Eun Ju Cho ABE workshop 2007

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 PDI1/pET-25b(+) NdeI BamH1 T7 promoter lac operator rbs His-tag PDI1 T7 terminator

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

8. E.coli overexpression Inoculate a single colony into LB medium (+ antibiotics) Incubate with shaking at 37℃ until the OD600 reaches 0.6 Add 1mM IPTG to the cultures and induce at 37℃ for 3hrs. Harvest cell from liquid culture by centrifugation • The lac operon inducer IPTG (isopropyl-b-D- 1-thiogalactopyranoside) is recommended for blue/white screening by lacZα- 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 (a) SDS-PAGE (b) Western blot analysis IPTG - + IPTG - + kDa M pET-15b PDI1 PDI1 250 kDa pET-15b PDI1 PDI1 105 75 105 67KDa 75 50 50 35 35 25 1st Ab : anti-PDI1 (1:1000) 2nd Ab : anti-rabbit (1:5000) 15 10

10. Preparation of cleared bacterial lysates using Bugbuster/Benzonase (Novagen) • 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 Nucleaseper 1ml Bugbuster used for resuspension. • c) Add 1KU rLysozyme Solution per 1ml BugBuster. • 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.