Protocol describes expression and purification of SH2 tensin fragment from bacterial culture.
Method Article
Tensin-SH2 expression and purification
https://doi.org/10.1038/nprot.2009.42
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tensin
SH2
Protocol describes expression and purification of SH2 tensin fragment from bacterial culture.
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
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 4hr 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 sup and pellet for gel.
Meanwhile, wash 2ml Ni-NTA beads (for 4L of SH2c cells) 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 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.
Dialize flow through in FPLC Buffer A.
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
Load 2ml protein (~3mg/ml)
Elute at: Flow rate 1ml/min
Gradient 0-50% FPLC Buffer B
Elute 40min
Run SDS PAGE
Dialize pure peak into buffer2. Do concentration. LN2 freeze.
FPLC: If separation of contaminants is not perfect lengthen the elution or shorten the gradient.
This protocol has been posted on Protocol Exchange, an open repository of community-contributed protocols sponsored by Nature Portfolio. These protocols are posted directly on the Protocol Exchange by authors and are made freely available to the scientific community for use and comment.
posted
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