Expression

1. Prepare the one sterile 15mL centrifuge tube with 5mL selective LB medium for each transformed expression strain.

Remember to include E/I₀N cultures for cleavage!

2. Incubate the cultures at 37°C overnight.

3. Use each culture to seed 500mL of selective TB medium in a 1L erlenmeyer flask.

4. Continue culturing at 37°C until the optical density (λ=600nm) reaches approximately 0.8.

This step should take approximately 3-4 hours.

5. After the OD600 reaches ~0.8, induce expression using IPTG at a final concentration of 0.1mM.

See IPTG Induction for calculations.

6. Continue culturing at 37°C for 3 hours.

Purification

7. Harvest cells by centrifugation (2,700xg, 4°C, 10min).

At this point cells may be frozen at -80°C for storage and later extraction.

Fong et al. 2009 cites ~1g wet cell paste at this step, however E/I₀C and E/I₀N were purified separately.

8. Resuspend cells in 20%(w/v) "low-salt lysis buffer".

At this point, E/I₀C and E/I₀N cells can be combined into a single lysis reaction!

Shi et al. cites E/I₀N:EI0C protein cleavage of the both the separate and combined lysis/purification steps at a 2:1 molar ratio.

9. Lyse cells through 2-4 pulses of sonication at 0.5 W RMS for 20s each.

10. Centrifuge lysate solutions (2,100xg, 4°C, 10min). Transfer supernatant to a fresh tube.

11. Precipitate the ELP fusion proteins by adding ammonium sulfate to a final concentration of 0.4M.

Fong et al. 2009 cites 200 μL clarified cell lysate at this step, however E/I₀C and E/I₀N were purified separately.

12. Incubate for 10 min at room temperature.

13. Recover precipitate via centrifugation (1,600xg, room temperature, 6min). Discard supernatant.

14. Again, dissolve the precipitate in "low-salt lysis buffer" and repeat steps 11-13.

This step should completely remove remaining contaminant host cell proteins.

15. Dissolve the precipitate in "low-pH cleavage buffer"

Fong et al. 2009 cites 150 μL "ice-cold" cleavage buffer at this step, however E/I₀C and E/I₀N were purified separately.

16. If performing separate E/I₀C & E/I₀N purifications, combine them now.

17. Incubate clevage reaction at room temperature for ~7 h.

At this point, the target expression product should be cleaved from the E/I₀C fusion proteins.

The reaction can be incubated overnight to increase yield.

18. Precipitate the ELP fusion proteins by adding ammonium sulfate to a final concentration of 0.4M.

Precipitation product should contain only E/I₀N and E/I₀C(sans target product).

19. Incubate for 10 min at room temperature.

20. Centrifuge sample (1,600xg, room temperature, 6min). Transfer supernatant to fresh tube.

21. Store at 4°C for short term or -20°C long term.

If storing long term, may want to supplement solution with glycerol etc.