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Use of gradient gel filtration as a refolding, buffer exchange and fine‐tuning step in denaturing protein purification
Author(s) -
Tovar Carmel N.,
Nguyen Samantha K.,
Odunuga Odutayo O.
Publication year - 2018
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.2018.32.1_supplement.535.1
Subject(s) - size exclusion chromatography , chemistry , guanidine , native state , circular dichroism , denaturation (fissile materials) , protein aggregation , recombinant dna , chromatography , inclusion bodies , protein purification , protein tertiary structure , biochemistry , nuclear chemistry , gene , enzyme
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 are either 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 analysis using circular dichroism and fluorescence microscopy coupled with functional assays confirmed that these proteins were correctly refolded and biologically active. This strategy provides a robust approach to refolding denatured proteins that can also be used simultaneously as buffer exchange and additional purification steps. Support or Funding Information Welch Foundation Grant # AN‐0008 This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .