Preparing a Selenomethionyl Proteinby Sara Lindgren, 01/22/2000 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

• LB media
• antibiotics (1000x conc.)
• 1M IPTG
• M9 media (minimal media) 1 Liter 5x M9 media: (sterile filtered)
  • 30g Na2 HPO4 or 64g Na2 HPO4 -7H2 O
  • 15g KH2 PO4
  • 5g NH4 Cl
  • 2.5g NaCl
  Dilute and autoclave before use.
• 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)

Procedure Day 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. 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. Day 2 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. 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 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.