Mutation of surface-exposed histidine residues of recombinant human granulocyte-colony stimulating factor (Cys17Ser) impacts on interaction with chelated metal ions and refolding in aqueous two-phase systems.
Site directed mutagenesis of Cys17-->Ser17 form of recombinant human granulocyte colony stimulating factor (rhG-CSF C17S) for sequential replacing of surface His(43) and His(52) with alanine was used to identify residues critical for the protein interaction with metal ions, in particular Ni(2+) chelated by dye Light Resistant Yellow 2 KT (LR Yellow 2KT)-polyethyleneglycol (PEG), and refolding after partitioning of inclusion bodies in aqueous two-phase systems. Strong binding of rhG-CSF (C17S) to PEG-LR Yellow 2KT-Cu(II) complex allowed for the adoption of affinity chromatography on Sepharose-LR Yellow 2KT-Cu(II) that appeared to be essential for the rapid isolation of mutated forms of rhG-CSF. Efficiency of that purification stage is exemplified by isolation of rhG-CSF (C17S, H43A) and rhG-CSF (C17S, H43A, H52A) mutants in correctly folded and highly purified state. Affinity partitioning of rhG-CSF histidine mutants was studied in aqueous two-phase systems containing Cu(II), Ni(II) and Hg(II) chelated by LR Yellow 2KT-PEG at pH 7.0 and Cu(II)-at pH 5.0. It was determined, that affinity of rhG-CSF mutants for metal ions decreased in the order of C17S>C17S, H43A>C17S, H43A, H52A for Cu(II), and C17S=C17S, H43A>C17S, H43A, H52A for Ni(II) ions, while affinity of all rhG-CSF mutants for Hg(II) ions was of the same order of magnitude. Influence of His(43) and His(52) mutation on protein refolding was studied by partitioning of the respective inclusion body extract in aqueous two-phase systems containing Ni(II) and Hg(II) ions. Data on rhG-CSF histidine mutant partitioning and refolding indicated, that His(52) mutation is crucial for the strength of protein interaction with chelated Ni(II) ions and refolding efficiency.
Zaveckas
Mindaugas
M
Institute of Biotechnology, Graiciūno 8, LT-2028, Vilnius, Lithuania. mzaveck@ibt.lt
Luksa
Virginijus
V
Zvirblis
Gintautas
G
Chmieliauskaite
Valerija
V
Bumelis
Vladas
V
Pesliakas
Henrikas
H
D002614:Chelating Agents / Q000378:metabolism*; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D016179:Granulocyte Colony-Stimulating Factor / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006639:Histidine / Q000737:chemistry / Q000235:genetics*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007093:Imidazoles / Q000737:chemistry; D007525:Isoelectric Focusing; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D002614:Chelating Agents; D007093:Imidazoles; D011994:Recombinant Proteins; D016179:Granulocyte Colony-Stimulating Factor; D006639:Histidine; C029899:imidazole