PRACTICAL CLASS 4
GTB 204
MOLECULAR BIOLOGY
TECHNIQUE
EXPRESSION OF CLONED GENES
IN E. Coli USING IPTG INDUCIBLE
PROMOTERS
Prof. Dr. Shaharum
Shamsuddin
[email protected]Modified by:
AP Dr. Nurul Asma
Abdullah
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Schematic diagram of a fusion vector
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1. Introduction to IPTG inducible promoters
Plasmid carrying IPTG-inducible promoters are capable of expressing proteins at
levels that exceed 30% of total mass of bacterial protein. These plasmids are well-suited
for small scale laboratory experiments, but the high cost of IPTG prevent their use for
large scale production of foreign proteins.
Vector system Fusion partner Comments Reference
pUC, pSK, Β-Galactosidase Expression under control www.stratagene.com
pBluescipt, of the lac promoter- www.lifetech.com
pGEM operator system
pGEX series GST IPTG-inducible Smith & Johnson
promoter, available with 1998
cleavage sequences Pharmacia
PMAL MBP IPTG-inducible Di Guan et al (1988)
promoter, MBP signal Maina et al (1988)
sequence facilitates www.neb.com
export to periplasm
pTrx, pTrxFus Trx IPTG-inducible LaVallie et al 1993
promoter, available with www.invitrogen.com
enterokinase cleavage
sequences
pET series Poly-His tag, selected T7 promoter (IPTG- Studier et al 1990
vectors also carry inducible), available www.novagen.com
tags for GST, Trx, sites for chemical, www.promega.com
DsbA and DsbC, enzymatic cleavage.
CBD
Table 1 : List of IPTG-induced plasmid vector
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Figure 1. The pET16b expression vector carries an N-terminal His.Tag sequence followed
by a factor Xa site and three cloning sites (shown in bold). Unique sites are shown on the
circular map. The sequence landmarks are as follows: T7 promoter (466-482), T7
transcription start (465), His.Tag coding sequence (360-389), MCS (319-335),T7 terminator
(213-259,) lacI coding sequence (869-1948), pBR322 origin (3885), bla coding sequence
(4646-5503). The box below shows the pET16b cloning/expression region.
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Figure 2. Plasmid map the parental vector pET16bSH3, showing some of the rare and
important sites. Note that the additional of Age I site (near Xho I) for the cloning
purposes. The N terminal domain of human CTCF is inserted into the multiple cloning site
via Nde I-Age I.
* Blp I is isoschizomer of Bpu 1102.
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1. Expression of recombinant protein using pET bacterial system
In this practical, the pET system (Novagen, R & D System, Europe) is used to
express the individual/truncated CTCF domains. The recombinant plasmids are transformed
into E. coli strains *BL 21 (DE3) and plated on the LB Plates containing 50µg/mL ampicilin.
Freshly transformed colonies are selected, inoculated into 5.0 mL of L-Broth (with 50µg/mL
ampicilin) and incubated for 12 hours at 37oC with shaking. These cultures are used as initial
stocks to produce bacterial from 500mL of LB. The 500mL cultures are incubated in the
shaking incubator for 3 hours at 37oC. After 3 hours incubation, the cells are induced with
IPTG (to a total concentration of 1.0 mM) and continued to grow at 37oC for 3 more hours.
After incubation, the culture is incubated on ice for 5 minutes and the cells are
pelleted by centrifugation at 5,000 rpm, at +4oC (Sorvall rotor JA 14). The supernatant is
removed and the pellet gently resuspended in 100 mL of ice-cold sterile Phosphate Buffered
Saline (2.7 mM KCl, 137.20 mM NaCl, pH 7.4). The bacterial suspension is spun at 5,000
rpm, 4oC for 10 minutes and this washing step is repeated twice.
The final pellet is lysed in 80 mL of lysis buffer (7 M urea, 20.0 mM HEPES pH
7.0) by vigorous vortexing to Resuspend the bacterial pellet. The lysis suspension is
incubated on ice for 30 minutes followed by 1-2 minutes vortexing to maximize the lysis.
Finally the suspension is centrifuged at 10,000 rpm, 4oC (Sorvall, rotor JA 14) for 20
minutes. The supernatant containing the expressed protein is transferred into a sterile 50 mL
tube and stored at -20oC for future SDS-PAGE/Western assay.
