refolding of inclusion body proteins from e. coli
TRANSCRIPT
Refolding of Inclusion Body Proteins from E. Coli
Inclusion Bodies (IBs)
Inclusion Bodies
Aggregation
How to obtain native activity and structure of the IBs ?
How Protein Folding in vivo?
Cytoplasm Crowding and Confinement
What does Correct Protein Folding Need?
Molecular Chaperon
What does Correct Protein Folding Need?
Protein Disulfide Isomerase (PDI)
What does Correct Protein Folding Need?
ER Quality Control System
What does Correct Protein Folding Need?
Free-energy Gradient
What does Correct Protein Folding Need?
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
IBs Refolding Procedure
Releasing IBs from E. coli cells
Separated IBs from the cell homogenate
IBs solubilization
Purify IBs
Refolding
IBs Refolding Procedure
Releasing IBs from E. coli cells
Separated IBs from the cell homogenate
IBs solubilization
Purify IBs
Refolding
Releasing IBs from E. coli
On the laboratory scale:
Sonication
French press
Enzymatic or chemical cleavage of the cells
Freeze–thawing
Releasing IBs from E. coli
For larger scale:
High pressure homogenization
Bead milling
IBs Refolding Procedure
Releasing IBs from E. coli cells
Separated IBs from the cell homogenate
IBs solubilization
Purify IBs
Refolding
Separated IBs
Elaborate washing procedures
Variety of buffer compositions
Differential centrifugation:
Separated IBs
Size-exclusion chromatography (SEC)
IBs Refolding Procedure
Releasing IBs from E. coli cells
Separated IBs from the cell homogenate
IBs solubilization
Purify IBs
Refolding
“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.
IBs Refolding Procedure
Releasing IBs from E. coli cells
Separated IBs from the cell homogenate
IBs solubilization
Purify IBs
Refolding
IBs Purification
Size-exclusion chromatography (SEC)
Ion exchange chromatography
Columns with solid-phase
…
IBs Refolding Procedure
Releasing IBs from E. coli cells
Separated IBs from the cell homogenate
IBs solubilization
Purify IBs
Refolding
Refolding Method
Two problems: Aggregation and Misfolding
Refolding Method
Refolding Method
Intermediate structures
Molten Globules
Refolding Method
Refolding Method
Subsequent
Concentration
Control protein concentrationTime-consumingBuffer-consuming
Renaturation
buffers
“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.
Size-exclusion Chromatography (SEC)
Denatured Protein
Loading Solution
ElutionSolution
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
Hydrophobic-Interaction Chromatography (HIC)
HIC can directly deal with guanidine-HCl denatured proteins
Other Chromatographic Techniques
Immobilized metal-ion affinity chromatography (IMAC)
Affinity chromatography (AFC), such as chromatography using chaperones GroEL and GroESas ligands
Liposome chromatographyβ-Cyclodextrin chromatographyTriton X-100 chromatography…
Contact Information:
Phone: 1-631-559-92691-631-448-7888
Fax: 1-631-938-8127 Address: 45-1 Ramsey Road, Shirley,
NY 11967, USA Email: [email protected]
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