Use of gradient gel filtration to simultaneously exchange buffer, purify and refold denatured proteins

Purification of recombinant proteins in denaturing conditions, 8 M urea or 6 M guanidine hydrochloride (GuHCl), has many practical advantages. Recombinant proteins can be solubilized from inclusion bodies in the presence of urea or GuHCl in buffers. Proteins purified in denaturing buffers generally contain fewer contaminants compared to their counterparts purified in native conditions; thus reducing the number of steps in the purification scheme. Denatured proteins can be stored for long time in buffers containing denaturants and then refolded as required. However, one major disadvantage of denaturing protein purification is the laborious refolding step, where the denaturant is carefully diluted out to allow the denatured protein to fold back into its native state. This step is not only time-consuming but can also result in significant loss of the protein through aggregation. Using a spectrum of proteins with measurable functional activities, we report the use of a modified gel filtration technique, gradi...