* Genotype for E. coli BL 21 (DE3) : F- ompT hsdSB (r -,m -) gal dcm (DE3) – Deficient
B B
in lon and ompT proteases.
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2. Polyacrylamide Gels for Protein Separation
Description
It often useful to resolve individual protein from a heterogenous mixture based on
their molecular weight in order to visualize their overall composition or to evaluate protein
purity from an immunoprecipitation. This method described the separation of proteins in an
electric field by inducing their movement through an acrylamide matrix.
Special equipment & solutions
1. Protein gel electrophoresis apparatus, plates, spacers, and so on (available by the
suppliers).
2. Gel dryer.
3. Solution for SDS-PAGE (see table 2).
The formation Polyacrylamide gel from Acrylamide and Bis-acrylamide.
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Stock Solution Description for recipe
Monomer solution For 200 mL preparation:
(30.8% Acrylamide + 2.7 % 60g Acrylamide (FW : 71.08)
Bis-Acryl.) 1.6g Bis-acrylamide (FW : 154.2)
Top up with ddH2O to 200 mL
4X Running Gel Buffer For 200 mL preparation:
(1.5 M Tris-Cl pH 8.8) 36.6g Tris (FW : 121.1)
Add 150 mL ddH2O and adjust to pH 8.8 with HCl
(concentrated) and top up with ddH2O to 200 mL
4X Stacking Gel Buffer For 200 mL preparation:
(0.5 M Tris-Cl pH 6.8) 12.0g Tris (FW : 121.1)
Add 150 mL ddH2O and adjust to pH 6.8 with HCl
(concentrated) and top up with ddH2O to 200 mL
10% (w/v) Sodium Dodecyl 10g SDS
Sulphate Add ddH2O to 100 mL
Can be store up to 6 months at room temperature
10% (w/v) Ammonium 1.0g Ammonium Persulphate
Persulphate (Initiator) Add ddH2O to 10 mL, aliquot into small tubes and keep at
-20oC until use. Always use fresh.
2X Treatment Buffer 0.125 M Tris-Cl, 4% SDS, 20% Glycerol,
(Loading Buffer) 0.2 M dithiotritol, 0.02% Bromophenol Blue, pH 6.8
Tank Buffer For 1 Liter :
Add 14.41g Glycine,
1g SDS, and 3.028g Tris (FW : 121.1)
Water Saturated Butanol 50 mL of n-butanol and 5 mL ddH2O – combine in a bottle
and shake. Use top phase to overlay gels. Store at room
temperature indefinitely.
Protein Standard Protein Standard : A molecular weight range of 6000 kDa
to 175,000 kDa from New England Biolab, USA.
TEMED Commercially available from SIGMA : Cat. No.
Coomassie Brilliant Blue 0.025% (w/v) Coomassie Brilliant Blue G-250 ;40% (v/v)
staining solution Methanol and 7% (v/v) Acetic Acid
Destaining solution I (for 40% (v/v) Methanol, 7% (v/v) Acetic Acid
Coomassie Brilliant Blue).
Destaining solution II (for 5% (v/v) Methanol, 7% (v/v) Acetic Acid
Coomassie Brilliant Blue).
Table 2. Stock solution for SDS-PAGE preparation (Laemmli,1970)
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Running/Separating Volume of Different percentage of Gels (in mL)
Gel Components 5% 7.5% 10% 12.5% 15%
Monomer solution 10 15 20 25 30
4x Running Buffer 15 15 15 15 15
10% APS 0.6 0.6 0.6 0.6 0.6
ddH2O 34.1 29.1 24.1 19.1 14.1
TEMED 0.02 0.02 0.02 0.02 0.02
Total Mixture (mL) 60 60 60 60 60
Table 3 : Formulation for SDS-PAGE Separating Gels [Recipes prepared solution
sufficient for 2 minigels, 10 mL each (1.5 mm thick)].
Stacking Gel Component 4% Acrylamide
Monomer Solution 625 µL
4x Stacking gel buffer 1250 µL
10% SDS 50 µL
DdH2O 3040 µL
10% APS 25 µL
TEMED 5 µL
Total Mixture 5000 µL
Table 4 : Stacking gel recipe (for 2 gels preparation).
