TITLE: Periplasmic Bacterial Expression and Purification of Elastin-like Polymers - high pH
AUTHORS: Eva Rose Balog

[DESCRIPTION]
This protocol describes the expression and purification procedures used in the Balog lab at the University of New England for producing recombinant elastin-like polymers (ELPs) in E. coli. It has been adapted from protocols used in the laboratory of Jennifer S. Martinez at Los Alamos National Laboratory, where it was originally developed based on the method of Hassouneh, Wafa et al. “Elastin-like polypeptides as a purification tag for recombinant proteins” Current protocols in protein science vol. Chapter 6 (2010): Unit 6.11. 

This fork of the original protocol has been modified to carry out the purification at pH 11.2 to decrease the transition temperature of basic ELPs. 

[GUIDELINES]
The protocol workflow is as follows: 

Day 1. Transformation 
Day 2. Growth 
Day 3. Harvest cells and begin purification 
Day 4. Finish purification and dialyze protein
Day 5. Freeze-dry protein
Day 6. Store dried protein 

A note about scale: 
This protocol is written for the growth of 1 L of final culture. Volumes can generally be scaled up or down proportionally. 

A note about expression conditions: 
The expression conditions reported here are generally a useful starting point, but each combination of ELP gene, vector backbone, and bacterial strain is unique. When starting work on a new construct, we always recommend performing small-scale test expressions to optimize choice of media, expression time, growth temperature, and IPTG induction methods. We have seen reproducibly good results using fresh transformants and 2-4 h starter cultures. Others have adapted this protocol to use glycerol stocks and overnight starter cultures. 

A note about keeping your protein happy: 
While there are many good "pause points" in this protocol, I recommend only leaving protein overnight for one night at most at 4 °C before the dialysis step. Highly concentrated protein has been known to aggregate irreversibly even at 4 °C.

A note about SDS-PAGE: 
If your ELP has no basic residues, it will not absorb Coomassie-based stains well. Image your gel soon after staining and destaining. For constructs that fail to bind Coomassie entirely, copper (II) chloride staining is recommended.  


[STEPS]
SECTION: Transformation
1. In a pre-chilled Eppendorf tube, mix 10-100 ng plasmid (typically 1-2 μL of mini-prep DNA) with 50-100 μL BL21(DE3) competent cells on ice. Let sit on ice for 30 min. 

SECTION: Transformation
2. Heat shock at 42 ºC in a water bath for 1 min. 
SECTION: Transformation
3. Allow cells to recover on ice for 2-3 min. 
SECTION: Transformation
4. Add 1 mL sterile nutrient-rich liquid medium such as SOC or 2XYT. 
SECTION: Transformation
5. Incubate 1 h at 37 ºC. 
SECTION: Transformation
6. Centrifuge at 7000 rpm for 1 min at room temperature. 
SECTION: Transformation
7. Remove all but ~50-100 μL of supernatant. Resuspend pellet in remaining supernatant. 
SECTION: Transformation
8. Pipette resuspended cells on 2XYT + agar + carbenicillan plate and use sterile glass beads to spread. 
SECTION: Transformation
9. Remove beads and store plate at 37 ºC overnight. 

SECTION: Expression
10. Inoculate 15 mL starter culture using a sterile loop to harvest several swipes of colonies from the transformation plate. Add 15 μL 1000X carbenicillan. Place flask in incubated shaker and shake at 200 rpm at 37 ºC until visibly cloudy (typically 2-4 h).
SECTION: Expression
11. Inoculate 1 L SuperBroth with full volume of starter culture. Add 1 mL 1000X carbenicillan. Return 1 L culture to shaking at 200 rpm at 37 ºC for 24 h. 
SECTION: Expression
12. Harvest cells by centrifugation at 4000 rpm for 20 min at 4 ºC. 
 

