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Granulocyte colony-stimulating factor (GCSF) fused with Fc Domain produced from E. coli is less effective than Polyethylene Glycol-conjugated GCSF.

ABSTRACT: Human granulocyte colony-stimulating factor (GCSF) is a well-known cytokine for neutropenia treatment. However, daily injections are required due to the short circulating half-life of the protein. To overcome this bottleneck, we fused GCSF with the Fc domain of IgG1 at the C terminus (GCSF-Fc) and with the maltose binding protein (MBP) tag at the N-terminus and expressed it as a soluble protein in the cytoplasm of E. coli. We also conjugated PEG aldehyde to GCSF to make PEG-GCSF. The bioactivities of GCSF-Fc and PEG-GCSF were similar to native GCSF using the mouse M-NFS-60 myelogenous leukemia cell line. The EC₅₀ dose-response curves for GCSF, GCSF-Fc and PEG-GCSF were 37 ± 12 pM, 75 ± 13.5 pM and 46 ± 5.5 pM, respectively. When the proteins were injected into neutropenic rats, the group injected with PEG-GCSF showed the highest and fastest recovery of neutrophils, followed by GCSF-Fc and GCSF. ELISA assay revealed the PEG-GCSF had the longest plasma circulation (>72 h), followed by GCSF-Fc (>48 h) and GCSF (~24 h), which is consistent with the in vivo activities of the proteins. In summary, the GCSF-Fc purified from E. coli was not as efficient as PEG-GCSF in treating neutropenic rats.

SUBMITTER: Do BH

PROVIDER: S-EPMC5526978 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Expression of granulocyte colony stimulating factor (GCSF) in Hansenula polymorpha.

Project description:Background and objectivesDuring past decades Hansenula polymorpha has attracted global attention for the expression of recombinant proteins due to its high growth rate, minimal nutritional porequirements and use of methanol as a low cost inducer.Materials and methodsThe corresponding nucleotide sequences for the expression of heterologous genes in Hansenula poylmorpha were extracted and assembled in an E. coli vector. The constructed expression cassette included formate dehydrogenase promoter (pFMD), a secretory signal sequence, a multiple cloning site (MCS) and methanol oxidase (MOX) terminator. Zeocin resistance gene fragment and complete cDNA encoding granulocyte colony stimulating factor (GCSF) were cloned downstream of the expression cassette in-frame with signal sequence. Restriction mapping and sequence analysis confirmed the correct cloning procedures. Final vector was transformed into Hansenula and recombinant host was induced for the expression of GCSF protein by adding methanol. SDS-PAGE and immuno-blotting were performed to confirm the identity of r-GCSF.ResultsThe expression cassette containing gcsf gene (615bp) and zeocin resistance marker (sh-ble, 1200bp) was prepared and successfully transformed into competent Hansenula polymorpha cells via electroporation. Zeocin resistant colonies were selected and GCSF expression was induced in recombinant Hansenula transformants using 0.5% methanol and an approximately 19kDa protein was observed on SDS-PAGE. Western blot analysis using serum isolated from GCSF-treated rabbit confirmed the identity of the protein.ConclusionsMolecular studies confirmed the designed expression cassette containing gcsf gene along with pFMD and signal sequence. The expressed 19kDa protein also confirmed the ability of designed vector in expressing heterologous genes in Hansenula cells.

| S-EPMC4833737 | biostudies-literature

Homodimeric cross-over structure of the human granulocyte colony-stimulating factor (GCSF) receptor signaling complex.

Project description:A crystal structure of the signaling complex between human granulocyte colony-stimulating factor (GCSF) and a ligand binding region of GCSF receptor (GCSF-R), has been determined to 2.8 A resolution. The GCSF:GCSF-R complex formed a 2:2 stoichiometry by means of a cross-over interaction between the Ig-like domains of GCSF-R and GCSF. The conformation of the complex is quite different from that between human GCSF and the cytokine receptor homologous domain of mouse GCSF-R, but similar to that of the IL-6/gp130 signaling complex. The Ig-like domain cross-over structure necessary for GCSF-R activation is consistent with previously reported thermodynamic and mutational analyses.

| S-EPMC1413920 | biostudies-literature

Efficient process development of recombinant human granulocyte colony-stimulating factor (rh-GCSF) production in Escherichia coli.

