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Use of gradient gel filtration to simultaneously exchange buffer, purify and refold denatured proteins
Author(s) -
Chaudhry Saad,
Burnett Marianne,
Odunuga Odutayo
Publication year - 2015
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.29.1_supplement.882.9
Subject(s) - chemistry , size exclusion chromatography , guanidine , chromatography , denaturation (fissile materials) , recombinant dna , native state , urea , circular dichroism , inclusion bodies , protein purification , protein aggregation , biochemistry , enzyme , gene , nuclear chemistry
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, gradient gel filtration, to successfully refold these aggregation‐prone proteins back to their native states during denaturing purification. Some of the proteins chosen are well‐known to precipitate significantly during refolding. However, using this technique, precipitation was either so minimal or non‐existent. Circular dichroism, enzymatic assays and dynamic molecular mass determination confirmed that the proteins were correctly refolded and biologically active. This study provides a robust approach to refolding denatured proteins that can also be used simultaneously as buffer exchange and additional purification step.