Protocol

Abbreviation

uva_expression_semet_1

Name

typical semet expression protocol at Wladek Minor group

Laboratory name

University of Virginia

Type

expression

Description

Expression of Seleno-Methionine-Labelled APC Proteins

The general procedure is to grow a small seed culture (10mL) of a methionine auxotrophic strain in M9 minimal media plus Met. This overnight culture is then used to seed a full-size culture (1L) with a limited amount of Met. The full cultures will use up all of the Met at an A595 appropriate for induction and will then be spiked with an excess of Se-Met. When the cells reenter log phase, they are induced with IPTG and allowed to express overnight at reduced temperature.

Note: Seleno-methionine is a very toxic compound. Lab gloves and a lab coat should be used when working with Se-Met and no solutions that contain Se-Met should be disposed of down the drain. There are large waste bottles in the fume hood for Se-Met waste, most notably the spent growth media.

Preparation (within 1-3 days prior)
1)Transform the vectors of interest into B834(DE3) (or derived) cells OR plate frozen B834(DE3)-transformed cell stocks on fresh plates. The vectors may be tranformed in advance and stored as glycerol stocks at -80 degree (though we have not yet tested this). In any case, the overnight cultures should start from plates rather than directly from frozen stock.
2)Autoclave water in large baffled expression flasks and confirm the other M9 minimal media components have been prepared in sufficient amounts. We will grow 1L of culture in each 2.5L flask.
3)Autoclave small flasks to grow the overnight seed cultures.

Day 1 (1 hour in the evening)
1)Per 1L of final growth culture, seed 10mL of M9 minimal media + 50ug/mL methionine with cells from the fresh plate. This is 10uL of the 50mg/mL Met stock solution. Generally, the working concentration of both Met and Se-Met is 50ug/mL.
2)Incubate the seed cultures overnight at 37 degree with shaking in 200mL (or larger) Erlenmeyer flasks.

Day 2 (8-11 hours; this is the long day)
1)Turn on the incubator and set it to 37 degree.
2)Prepare the large flask(s) of M9 minimal media according to the M9 minimal directions, with a final concentration of 8ug/mL methionine (80uL of the 50mg/mL Met stock solution). This is intentionally less than the working concentration of Met, which is 50ug/mL as mentioned above. At this concentration, at an A595 of 0.6-08, the cells will exhaust the supply of methionine in the culture.
3)Allow the large flasks to warm in the incubator for 30-60 minutes.
4)Seed the large 1L cultures with the 10mL overnight seed cultures, record the time, and set to incubate with shaking at 37 degree.
5)Monitor the growth over the next 7-10 hours by monitoring the absorbance at 595nm. The cultures should eventually enter log phase, then level out at an A595 of 0.6-08.
6)Prepare a few milliliters of 1M IPTG if none is already available. This should be stored in the -20 degree freezer.
7)After the cells have levelled out for 1-2 hours, add seleno-methionine to the large cultures to a final concentration of 50ug/mL (2mL of the 25mg/mL Se-Met stock solution). The cells should reenter log phase as they begin to take up the Se-Met.
8)Wait 15-30 minutes, than induce by adding IPTG to a final concentration of 1mM (1mL of the 1M IPTG stock).
9)Change the shaker temperature to 16 degree and allow the induced cultures to express overnight.

Day 3 (3 hours in the morning)
1)After the cultures have expressed overnight, remove the large flasks from the incubator and set on ice. Save a small sample to test the final A595. The final absorbance should be about 1.3-1.4. Ideally, the total length of the overnight induction should be less than 10 hours, but I have allowed these to go longer without incident so far.
2)Weigh the liter centrifuge bottles used to pellet the cells. We have been borrowing these from the Nakamoto lab, since we use their Avanti J-20XP to do the actual pelleting.
3)Pellet the cells in the JLA8.1000 rotor in the J-20XP, with a spin of 15 minutes at 7000 rpm at 4 degree.
4)Carefully decant the supernatant and reweigh the bottles to determine the weight of the pellet. It should weigh 4-8g. The supernatants from the spins should be disposed of in the waste bottles in the hood.
5)Resuspend the pellet in 25-35 mL of filtered resuspension buffer (500mM NaCl / 50mM HEPES pH=7.5 / 5% glycerol) on ice.
6)At this point, the cells may be
1.Lysed and purified immediately. See the APC Protein Purification protocol.
2.Flash-frozen in liquid N2 in Falcon tubes and stored at -80 degree.

The seleno-methionine incorporation protocol is adapted from a method originally developed by Arun Mohanty and David Chimento at U.Va. The native expression protocol of APC proteins was developed by the lab of Aled Edwards at U.Toronto.
Matthew Zimmerman / 15 Feb 2004