Protein expression with unnatural amino acids in Pseudomonas aeruginosa

Protein expression including unnatural amino acid in Pseudomonas aeruginosa PAO1 and cells lysis

Materials and Equipment

1. Pseudomonas aeruginosa PAO1 harboring expression plasmid*.

2. Sterile LB Miller.

3. Proper antibiotics corresponding to plasmid selection.

4. Unnatural amino acid for incorporation.

5. Bacterial incubator set for 37^oC.

6. 100 mM phosphate buffer (PB) (pH 7).

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.

12. Cooled centrifuge at 10000 g.

*  The expression plasmid requires several genetic elements, such as in the pPaGE plasmid (addgene plasmid #160089). Apart from a Pseudomonas aeruginosa-responsive selection gene, an origin of replication and expression promoter (such as endogenous promoter fliC), the plasmid requires a Methanosarcina mazei pyrrolysyl orthogonal translation system.

Regarding the expression gene aimed for unnatural amino acid incorporation, the reading-frame should contain a TAG mutation in the desired location for incorporation.

Procedure

1. Prepare LB Miller 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 another 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, sediment 1 mL of culture and wash with 1 mL of 100 mM phosphate buffer (PB) (pH 7).

5. Sediment 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 reaction, SDS-PAGE, fluorescent imaging, and Western blot.