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…

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