Presentation on theme: "Recombinant Expression of PDI in E. coli"— Presentation transcript:
1 Recombinant Expression of PDI in E. coli Recombinant Expression of PDI in E. coliNatasha CortezABE Summer Workshop 2007
2 OverviewTo 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..
3 Process of Recombinant Expression Prepare pET Vectorand Insert DNAClone Insert into pETVectorTransform CellsInduce Expression of TargetProteinExtract Target ProteinSDS PageWestern Blot
4 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.NdelBamHINdelBamHi
5 TransformationA process that allows E. coli to be able to uptake the vector containing the foreign DNAWeaken 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 swellQuick chill to make vectors transmit into cells.
6 To Induce Expression we must use IPTG T7 promoter lac operon rbs his-tag PDI T3 terminatorRepressorAdding IPTG to our cultures allows our target genes to be translated.
7 Induce ExpressionInnoculate a single colony of E. coli onto LB media with antibioticsIncubate with shaking at RT until OD reaches 0.6Add IPTG to cultures and induce at 37 degrees for 3 hoursHarvest cells from liquid cultures by centrifuging.
8 Preparation of Bacterial Lysates Empty Vector+IPGT-IPTGResuspend each pellet in Bugbbuster protein extraction bufferBenzoase 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 min at RT.Centrifuge to pelletCollect supernatant.
9 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.
10 SDS Page Done on a 10% polyacrylimide gel SDS PageDone on a 10% polyacrylimide gelDenaure proteins so that they are linear and able to migrate through the gelCoat with SDS so that all molecules will have a negative charge and will migrate through the gel towards the positive electrode according to size.
11 Coomassie StainStain the gel with filtered Coomassie at RT with shaking for 2 hoursDestain with destaining buffer to further absorb CoomassieSandwich the gel between drying film.
12 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 v
13 Western Blot Block with TBST w/5% nonfat milk Block with TBST w/5% nonfat milkIncubate with primary Antibody ( Rabbit anti-PDI) diluted to 1:1000 in blocking agentWash with TSBTIncubate with secondary Antibody (anti Rabbit congugated to HRP) diluted to 1:5000 in blocking agent.Wash with TBSTApply Luminol substrateECL Detection
14 ResultsIn the –IPTG cells our inserted PDI gene would have not been translatedThe three bands similar in size is probably the vector.PDIvector