Isolation Purification and Separation

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Authors: Robert Liddington & Andrey Bobkov

Introduction

Protocol describes expression and purificaton of PTB domain from tensin in bacterial culture.

Reagents

  • pET15b-His-Tensin-PTBc (aa1653-1786)
  • MW 15.5KD
  • Ext Coef 0.484 (or 7090 to get µM)

  • Binding Buffer (High Salt)

    • 5mM Imidazole
    • 500mM NaCl
    • 20mM Tris-HCl pH 7.9
    • 1mM DTT
  • Elution Buffer (Low Salt)

    • 300mM Imidazole
    • 100mM NaCl
    • 20mM Tris-HCl pH 7.9
    • 1mM DTT
  • Wash Buffer (Low Salt)

    • 30mM Imidazole
    • 100mM NaCl
    • 20mM Tris-HCl pH 7.9
    • 1mM DTT
  • Dialysis Buffer

    • 20mM Tris-HCl pH 7.9
    • 100mM NaCl
    • 1mM DTT

Procedure

Expression

  1. 100ml o/n LB/amp culture
  2. Next day seed 700ml LB/amp with 100ml o/n culture, 37°C.
  3. Grow cells until OD600 = 0.6-1.2
  4. Induce with 1mM IPTG. Grow for 3hr 37°C.
  5. Harvest 8krpm, 4 °C, 10min.
  6. Resuspend 800ml cell culture pellet in 20ml Ni-NTA binding buffer. LN2 freeze.

Purification

  1. Thaw 1L cells. Add PI tablet w/o EDTA. Add 2mM PMSF.
  2. Add 0.4mg/ml lysozyme. Rock 15-30min until thick.
  3. Add 10mM MgCl2. Add 0.4µg/ml DNaseI. Rock 15-30min until loose.
  4. Add 0.1% Tx-100. Rock 15min. Save 10µl for SDS-PAGE.
  5. Spin 16krpm, 4°C, 15min. Save some supernatant and pellet for gel.
  6. Meanwhile, wash 2ml Ni-NTA beads with binding buffer.
  7. Load supernatant onto beads.
  8. Wash with 50ml binding buffer.
  9. Wash with 50ml wash buffer low salt.
  10. Elute with elution buffer low salt.
  11. Dialize into dialysis buffer while digesting off His tag with biotinylated thrombin. Digest for 6hr RT. Capture biotinylated thrombin with streptavidin agarose.
  12. Run SDS PAGE on purification. Determine if need to do further purification on S75 or S200. Do a quick protein concentration check. Concentrate if necessary.

Author information

Robert Liddington & Andrey Bobkov, The Burnham Institute

Source: Protocol Exchange (2009) doi:10.1038/nprot.2009.41. Originally published online 5 February 2009.

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