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](http://brie.bmsc.washington.edu/scatter/). 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 - antibiotics (1000x conc.) - 1M IPTG - M9 media (minimal media) - 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. - 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** 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** 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.) - 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 1. 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](http://brie.bmsc.washington.edu/scatter/): Principles, WebTools, and Related Links provided by Ethan A. Merritt at University of Washington.