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

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

“…PEG 6000-LR Yellow 2KT derivative was synthesized by coupling of reactive dye LR Yellow 2KT to PEG 6000 [28]. PEG 6000-LR Yellow 2KT-Ni(II) and Hg(II) complexes were prepared from demetallized PEG-dye and relevant metal salt [23].…”

“…We examined the partitioning of purified, correctly folded rhG-CSF (C17S, H43A, H170A) and (C17S, H43A, H52A, H156A) mutants and their refolding from inclusion bodies in two-phase systems containing chelated metal ions. Partitioning and refolding results for these mutants were further compared with those for rhG-CSF (C17S, H43A) and (C17S, H43A, H52A) from our previous work [23]. We report that surface histidine residues of rhG-CSF play a critical role in the interaction of protein with Ni(II) and refolding in aqueous two-phase systems, containing chelated Ni(II) ions.…”

“…Cloning, DNA preparation, transformation and expression of modified hG-CSF variants in E. coli were performed similarly as described in [23]. Isolation of inclusion bodies from harvested E. coli cells, refolding and purification of rhG-CSF mutants were performed according to [23], with several modifications. Three grams of inclusion bodies were solubilized in 100 ml, 10 mM Tris-HCl buffer (pH 7.0), containing 7 M GdmHCl by stirring overnight at 4 • C. The solution was centrifuged at 40,000 × g for 25 min.…”

“…The site-directed mutagenesis of more stable C17S variant of rhG-CSF was used for sequential replacing of His43 and His52 residues to alanine. We have found that both histidine mutations caused only a slight effect on partitioning and refolding of rhG-CSF (C17S) in two-phase systems containing Hg(II) ions [23]. However, the mutation of His52 resulted in a considerable decrease in both protein affinity to Ni(II) and its refolding efficiency in two-phase systems containing chelated Ni(II) ions.…”

“…However, little is known about the influence of the interaction strength between protein and chelated metal ion on refolding efficiency. Since histidine residues dominate this interaction, we have recently evaluated the influence of two surface histidines of rhG-CSF on protein interaction with chelated metal ions and refolding in aqueous two-phase systems [23]. RhG-CSF was selected as a model protein for these studies since the visualization of its NMR structure [24] with RasMol viewer [25] revealed that four histidine residues -His43, His52, His156 and His170 -are surface-exposed.…”