Recombinant Expression of PDI in E. coli

1. 0 Recombinant Expression of PDI in E. coli Natasha Cortez ABE Summer Workshop 2007

2. 0 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..

3. 0 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

4. 0 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

5. 0 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.

6. 0 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

7. 0 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.

8. Preparation of Bacterial Lysates 0 Empty Vector +IPGT -IPTG • 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.

9. 0 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. 0 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.

11. 0 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.

12. 0 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

13. 0 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

14. 0 Results • In the –IPTG cells our inserted PDI gene would have not been translated • The three bands similar in size is probably the vector. PDI vector