Dual ELP-tagged Split Intein System Expression and Purification
Introduction
Materials
- Consumables
- ammonium sulfate
- "Low-pH Cleavage Buffer"
- "Low-Salt Lysis Buffer"
- Luria-Bertani (LB) selective medium (100 μg/mL Ampicillin)
- Terrific Broth (TB) selective medium (100 μg/mL Ampicillin)
- IPTG
- Disposables
- pipet tips
- cuvettes
- centrifuge tubes (15mL)
- Equipment
- Erlenmeyer flasks
- spectrophotometer
- sonication cell disruptor
- centrifuge
Procedure
Expression
- 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!
- Incubate the cultures at 37°C overnight.
- Use each culture to seed 500mL of selective TB medium in a 1L erlenmeyer flask.
- Continue culturing at 37°C until the optical density (λ=600nm) reaches approximately 0.8.
This step should take approximately 3-4 hours.
- After the OD600 reaches ~0.8, induce expression using IPTG at a final concentration of 0.1mM.
See IPTG Induction for calculations.
- Continue culturing at 37°C for 3 hours.
Purification
- 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.
A A | B B | |
1 1 | EI0C Wet Cell Weight (g)* | 1 |
2 2 | EI0N Wet Cell Weight (g)* | 2 |
3 3 | Low-salt lysis buffer (mL) | 15 |
- 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.
- Lyse cells through 2-4 pulses of sonication at 0.5 W RMS for 20s each.
- Centrifuge lysate solutions (2,100xg, 4°C, 10min). Transfer supernatant to a fresh tube.
A A | B B | |
1 1 | Ammonium sulfate Stock Concentration (M)* | 1.6 |
2 2 | Clarified Lysate Volume (mL)* | 3 |
3 3 | Ammonium Sulfate Volume (mL) | 1 |
- 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.
- Incubate for 10 min at room temperature.
- Recover precipitate via centrifugation (1,600xg, room temperature, 6min). Discard supernatant.
- Again, dissolve the precipitate in "low-salt lysis buffer" and repeat steps 11-13.
This step should completely remove remaining contaminant host cell proteins.
- 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.
- If performing separate E/I0C & E/I0N purifications, combine them now.
- 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.
- 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).
- Incubate for 10 min at room temperature.
- Centrifuge sample (1,600xg, room temperature, 6min). Transfer supernatant to fresh tube.
- Store at 4°C for short term or -20°C long term.
If storing long term, may want to supplement solution with glycerol etc.
This protocol describes how to express and purify proteins using the ELP-tagged split intein system described in Shi et al. Briefly, this system exploits a complementary pair of ELP-intein and ELP-intein-product fusions, which when when both are present will act to cleave the product from the fusion and allow for simple purification.
Abbreviations:
(p)E/I0C - ELP-intein-product fusion. The expression product is ELP-tagged C-terminal portion of the intein fused to the desired expression product. Plasmid confers Ampicillin resistance.
(p)E/I0N - ELP-intein fusion. This ELP-tagged N-terminal portion of the intein which can complement the C-terminal portion and restore functionality, causing cleavage of E/I0C at the intein-product transition point. Plasmid confers Ampicillin resistance.
Typically this vector-system is used in Escherichia coli BLR(DE3).
See: TSS Competent Cell Preparation & Transformation for proven methods of BLR(DE3) strain construction.
Shi, C., Meng, Q. & Wood, D. (2013). A dual ELP-tagged split intein system for non-chromatographic recombinant protein purification. Appl Microbiol Biotechnol 97, 829-835.