title: Efficient refolding of a hydrophobic protein with multiple S-S bonds by on-resin immobilized metal affinity chromatography.
abstract: 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 26 kDa 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.
authors.affiliation: Moscow Research Institute of Medical Ecology, Simpheropolski blvd.8, Moscow, 117638, Russia.
authors.forename: Olga A
authors.initials: OA
authors.lastname: Sharapova
authors.forename: Maria S
authors.initials: MS
authors.lastname: Yurkova
authors.forename: Daniela K
authors.initials: DK
authors.lastname: Laurinavichyute
authors.forename: Svetlana M
authors.initials: SM
authors.lastname: Andronova
authors.forename: Alexey N
authors.initials: AN
authors.lastname: Fedorov
authors.forename: Sergey E
authors.initials: SE
authors.lastname: Severin
authors.forename: Evgeny S
authors.initials: ES
authors.lastname: Severin
mesh_terms: D002846:Chromatography, Affinity / Q000295:instrumentation / Q000379:methods*; D004220:Disulfides / Q000737:chemistry*; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D008670:Metals / Q000737:chemistry; D011485:Protein Binding; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification; D012117:Resins, Synthetic / Q000737:chemistry*; D000509:alpha-Fetoproteins / Q000737:chemistry* / Q000302:isolation & purification*
chemical_list: D004220:Disulfides; D008670:Metals; D011994:Recombinant Proteins; D012117:Resins, Synthetic; D000509:alpha-Fetoproteins