* To determine/optimize the pore size of the acrylamide gel to give the best separation and
resolution for the molecular of interest, percentage of crosslinker (%C) was calculated
using the formula below (Bio-Rad manual) :
The weight percentage = grams of crosslinker x 100
of crosslinker (% C) grams acrylamide + grams crosslinker
Ratio %C Common application
19 : 1 5 DNA sequencing
29 : 1 3.3 Protein sequencing
37.5 : 1 2.6 Protein sequencing
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Methods
1. Attach clean glass plates to each other, separated by lightly greased spacer. Different
gel system may have slight variation. Follow the manufacturer’s instructions.
2. Add distilled water into the gel system to check the leaking.
3. Add 30 µL TEMED to catalyze polymerization of monomer (monomer preparation
is shown in table 3). Pour the mixture into the prepared plates and gently layer a few
millimeters of 2x stacking gel buffer on top to form a flat surface while acrylamide
polymerizes with water saturated butanol.
4. After 1 hour, pour the water saturated butanol out. Prepare a top stacking gel. Mix 1 mL
of 30 % acrylamide/bisacrylamide (29:1), 4 mL of H2O, 5 mL of 2x stacking gel buffer,
10 µ L ammonium persulfate, and 5 µL TEMED. Pour enough of this stacking gel
mixture to fill the upper of the gel. Insert sample position forming comb, and allow
stacking gel to polymerize. The additional of the stacking gels sharpens the bands and
increases the resolution.
5. Mix 10 µ L of sample with 10 µL of 2x sample buffer. Heat to 95oC for 3 minutes and
load sample on gel. Include molecular weight standard in one and two lanes.
6. Run gels until blue dye front just run out of gel. The typical setting is 100 constant V,
which should run in 2 hours. Time and voltage with depend on apparatus and gel
thickness.
7. Gel will be stained with Coomassie brilliant blue for at least 2 hours.
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Western blot analysis
Description
This method allows the investigator to identify specific proteins resolved by SDS
Polyacrylamide gel electrophoresis by binding with specific antibody. Protein resolved on
acrylamide gel is transferred to an PVDF membrane that is incubate with the antibody. The
primary antibody specifically binds its epitope and the bound antibody is detected with a
secondary antibody species.
Special equipment
Electrophoresis transfer apparatus
Plastic bag sealer
Rocking apparatus
Reagent
100 % methanol
CAP transfer buffer (10 mL 0.5 M CAPS, 12.5 mL Methanol. 477.5 ddH20)
Blocking solution (5% fat free milk, 0.1% Tween 20, 1x TBS dissolve in sterile water)
Washing buffer (1x TBS, 0.05% Tween 20)
Primary antibody (α-His Tag 1:1000)
Secondary antibody (α-mouse 1:1000)
Nitrocellulose paper
Methods
1. Prepare Polyacrylamide-SDS gel for separation of proteins.
2. Electrophoretically transfer protein in Polyacrylamide gel in NC paper.
a. Rinse both the gel and the PVDF membrane in the CAPS transfer buffer.
Smooth filter over gel by rolling with 5-1 glass pipette to remove air
bubbles.
b. Wrap pieces of Whatman paper (prewetted with CAPS transfer buffer)
around gel/PVDF membrane to make a sandwich. Keep them wet and avoid
bubbles.
c. Paper/gel/PVDF membrane paper sandwich is placed in electrophoretic
transfer apparatus, as suggested by manufacturer (Check the diagram
below).
d. Connect to the power supply and run the electrophoretic transfer, as suggest
by manufacturer.
e. After transfer is completed, remove the filter and gel from unit. Discard gel.
3. Block the PVDF membrane in blocking solution for 30 minutes on the rocking
platform.
4. Wash the PVDF membrane in washing buffer for 3 times (10 minutes each).
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Antibody Reaction
1. Incubate the PVDF membrane with primary antibody for 1 hour; Place it on the
rocking platform for distribution.
2. Decant the antibody into sterile tube (the antibody can be reuse for next reaction).
3. Wash the PVDF membrane again with washing buffer for 3 times on rocking
platform. (10 minutes each).
4. The PVDF membrane again will incubate with secondary antibody for 1 hour on
rocking platform.
5. The PVDF membrane will again wash with the washing buffer for 3 times. (10
minutes each).
6. The PVDF membrane then will use for film development to get the result through
the chemiluminescence film developing.
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