| 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.
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