Biochemistry

scientificprotocols authored almost 4 years ago

Author: Sara Lindgren

Purpose

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.

Materials

  1. LB media
  2. antibiotics (1000x conc.)
  3. 1M IPTG
  4. M9 media (minimal media)
  5. 1 Liter 5x M9 media: (sterile filtered)
    • 30g Na2HPO4 or 64g Na2HPO4-7H2O
    • 15g KH2PO4
    • 5g NH4Cl
    • 2.5g NaCl
      • Dilute and autoclave before use.
  6. 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.
  7. 20% glucose (sterile filtered or autoclaved)
  8. 1M MgSO4 (sterile filtered)
  9. 2M CaCl2 (sterile filtered)
  10. 0.5% (w/v) Thiamine Solution (sterile filtered)

Procedure

Day 1

  1. 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.
  2. Prepare M9 stock media. Dilute and autoclave before use.
  3. Prepare amino acid 50x Stock.
  4. Prepare a 150mL overnight culture consisting of 150mL LB, 150uL antibiotics (1000x conc.), and 150uL of day culture. Grow at 37°C overnight.

Day 2

  1. 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.)
  2. Inoculate M9 with 50mL overnight culture and grow until an OD600=0.5-0.6. (~2.0 - 2.5 hours)
  3. Add 100mg threonine, lysine hydrochloride, phenylalanine to the culture.
  4. Add 50mg leucine, isoleucine, valine to the culture (all as solid powders).
  5. Add 120mg DL-Se-Met or 60mg L-Se-Met to the culture (as a solid powder).
  6. Continue to grow the culture for 15 minutes.
  7. Induce with 1mL 1M IPTG (final concentration = 1mM).
  8. Grow about 6-8 hours (whatever is optimal for the protein of interest).
  9. Collect cells as usual and proceed to purification steps.

Expected Results

  • 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.

References

  1. Doublie, S. (1997) Preparation of Selenomethionyl Proteins for Phase Determination. Methods in Enzymology 276, 523-530.
  2. Deacon, AM., Ealick SE. (1999) Selenium-based MAD phasing: setting the sites on larger structures. Structure 7, R161-R166
  3. Protocol originally obtained from Qing Fan at Don Wiley's Lab.
  4. X-ray Anomalous Scattering: Principles, WebTools, and Related Links provided by Ethan A. Merritt at University of Washington.

DOI

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