Development of a gel filtration protocol that eliminates precipitation during refolding of chemically denatured proteins

Abstract

Purification of recombinant proteins in denaturing conditions has many practical advantages. Chemical denaturants such as urea and guanidine hydrochloride are used to solubilize recombinant proteins from inclusion bodies. Proteins purified in denaturing conditions generally contain fewer contaminants compared to their counterparts purified in native buffers; thus reducing the number of steps in the purification scheme. Chemically denatured proteins can be stored for long time in buffer containing the denaturant and then refolded as required. Refolding is commonly performed by equilibrium dialysis and sometimes on metal affinity columns by diluting out the denaturant and allowing the protein to fold back into its native state. The refolding steps often result in significant loss of protein through aggregation. Using a modified gel filtration technique, we successfully refolded a spectrum of proteins that either are highly aggregation‐prone or contain several disulfide bonds or both, back to their native states. Some of the proteins are well known to precipitate significantly during refolding. However, using this technique, precipitation was either minimal or non‐existent. Secondary and tertiary structural analyses coupled with functional assays confirmed that these proteins were correctly refolded and biologically active. This strategy provides a robust approach to refolding denatured proteins, and simultaneously performing buffer exchange and additional purification steps.Support or Funding InformationRobert A. Welch Foundation Grant #AN‐0008