Project description:BACKGROUND:The protein hormone granulocyte colony-stimulating factor (GCSF) stimulates the production of white blood cells and plays an important role in medical treatment of cancer patients. METHODS:An efficient process was developed for heterologous expression of the human GCSF in E. coli BL21 (DE3). The feeding rate was adjusted to achieve the maximum attainable specific growth rate under critical value. In this method, specific growth rate was maintained at the maximum value of 0.55 h?¹ at the beginning of feeding to 0.4 h-1 at the induction time. Recombinant human GCSF (rh-GCSF) was produced as inclusion body. At first, inclusion bodies were released by cell disruption and then washed, solubilized and refolded. Finally, the rh-GCSF was purified by cation exchange chromatography. RESULTS:Obviouly, higher specific growth rate decreases process time and consequently increases productivity. The final concentration of biomass and GCSF was achieved 126 g DCW.l?¹ and 32.1 g.l?¹. Also, the final specific yield (YP/X) and total productivity of rh-GCSF were obtained 254 mg.g?¹ DCW and 1.83 g.l?¹.h?¹, respectively. According to the available data, this is one of the highest YP/X and productivity that has been reported for any human protein which is expressed in E. coli. Recovery yield of purification process was %40 and purity of recombinant protein was over than 99%. The circular dichroism spectra of purified rh-GCSF, Neupogen and PD-Grastim showed that all proteins have a similar secondary structure. CONCLUSION:Modified exponential feeding strategy for fed-batch cultivation of recombinant E. coli, results in minimum fed-batch duration and maximum productivity.

| S-EPMC4412921 | biostudies-literature

Human Granulocyte-Macrophage Colony-Stimulating Factor Fused to Elastin-Like Polypeptides Assembles Biologically-Active Nanoparticles.

Project description:Human granulocyte-macrophage colony-stimulating factor (hGMCSF) is crucial in the immune system as it stimulates survival, proliferation, differentiation, and functional activation of myeloid hematopoietic cells. hGMCSF is integral to approved therapies, including monoclonal antibodies against checkpoint inhibitors, chimeric antigen receptors, and prevention of chemotherapy-induced neutropenia. Recombinant hGMCSF can be purified from Escherichia. coli; however, it forms inclusion bodies that require solubilization and refolding. Alternatively, this manuscript describes its fusion with an elastin-like polypeptide (ELP). Previously reported as purification tags and solubility enhancers, ELPs are recombinant polypeptides that undergo reversible temperature-dependent phase separation. This report is the first to show that fusion to an ELP enables direct purification of hGMCSF fusions from the soluble fraction of bacterial lysate. Surprisingly, these ELP-fusions assemble stable, small, spherical nanoparticles that maintain pro-mitotic activity of hGMCSF. These nanoparticles exhibit ELP-mediated phase separation; however, nanoparticle assembly significantly increases the entropic and enthalpic cost of phase separation compared to ELP alone. The attachment of a high molecular weight ELP to a difficult-to-express protein, like hGMCSF, appears to be a useful strategy to stabilize bioactive, protein-based nanoparticles, which may have broad applications in medicine and biology.

| S-EPMC7489497 | biostudies-literature

Semisynthesis of Homogeneous, Active Granulocyte Colony-Stimulating Factor Glycoforms.

Project description:Granulocyte colony stimulating factor (G-CSF) is a cytokine used to treat neutropenia. Different glycosylated and non-glycosylated variants of G-CSF for therapeutic application are currently generated by recombinant expression. Here, we describe our approaches to establish a first semisynthesis strategy to access the aglycone and O-glycoforms of G-CSF, thereby enabling the preparation of selectively and homogeneously post-translationally modified variants of this important cytokine. Eventually, we succeeded by combining selenocysteine ligation of a recombinantly produced N-terminal segment with a synthetic C-terminal part, transiently equipped with a side-chain-linked, photocleavable PEG moiety, at low concentration. The transient PEGylation enabled quantitative enzymatic elongation of the carbohydrate at Thr133. Overall, we were able to significantly reduce the problems related to the low solubility and the tendency to aggregate of the two protein segments, which allowed the preparation of four G-CSF variants that were successfully folded and demonstrated biological activity in cell proliferation assays.

| S-EPMC9804750 | biostudies-literature

RUNX1 haploinsufficiency results in granulocyte colony-stimulating factor hypersensitivity.

Project description:RUNX1/AML1 is among the most commonly mutated genes in human leukemia. Haploinsufficiency of RUNX1 causes familial platelet disorder with predisposition to myeloid malignancies (FPD/MM). However, the molecular mechanism of FPD/MM remains unknown. Here we show that murine Runx1(+/-) hematopoietic cells are hypersensitive to granulocyte colony-stimulating factor (G-CSF), leading to enhanced expansion and mobilization of stem/progenitor cells and myeloid differentiation block. Upon G-CSF stimulation, Runx1(+/-) cells exhibited a more pronounced phosphorylation of STAT3 as compared with Runx1(+/+) cells, which may be due to reduced expression of Pias3, a key negative regulator of STAT3 signaling, and reduced physical sequestration of STAT3 by RUNX1. Most importantly, blood cells from a FPD patient with RUNX1 mutation exhibited similar G-CSF hypersensitivity. Taken together, Runx1 haploinsufficiency appears to predispose FPD patients to MM by expanding the pool of stem/progenitor cells and blocking myeloid differentiation in response to G-CSF.

| S-EPMC4742622 | biostudies-literature

Structural Analysis of the Colony-Stimulating Factor 3 Gene of Granulocyte Colony-Stimulating Factor-Producing Urothelial Cancer.

