Overview of Protein Refolding

Recombinant protein expression technology is widely used in protein functional research. Heterologous gene expression commonly leads to production of inclusion bodies. To obtain soluble protein, inclusion bodies must be refolded into an active form through refolding methods. Refolding is initiated by reducing the concentration of denaturant to solubilize inclusion bodies.

Protein Refolding

What is the inclusion body?

Inclusion bodies(IBs) are the formation of insoluble protein particles which are caused by expression of heterologous genes, especially in highly efficient expression in Escherichia coli cells. The protein with biological activity in cells are usually soluble. IBs are aggregates of unfolded and with no biological activity. Therefore, IBs must be dissolved by denaturing agent and refolding methods.

Formation of inclusion body

1) Over expression is more likely to cause inclusion bodies formation
2) The amino acid composition of recombinant proteins: the more with sulfur amino acid, the easy to form
3) The expression environment of recombinant protein: over high fermentation temperature
4) In eukaryotes, the enzymes of post-translational modification are lacked, which results in the accumulation of intermediates. So IBs are easy to form in cells

Refolding Methods

    • Dialysis, dilution and ultrafiltration - These three methods are traditional and generally used in the protein refolding. The recovery rate is very low and it is difficult to be separated from hybrid proteins. Dilution method is time-consuming and easy to form aggregates of inactive proteins, not suitable for industrial production.
    • Gel filtration chromatography - Gel filtration chromatography is used for refolding high concentration of protein. The recovery rate is higher than dilution method and this method can also be applied to purification of protein.
    • Add accelerant - The function of accelerant can stabilize the natural structure of folding protein, increase the solubility of refolding intermediate or increase the unfolded protein solubility.
    • LC (liquid chromatography) - Liquid chromatography is one of the effective methods for purifying proteins, which becomes an essential tool for recombinant protein purification, like hydrophobic interaction chromatography (HIC), ion exchange chromatography (IEC), gel exclusion chromatography (SEC), affinity chromatography (AFC), etc. Compared with the traditional dilution and dialysis method, liquid chromatography has the below advantages:
      1) Denaturing agent can be quickly removed.
      2) The adsorption of denatured protein are obviously reduced, therefore to avoid the generation of precipitation.
      3) It can separate the protein of interest from hybrid proteins, and make renaturation and purification at the same time.

How to validate refolding

1) Gel electrophoresis
2) Spectroscopic method - It only be used for refolding process in research. Ultraviolet difference spectra , Fluorescence spectra and Circular Dichroic Spectroscopy are included.
3) Chromatographic process - IEX, RP-HPLC, CE, etc
4) Immunological methods - Elisa, Western blot