1. Introduction recombinant expression of protein disulfide isomerase (PDI) using the model plant Arabidopsis thaliana Eun Ju Cho ABE workshop 2007

2. What is PDI? PDI : Protein Disulfide Isomerase Schematic model of PDI functions Catalyzes the formation, reduction and isomerization of disulfide bonds in proteins

3. PDI functions

4. Overview of the Recombinant protein Process Choose a pET Vector Prepare pET Vector Prepare Insert DNA Clone Insert into pET Vector Transform into Expression Host Induce an Optimize Expression of Target Protein Scale-up culture size Extract Target Protein

5. pET-15b Vector

6. pET-15b vector for recombinant expression of His· tag proteins PDI 1/ pET-25b(+) NdeI BamH1 T7 promoter lac operator rbs His-tag PDI1 T7 terminator

7. Gene cloning of PDI 1 5’ 3’BamHI NdeI BamHI pET-15b Electrophoresis Vector ligation Gel elution & purification Colony correction (using PCR) Colony culture and Confirm by sequencing E.coli overexpression ( IPTG induction) SDS-PAGEWestern blot RT-PCR (using Arabidopsis RNA) PCR (using NdeI, BamHI linker primer) 5’ 3’ BamHI NdeI Cut (NdeI, BamHI) Transformation to DH5a vector Cut (NdeI, BamHI) M PDI1-2 PDI1-4 PDI9-2 Primer : T7 (P/T) PDI 1: 1704bp Transformation to expression host (BL21(DE3)) Gel elution & purification Electrophoresis

8. Inoculate a single colony into LB medium (+ antibiotics) Incubate with shaking at 37 ℃ until the OD 600 reaches 0.6 Add 1mM IPTG to the cultures and induce at 37 ℃ for 3hrs. E.coli overexpression Harvest cell from liquid culture by centrifugation The lac operon inducer IPTG (isopropyl-b-D- 1-thiogalactopyranoside) is recommended for blue/white screening by lacZ α- complementation with appropriate vectors and host strains, and for protein expression and other lac promoter-controlled expression system.

9. Western blot of PDI1 overexpression in E. Coli kDa 50 75 105 35 kDa 250 15 50 75 10 25 105 35 M pET-15b PDI1 PDI1 pET-15b PDI1 PDI1 67KDa (a) SDS-PAGE(b) Western blot analysis 1 st Ab : anti-PDI1 (1:1000) 2 nd Ab : anti-rabbit (1:5000) IPTG - + IPTG

10. Preparation of cleared bacterial lysates using Bugbuster/Benzonase (Novagen) 1. Harvest cell from liquid culture by centrifugation at 10,000 x g for 10min. Decant the supernatant. Determine the wet weight of the pellet. 2. Completely resuspend the cell pellet in room temperature BugBuster Protein Extraction Reagent Mix by pipetting or gentle vortexing. * BugBuster Protein Extraction Reagent Mix a) Use 5ml reagent per gram of wet cell paste. b) Add 1ul Benzonase Nuclease per 1ml Bugbuster used for resuspension.Benzonase Nuclease c) Add 1KU rLysozyme Solution per 1ml BugBuster.rLysozyme d) Benzonase and rLsozyme can be pre-mixed with BugBuster for rapid sample processing. Pre-mixed Benzonase, rLysozyme and BugBuster should be prepared immediately before use and stored at 4°C. 3. Incubate the cell suspension on a shaking platform or rotating mixer at a slow setting for 10-20min at room temperature. 4. Remove insoluble cell debris by centrifugation at 16,000 X g for 20 min at 4°C. 5. Transfer supernatant to a fresh tube. Maintain clarified extracts on ice for short term storage (a few h) or freeze at -20°C until needed.

11. Benzonase Nuclease : It degrades all forms of DNA and RNA (single stranded, double stranded, linear and circular) while having no proteolytic activity. The enzyme completely digests nucleic acids to 5′-monophosphate terminated oligonucleotides 2 to 5 bases in length (below the hybridization limit), which is ideal for removal of nucleic acids from recombinant proteins, enabling compliance with FDA guidelines for nucleic acid contamination. rLysozyme™ Solution contains a highly purified and stabilized recombinant lysozyme that can be used for lysis of Gram-negative bacteria, such as E. coli. The enzyme catalyzes the hydrolysis of N-acetylmuramide linkages in bacterial cell walls.

12. Thank you for your attention.