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Protocol for Purifying hed KS-CLF protein from Streptomyces
NOTE:
This protocol is typically for purifying hed KS-CLF heterodimer from 1 L (2 X 500 mL) of Streptomyces culture. The same protocol can be scaled up whenever necessary to purify more protein. The strain used is CH999/pBOOST*/pAD212. CH999 is a re-engineered Streptomyces coelicolor strain which lacks the act cluster. pAD212 is a pRM5 derived shuttle vector carrying minimal hed PKS (hed C, hed D, hed E). hed D (CLF) contains N-terminal FLAG tag which makes it easier to purify the KS-CLF heterodimer by Anti-FLAG antibody affinity column chromatography.
Materials
- R5 plates containing the appropriate Streptomyces strain.
- 1 x 250 mL Ultra Yield Flask (Eliminates Springs) and Enhanced AirOtop Seals (Eliminates Springs). 2 x 2.5 L Ultra Yield Flask (No need to insert springs with Ultra Yield Flasks) and Enhanced AirOtop Seals.
- Autoclaved water
- Autoclaved absorbent cotton balls
- Antibiotics. 50mg/mL Thiostrepton in DMSO (don't filter sterilize) and 100mg/mL Apramycin Sulfate in water (filter sterilize).
- Liquid R5 medium. Prepare about 500 mL of R5 liquid media in a 500 mL bottle and autoclave. Prepare and autoclave the 4 solutions that you need to add after autoclaving the media. Store the R5 media at 4°C for long term use without adding the four solutions.
- Stroud Minimal Media. (See fig 1 after Method)
- Disruption Buffer: 200mM Na2HPO4, 200mM NaCl, 2.5mM DTT, 1.5mM Benzamidine, 2.5mM EDTA, 2mg/mL Pepstatin, 2mg/mL Leupeptin, 30% v/v glycerol, pH 7.0
- Sonicator
- 4% PEI (polyethyleneimine) solution stored at 4°C. Buy PEI and dilute in autoclaved water.
- Ammonium Sulfate (solid crystalline)
- Chromatography columns appropriate for 2mL.
- ANTI-FLAG M2 monoclonal antibody bound to agarose on the ANTI-FLAG M2 agarose affinity gel (Sigma)
- TBS buffer: 50mM Tris-HCl, 150mM NaCl, pH 7.4
- TBS/A buffer: 50mM Tris-HCl, 150mM NaCl, 50% glycerol, 0.02% sodium azide, pH 7.4
- 0.1 M glycine HCl, pH 3.5
- FLAG peptide stored at -20°C (buy from Genscript, it's cheaper!). Make a stock solution of FLAG peptide in TBS buffer at 5mg/mL, make aliquots of 200μl and store at -20°C. NOTE: You can also buy 3X FLAG peptide from Sigma.
Method
- Prepare R5 plate and streak the cells from another plate homogeneously throughout the R5 plate and incubate at 30°C for 7 days. NOTE: Plan your experiment accordingly as seed culture can be started after 7 days. It's better to start the seed culture from a fresh plate rather than frozen spore stock or age-old plates.
- Take out an R5 plate having confluent lawn of the strain from the incubator. Pour 10ml of autoclaved water. Take a small piece of autoclaved cotton and make it wet. Collect the spore by moving the cotton around the plate and syringe in all of the liquid though the cotton. Spin down the solution for 10 min and discard the supernatant.
- Start 50ml of seed culture in 250 mL Ultra Yield Flask (Eliminates Springs) and Enhanced AirOtop Seals containing R5 liquid media. Transfer the collected spore and incubate in 30°C shaker @ 240 rpm for 2 days. NOTE: Stop the culture just when the solution starts turning brownish or reddish.
- Transfer 25ml of the seed culture to 500mL of SMM media in 2.5 L Ultra Yield Flask and Enhanced AirOtop Seals; shake at 30°C @ 250 rpm for 48 hours. NOTE: Best indication of the protein expression is when the mycelia turns grayish and color of the solution turns brownish which may happen even before 48 hrs. The cells should be harvested just at this point.
Fig 1 (Stroud Minimal Media)
| Total Volume | 500mL |
|---|---|
| H2O | 425mL |
| PEG 8000 | 31.25g |
| TES Buffer (0.25M, pH7.2) | 50mL |
| Casamino Acid (20%) | 12.5mL |
| Glucose (50%) | 10mL |
| Phosphate Solution | 1.25mL |
| Trace Element Solution | 0.625mL |
