Efficient refolding of a hydrophobic protein with multiple S-S bonds by on-resin immobilized metal affinity chromatography.
Summary of "Efficient refolding of a hydrophobic protein with multiple S-S bonds by on-resin immobilized metal affinity chromatography."
The efficient refolding of recombinant proteins produced in the form of inclusion bodies (IBs) in Escherichia coli still is a complicated experimental problem especially for large hydrophobic highly disulfide-bonded proteins. The aim of this work was to develop highly efficient and simple refolding procedure for such a protein. The recombinant C-terminal fragment of human alpha-fetoprotein (rAFP-Cterm), which has molecular weight of 26kDa and possesses 6 S-S bonds, was expressed in the form of IBs in E. coli. The C-terminal 7× His tag was introduced to facilitate protein purification and refolding. The refolding procedure of the immobilized protein by immobilized metal chelating chromatography (IMAC) was developed. Such hydrophobic highly disulfide-bonded proteins tend to irreversibly bind to traditionally used agarose-based matrices upon attempted refolding of the immobilized protein. Indeed, the yield of rAFP-Cterm upon its refolding by IMAC on agarose-based matrix was negligible with bulk of the protein irreversibly stacked to the resin. The key has occurred to be using IMAC based on silica matrix. This increased on-resin refolding yield of the target protein from almost 0 to 60% with purity 98%. Compared to dilution refolding of the same protein, the productivity of the developed procedure was two orders higher. There was no need for further purification or concentration of the renatured protein. The usage of silica-based matrix for the refolding of immobilized proteins by IMAC can improve and facilitate the experimental work for difficult-to-refold proteins.
Affiliation
Moscow Research Institute of Medical Ecology, Simpheropolski blvd.8, Moscow, 117638, Russia.
Journal Details
This article was published in the following journal.
Name: Journal of chromatography. A
ISSN: 1873-3778
Pages: 5115-9
Links
- PubMed Source: http://www.ncbi.nlm.nih.gov/pubmed/21676401
- DOI: http://dx.doi.org/10.1016/j.chroma.2011.05.075
Medical and Biotech [MESH] Definitions
Protein Denaturation
The exposure of protein to chemicals, or heat, which disrupt the non-covalent bonds and/or disulfide bonds responsible for maintaining the three-dimensional shape and activity of the native protein, while leaving the peptide bonds intact.
Protein Refolding
Conformational transitions of a protein from unfolded states to a more folded state.
Proline-rich Protein Domains
Protein domains that are enriched in PROLINE. The cyclical nature of proline causes the peptide bonds it forms to have a limited degree of conformational mobility. Therefore the presence of multiple prolines in close proximity to each other can convey a distinct conformational arrangement to a peptide chain.
Cholestyramine Resin
A strongly basic anion exchange resin whose main constituent is polystyrene trimethylbenzylammonium Cl(-) anion.
Pulmonary Surfactant-associated Protein C
A pulmonary surfactant associated protein that plays a role in alveolar stability by lowering the surface tension at the air-liquid interface. It is a membrane-bound protein that constitutes 1-2% of the pulmonary surfactant mass. Pulmonary surfactant-associated protein C is one of the most hydrophobic peptides yet isolated and contains an alpha-helical domain with a central poly-valine segment that binds to phospholipid bilayers.
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