Preparation of Seleno-Methionine labeled protein

(adopted from original protocol found in:

VanDuyne G. D. et. Al. (1993) J. Mol. Biol. Vol. 229 pp. 105-124)

WARNING!

SeMet is TOXIC. The culture supernatant should be collected for disposal as hazardous waste. Fill the waste bottles to shoulder-height, not all the way to the screw cap. Use the Online Tag Program to generate hazardous waste tags as soon as you start generating the hazardous waste and affix the labels to the waste bottles immediately.

Refer to your lab-specific Standard Operating Procedure (SOP) for the proper way to work with SeMet. At a minimum you will need to use the appropriate Personal Protective Equipment (PPE) which includes the use of nitrile gloves, lab coat, and full-coverage shoes.

Make 5x M9 salts

To 800ml H₂O add:

64g Na₂HPO₄-7H₂O (make sure it is heptahydrate, 7 H₂O. Adjust for MW otherwise)

15g KH₂PO₄

2.5g NaCl

5.0g NH₄Cl

Stir until dissolved

pH to 7.0 ~ 7.4 (I pH to 7.2)

Adjust to 1000ml with distilled H2O.

Sterilize by autoclaving.

To make M9 Minimal Media:

Autoclave 780ml deionized water in 3-liter flask.

Add 200ml of 5x M9 salts.

Add 20 ml of 20% glucose (or other carbon source; filter sterilize glucose solutions)

Add 2ml of 1M MgSO₄ (filter sterilize 1M stock solution)

Add 100ul of 1M CaCl₂ (do not worry if you see a cloud of precipitation when you add)

Optional: Add 100ul of 0.5% thiamine (I usually don’t and it does not seem to matter).

Note: It seems that the ingredients have enough trace metals that you don't have to worry about making a trace metals solution.

Grow the overnight culture in minimal media:

I usually start a LB culture (2~5ml) first thing in the morning from a colony or glycerol stock, and inoculate 50ml (or 100ml) of minimal media in a 250ml flask (or 500ml flask) with 2ml (or 4ml) LB culture (should be very turbid) before I leave for the day. So far in my experience, the overnight minimal media cultures were always saturated or near saturation the following morning. If you can, pre-warm and pre-aerate the liters of minimal media overnight (I find it makes a noticeable difference in the initial growth rates).

Note: I have only used BL21 derived cell lines. Unless you are using an amino acid auxotroph, for which you should talk to Mark Arbing, it shouldn't matter. The purpose of growing the overnight culture in minimal media is so that the cells aren't shocked. The original protocol had you spinning down the cells grown in LB and resuspending in minimal media before inoculating the large minimal media culture.

Inoculate minimal media with overnight culture:

I have been inoculating 1 liter of minimal media with 20ml of overnight culture. If you want to be a stickler, you should inoculate with 5-10ml. Doubling times have generally been around an hour, but once it took around 10 hours to get the OD to 0.5.

Add amino acid mix to inhibit methionine synthesis and to supply selenomethionine

Amino acid mix: 100mg K, F, and T; 50mg I, L, and V; and 60mg SeMet per liter.

Add at OD 0.5~0.6 and grow for an additional 15 minutes

Induction, Expression, and Harvesting

Induction, expression, and harvesting from this point on are supposed to be the same as for your regular sulfur-methionine protein. Since I have been expressing at 25ºC, I have been lowering the temperature to 25ºC 10 minutes after adding the amino acid mix, and inducing to a final [IPTG] of 1 mM and expressing overnight (16~18 hours in practice). My SeMET expression times have been similar to my Sulfur-MET expression times, and the yields have been comparable. If you express at 37ºC, you might want to express for an additional hour or so to optimize yields. It will probably depend on your protein. Remember to collect the supernatant in screw-cap bottles and to dispose as hazardous waste!