Expression and purification of Centrin
This protocol is optimized for CRCN, but should work well for the other constructs
as well.
Abbreviations:
CT = centrin (aka caltractin)
CRC = Chlamydomonas reinhardtii centrin
CRCN = N-terminal half fragment of CRC
CRCC = C-terminal half fragment of CRC
pRSETA-t = a truncated version of the pRSET A vector which can be purchased
from Invitrogen. The vector was modified to use a thrombin cleavage site for
removal of the N-terminal (His)6 tag. After cleavage there only remains a Gly-Ser
sequence N-terminal to the start of the protein.
Note: CT plasmids in the pRSETA-t vector tend to get kicked out. It is advisable
to work from fresh transformants with each prep, and to check expression before
proceeding to large-scale labeled growths.
Preparation of unlabeled Centrin constructs
Day 1
Transform BL21 (DE3) or BL21 (DE3) pLysS cells with plasmid. Use a 2 minute
heat shock.
Plate transformation reaction on LB agar containing carbenicillin (75µg/ml) (and
chloramphenicol (34µg/ml) if using pLysS cells). Grow overnight at 37°C.
Day 2
Inoculate 10ml LB culture (with appropriate antibiotics) with a single colony from
the transformation plate. It is wise to set up multiple cultures at all steps, in case
some do not grow well. Grow overnight at 37°C while shaking.
Day 3
Inoculate each 1 L culture (in 2800ml Fernbach flasks, with appropriate
antibiotics) with 10ml overnight culture. Grow at 37°C while shaking. Induce
cultures using 1mM IPTG (final concentration) when absorbance at 600nm reads
between 0.6 and 0.8 O.D. Remember to save a pre-induction sample for SDSPAGE. Allow induced cultures to shake at 37°C for 3 to 4 hours. Harvest cells
by centrifugation at 6,000 rpm for 30 minutes. Discard supernatant and either
proceed with sonication and NiNTA chromatography, or freeze the pellet at -80°C
until ready to purify protein. See protocols below for NiNTA and MonoQ
purification.
Preparation of labeled (13C and/or 15N) Centrin constructs
Day 1
Transform BL21 (DE3) or BL21 (DE3) pLysS cells with plasmid. Use a 2 minute
heat shock.
Plate transformation reaction on LB agar containing carbenicillin (75µg/ml) (and
chloramphenicol (34µg/ml) if using pLysS cells). Grow overnight at 37°C.
Day 2
Inoculate 10ml LB culture (with appropriate antibiotics) with a single colony from
the transformation plate. It is wise to set up multiple cultures at all steps, in case
some do not grow well. Grow overnight at 37°C while shaking.
Day 3
Add 5µl of saturated LB culture to 10ml of M9 minimal media containing required
isotopes and antibiotics. Grow overnight at 37°C while shaking.
Day 4
Remove 500µl from saturated minimal culture, centrifuge at 2500rpm for 1
minute, discard supernatant. Resuspend pellet in fresh M9 minimal media
containing required isotopes and antibiotics and add to 50 ml flask. Incubate 4
hours at 37°C while shaking.
If cultures are saturated at this point, place them on the benchtop at room
temperature until the next day. Meanwhile, remove 1 ml from each 50ml culture
and induce expression with 1mM IPTG (final concentration). Shake induced 1ml
cultures at 37°C for 3 hours, then check expression levels by SDS-PAGE. Use
the best expresser(s) to inoculate the 1L cultures.
For each 1 L culture, remove 10 ml of saturated 50 ml culture and centrifuge at
2500 rpm for 15 minutes (longer if necessary to recover all cells from
supernatant). Resuspend each pellet in fresh M9 minimal media containing
required isotopes and antibiotics and add to the 1L flask. Grow overnight at 37°C
while shaking. It may take between 12 and 36 hours for the cultures to reach
induction stage.
Day 5
Induce cultures using 1mM IPTG (final concentration) when absorbance at
600nm reads between 0.6 and 0.8 O.D. Remember to save a pre-induction
sample for SDS-PAGE. Allow induced cultures to shake at 37°C for 3 to 4 hours.
Harvest cells by centrifugation at 6,000 rpm for 30 minutes. Discard supernatant
and either proceed with sonication and NiNTA chromatography, or freeze the
pellet at -80°C until ready to purify protein.
