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Eun Ju Cho ABE workshop 2007

Introduction recombinant expression of protein disulfide isomerase (PDI) using the model plant Arabidopsis thaliana. Eun Ju Cho ABE workshop 2007. What is PDI? PDI : Protein Disulfide Isomerase Schematic model of PDI functions Catalyzes the formation, reduction and isomerization

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Eun Ju Cho ABE workshop 2007

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  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.

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