Protein expression in PAO1 and cells lysis

Proteins expression with an incorporated unnatural amino acid in Pseudomonas aeruginosa PAO1 and cells lysis

Written by Dr. Eden Ozer

Materials and Equipment

1. Pseudomonas aeruginosa PAO1 harboring expression plasmids.
2. Sterile LB Miller broth.
3. Proper antibiotics corresponding to plasmid selection markers.
4. Unnatural amino acid for incorporation.
5. Bacterial incubator set for 37^oC.
6. 100 mM phosphate buffer (PB) (pH 7.0).
7. BugBuster 10X protein extraction reagent (Merck, Billerica, MA, USA).
8. Turbonuclease (Sigma-Aldrich, St. Louis, MO, USA).
9. lysozyme (Sigma-Aldrich, Rehovot, Israel).
10. Protease inhibitor (Merck, Darmstadt, Germany).
11. Room temperature shaker/incubator.
12. Cooled centrifuge at 10000 g.

Procedure

1. Prepare sterilized LB Miller broth with proper antibiotics.
2. Grow initial culture of PAO1 harboring plasmid at 37°C for 24 hours.
3. Dilute the initial culture 1:100 for additional 24 hours of growth at 37^oC. In case of unnatural amino acid incorporation, supplement culture with a final concentration of 1 mM unnatural amino acid.
4. Following 24 hours, precipitate 1.0 mL of culture and wash with 1.0 mL of 100 mM phosphate buffer (PB) (pH 7.0).
5. Precipitate the cells once again and resuspend with 100 μL of lysis solution composed of 90% (v/v) PB, 10% (v/v) BugBuster 10X protein extraction reagent (Merck, Billerica, MA, USA), Turbonuclease (Sigma-Aldrich, St. Louis, MO, USA), lysozyme (Sigma-Aldrich, Rehovot, Israel), and Protease inhibitor (Merck, Darmstadt, Germany).
6. Incubate cells with lysis solution for 30 min. at room temperature while shaking.
7. Once lysis is done, centrifuge at 4^oC 10,000 g for 10 min.
8. Supernatant contains soluble protein fraction and is considered as lysate.
9.  Lysate was used for downstream analyses in the form of click reactions to a fluorophore, SDS-PAGE, fluorescent imaging, and Western blotting using anti-His antobody.