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Lee, Ah Young; Chung, Hye Kyung; Bae, Eun Kyong; Hwang, Jung Suk; Sung, Bong Whan; Cho, Chung Won; Kim, Jin Kyoo; Lee, Kyung Hwa; Han, Jae Yong; Lee, Choon Taek; Youn, Heejung

doi:10.1023/a:1022346800375

Cited by 11 publications

(4 citation statements)

References 19 publications

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“…Flexible glycine‐rich regions have been known to be natural linkers in proteins, as they consist of loops that connect domains in multi‐domain proteins (Reddy Chichili et al, 2013 ). A GS linker was earlier inserted between GM‐CSF and interleukin (IL)‐3 to provide conformational flexibility, and consequently enhance the folding of GM‐CSF and IL‐3 to their native form (Curtis et al, 1991 ; A. Y. Lee et al, 2003 ).…”

Section: Resultsmentioning

confidence: 99%

Synthetic pro‐peptide design to enhance the secretion of heterologous proteins by Saccharomyces cerevisiae

Cho

Jang

et al. 2022

MicrobiologyOpen

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Heterologous protein production in Saccharomyces cerevisiae is a useful and effective strategy with many advantages, including the secretion of proteins that require posttranslational processing. However, heterologous proteins in S. cerevisiae are often secreted at comparatively low levels. To improve the production of the heterologous protein, human granulocyte colony‐stimulating factor (hG‐CSF) in S. cerevisiae , a secretion‐enhancing peptide cassette including an hIL‐1β‐derived propeptide, was added and used as a secretion enhancer to alleviate specific bottlenecks in the yeast secretory pathway. The effects of three key parameters— N ‐glycosylation, net negative charge balance, and glycine‐rich flexible linker—were investigated in batch cultures of S. cerevisiae . Using a three‐stage design involving screening, selection, and optimization, the production and secretion of hG‐CSF by S. cerevisiae were significantly increased. The amount of extracellular mature hG‐CSF produced by the optimized propeptide after the final stage increased by 190% compared to that of the original propeptide. Although hG‐CSF was used as the model protein in the current study, this strategy applies to the enhanced production of other heterologous proteins, using S. cerevisiae as the host.

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Section: Resultsmentioning

confidence: 99%

Synthetic pro‐peptide design to enhance the secretion of heterologous proteins by Saccharomyces cerevisiae

Cho

Jang

et al. 2022

MicrobiologyOpen

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“…33 Extensive efforts were also made to engineer more effective forms of the myeloid growth factor by combining G-CSF with IL-3, 34 GM-CSF with IL-3, 35,36 and G-CSF with GM-CSF. 37 In general, these agents initially looked promising but have proven to be immunogenic and therefore are not appropriate for full clinical development and medical applications.…”

Section: Other Modificationsmentioning

confidence: 99%

Neutrophil Biology and the Next Generation of Myeloid Growth Factors

Dale¹

2009

J Natl Compr Canc Netw

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Neutrophils are the body's critical phagocytic cells for defense against bacterial and fungal infections. The bone marrow must produce approximately 10 × 109 neutrophils/kg/d to maintain normal blood neutrophil counts. Production of neutrophils depends on myeloid grow factors, particularly granulocyte colony-stimulating factor (G-CSF). After the original phase of development, researchers modified these growth factors to increase their size and delay their renal clearance, increase their biologic potency, and create unique molecules for business purposes. Pegylated G-CSF is a successful product of these efforts. Researchers have also tried to identify small molecules to serve as oral agents that mimic the parent molecules, but these programs have been less successful. In 2006, the European Medicines Agency established guidelines for the introduction of new biologic medicinal products claimed to be similar to reference products that had previously been granted marketing authorization in the European community. In Europe these products are called bio-similars. Throughout the world, new version and copied version of G-CSF and other myeloid growth factors are now appearing. Some properties of the myeloid growth factors are similar to other agents, offering opportunities for the development of alternative drugs and treatments. For example, recent research shows that hematopoietic progenitor cells can be mobilized with a chemokine receptor antagonist, chemotherapy, G-CSF, and granulocyte macrophage colony-stimulating factor. Advances in neutrophil biology coupled with better understanding and development of the myeloid growth factors offer great promise for improving the care of patients with cancer and many other disorders.

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“…The synergistic activity of cytokines promoted the development of fusion proteins (Schuh & Morrissey, 1999;Lee et al, 2003;Chen et al, 2005;Ng & Galipeau, 2015). Fusion cytokines are attractive for therapeutic applications because their concerted activity enhances the effect of the separate moieties and even confer novel functions.…”

Section: Introductionmentioning

confidence: 99%

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

Mickiene

Dalgėdienė

Zvirblis

et al. 2020

PeerJ

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Background Stem 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. Methods The 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. Results The 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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Cited by 11 publications

References 19 publications

Synthetic pro‐peptide design to enhance the secretion of heterologous proteins by Saccharomyces cerevisiae

Synthetic pro‐peptide design to enhance the secretion of heterologous proteins by Saccharomyces cerevisiae

Neutrophil Biology and the Next Generation of Myeloid Growth Factors

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

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