![Page 1: Recombinant Expression of PDI in E. coli Natasha Cortez ABE Summer Workshop 2007](https://reader036.vdocuments.us/reader036/viewer/2022062417/551b56da5503465c7e8b5d3c/html5/thumbnails/1.jpg)
Recombinant Expression of PDI in E. coli
Natasha Cortez
ABE Summer Workshop 2007
![Page 2: Recombinant Expression of PDI in E. coli Natasha Cortez ABE Summer Workshop 2007](https://reader036.vdocuments.us/reader036/viewer/2022062417/551b56da5503465c7e8b5d3c/html5/thumbnails/2.jpg)
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..
![Page 3: Recombinant Expression of PDI in E. coli Natasha Cortez ABE Summer Workshop 2007](https://reader036.vdocuments.us/reader036/viewer/2022062417/551b56da5503465c7e8b5d3c/html5/thumbnails/3.jpg)
Process of Recombinant Expression
Prepare pET Vector and Insert DNA
Clone Insert into pETVector
Transform Cells
Induce Expression of TargetProtein
Extract Target Protein
SDS Page
Western Blot
![Page 4: Recombinant Expression of PDI in E. coli Natasha Cortez ABE Summer Workshop 2007](https://reader036.vdocuments.us/reader036/viewer/2022062417/551b56da5503465c7e8b5d3c/html5/thumbnails/4.jpg)
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.
BamHINdel
Ndel BamHi
![Page 5: Recombinant Expression of PDI in E. coli Natasha Cortez ABE Summer Workshop 2007](https://reader036.vdocuments.us/reader036/viewer/2022062417/551b56da5503465c7e8b5d3c/html5/thumbnails/5.jpg)
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.
![Page 6: Recombinant Expression of PDI in E. coli Natasha Cortez ABE Summer Workshop 2007](https://reader036.vdocuments.us/reader036/viewer/2022062417/551b56da5503465c7e8b5d3c/html5/thumbnails/6.jpg)
To Induce Expression we must use IPTG
Adding IPTG to our cultures allows our target genes to be translated.
T7 promoter lac operon rbs his-tag PDI T3 terminator
Repressor
![Page 7: Recombinant Expression of PDI in E. coli Natasha Cortez ABE Summer Workshop 2007](https://reader036.vdocuments.us/reader036/viewer/2022062417/551b56da5503465c7e8b5d3c/html5/thumbnails/7.jpg)
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.
![Page 8: Recombinant Expression of PDI in E. coli Natasha Cortez ABE Summer Workshop 2007](https://reader036.vdocuments.us/reader036/viewer/2022062417/551b56da5503465c7e8b5d3c/html5/thumbnails/8.jpg)
• 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.
+IPGT -IPTG Empty Vector
Preparation of Bacterial Lysates
![Page 9: Recombinant Expression of PDI in E. coli Natasha Cortez ABE Summer Workshop 2007](https://reader036.vdocuments.us/reader036/viewer/2022062417/551b56da5503465c7e8b5d3c/html5/thumbnails/9.jpg)
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.
![Page 10: Recombinant Expression of PDI in E. coli Natasha Cortez ABE Summer Workshop 2007](https://reader036.vdocuments.us/reader036/viewer/2022062417/551b56da5503465c7e8b5d3c/html5/thumbnails/10.jpg)
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.
![Page 11: Recombinant Expression of PDI in E. coli Natasha Cortez ABE Summer Workshop 2007](https://reader036.vdocuments.us/reader036/viewer/2022062417/551b56da5503465c7e8b5d3c/html5/thumbnails/11.jpg)
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.
![Page 12: Recombinant Expression of PDI in E. coli Natasha Cortez ABE Summer Workshop 2007](https://reader036.vdocuments.us/reader036/viewer/2022062417/551b56da5503465c7e8b5d3c/html5/thumbnails/12.jpg)
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
![Page 13: Recombinant Expression of PDI in E. coli Natasha Cortez ABE Summer Workshop 2007](https://reader036.vdocuments.us/reader036/viewer/2022062417/551b56da5503465c7e8b5d3c/html5/thumbnails/13.jpg)
Western Blot• 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
![Page 14: Recombinant Expression of PDI in E. coli Natasha Cortez ABE Summer Workshop 2007](https://reader036.vdocuments.us/reader036/viewer/2022062417/551b56da5503465c7e8b5d3c/html5/thumbnails/14.jpg)
Results
vector
PDI
• In the –IPTG cells our inserted PDI gene would have not been translated
•The three bands similar in size is probably the vector.