title: Effect of surface histidine mutations and their number on the partitioning and refolding of recombinant human granulocyte-colony stimulating factor (Cys17Ser) in aqueous two-phase systems containing chelated metal ions.
abstract: High-level expression of recombinant proteins in Escherichia coli frequently leads to the formation of insoluble protein aggregates, termed inclusion bodies. In order to recover a native protein from inclusion bodies, various protein refolding techniques have been developed. Column-based refolding methods and refolding in aqueous two-phase systems are often an attractive alternative to dilution refolding due to simultaneous purification and improved refolding yields. In this work, the effect of surface histidine mutations and their number on the partitioning and refolding of recombinant human granulocyte-colony stimulating factor Cys17Ser variant (rhG-CSF (C17S)) from solubilized inclusion bodies in aqueous two-phase systems polyethylene glycol (PEG)-dextran, containing metal ions, chelated by dye Light Resistant Yellow 2KT (LR Yellow 2KT)-PEG derivative, was investigated. Human G-CSF is a growth factor that regulates the production of mature neutrophilic granulocytes from the precursor cells. Initially, the role of His156 and His170 residues in the interaction of rhG-CSF (C17S) with Cu(II), Ni(II) and Hg(II) ions, chelated by LR Yellow 2KT-PEG, was investigated at pH 7.0 by means of affinity partitioning of purified, correctly folded rhG-CSF (C17S) mutants. It was determined that both His156 and His170 mutations reduced the affinity of rhG-CSF (C17S) for chelated Cu(II) ions at pH 7.0. His170 mutation significantly reduced the affinity of protein for chelated Ni(II) ions. However, histidine mutations had only a small effect on the affinity of protein for Hg(II) ions. The influence of His156 and His170 mutations on the refolding of rhG-CSF (C17S) from solubilized inclusion bodies in aqueous two-phase systems PEG-dextran, containing chelated Ni(II) and Hg(II) ions, was investigated. Reversible interaction of protein mutants with chelated metal ions was used for refolding in aqueous two-phase systems. Both histidine mutations resulted in a significant decrease of protein refolding efficiency in two-phase systems containing chelated Ni(II) ions, while in the presence of chelated Hg(II) ions their effect on protein refolding was negligible. Refolding studies of rhG-CSF variants with different number of histidine mutations revealed that a direct correlation exists between the number of surface histidine residues and refolding efficiency of rhG-CSF variant in two-phase systems containing chelated Ni(II) ions. This method of protein refolding in aqueous two-phase systems containing chelated metal ions should be applicable to other recombinant proteins that contain accessible histidine residues.
authors.affiliation: Institute of Biotechnology, Graiciūno 8, LT-02241 Vilnius, Lithuania. mzaveck@ibt.it
authors.forename: Mindaugas
authors.initials: M
authors.lastname: Zaveckas
authors.forename: Aurelija
authors.initials: A
authors.lastname: Zvirbliene
authors.forename: Gintautas
authors.initials: G
authors.lastname: Zvirblis
authors.forename: Valerija
authors.initials: V
authors.lastname: Chmieliauskaite
authors.forename: Vladas
authors.initials: V
authors.lastname: Bumelis
authors.forename: Henrikas
authors.initials: H
authors.lastname: Pesliakas
mesh_terms: D000818:Animals; D045744:Cell Line, Tumor; D002614:Chelating Agents / Q000737:chemistry*; D003545:Cysteine / Q000737:chemistry*; D004591:Electrophoresis, Polyacrylamide Gel; D016179:Granulocyte Colony-Stimulating Factor / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D006639:Histidine / Q000235:genetics*; D008670:Metals / Q000737:chemistry*; D051379:Mice; D009154:Mutation; D017510:Protein Folding; D011994:Recombinant Proteins; D012694:Serine / Q000737:chemistry*; D014867:Water
chemical_list: D002614:Chelating Agents; D008670:Metals; D011994:Recombinant Proteins; D014867:Water; D016179:Granulocyte Colony-Stimulating Factor; D012694:Serine; D006639:Histidine; D003545:Cysteine