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Characterization of recombinant-derived granulocyte-colony stimulating factor (G-CSF).

ABSTRACT: Human granulocyte colony-stimulating factor (G-CSF), and a mutant having a Ser for Cys substitution at residue 18 were produced in Escherichia coli strain W3110. About 60 mg of pure protein was obtained from 50 g of wet cells with a recovery of about 20%. The proteins were characterized physically and chemically, including determination of disulphide bonds, which were found to exist between residues 37-43 and 65-75. Cys-18 is not involved in disulphide bond formation and was substituted by Ser with no effects on gross protein conformation or biological activity. Both the wild-type and the mutant recombinant-derived proteins, although not glycosylated, possess colony-stimulating activities. In a bioassay using the murine myelomonocytic leukaemic cell line WEH1 3B D+, activities were obtained which were similar to those of natural G-CSF and of a glycosylated recombinant-derived human G-CSF produced in monkey cells.

SUBMITTER: Wingfield P

PROVIDER: S-EPMC1135389 | biostudies-other | 1988 Nov

REPOSITORIES: biostudies-other

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Project description:BACKGROUND:Biopharmaceutical drugs are mainly recombinant proteins produced by biotechnological tools. The patents of many biopharmaceuticals have expired, and biosimilars are thus currently being developed. Human granulocyte colony stimulating factor (hG-CSF) is a hematopoietic cytokine that acts on cells of the neutrophil lineage causing proliferation and differentiation of committed precursor cells and activation of mature neutrophils. Recombinant hG-CSF has been produced in genetically engineered Escherichia coli (Filgrastim) and successfully used to treat cancer patients suffering from chemotherapy-induced neutropenia. Filgrastim is a 175 amino acid protein, containing an extra N-terminal methionine, which is needed for expression in E. coli. Here we describe a simple and low-cost process that is amenable to scaling-up for the production and purification of homogeneous and active recombinant hG-CSF expressed in E. coli cells. RESULTS:Here we describe cloning of the human granulocyte colony-stimulating factor coding DNA sequence, protein expression in E. coli BL21(DE3) host cells in the absence of isopropyl-beta-D-thiogalactopyranoside (IPTG) induction, efficient isolation and solubilization of inclusion bodies by a multi-step washing procedure, and a purification protocol using a single cationic exchange column. Characterization of homogeneous rhG-CSF by size exclusion and reverse phase chromatography showed similar yields to the standard. The immunoassay and N-terminal sequencing confirmed the identity of rhG-CSF. The biological activity assay, in vivo, showed an equivalent biological effect (109.4%) to the standard reference rhG-CSF. The homogeneous rhG-CSF protein yield was 3.2 mg of bioactive protein per liter of cell culture. CONCLUSION:The recombinant protein expression in the absence of IPTG induction is advantageous since cost is reduced, and the protein purification protocol using a single chromatographic step should reduce cost even further for large scale production. The physicochemical, immunological and biological analyses showed that this protocol can be useful to develop therapeutic bioproducts. In summary, the combination of different experimental strategies presented here allowed an efficient and cost-effective protocol for rhG-CSF production. These data may be of interest to biopharmaceutical companies interested in developing biosimilars and healthcare community.

Project description:BackgroundStem cell factor (SCF) and granulocyte-colony stimulating factor (G-CSF) are well-characterized vital hematopoietic growth factors that regulate hematopoiesis. G-CSF and SCF synergistically exhibit a stimulatory effect on hematopoietic progenitors. The combination of G-CSF and SCF has been used for mobilization of peripheral blood progenitor cells in cancer and non-cancerous conditions. To overcome challenges connected with the administration of two cytokines, we developed two fusion proteins composed of human SCF and human G-CSF interspaced by an alpha-helix-forming peptide linker.MethodsThe recombinant proteins SCF-Lα-GCSF and GCSF-Lα-SCF were purified in three steps using an ion-exchange and mixed-mode chromatography. The purity and quantity of the proteins after each stage of purification was assessed using RP-HPLC, SDS-PAGE, and the Bradford assays. Purified proteins were identified using high-performance liquid chromatography/electrospray ionization mass spectrometry (HPLC/ESI-MS) and the Western blot analyses. The molecular weight was determined by size exclusion HPLC (SE-HPLC). The activity of heterodimers was assessed using cell proliferation assays in vitro. The capacity of recombinant fusion proteins to stimulate the increase of the absolute neutrophil count in rats was determined in vivo. The binding kinetics of the proteins to immobilized G-CSF and SCF receptors was measured using total internal reflection ellipsometry and evaluated by a standard Langmuir kinetics model.ResultsThe novel SCF-Lα-GCSF and GCSF-Lα-SCF proteins were synthesized in Escherichia coli. The purity of the heterodimers reached >90% as determined by RP-HPLC. The identity of the proteins was confirmed using the Western blot and HPLC/ESI-MS assays. An array of multimeric forms, non-covalently associated dimers or trimers were detected in the protein preparations by SE-HPLC. Each protein induced a dose-dependent proliferative response on the cell lines. At equimolar concentration, the heterodimers retain 70-140% of the SCF monomer activity (p ≤ 0.01) in promoting the M-07e cells proliferation. The G-CSF moiety in GCSF-Lα-SCF retained 15% (p ≤ 0.0001) and in SCF-Lα-GCSF retained 34% (p ≤ 0.01) of the monomeric G-CSF activity in stimulating the growth of G-NFS-60 cells. The obtained results were in good agreement with the binding data of each moiety in the fusion proteins to their respective receptors. The increase in the absolute neutrophil count in rats caused by the SCF-Lα-GCSF protein corresponded to the increase induced by a mixture of SCF and G-CSF.

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Characterization of recombinant-derived granulocyte-colony stimulating factor (G-CSF).

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