Periplasmic protein expression – FimH truncate expression in E

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Periplasmic protein expression – FimH truncate expression in E. coli
Sunday: Start overnight cultures of cells containing the FimH truncate expression-vector from
glycerol stocks, (Gelareh)
May 12th, Monday
Preparation of large culture of FimHtr:
NB: Steps 1. and 2. have to be carried out under sterile conditions. There is a
great risk of phage contamination in the lab, which would lead to cell death and
NO RESULTS! So, please prepare everything with outmost care!
1. Inoculation: Prepare two baffled flasks with a final volume of no more than 800 ml
in each. Add 40 ml of the overnight culture to 760 ml of fresh minimal media#
(containing 50 mg/l ampicillin, i.e. 400 ul of the 100 mg/l stock solution). Grow
the cultures for 3-4 h in a shaker at 37 C, until the optical density of the culture
have reached approximately 0.6 units (OD600). To make sure that the cultures are
ok, check the growth progress by measuring OD600 every hour.
#
Minimal media: (800 ml culture) 40 ml 20xM9 salt, 700 ml sterile water, 20 ml 20% Glucose, 100 ul
CaCl2, 800 ul MgSO4. FimH shows strong binding affinity for mannose derivatives. The lectin picks
up a butyl -D-mannoside from the Luria-Bertani (LB) medium during expression, which is why
minimal media is used instead. This provides proteins with an available carbohydrate binding site for
later binding studies.
2. Induction: Add IPTG, to a final concentration of 5 mM (i.e 400 ul of the 1M IPTG
stock) and grow for another 2 h.
3. Harvesting: Spin down the cells at 4500rpm (swing out rotor) for 20 min. Weigh
the flasks (with lid) before the cultures are added. Balance your flasks (two flasks per
group, mark them well so that you don’t mix them up) and run the centrifuge together
with the other groups.
While waiting – Add Jododpax to the baffled culture flasks. After the centrifugation,
empty the supernatant into the flasks and fill them up with water. It is very important
to kill off all bacteria and other possible contaminants as soon as possible, in order to
prevent a contamination for everyone else in the lab.
4. Periplasmic preparation:
i) Weigh the cells. Resuspend cells in 20 mM Tris-HCl pH 8.0, 30 % sucrose,
5 mM EDTA. Take 4 ml of sucrose mix per gram cell. Transfer sample to 50
ml Falcon tubes and incubate on ice for 20 min.
ii) Spin down the samples at 8500 xg (fixed angle rotor) for 25 min. Discard
the supernatant, but save it for further analysis (this is called the sucrose
fraction).
iii) Extract the periplasmic content by osmotic shock. Dissolve the pellet in 5
mM MgCl2 (4-5 ml per gram cell) and half a tablet of protease inhibitor
cocktail (Roche). Incubate on ice for 15 min.
iv) Spin down the samples at 8500 xg (fixed angle rotor) for 25 min. Take 50
ul of the supernatant (periplasmic fraction) for analysis. Dialyze the
supernatant against 20 mM sodium phosphate buffer pH 7.8, in cold room
under continuous stirring, over night.
5. SDS-PAGE to analyze the expression: Prepare a 15 % polyacrylamide SDS gel,
using the Bio Rad system. Prepare the sucrose, pellet and periplasmic fractions
according to the following: add 6 ul of 4x SDS loading buffer to 18 ul of the
sucrose and periplasmic fractions. Dissolve some of the pellet in 18 ul of water and
add 6 ul of the loading buffer. Boil the samples and a low molecular weight marker
for 10 min at 95 C. Add 5 % -mercaptoethanol to each and spin down. Mount the
gel and add buffer. NB: Check for leakage! Load approximately 15 ul of each
sample and run at 200 V for approximately 40 min. Stain in Commasie blue stain.
From Wet Lab 1:
SDS-PAGE
Prepare the gels like this:
15% separating gel, pH 8.8 ml
88% Glycerol
0.525
1M Tris-HCl, pH 8.8
1.87
30% AA-bisAA (30:1)
2.5
10% SDS
0.05
10% PSA
0.05
TEMED
0.005
5 ml
4% stacking gel, pH 6.8
H2O
1M Tris-HCl, pH 6.8
30% AA-bisAA (30:1)
10% SDS
10% PSA
TEMED
ml
1.84
0.312
0.325
0.025
0.0125
0.005
2.5 ml
Assemble the glass plates and spacers.
