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
- 100ml o/n LB/amp culture
- Next day seed 700ml LB/amp with 100ml o/n culture, 37°C.
- Grow cells until OD600 = 0.6-1.2
- Induce with 1mM IPTG. Grow for 3hr 37°C.
- Harvest 8krpm, 4 °C, 10min.
- Resuspend 800ml cell culture pellet in 20ml Ni-NTA binding buffer. LN2 freeze.
Purification
- Thaw 1L cells. Add PI tablet w/o EDTA. Add 2mM PMSF.
- Add 0.4mg/ml lysozyme. Rock 15-30min until thick.
- Add 10mM MgCl2. Add 0.4µg/ml DNaseI. Rock 15-30min until loose.
- Add 0.1% Tx-100. Rock 15min. Save 10µl for SDS-PAGE.
- Spin 16krpm, 4°C, 15min. Save some supernatant and pellet for gel.
- Meanwhile, wash 2ml Ni-NTA beads with binding buffer.
- Load supernatant onto beads.
- Wash with 50ml binding buffer.
- Wash with 50ml wash buffer low salt.
- Elute with elution buffer low salt.
- Dialize into dialysis buffer while digesting off His tag with biotinylated thrombin. Digest for 6hr RT. Capture biotinylated thrombin with streptavidin agarose.
- 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.