' 

Tris-HCl, pH 8.0, 100 mM NaCl, 0.1 mM EDTA,  50% glycerol 

 

'''Storage Buffer B:''' 

100 mM DTT, 10% Tween20, 10% Nonidet P-40 

 

'''DO NOT USE the following protocols.'''

== '''Purification Protocol 2 ''' ==

1. Lyse cells. 

2. Add NaCl to 1.5 M to dissolve polymerases 

3. Heat the mixture  75° C for 30 min on a water bath. 

4. Spin 

5. Dialyze against B100 buffer (25 mM Tris HCl pH 8.5, 0.1 mM EDTA, 5% glycerol, 1 mM DTT, 100 mM NaCl). 

6.  Purify on a heparin sepharose column using 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). 

7. Dialysis Taq279-containing fraction against B50. 

8.  Purify on 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). 

9. Dialyse Taq279 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 3''' ==

1. Suspend 5 g sedimented cells 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) 

2. lyse by freeze-thaw then ultrasound. 

3. Add DNase I to a final concentration of 20 mg/ml and MgCl2 to a final concentration of 4 mM. 

4. Incubate  at 25° C. for 30 min. 

5. Heat incubation for 30 min at 72° C. 

6. Spin (22,000g). 

7. Collect supernatant. 

8. Load to a 10 ml Ni-NTA superflow column (Qiagen). 

9. Wash column  with two volumes of buffer A (50 mM NaH2PO4 , 300 mM NaCl, 10 mM imidazole, pH 8.0). 

10. Eluate Taq 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). 

11. The His-tagged Taq279 is further purified by anion exchange chromatography with Q sepharose. 

12. Dialyse the fraction containing the purified His-tagged Taq279 polypeptide 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). 

[[|]] 

 

1. 10X Taq Buffer with KCl 100mM Tris-HCl (pH 8.8 at 25°C), 500mM KCl, 0.8% Nonidet P40.

2. 10X Taq Buffer with KCl and 15mM MgCl2 100mM Tris-HCl (pH 8.8 at 25°C), 500mM KCl, 0.8% Nonidet P40, 15mM MgCl2.

3. 10X Taq Buffers with KCl Set 100mM Tris-HCl (pH 8.8 at 25°C), 500mM KCl, 0.8% Nonidet P40, MgCl2 different in each buffer (12.5-30mM)

4. 10X Taq Buffer with (NH4)2SO4 750mM Tris-HCl (pH 8.8 at 25°C), 200mM (NH4)2SO4, 0.1% Tween 20.

5. 10X Taq Buffer with (NH4)2SO4 and 20mM MgCl2 750mM Tris-HCl (pH 8.8 at 25°C), 200mM (NH4)2SO4, 0.1% Tween 20, 20mM MgCl2.

6. 10X Taq Buffer without Detergent 100mM Tris-HCl (pH 8.8 at 25°C), 500mM KCl.

7. 10X Pfu Buffer with 20mM MgSO4 200mM Tris-HCl (pH 8.8 at 25°C), 100mM (NH4)2SO4, 100mM KCl, 1% (v/v) Triton X-100, 1mg/ml BSA and 20mM MgSO4.

8. 10X Pfu Buffer 200mM Tris-HCl (pH 8.8 at 25°C), 100mM (NH4)2SO4, 100mM KCl, 1% (v/v) Triton X-100, 1mg/ml BSA 

 

== 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. 

 

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

Taq377 Purification - Revision history

Paul.xu at 21:57, 16 March 2012