Procedure for Expressing and Purifying Bni1 P
Amity Manning 4/1/03
Expected Yield is approximately 200 ug per 1 L culture.
Expression:
Bni1 P is overexpressed with a GST plasmid in E. coli Bl21 DE3 RP
The plasmid is marked with KAN resistance, the strain is marked with
Chloramphenicol resistance.
Cultures should be grown in volumes no more that one fourth the volume of the
flask (i.e.: 1 L culture in 4 L flask).
Inoculate several fresh colonies (transformed the previous night) into selective
media. Grow Culture at 37ºC, shaking to an OD600=0.8-1.2
Once desired OD is reached, cool flask and media quickly by placing on ice for
approximately 5 minutes.
IPTG induce the culture with an addition of 0.4 mM IPTG (1 mL of stock IPTG
(0.4 M) for 1 L culture). Place flask at 20ºC and continue growing overnight.
Pellet the cells, wash with cold PBS, resuspend in cold PBS (resuspend in 10 mL
per liter of culture).
Freeze in oakridge tube at -80º.
Purification:
Unless specified otherwise, Cell extracts and Protein should be kept at 4ºC
throughout the purification process.
Thaw pellet quickly by warming tube in hands or under warm water. Once the
pellet begins to thaw, add protease inhibitors and keep the tube on ice
Sonicate the thawed pellet 3-4 times for 30 seconds each, allowing to cool on ice
between each sonication. Add PMSF after first round of soncation.
Generate medium speed supernatant (MSS) by centrifuging 15 minutes, 12,000
rpm, 4ºC in SS34 rotor.
Harvest supe (~10 mL for 1 liter culture) and add 0.5 mL beads (amount of beads
is determined based on gel run of extract or MSS). Fill tube to volume with cold
PBS.
Allow beads to bind protein while rocking in cold room 2 hrs.
Spin down and transfer beads to 15 mL falcon tube (if not already in one) and
wash beads 5 times with cold PBS. Wash beads 3 times with cold HEK buffer.
Transfer beads to 1.5 mL eppendorf tube and remove excess buffer. (Total
volume ~ 500 uL)
Run gel of MSS, Sup After Beads (SAB), and Beads Before TEV digestion
(BBTev) and coomassie Stain to evaluate progress of prep so far. GST-Bni1 P
runs just above the 66 kDa, Bni1 P runs just below the 45 kDa.
Add 5 uL of TEV and MIX WELL. Digest overnight at 16ºC.
Run gel of beads after digest (BAD) and supe after digest (SAD) and coomasie
stain to determine effectiveness of the digest.
Spin down beads and harvest supe. Wash beads with 100 uL HEK, spin down
beads and harvest the remaining supernatant through a hole punctured in the
bottom.
Pool collected supernatant and dilute supe 1:4 with HE buffer. Load sample onto
monoQ column.
Run 20 mL gradient (0-300 mM KCl) HEK buffer at 1 mL /min and collect 0.5 mL
fractions. Bni1 P elutes in 200 mM KCl.
Run 10 uL of peak fractions on 12%gel, coomasie stain and identify protein
containing fractions.
Pool desired fractions and concentrate in cen10 to about 200 uL, dilute to 400 uL
with HE, concentrate to 200 uL, dilute to 400 uL with HE, concentrate to 1-5 uM.
Aliquot and snap freeze in liquid nitrogen.
Buffers Used:
PBS buffer
HE buffer:
20 mM HEPES pH 7.5
1 mM EDTA
HEK buffer:
20 mM HEPES pH 7.5
50 mM KCl
1 mM EDTA
FPLC Buffers:
Buffer A: HEK
Buffer B: HEK + 1M KCl