SECTION: Purification
13. Thaw cell pellets in water and immediately transfer to ice. Resuspend cell pellets thoroughly by pipetting and vortexing in 80 mL of ice-cold 10 mM CAPS pH 11.2, 150 mM NaCl, 20% sucrose. Ice for 15 min.
 

SECTION: Purification
14. Centrifuge at 4200 rpm for 10 min at 4 ºC. Remove supernatant to a fresh pre-chilled beaker or bottle on ice. This sample is Periplasmic Fraction 1. 
SECTION: Purification
15. Resuspend pellets thoroughly by pipetting and vortexing in 50 mL cold, sterile 10 mM CAPS pH 11.2. Ice for 15 min. 

 

SECTION: Purification
16. Centrifuge at 4200 rpm for 10 min at 4 ºC. The supernatant after this spin is Periplasmic Fraction 2. 
SECTION: Purification
17. Pool Periplasmic Fraction 1 and Periplasmic Fraction 2 and measure the volume (which should be ~130 mL). This is your Pooled Periplasmic Fraction. 
SECTION: Purification
18. Allow the pooled periplasmic fraction to warm to room temperature. Add dry NaCl to a final concentration of 3 M. For 130  mL of pooled periplasmic fraction this is ~22 g of NaCl. 
 

SECTION: Purification
19. Centrifuge at 14,000 rpm for 15 min at room temperature. 
 

SECTION: Purification
20. Carefully remove the supernatant. This is your Salt Supernatant sample, which you expect should not contain ELP. Resuspend pellets in 15 mL ice-cold, sterile 10 mM CAPS pH 11.2, 150 mM NaCl. Incubation on ice and gentle vortexing will assist with resuspension. Pipetting is not recommended as the protein is quite sticky and will be lost by sticking to pipettes. 
 

SECTION: Purification
21. While still keeping resuspended protein cold, split volume into an even number of pre-chilled 1.5 mL Eppendorf tubes (~1 mL per tube). 
SECTION: Purification - Temperature Cycling
24. Add ~100 µL 5 M NaCl to each tube. Mix by inversion. If solution does not turn cloudy, add another 100 µL 5 M NaCl. 
SECTION: Purification - Temperature Cycling
25. Centrifuge at 14,000 rpm for 10 min at room temperature. Grey-white protein pellets should form. 
SECTION: Purification - Temperature Cycling
26. Add 600 µL cold, sterile 10 mM CAPS pH 11.2, 150 mM NaCl to each tube. Resuspend by alternating vortexing and incubating on ice or by rotating at 4 ºC. 
 

SECTION: Purification
22. Centrifuge Eppendorf tubes at 16,000 rpm for 10 min at 4 ºC. A brown contamination pellet should form. 
SECTION: Purification
23. Transfer supernatants to fresh Eppendorf tubes and return protein to room temperature. This sample is Cold Supernatant 1, representing protein purity after one round of inverse transition cycling. 
SECTION: Purification - Temperature Cycling
27. Centrifuge at 14,000 rpm for 10 min at 4 ºC. A brown contamination pellet may form. 
SECTION: Purification - Temperature Cycling
28. Transfer supernatants to half the number of fresh Eppendorf tubes by pooling two supernatants per fresh tube. The supernatant at this step is Cold Supernatant 2.
SECTION: Purification - Temperature Cycling
29. Repeat steps 24-28 until a contamination pellet is no longer visible after step 27 (cold spin), plus one more time for good measure. 
SECTION: Post-purification
30. Analyze samples by SDS-PAGE. Suggested samples: 
Periplasmic Fraction 1
Periplasmic Fraction 2 or Pooled Periplasmic Fraction 
Salt Supernatant 
Cold Supernatant 1
Cold Supernatant 2 
Cold Supernatant (...) 
SECTION: Post-purification
31. Dialyze purified protein against 4 L of pre-chilled deionized water overnight at 4 ºC. 
SECTION: Post-purification
32. Store dialyzed protein at -80 ºC or flash freeze in liquid nitrogen. Lyophilize for long-term storage at -20 ºC.