Project description:Background Granulocyte colony-stimulating factor (G-CSF) is a member of the CSF family of glycoproteins that regulate the proliferation, differentiation, and mobilization of neutrophils. G-CSF-producing malignant cancers have been reported to occur in various organs and are mostly associated with poor clinical prognosis. Here, we analyzed the structure of the CSF3 gene encoding the G-CSF protein to delineate the mechanism of G-CSF production by the cancer cells. Methodology Two cases of G-CSF-producing urothelial cancers and three cases of G-CSF-nonproducing bladder cancers were enrolled for genetic analysis. Results In one case of G-CSF-producing bladder cancer, six somatic mutations were detected in the 5'- upstream region of the CSF3 gene. No somatic mutations in the CSF3 gene were detected in another case of G-CSF-producing renal pelvic cancer and G-CSF-nonproducing bladder cancers. Copy numbers of the CSF3 gene were not increased in G-CSF-producing urothelial cancers. Conclusions Somatic mutations in the 5'- upstream region of the CSF3 gene may cause G-CSF protein overproduction.

| S-EPMC10516146 | biostudies-literature

Granulocyte and granulocyte-macrophage colony stimulating factors for newly diagnosed patients with myelodysplastic syndromes.

Project description: Not available

| S-EPMC10405220 | biostudies-literature

Granulocyte colony-stimulating factor is not pathogenic in lupus nephritis.

Project description:Systemic lupus erythematosus (lupus) is an autoimmune disease characterized by autoantibodies that form immune complexes with self-antigens, which deposit in various tissues, leading to inflammation and disease. The etiology of disease is complex and still not completely elucidated. Dysregulated inflammation is an important disease feature, and the mainstay of lupus treatment still utilizes nonspecific anti-inflammatory drugs. Granulocyte colony-stimulating factor (G-CSF) is a growth, survival, and activation factor for neutrophils and a mobilizer of hematopoietic stem cells, both of which underlie inflammatory responses in lupus. To determine whether G-CSF has a causal role in lupus, we genetically deleted G-CSF from Lyn-deficient mice, an experimental model of lupus nephritis. Lyn-/- G-CSF-/- mice displayed many of the inflammatory features of Lyn-deficient mice; however, they had reduced bone marrow and tissue neutrophils, consistent with G-CSF's role in neutrophil development. Unexpectedly, in comparison to aged Lyn-deficient mice, matched Lyn-/- G-CSF- /- mice maintained neutrophil hyperactivation and exhibited exacerbated numbers of effector memory T cells, augmented autoantibody titers, and worsened lupus nephritis. In humans, serum G-CSF levels were not elevated in patients with lupus or with active renal disease. Thus, these studies suggest that G-CSF is not pathogenic in lupus, and therefore G-CSF blockade is an unsuitable therapeutic avenue.

| S-EPMC8342225 | biostudies-literature

Neutralizing Anti-Granulocyte Macrophage-Colony Stimulating Factor Autoantibodies Recognize Post-Translational Glycosylations on Granulocyte Macrophage-Colony Stimulating Factor Years Before Diagnosis and Predict Complicated Crohn's Disease.

Project description:Background & aimsAnti-granulocyte macrophage-colony stimulating factor autoantibodies (aGMAbs) are detected in patients with ileal Crohn's disease (CD). Their induction and mode of action during or before disease are not well understood. We aimed to investigate the underlying mechanisms associated with aGMAb induction, from functional orientation to recognized epitopes, for their impact on intestinal immune homeostasis and use as a predictive biomarker for complicated CD.MethodsWe characterized using enzyme-linked immunosorbent assay naturally occurring aGMAbs in longitudinal serum samples from patients archived before the diagnosis of CD (n = 220) as well as from 400 healthy individuals (matched controls) as part of the US Defense Medical Surveillance System. We used biochemical, cellular, and transcriptional analysis to uncover a mechanism that governs the impaired immune balance in CD mucosa after diagnosis.ResultsNeutralizing aGMAbs were found to be specific for post-translational glycosylation on granulocyte macrophage-colony stimulating factor (GM-CSF), detectable years before diagnosis, and associated with complicated CD at presentation. Glycosylation of GM-CSF was altered in patients with CD, and aGMAb affected myeloid homeostasis and promoted group 1 innate lymphoid cells. Perturbations in immune homeostasis preceded the diagnosis in the serum of patients with CD presenting with aGMAb and were detectable in the noninflamed CD mucosa.ConclusionsAnti-GMAbs predict the diagnosis of complicated CD long before the diagnosis of disease, recognize uniquely glycosylated epitopes, and impair myeloid cell and innate lymphoid cell balance associated with altered intestinal immune homeostasis.

| S-EPMC10127946 | biostudies-literature

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