Generation of recombinant protein

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Mammalian TAP kit

Intein fusion kit

[edit]

Purification of intein fusion from E. coli

Purify recombinant proteins under a native condition using chitin affinity chromatography and induced on column cleavage of intein acyl shifted intermediate.

  1. Inoculate 50 mL 2X YT media (+Amp)
  2. Seed 2 2L flask containing 1L 2X YT media (+Amp, +Cm) with 25mL of the O/N inoculum
  3. 37°C shaker
  4. at OD = 1.0, induced with 1mM IPTG (1mL 1M IPTG per 1L of culture)
  5. 3 hours @ 30°C shaker or 6 hours at RT
  6. harvest cells by centrifugation in J6 12 min @ 4K rpm
  7. Wash pellet with 25mL 1X PBS
  8. Transfer to 50mL conical
  9. Spin in J6 4K rpm 12min
  10. Store cell pellet in –80°C
  11. Thaw cell pellet on ice
  12. Resuspend the pellet in 25mL A500 (10mM Tris pH 8.0; 500mM NaCl; 0.1% NP-40 + protease inhibitors) with 0.5mg/mL Lysozyme
  13. Lyse cells by sonication (3 times at setting 7 40 seconds each time on a Branson sonifier)
  14. Clarify lysate by centrifugation at 12,000 rpm for 30 min (in oakridge tubes)
  15. Equilibriate 3mL chitin-agarose with A500
  16. Bind in batch supernatant with resin 2 hours.
  17. Wash the resin 4X with 50mL A500
  18. 10mL of A150 + 200mM DTT + protease inhibitor at 4°C O/N
  19. 10% SDS-PAGE - 20uL sample + 10uL 2X SB loaded
[edit]

Purification of native his tag fusion from E. coli

  1. Inoculate 50 mL 2X YT media (+Amp)
  2. Seed 2 2L flask containing 1L 2X YT media (+Amp, +Cm) with 25mL of the O/N inoculum
  3. 37°C shaker
  4. at OD = 1.0, induced with 1mM IPTG (1mL 1M IPTG per 1L of culture)
  5. 3 hours @ 30°C shaker or 6 hours at RT
  6. harvest cells by centrifugation in J6 12 min @ 4K rpm
  7. Wash pellet with 25mL 1X PBS
  8. Transfer to 50mL conical
  9. Spin in J6 4K rpm 12min
  10. Store cell pellet in –80°C
  11. Thaw cell pellet on ice
  12. Resuspend the pellet in 25mL A500 (10mM Tris pH 8.0; 500mM NaCl; 0.1% NP-40 + protease inhibitors) with 0.5mg/mL Lysozyme
  13. Lyse cells by sonication (3 times at setting 7 40 seconds each time on a Branson sonifier)
  14. Clarify lysate by centrifugation at 12,000 rpm for 30 min
  15. Equilibriate 2mL 50% Ni-NTA resin with A500
  16. Bind in batch supernatant with resin 2 hours.
  17. Wash the resin 4X with 50mL A500
  18. transfer resin to econocolumn
  19. wash column with 10mL A150 (10mM Tris pH 8.0; 150mM NaCl; 0.1% NP-40 + protease inhibitors)
  20. elute 1mL fractions with A150 + 100mM imidazole + protease inhibitor - collect 10 fractions
  21. analyze eluted fractions by BCA and 10% SDS-PAGE - 20uL sample + 10uL 2X SB loaded
  22. pool peak fractions and dialyze > 4 hrs in 25mM Hepes; pH8, 150mM NaCl; 5mM DTT; 20% glycerol at 4°C
[edit]

Purification of denatured his tag fusion from E. coli

  1. Inoculate 50 mL 2X YT media (+Amp)
  2. Seed 2 2L flask containing 1L 2X YT media (+Amp, +Cm) with 25mL of the O/N inoculum
  3. 37°C shaker
  4. at OD = 1.0, induced with 1mM IPTG (1mL 1M IPTG per 1L of culture)
  5. 3 hours @ 30°C shaker or 6 hours at RT
  6. harvest cells by centrifugation in J6 12 min @ 4K rpm
  7. Wash pellet with 25mL 1X PBS
  8. Transfer to 50mL conical
  9. Spin in J6 4K rpm 12min
  10. Store cell pellet in –80°C
  11. Thaw cell pellet on ice
  12. Resuspend the pellet in 25mL A150U (10mM Tris, pH 8.0; 150mM NaCl; 8M Urea +protease inhibitors) with 0.5mg/mL Lysozyme
  13. Lyse cells by sonication (3 times at setting 7 40 seconds each time on a Branson sonifier)
  14. Clarify lysate by centrifugation at 12,000 rpm for 30 min
  15. Equilibriate 2mL 50% Ni-NTA resin with A150U
  16. Bind in batch supernatant with resin 2 hours.
  17. Wash the resin 4X with 50mL A150U
  18. transfer resin to econocolumn
  19. wash column with 10mL A150U (10mM Tris pH 8.0; 150mM NaCl; 0.1% NP-40 + protease inhibitors)
  20. elute 1mL fractions with A150U + 100mM imidazole + protease inhibitor - collect 10 fractions
  21. analyze eluted fractions by BCA and 10% SDS-PAGE - 20uL sample + 10uL 2X SB loaded
  22. pool peak fractions
  23. step dialyze the sample in 25mM Hepes; pH8, 150mM NaCl; 1mM DTT with progressive lower Urea concentration.
    1. dialyze sample in 500mL 6M for 2hours - O/N
    2. add 250mL fresh buffer without Urea to [Urea]=4M and dialyze for 2 hours
    3. add 250mL fresh buffer without Urea to [Urea]=3M and dialyze for 2 hours
    4. transfer the sample to 500mL fresh buffer with 2M Urea and dialyze for 2 hours
    5. add 500mL fresh buffer without Urea to [Urea]=1M and dialyze for 2 hours
    6. transfer the sample to 500mL fresh buffer without Urea but with 10% glycerol and dialyze 4hours - O/N
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