Two protocols have been applied in our lab to purify proteins from inclusion bodies: Sarkosyl Method or Lin-Cheng Method.
Sarkosyl Method:
In this method cells lysed by sonication in the presence of lysosyme. After centrifugation pelleted protein (inclusion bodies) is solublized with 1 % sarkosyl.
Materials:
STE Buffer 100 mM NaCl
10 mM Tris-HCl pH 8.0
1 mM EDTA
STE-S STE Buffer (1 % Sarkosyl)
STE-T STE Buffer (1 % Triton X-100)
Procedure:
This procedure is written for a 150 mL culture you may scale it up or down accordingly.
1. Cells from 150 mL culture were harvested in the Beckmann J2-21 in a JA-20 rotor at 5000 rpm for 15 minutes @ 4°C.
2. Resuspend the resulting pellet in 3 mL of STE buffer.
3. Add lysozyme to a final concentration of 150 µg/mL and place on ice for 30 minutes.
4. Sonicate on ice, using a 40 second sonication and a 20 second rest period (set to 40 and use the small probe). Repeat an additional three times.
5. Aliquot into two 1.5 mL microfuge tubes.
6. Spin for 5 minutes @ 4°C on maximum. (Keep the supernatant for further analysis).
7. Resuspend the pellet in 0.1 mL of STE-S. This will require vigorous pipetting (the pellet may not be completely soluble).
8. Centrifuge for 2 minutes at 4°C and transfer the supernatant to a 1.5 mL microfuge tube.
9. Add 9 volumes of STE-T to the supernatant (to form a mixed-micelle with sarkosyl).
10. Samples can be frozen on dry ice or liquid nitorogen and stored in the -80°C freezer until required.
