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Refolding of inclusion body proteins from e. coli

This ppt explains how protein fold in vivo and inclusion bodies refold procedure. It also introduces IBs refolding methods, such as dilution and chromatography.

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Refolding of inclusion body proteins from e. coli

  1. 1. Refolding of Inclusion Body Proteins from E. Coli
  2. 2. Inclusion Bodies (IBs) Inclusion Bodies Aggregation
  3. 3. How to obtain native activity and structure of the IBs ?
  4. 4. How Protein Folding in vivo?
  5. 5.  Cytoplasm Crowding and Confinement What does Correct Protein Folding Need?
  6. 6.  Molecular Chaperon What does Correct Protein Folding Need?
  7. 7.  Protein Disulfide Isomerase (PDI) What does Correct Protein Folding Need?
  8. 8.  ER Quality Control System What does Correct Protein Folding Need?
  9. 9.  Free-energy Gradient What does Correct Protein Folding Need?
  10. 10. Why we still use IB expression? How to avoid IBs:  Expression systems  Promoter  Low copy number plasmid  Host  pH  Minimal media  IPTG concentration  Induction temperature  … Benefit of IBs:  Easily separate from soluble impurities  Higher expression level
  11. 11. IBs Refolding Procedure Releasing IBs from E. coli cells Separated IBs from the cell homogenate IBs solubilization Purify IBs Refolding
  12. 12. IBs Refolding Procedure Releasing IBs from E. coli cells Separated IBs from the cell homogenate IBs solubilization Purify IBs Refolding
  13. 13. Releasing IBs from E. coli On the laboratory scale:  Sonication  French press  Enzymatic or chemical cleavage of the cells  Freeze–thawing
  14. 14. Releasing IBs from E. coli For larger scale:  High pressure homogenization  Bead milling
  15. 15. IBs Refolding Procedure Releasing IBs from E. coli cells Separated IBs from the cell homogenate IBs solubilization Purify IBs Refolding
  16. 16. Separated IBs  Elaborate washing procedures  Variety of buffer compositions Differential centrifugation:
  17. 17. Separated IBs Size-exclusion chromatography (SEC)
  18. 18. IBs Refolding Procedure Releasing IBs from E. coli cells Separated IBs from the cell homogenate IBs solubilization Purify IBs Refolding
  19. 19. “ The vast majority of solubilization procedures rely on strong denaturants such as 6 M guanidine hydrochloride or 8 M urea. If the protein contains disulfide bonds, a reducing agent such as DTT should be added.
  20. 20. IBs Refolding Procedure Releasing IBs from E. coli cells Separated IBs from the cell homogenate IBs solubilization Purify IBs Refolding
  21. 21. IBs Purification Size-exclusion chromatography (SEC) Ion exchange chromatography Columns with solid-phase …
  22. 22. IBs Refolding Procedure Releasing IBs from E. coli cells Separated IBs from the cell homogenate IBs solubilization Purify IBs Refolding
  23. 23. Refolding Method Two problems: Aggregation and Misfolding
  24. 24. Refolding Method
  25. 25. Refolding Method Intermediate structures Molten Globules
  26. 26. Refolding Method
  27. 27. Refolding Method Subsequent Concentration Control protein concentration Time-consuming Buffer-consuming Renaturation buffers
  28. 28. “In chromatographic refolding, the solid medium acts as a kind of chaperone or assistant to help the protein refolding occur in a correct way, which minimizes misfolding and aggregation. The feed solution containing denatured protein and denaturant is loaded into the column packed with porous microspheres. Renaturation buffers are introduced to elute the denatured protein to move through the column. During this process, simultaneous refolding and adsorption take place. The solid phase helps the correct folding of the protein. At the column outlet, the protein may exit in the correct form.
  29. 29. Size-exclusion Chromatography (SEC) Denatured Protein Loading Solution Elution Solution
  30. 30. Ion-Exchange Chromatography (IEC)  Adsorption of protein could separate individual molecules, thus the chance of aggregation is minimized.  Optimize denaturant concentration and pH simultaneously is difficult Dual-gradient IEC process:  High pH from pI: Prevent aggregation  Low pH near pI: Biologically active conformation
  31. 31. Hydrophobic-Interaction Chromatography (HIC) HIC can directly deal with guanidine-HCl denatured proteins
  32. 32. Other Chromatographic Techniques Immobilized metal-ion affinity chromatography (IMAC) Affinity chromatography (AFC), such as chromatography using chaperones GroEL and GroES as ligands Liposome chromatography β-Cyclodextrin chromatography Triton X-100 chromatography …
  33. 33. Contact Information: Phone: 1-631-559-9269 1-631-448-7888 Fax: 1-631-938-8127 Address: 45-1 Ramsey Road, Shirley, NY 11967, USA Email: info@creative-biomart.com
  34. 34. Thanks! Any questions? You can find me at contact@creative-biomart.com

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This ppt explains how protein fold in vivo and inclusion bodies refold procedure. It also introduces IBs refolding methods, such as dilution and chromatography.

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