Expression is leaky even in pLysS cells:
CRCN-post Induction
Purification
Buffers:
Sonication Buffer
25 mM Tris HCl
(pH=8)
300 mM NaCl
1 mM PMSF
Roche Mini Complete EDTA-free protease inhibitor cocktail
(1 tablet per 20ml buffer)
NiNTA Buffer 10
25 mM Tris HCl
(pH=8)
300 mM NaCl
10 mM Imidazole
1 mM PMSF
Roche Mini Complete EDTA-free protease inhibitor cocktail
(1 tablet per 20ml buffer)
NiNTA Buffer 50
25 mM Tris HCl
(pH=8)
300 mM NaCl
50 mM Imidazole
1 mM PMSF
Roche Mini Complete EDTA-free protease inhibitor cocktail
(1 tablet per 20ml buffer)
NiNTA Buffer 150
25 mM Tris HCl
(pH=8)
300 mM NaCl
150 mM Imidazole
1 mM PMSF
Roche Mini Complete EDTA-free protease inhibitor cocktail
(1 tablet per 20ml buffer)
NiNTA Buffer 300
25 mM Tris HCl
(pH=8)
300 mM NaCl
300 mM Imidazole
1 mM PMSF
Roche Mini Complete EDTA-free protease inhibitor cocktail
(1 tablet per 20ml buffer)
MonoQ Buffer A
25 mM Tris HCl
(pH=8)
25 mM NaCl
5 mM EDTA
MonoQ Buffer B
25 mM Tris HCl
(pH=8)
1 M NaCl
All buffers should be ice cold before use.
Sonication:
Lyse pellet from 1L culture in 20 ml of ice cold sonication buffer. Use the Fisher
Sonic Dismembranator Microtip (power 5): 10 minute total process time, 20
seconds ON, 30 seconds OFF.
note: do not use lysozyme or DNAse.
Centrifuge lysate in chilled tube and rotor for 30 minutes at 18,000 rpm.
NiNTA Chromatography:
Filter supernatant (0.22µm), and apply to 10ml NiNTA column (equilibrated in
NiNTA Buffer 10). Collect fall-through in a single tube. Prepare fraction tubes by
adding 100 µl of 100mM PMSF to each tube.
Wash and elute as follows:
3 X 10ml Buffer 10
3 X 10ml Buffer 50
3 X 10ml Buffer 150
Collect each 10ml as a separate fraction. Put tubes on ice as soon as each
collection is complete. CRCN will begin to elute with the last aliquot of Buffer 50,
and completely elute in Buffer 150. Check fractions by SDS-PAGE:
CRCN
Use Buffer 300 to elute higher molecular weight contaminants, so that NiNTA
resin will be ready to use again. Wash resin in water and 20% ethanol, seal
column and store at 4°C for later use.
Dialyze the CRCN containing fractions in ice-cold buffer MonoQ-A (use 2 x 4L,
one overnight.)
Thrombin-cleave dialyzed protein solution at room temperature for one hour
while mixing (use 10 units of Thrombin per 1L pellet).
MonoQ Chromatography
Filter supernatant (0.22µm), and apply to MonoQ column set up on Akta FPLC.
Wash with 2-3 CV of MonoQ Buffer A, followed by gradient elution with MonoQ
Buffer B (0-100%B over 8CV). Collect 4 ml fractions, check by SDS-PAGE.
MonoQ10CRCN001:1_UV
MonoQ10CRCN001:1_Inject
MonoQ10CRCN001:1_Cond
MonoQ10CRCN001:1_Logbook
MonoQ10CRCN001:1_Cond%
MonoQ10CRCN001:1_Conc
MonoQ10CRCN001:1_Fractions
mAU
160
140
120
100
80
60
40
20
0
1
2
3
4
0
5
6
7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53
50
100
150
ml
CRCC elutes at 200-300 mM NaCl or 20-30%B
CRCN has a charge of -1, and elutes at 200mM NaCl
or 20%B.
CRCN-(His)6 elutes at 120-150mM NaCl
CRC-wt elutes at 230-300mM NaCl
CRCN
6His
Pool pure fractions and dialyze into ice-cold buffer of choice. Store purified
protein at -80°C.
Some Useful Hints:
Each liter of LB culture should produce about 5 g of wet cell pellet, which should
yield about 25 mg of protein. Minimal media yields will be less, on the order of
10 mg per liter.
CRCN can precipitate in low salt buffers. It is most stable in 150mM NaCl and
10mM CaCl2.
If CRC-wt precipitates in dialysis, add small amounts of EGTA up to 1mM. You
may have to add more CaCl2 afterward to allow the Thrombin reaction to work.
Follow standard procedures for cleaning MonoQ column after use.
Last Modified by S.Meyn
10-Oct-03