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

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.     

<Emphasis Type="SmallCaps">l</Emphasis>-Arginine Suppresses Aggregation of Recombinant Growth Hormones in Refolding Process from <Emphasis Type="Italic">E. coli</Emphasis> Inclusion Bodies

l-Arginine was used to suppress the aggregation of recombinant mink and porcine growth hormones in the refolding process from E. coli inclusion bodies by solubilization–dilution protocol at high protein concentration and pH 8.0. The influence of l-arginine concentration on the renaturation yield of both proteins was investigated. l-Arginine effectively suppressed the precipitation of growth hormones during dilution, but did not inhibit soluble oligomers formation. The results of mink and porcine growth hormones purification from 4 g of biomass are presented.     

Congo red interaction with alpha-proteins

The ability of Congo red to form complexes with alpha-proteins, human growth hormone and human interferon-alpha2b, was found by absorption difference spectroscopy. A human growth hormone-Congo red complex was isolated by gel-permeation chromatography, and its visible absorption spectrum was registered in comparison to free dye. The ability of Congo red to induce dimerization of human growth hormone was demonstrated using chemical cross-linking agents 1,3,5-triacryloyl-hexahydro-s-triazine and ethylene glycol bis(succinimidylsuccinate).     

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.     

Dimerization of human growth hormone in the presence of metal ions

Zn(2+) and Co(2+) ions are known to promote human growth hormone reversible dimerization. In these studies, dimerization was also shown to be initiated by nine other metal ions: Cd(2+), Hg(2+), Cu(2+), Ag+, Au(3+), Au+, Pd(2+), Ni(2+), and Pt(4+). In some cases (Hg(2+), Ag(+), Au(3+), and Ni(2+)) formation of higher oligomers also took place. In addition further detailed investigation of dimerization in the presence of Zn(2+) ions was carried out.     

Refolding of porcine growth hormone from inclusion bodies of Escherichia coli

Escherichia. coli cells expressing porcine growth hormone were grown in a batch fermentation process. The expression level was estimated to be nearly 40% of the total cellular protein after 2–3 h of induction with 1?mM isopropyl β-d-thiogalactoside. Porcine growth hormone expressed as inclusion bodies was solubilized in 8 M urea. Refolding conditions following a dilution protocol in the presence of β-mercaptoethanol or using a glutathione pair were tested. Reverse phase-HPLC was applied to distinguish oxidized, misfolded and reduced forms of the hormone. A ratio of reduced to oxidized glutathione equal to 2/1 was chosen to avoid the formation of misfolded forms at high protein concentration.     

Anti-aggregatory effect of cyclodextrins in the refolding process of recombinant growth hormones from Escherichia coli inclusion bodies

Cyclodextrins with different ring size and ring substituents were tested for recombinant mink and porcine growth hormones aggregation suppression in the refolding process from Escherichia coli inclusion bodies. Methyl-β-cyclodextrin and 2-hydroxypropyl-β-cyclodextrin show a positive effect on the aggregation suppression of both proteins. The influence of different methyl-β-cyclodextrin and 2-hydroxypropyl-β-cyclodextrin concentrations on the renaturation yield of both growth hormones was investigated. Moreover, methyl-β-cyclodextrin and 2-hydroxypropyl-β-cyclodextrin suppress not only folding-related, but also temperature-related aggregates formation of both proteins. Circular dichroism experiments (monitoring of protein solution turbidity by registering high tension voltage) showed that the onset temperature of aggregation of both growth hormones increased with increasing 2-hydroxypropyl-β-cyclodextrin concentration. In conclusion, cyclodextrins have perspectives in biotechnology of veterinary growth hormones not only for protein production, but also for its storage.     

The influence of different glycosylation patterns on factor VII biological activity

In this study the bioactivity of three differently glycosylated blood coagulation factor VII (FVII) variants (human plasma FVII, recombinant human FVII produced in CHO and BHK cell cultures) were analyzed and compared. Surface plasmon resonance studies of FVII interaction with soluble and full length TF together with FVII autoactivation assays revealed that BHK-derived FVII has the highest bioactivity, while human plasma and CHO-derived FVII showed very similar bioactivity. The affinity of FVII variants to TF correlates with FVII autoactivation rates – the higher the affinity, the faster the autoactivation rate.     

Divinyl Sulfone as a Crosslinking Reagent for Oligomeric Proteins

The kinetics of the nucleophilic addition reactions of divinyl sulfone to amino groups of glycine and model proteins was studied in aqueous solution at 30°C. The rate constants for glycine, bovine serum albumin, and ₁-casein were (4.84 ± 0.58) × 10^–1, (2.97 ± 0.31) × 10^–2, and (2.38 ± 0.49) × 10^–2 M^–1 s^–1, respectively. Divinyl sulfone was proposed as a crosslinking reagent for the qualitative detection of protein association in solution. The crosslinking capacity of divinyl sulfone was compared to that of 1,3,5-triacryloylhexahydro-s-triazine.     

Production of recombinant mink growth hormone in <Emphasis Type="Italic">E. coli</Emphasis>

Escherichia coli cells expressing mink (Mustela vison) growth hormone were grown in a batch fermentation process. The expression level was estimated to be 27% of the total cellular protein after 3 h of induction with 1 mM isopropyl β-d-thiogalactoside (IPTG). If the expression of mink growth hormone (mGH) was induced with 0.2 mM IPTG, the concentration of target protein was slightly lower and was found to be 23% at the same time after induction. mGH expressed as inclusion bodies was solubilized in 8 M urea and renatured by dilution protocol at a protein concentration of 1.4–2.1 mg/ml in the presence of glutathione pair in a final concentration of 11.3 mM. [GSH]/[GSSG] ratio equal to 2/1 was used. Two-step purification process comprising of ion-exchange chromatography on Q-Sepharose and hydrophobic chromatography on Phenyl-Sepharose was developed. Some 25–30 mg of highly purified and biologically active mGH was obtained from 4 g of biomass. The method presented in this study allows producing large quantities of mGH and considering initiation of scientific investigation on mGH effect on mink in vivo and availability in fur industry.