Protocol

Abbreviation

uva_expression_native_1

Name

typical native expression protocol at Wladek Minor group

Laboratory name

University of Virginia

Type

expression

Description

Expression of Native APC Proteins

Preparation (within 1-3 days prior)
1)Transform the vectors of interest into the BL21(DE3)-derived expression strain OR plate frozen expression stocks on fresh plates. The vectors may be transformed in advance and stored as glycerol stocks at -80 degree. 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 TB components have been prepared in sufficient amounts. We will grow 1L of culture in each 2.5L flask.
3)Autoclave small flasks (250mL or 500mL, preferably baffled) in which to grow the seed cultures.

Day 1 (8-11 hours; this day is the long day)
1)Turn on the shaker and set it to 37 degree.
2)Prepare 25mL of TB media (plus the appropriate antibiotics) in the small flasks. Pick 5-10 colonies from the fresh plates and inoculate. Set the seed cultures in the shaker at 37 degree at ~240rpm (or so).
3)Prepare the large flask(s) of TB media according to the TB directions. Add antifoam A and the appropriate antibiotics. Set them in the shaker to warm up to 37 degree.
4)Prepare the 1M IPTG stock (if necessary). This should be stored at -20 degree.
5)When there is noticeable growth in the small cultures (3-4 hours), transfer the small cultures into the large flasks. Monitor the resulting growth by A595 every 30-45 minutes.
6)When the large cultures have reached an A595 of ~2.0 (~4 hours), induce the large cultures with 1mM IPTG (1mL of the 1M stock).
7)Decrease the shaker temperature to 16 degree and allow to express overnight.

Day 2 (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. 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 6-10g. The supernatants may be disposed of down the drain.
5)Resuspend the pellet in 25-35 mL of filtered suspension buffer (500mM NaCl / 50 mM 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.

Matthew Zimmerman / 16 Feb 2004