Presentation on theme: "Recombinant Expression of PDI in E. coli Natasha Cortez ABE Summer Workshop 2007."— Presentation transcript:
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. 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 Adding IPTG to our cultures allows our target genes to be translated. T7 promoter lac operon rbs his-tag PDI T3 terminator Repressor
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.
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
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 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 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.