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Recombinant DNA Techniques (DNA Cloning)

生命科學實驗. Plasmid DNA miniprep . PCR R. E. digestion Gel purification, Ligation, Transformation Preparation of competent cells. Gene Plasmid b.p . ORF E.coli strain. Recombinant DNA Techniques (DNA Cloning). GST-LEU2 SDS-PAGE Coomasie Blue ( Green Angel) Staining

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Recombinant DNA Techniques (DNA Cloning)

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  1. 生命科學實驗 Plasmid DNA miniprep. PCR R. E. digestion Gel purification, Ligation, Transformation Preparation of competent cells Gene Plasmid b.p. ORF E.coli strain Recombinant DNA Techniques (DNA Cloning) GST-LEU2 SDS-PAGE Coomasie Blue (Green Angel) Staining O.D.280, Bradford and Bicinchoninic acid assays (BCA) Expression, Purification and quantification of recombinant protein M.W. Ab Detection of recombinant protein Immunological (Western) Analysis Application of recombinant protein Enzyme assay (DHase) of GST-LEU2

  2. Outline for expression and purification of GST-fusion protein Clone gene into pGEX vector Week 1 -/ + IPTG induction SDS-PAGE Test expression of fusion protein (small scale), SDS-PAGE Week 2 Lysozyme, DNaseI/ sonication Culture (large scale), harvest and lyse cells Binding Washing Elution Week 3 GST-fusion protein purification, making new protein gel for week 4 Week 4 SDS-PAGE Coomasie Blue Staining Bradford assays Detection and quantification of GST fusion protein

  3. GST-fusion system • A one-step system to purify protein. • Developed by Smith and Johnson in 1988, using glutathione S-transferase (GST) protein from Schistosomajaponicum. • Protein of interest is usually fused at the C-terminus of GST. • GST binds to glutathione, an tripeptide antioxidant, with high affinity. beads GST protein glutathione

  4. IPTG Induction • A way to induce expression of large amount of protein in E.coli. tac promoter: hybrid promoter of trp and lac, strong expression upon induced X Lac repressor GST LEU2 P L :IPTG conformational change of Lac repressor P L GST LEU2

  5. SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) 聚丙烯胺膠體

  6. Expression of GST-LEU2 Protein GST GST-LEU2 GST-leu2 IPTG (h): 0 0 0 3 3 3 5 5 5 Green Angel Protein Staining 170 130 95 72 55 43 34 26 17

  7. GST-LEU2 protein purification (large scale) Lysate preparation (1.5 hours) Glutathione-beads binding (2 hours) Wash (40 mins) Elution (40 mins)

  8. GST-LEU2 lysatepreparation Lysis buffer: 50 mMNaCl: providing a osmotic shock and keep protein soluble 50 mMTris HCI, pH 7.5 1 mM EDTA: inhibits divalent cation-dependent proteases 1% (v/v) Triton X-100: detergent, increasing protein solubility Protease inhibitor cocktail PMSF (phenylmethylsulfonyl fluoride): serine protease inhibitor, rapidly degraded in water Lysozyme: a glycoside hydrolase of peptidoglycans, break the carbohydrate in the bacterial cell wall. DNase I: digest bacterial DNA to reduce viscosity of lysate Other lysis methods: Sonication: useful for a variety of cell types. Homogenization/ Glass bead/ French press: high pressure to force cells through a narrow space, thereby shearing the cell walls and membrane.

  9. GST-LEU2 purification beads + GST-LEU2 lysate beads GST-LEU2 after-binding washing beads GST-LEU2 wash

  10. GST-LEU2 elution beads GST-LEU2 free glutathione beads GST-LEU2 elute

  11. GST fusion protein purification Lane 1: whole cell lysate Lane 2: after binding Lane 3: wash Lane 4: elute

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