TITLE: Expression and purification of bacterial proteins (via  N-terminal His-tag)
AUTHORS: Verena Dederer, Sebastian Mathea, Stefan Knapp

[DESCRIPTION]
Recombinant protein production in bacteria provides an efficient system for obtaining high yields in a short time and at low cost. Although not every protein is suitable for expression in bacteria, it is usually the first choice to start expression trials. If successful, more elaborate protein purification protocols can be developed. Here we provide a two-step protocol for protein purification from bacterial cells. 

[STEPS]
SECTION: Plamid Transformation into E.coli Rosetta cells
1. Thaw 10 µL E.coli Rosetta cells gently on ice
SECTION: Large Scale Protein Expression (4L)
11. Next morning, dilute grown overnight culture 10 mL into 1000 mL terrific broth expression medium containing the appropriate antibiotic
SECTION: Large Scale Protein Expression (4L)
9. From a single colony of the LB plates inoculate50 mL containing the appropriate selection pressure 
SECTION: Plamid Transformation into E.coli Rosetta cells
3. Apply heatshock for 45 s in a 42 °C
SECTION: Plamid Transformation into E.coli Rosetta cells
2. Add 25 ng respective expression plasmid and keep on icefor 30 min
♦ DARPin E11 in pQE30 vector (Qiagen) - this encodes: N-terminal 8xHis-tag and a C-terminal 3xFLAG-tag
♦ Rab8 in pET28A vector (Novagen) - this encodes a N‑terminal TEV‑cleavable 6xHis-tag
SECTION: Plamid Transformation into E.coli Rosetta cells
4. Let cells recover for 5 minon ice
SECTION: Plamid Transformation into E.coli Rosetta cells
5. Add 100 µL and incubate cells for 30 minat 37 °C
SECTION: Plamid Transformation into E.coli Rosetta cells
6. Plate cell suspension on LB agar plates containing appropriate selection pressure  
SECTION: Plamid Transformation into E.coli Rosetta cells
8. Plates can be stored at 4 °C for several weeks or until further use
SECTION: Large Scale Protein Expression (4L)
13. Once OD600 reaches 1.0, reduce temperature to 18 °C and wait for another 30 min  before adding 500 millimolar (mM) 
SECTION: Large Scale Protein Expression (4L)
14. Incubate for 1080 minshaking at   
SECTION: Large Scale Protein Expression (4L)
15. Harvest cells by centrigufation with 1000 x g, 15 min, 4 °Cmin
SECTION: Large Scale Protein Expression (4L)
16. Freeze pellet and store -20 °C  until further use

SECTION: 2-Step Protein Purification
17. Thaw pellet and resuspend in 300 mLlysis buffer
 

SECTION: 2-Step Protein Purification
18. Lyse by sonication 3 rounds with 5 sec pulse, 10 sec pause, 3 min total pulse time on ice 
SECTION: 2-Step Protein Purification
19. Clear lysate by ultracentrifugation 1000000 x g, 45 min, 4 °C 
SECTION: 2-Step Protein Purification
20. Perform Ni-NTA
SECTION: 2-Step Protein Purification
21. Concentrate eluate to volume of 5 mLand subject to size-exclusion chromatography  on a S200 gel filtration column
 


SECTION: 2-Step Protein Purification
22. Pool fractions containing your protein of interest
SECTION: 2-Step Protein Purification
23. Measure protein concentration and do additional quality control measurements such as SDS PAGE with Coomassie staining, mass spectrometry to validate you sample
SECTION: 2-Step Protein Purification
20.1. Load supernatant onto pre-equilibrated Ni-NTA sepharose beads
SECTION: 2-Step Protein Purification
20.2. Wash beads rigorously with lysis buffer (50 CV)
SECTION: 2-Step Protein Purification
20.3. Elute protein using lysis buffer supplemented with 300 millimolar (mM)

SECTION: 2-Step Protein Purification
24. Flash freeze aliquots of your protein of interest and store at -80 °C 

SECTION: Plamid Transformation into E.coli Rosetta cells
7. Incubate 5 minat37 °C
SECTION: Large Scale Protein Expression (4L)
10. Incubate the liquid culture shaking at  
SECTION: Large Scale Protein Expression (4L)
12. Grow cells shaking at   until OD600 reached 1.0