Isolation Purification and Separation

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Authors: Robert Liddington & Andrey Bobkov

Introduction

Protocol describes expression and purification of SH2 tensin fragment from bacterial culture.

Reagents

  • pET15b-His-Tensin-SH2c (aa1514-1631)
  • MW 13.6KD
  • Ext Coef 0.801 (or 10930 to get µM)

  • Binding Buffer (High Salt)

    • 5mM Imidazole
    • 500mM 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
  • Elution Buffer (Low Salt)

    • 300mM Imidazole
    • 100mM NaCl
    • 20mM Tris-HCl pH 7.9
    • 1mM DTT
  • Dialysis Buffer1

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

    • 50mM Hepes pH 7.6
    • 1mM DTT
  • FPLC Buffer B

    • 50mM Hepes pH 7.6
    • 1M NaCl
    • 1mM DTT
  • Dialysis Buffer2

    • 20mM Tris-HCl pH 7.9
    • 50mM 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 4hr 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 sup and pellet for gel.
  6. Meanwhile, wash 2ml Ni-NTA beads (for 4L of SH2c cells) 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 buffer1 while digesting off His tag with biotinylated thrombin. Digest for 6hr RT. A lot of protein will ppt out of solution. Spin 40k, 4 °C, 20min. Capture biotinylated thrombin with streptavidin agarose.
  12. Dialize flow through in FPLC Buffer A.
  13. Run SDS PAGE on purification. Do a quick protein concentration check.

FPLC Purification

Use 1ml Qlinear 40mlCV FPLC program. This one gets around the computer bug that messes up the fraction collector.

Use a 1ml SP-Cation Exchange column

  1. Load 2ml protein (~3mg/ml)
  2. Elute at: Flow rate 1ml/min
    • Gradient 0-50% FPLC Buffer B
    • Elute 40min
  3. Run SDS PAGE
  4. Dialize pure peak into buffer2. Do concentration. LN2 freeze.

Troubleshooting

FPLC: If separation of contaminants is not perfect lengthen the elution or shorten the gradient.

Author information

Robert Liddington & Andrey Bobkov, The Burnham Institute

Source: Protocol Exchange (2009) doi:10.1038/nprot.2009.42. Originally published online 5 February 2009.

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