Preparing a Selenomethionyl Protein by Sara Lindgren, 01/22/2000
The protocol describes how to prepare selenomethionyl (Se-Met) protein using a regular E.coli strain. Selenium can be used for phase determination in multi-wavelength anomalous diffraction (MAD) method. Se-Met can often replace methionine residues in a protein without affecting the protein's properties, therefore producing a protein advantageous for crystal structure solving. Also, the X-ray absorption edge of selenium is easily accessible by synchrotron radiation, making a Se-Met crystal ideal for collecting anomalous X-ray diffraction data. The Se-Met proteins can also be prepared using insect cells and CHO cells, which will be described in separate protocols.
- LB media
- antibiotics (1000x conc.)
- 1M IPTG
- M9 media (minimal media)
1 Liter 5x M9 media: (sterile filtered)
Dilute and autoclave before use.
- 30g Na2HPO4 or 64g Na2HPO4-7H2O
- 15g KH2PO4
- 5g NH4Cl
- 2.5g NaCl
- Amino acid 50x stock
Use all amino acids EXCEPT Gly, Ala, Pro, Asn, Cys, and Met at a concentration of 2mg/ml
To help in dissolving the amino acids, autoclave for 10 minutes.
- 20% glucose (sterile filtered or autoclaved)
- 1M MgSO4 (sterile filtered)
- 2M CaCl2 (sterile filtered)
- 0.5% (w/v) Thiamine Solution (sterile filtered)
- Prepare a 2mL day culture consisting of 2mL LB media, 2uL antibiotics (1000x conc.), and a single E.coli colony. Grow at 37°C all day.
- Prepare M9 stock media. Dilute and autoclave before use.
- Prepare amino acid 50x Stock.
- Prepare a 150mL overnight culture consisting of 150mL LB, 150uL antibiotics (1000x conc.), and 150uL of day culture. Grow at 37°C overnight.
- To each liter M9 (1x conc.) add:
10mL 20% Glucose (sterile filtered or autoclaved)
2mL 1M MgSO4 (sterile filtered)
0.05mL 2M CaCl2 (sterile filtered)
0.1mL 0.5% (w/v) thiamine solution (sterile filtered)
1mL antibiotics (1000x conc.)
20mL amino acid 50x Stock (If precipitate is seen, heat to 60-70°C and shake.)
- Inoculate M9 with 50mL overnight culture and grow until an OD600=0.5-0.6. (~2.0 - 2.5 hours)
- Add 100mg threonine, lysine hydrochloride, phenylalanine to the culture.
Add 50mg leucine, isoleucine, valine to the culture (all as solid powders).
- Add 120mg DL-Se-Met or 60mg L-Se-Met to the culture (as a solid powder).
- Continue to grow the culture for 15 minutes.
- Induce with 1mL 1M IPTG (final concentration = 1mM).
- Grow about 6-8 hours (whatever is optimal for the protein of interest).
- Collect cells as usual and proceed to purification steps.
- Se-Met protein will show slightly larger MW than the native protein in mass spectrum.
- Se-Met protein may behave slightly differently from the native protein in purifications and crystallization.
- Doublie, S. (1997) Preparation of Selenomethionyl Proteins for Phase Determination. Methods in Enzymology 276, 523-530.
- Deacon, AM., Ealick SE. (1999) Selenium-based MAD phasing: setting the sites on larger structures. Structure, 7, R161-R166
- Protocol originally obtained from Qing Fan at Don Wiley's Lab.
- X-ray Anomalous Scattering: Principles, WebTools, and Related Links provided by Ethan A. Merritt at University of Washington.
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