Use of anionic denaturing detergents to purify insoluble proteins after overexpression

BMC Biotechnology

Table 2 Summary of the purification procedure

Step	Description	Purpose	Comments
1	Growth, induction and harvest of induced cells	Overexpression of target protein in E. coli	As described elsewhere [23]
2	Resuspension in 1% SDS in PBS	Lysis of cells	Works fine in 1/20 to 1/10 of the original culture volume. Other buffers also work (50 mM Tris/Cl pH 7.0, 100 mM NaCl and 1% SDS)
3	Sonication	Solubilisation of proteins from inclusion bodies	Until solution turns clear. Often faster than 2 minutes.
4	Incubation on ice for 30 minutes	Precipitation of SDS	Precipitation of SDS apparent after 5 minutes. 1 h incubations on ice possible.
5	Centrifugation	Removal of precipitated SDS	SS34 rotor, 13 krpm, 20 minutes, 4°C
6	Ni/NTA affinity purification	Capture and washing of hexahistidine tagged target protein	See M&M for buffer compositions and details.
7	Elution in 0.1% Sarkosyl	Elution in a dialyzable detergent that is compatible with refolding [24]	Other detergents could be used but were not tested.

Key steps in the procedure are described and their purpose briefly explained. The comments are based on the experiments described herein and additional purifications using the same principle but with slightly different protocols (altered buffers, volumes and incubation time on ice, data not shown).

ISSN: 1472-6750