Pour the separating gel to about 1 cm below the wells of the comb.
Pour the stacking gel and insert the comb immediately.
When the stacking gel has set, place in gel rig and immerse in buffer.
Prepare the samples:
Take 30 ul of each sample and mix with 10 ul 4x SDS loading buffer containing 0.1
M -mercaptoethanol (added fresh before use)
Heated the mixture at 100C for 5 min.
Load the samples into the wells ( no more than 15 ul).
Be sure to use markers.
Run with constant voltage (200V) for about 1 hr.
Staining the gel with Coomassie Blue:
Place the gel in a box, eg. An empty tip box.
Add the fix solution (30% EtOH, 10% HAc) and heat it to near boiling in the
microwave oven (10 – 20 s).
Pour the fix solution back for reuse the fix solution.
Add 50 ml Coomassie Blue solution and heat it to near boiling.
Incubate 5 min at room temperature; shake it every now and then.
Pour the Coomassie solution back into its Falcon tube for reuse (can be reused up to
10 times).
Rinse the gel with some water and heat it to near boiling.
Incubate 5 min or longer at room temperature, shake it every now and then.
Change the water and heat and incubate again until the background is low enough.
The gel is now ready for scanning!
May 13th, Tuesday
Protein purification by liquid chromatography, using a HiTrap chelating column.
1. To remove any precipitated proteins, before purification, centrifuge the dialyzed
periplasmic fraction in Falcon tubes at 8500 rpm (fixed angle rotor) for 30 min.
2. Prepare the buffers for the purification:
Buffer A: 20 mM sodium phosphate buffer pH 7.8 (equilibration, loading,
washing)
Buffer B: 20 mM sodium phosphate buffer pH 7.8, 500 mM imidazole (elution)
3. Prepare the column: load on Ni-chloride and wash off the excess with water. Wash
the system and the column with buffer A and B and finally equilibrate the column
with buffer A.
4. Filter the dialyzed supernatant before loading the sample onto the column, using
0.45 um sterile filters.
5. Create a purification program after discussing the purification steps with your labpartners and teacher. (Hint elute the protein with a sharp 0-500 mM imidazole
gradient.) Run the program.
6. Collect the FimHtr fractions and dialyze against 50 mM sodium acetate pH 5.25
(buffer A, which is going be used in the ion exchange step on Wednesday) under
continuous stirring, over night. Take off 50 ul of the sample for SDS-PAGE.
Column information
Name: HiTrap Chelating Column (GE)
Technique: Complex binding of the C-terminally His-tagged FimHtr with Nickel ion
coated sepharose column matrix
Column bed volume: 5 ml
Maximum sample volume: 12 mg pure His-tagged protein per ml/medium
Recommended flow rate: 5 ml/ min
Maximum pressure: 0.5 MPa
May 14th, Wednesday
Protein purification, using a MonoS ion exchange column. (Day 2)
1. Proceed as yesterday. Centrifuge the dialyzed protein sample. (Falcon tubes at 8500
rpm for 30 min)
2. Prepare buffers:
Buffer A: 50 mM sodium acetate pH 5.25 (equilibration, loading, washing)
Buffer B: 50 mM sodium acetate pH 5.25, 500 mM NaCl (elution)
3. Prepare column: wash the system and the column with buffer A and B and
equilibrate the column with buffer A.
4. Filter the dialyzed sample, using 0.45 um sterile filters, and measure the
concentration of the protein before loading it onto the column. Use the Nano-drop
spectrophotometer and the Abs280 program (absorbance measured at wavelength
280 nm).
5. Create a purification program after discussing the purification steps with your labpartners and teacher. (Hint: elute the protein with a 0-500 mM NaCl gradient.) Run
the program.
6. Finally, analyze the fractions on a 15 % SDS gel to check the quantity and the
purity of the protein sample after the final purification step. Include the sample
taken after the Ni Hitrap purification step.
7. Measure the final concentration of the protein.
Column information
Name: MonoS column (GE)
Technique: Cation exchanger, binding of the positively charged protein.
Column bed volume: 8 ml
Recommended flow rate: 2 ml/ min
Maximum pressure: 5 MPa