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Improved Procedure for Refolding Insoluble Proteins

IB-2004

   
     
 
Crystals obtained from two insoluble proteins refolded by on-column chemical refolding.
A) Crystals of 1105B, 300 x 150 x 150 microns.
B) Crystals of 1349B, 100 x 50 x 50 microns.
 
     

APPLICATIONS OF TECHNOLOGY:

  • Protein preparation for x-ray
  • Protein purification kits and reagents
  • Protein functional analysis crystallography and NMR

ADVANTAGES:

  • No filtration or concentration required
  • Applicable to a wide range of insoluble proteins

ABSTRACT:

Sung-Hou Kim, Rosalind Kim and Natalia Oganesyan at Berkeley Labs have developed an improved method for refolding insoluble proteins from inclusion bodies. This method is less time-consuming, does not require filtration or concentration, is compatible with a wide range of proteins, and is easily adaptable for high-throughput processing. The method is used in protein preparation for x-ray crystallography and NMR.

A major challenge in the purification of insoluble proteins lies in the refolding of proteins from inclusion bodies (insoluble protein aggregates). Current methods are time-consuming and require filtration and concentration steps that may reduce protein yields.

The Berkeley Lab method incorporates several improvements on current techniques. Solubilized His-tagged proteins are bound to Ni-NTA resin in the presence of urea and buffer-containing detergent. The resin is then washed with cyclodextrin to remove the detergent and promote correct folding. The eluted protein is then purified by ion-exchange and size exclusion chromatography. Finally, dynamic light scattering and circular dichroism spectroscopy are used to evaluate refolding. A high percentage of the refolded proteins tested were able to produce crystals.

STATUS:

  • Patent Pending. Available for licensing or collaborative research

FOR MORE INFORMATION SEE:

Oganesyan, N., Kim, S., Kim, R., "On-Column Chemical Refolding of Proteins." PharmaGenomics, Sept. 2004.

REFERENCE NUMBER: IB-2004

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