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 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. 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.

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

6His

CRCN

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 CaCl_2.

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