Comparative studies of recombinant human granulocyte-colony stimulating factor, its Ser-17 and (His)6-tagged forms interaction with metal ions by means of immobilized metal ion affinity partitioning. Effect of chelated nickel and mercuric ions on extraction and refolding of proteins from inclusion bodies.
The chelation capability of the reactive dye Light Resistant Yellow 2KT towards metal ions, particularly mercury(II) was evaluated in the pH range 5.0-7.0, and it was shown that the dye-Hg(II) complex has a free site for the interaction with human recombinant granulocyte-colony stimulating factor (rhG-CSF) from Escherichia coli. Affinity partitioning of three rhG-CSF forms--native, rhG-CSF[Cys17--->Ser17] and (His)6-rhG-CSF was studied in aqueous two-phase systems, which contained metal ions--Cu(II), Ni(II) and Hg(II)--chelated by dye-poly(ethylene glycol) at pH 5.0 and 7.0, in the presence or absence of many selected agents. It was determined, that chelated Ni(II) ions exhibited stronger interaction with the hexahistidine-tagged protein form, while the extraction power of Cu(II) ions was found to be of comparable order of magnitude for all three protein forms at pH 7.0. A comparative study of rhG-CSF and both its forms partitioning in the presence of chelated Hg(II) ions at pH 7.0 and 5.0 revealed possible direct interaction between Hg(II) ions and unpaired Cys-17 of rhG-CSF. The partitioning of three rhG-CSF forms inclusion body extract was studied in the presence of chelated Ni(II) and Hg(II) ions thus explaining the efficiency of targeted proteins renaturation gained upon their inclusion body forms interactions with chelated metal ions.
Zaveckas
M
M
Institute of Biotechnology, Vilnius, Lithuania.
Baskeviciŭte
B
B
Luksa
V
V
Zvirblis
G
G
Chmieliauskaite
V
V
Bumelis
V
V
Pesliakas
H
H
D002846:Chromatography, Affinity / Q000379:methods*; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel; D016179:Granulocyte Colony-Stimulating Factor / Q000737:chemistry*; D006639:Histidine / Q000737:chemistry*; D006801:Humans; D008628:Mercury / Q000737:chemistry*; D009532:Nickel / Q000737:chemistry*; D017510:Protein Folding; D011994:Recombinant Proteins; D012694:Serine / Q000737:chemistry*
D011994:Recombinant Proteins; D016179:Granulocyte Colony-Stimulating Factor; D012694:Serine; D006639:Histidine; D009532:Nickel; D008628:Mercury