Download Recombinant Expression of PDI in E. coli

Recombinant Expression of PDI
in E. coli
Natasha Cortez
ABE Summer Workshop 2007
Overview
To induce E. coli to express foregin DNA. Our gene of interest is PDI
(protein disulfide isomerase). By ligating PDI into a pET-15b vector (an
expression vector), and inserting this into E. coli PDI can be expressed after
addng IPTG..
Process of Recombinant Expression
Prepare pET Vector
and Insert DNA
Clone Insert into pET
Vector
Transform Cells
Induce Expression of Target
Protein
Extract Target Protein
SDS Page
Western Blot
Gene Clonig of PDI 1
-PDI 1 Gene is attained from RT-PCR and has Ndel and
BamHI sticky ends.
-pET-15b Vector is cut at the BamHI and Ndel sites
-This ensures that the correct reading frame is preserved
so that proteins will be translated correctly.
Ndel
BamHI
Ndel
BamHi
Transformation
• A process that allows E. coli to be able to uptake the
vector containing the foreign DNA
• Weaken cell walls. This can be done chemically (CaCl2
solution), or through electroporation. Ours were done
chemically.
• Heat Shock the cells for 30 seconds so that cells swell
• Quick chill to make vectors transmit into cells.
To Induce Expression we must use
IPTG
T7 promoter
lac operon
rbs
his-tag
PDI
T3 terminator
Repressor
Adding IPTG to our cultures allows our
target genes to be translated.
Induce Expression
• Innoculate a single colony of E. coli onto LB
media with antibiotics
• Incubate with shaking at RT until OD reaches
0.6
• Add IPTG to cultures and induce at 37 degrees
for 3 hours
• Harvest cells from liquid cultures by centrifuging.
Preparation of Bacterial Lysates
+IPGT
-IPTG
Empty Vector
• Resuspend each pellet in Bugbbuster protein extraction
buffer
• Benzoase Nuclease ( degrades all forms of DNA and
RNA)
• rLysozyme (contains lysozyme used for lysis of gram
negative bacteria like E. coli.)
• Incubate with shaking for 10-20 min at RT.
• Centrifuge to pellet
• Collect supernatant.
Protein Quantification
• Add 100 ul of reagent A and
800 ul of reagent B to +IPTG,
-IPTG, and empty vector
tubes and vortex.
• Do the same to BSA
concentrations of 0, 0.5, 1,
2.5, 5, and 10 as a standard
curve to determine protein
concentration.
SDS Page
• Done on a 10% polyacrylimide gel
• Denaure proteins so that they are linear and
able to migrate through the gel
• Coat with SDS so that all molecules will have a
negative charge and will migrate through the gel
towards the positive electrode according to size.
Coomassie Stain
• Stain the gel with filtered
Coomassie at RT with shaking
for 2 hours
• Destain with destaining buffer to
further absorb Coomassie
• Sandwich the gel between
drying film.
Gel Transfer to nitrocellulose
memebrane
• Close gel sandwich clamp.
• Put in box and fill with transfer buffer and ice with
spinning stir bar.
• Run at 60-100v
Western Blot
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Block with TBST w/5% nonfat milk
Incubate with primary Antibody ( Rabbit anti-PDI) diluted to 1:1000 in blocking
agent
Wash with TSBT
Incubate with secondary Antibody (anti Rabbit congugated to HRP) diluted to
1:5000 in blocking agent.
Wash with TBST
Apply Luminol substrate
ECL Detection
Results
• In the –IPTG cells our
inserted PDI gene would
have not been
translated
PDI
vector
•The three bands similar
in size is probably the
vector.