Construction, Purification, and Characterization of a Homodimeric Granulocyte Colony-Stimulating Factor

Abstract

Granulocyte colony-stimulating factor (G-CSF) has found widespread clinical application, and modified forms with improved biopharmaceutical properties have been marketed as well. PEGylation, the covalent modification of G-CSF with polyethylene glycol (PEG), has a beneficial effect on drug properties, but there are concerns connected to the immunogenicity of PEGylated compounds and bioaccumulation of the synthetic polymer. To overcome challenges connected with chemical modifications, we developed fusion proteins composed of two G-CSF molecules connected via different peptide linkers. Three different homodimeric G-CSF proteins were purified, and their in vitro and in vivo activities were determined. A G-CSF dimer, GCSF-Lα, was constructed using an alpha-helix-forming peptide linker, and it demonstrated an extended half-life in serum with a stronger neutrophil response as compared to the monomeric G-CSF protein. The GCSF-Lα protein, therefore, might be selected for further studies as a potential drug candidate.

Human granulocyte-colony stimulating factor (G-CSF)/stem cell factor (SCF) fusion proteins: design, characterization and activity

“…Figure 6 The proliferation of G-NFS-60 (A) and M-07e (B) cells induced by the purified SCF-La-GCSF and GCSF-La-SCF. The plots represent the specific biological activity of the heterodimers calculated from proliferation curves using the equations as described previously (Mickiene et al, 2017).…”

“…The scf gene was amplified to introduce the BamHI and HindIII sites at the 5′-and 3′-ends of the PCR fragment using primers 5′-TGGATCCGAAGGGATCTGCCGTAATCG and 5′-TAAGCTTAGGCTGCAACAGGGGG, respectively. The copy of the human g-scf gene was cut out with the enzymes BamHI and HindIII from the plasmid bearing two copies of the g-csf gene interspaced by the Lα sequence ( Mickiene et al, 2017 ). The BamHI/HindIII digested DNA fragment coding for SCF was fused with the DNA construct bearing the g-scf gene and the Lα linker sequence.…”

“…The in vitro biological activity of SCF-Lα-GCSF and GCSF-Lα-SCF was determined using the SCF-dependent human M-07e cell line ( Drexler, Zaborski & Quentmeier, 1997 ) and G-CSF-dependent mouse G-NFS-60 cell line ( Weinstein et al, 1986 ; Matsuda, Shirafuji & Asano, 1989 ) as described previously ( Mickiene et al, 2017 ). The SCF and G-CSF monomers were used as the reference standards.…”

“…The proliferation curves were constructed by plotting the log2 dilution of the proteins or standards against the absorbance value at 490 nm. The specific biological activity of the heterodimers was determined using the equations as described previously (Mickiene et al, 2017).…”

“…In this study, we described the development of two heterodimeric fusion proteins composed of human SCF and human G-CSF connected via a peptide linker, which ensured the activity of two monomers in the heterodimeric molecule (Arai et al, 2001;Mickiene et al, 2017;Balevicius et al, 2019). The heterodimers were purified, characterized, and compared with monomeric human G-CSF and SCF.…”