Purification and molecular cloning of prostacyclin-stimulating factor from serum-free conditioned medium of human diploid fibroblast cells.
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ABSTRACT:
SUBMITTER: Yamauchi T
PROVIDER: S-EPMC1137368 | biostudies-other | 1994 Oct
REPOSITORIES: biostudies-other
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Project description:Four forms of mouse granulocyte/macrophage colony-stimulating factor (GM-CSF) were purified 100,000-fold from mouse lung conditioned medium. Each of the CSF species stimulated the formation of both granulocyte and macrophage colonies, and half-maximal stimulation in the semi-solid mouse bone-marrow colony assay occurred at 1 pm. The four GM-CSF species exhibited similar charge microheterogeneity, focusing between pH 4.2 and pH 5.2. On SDS/polyacrylamide gels two of the GM-CSF sub-species had apparent Mr values of 23,000, and the other two, 21, 000. Treatment with neuraminidase decreased the Mr values of these two sets to 21,000 and 19,000 respectively. Incubation with endoglucosidase F decreased the charge heterogeneity and the Mr of all species to 16,500. A gas-phase radioiodination procedure was used to incorporate 2-3 atoms of 125I/molecule into purified GM-CSF without any loss of biological activity. The 125I-labelled GM-CSF was analysed on a microbore reversed-phase h.p.l.c. column to determine its specific radioactivity directly. This 125I-labelled GM-CSF molecule is suitable for cell-surface receptor-binding studies.
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Project description:Mesenchymal stem cells (MSCs) have exhibited great potential as a regenerative medicine, and MSC-derived paracrine effects, mainly including the secretion of various bioactive factors, play critical roles in MSC-based therapies. MSC-derived serum-free conditioned medium (MSC-CM) is defined as the secretome of MSC-derived bioactive factors and is considered a new cell-free therapeutic agent for disease treatment. However, the MSC-CM used in previous studies was prepared by a nearly disposable method that the MSCs were discarded after preparing MSC-CM, and the preparation time was variable; simultaneously, the viability changes of MSCs after MSC-CM preparation are still unknown. Therefore, this study takes MenSCs as a research project and aims to explore the suitable period of sustainable MenSC-CM preparation rather than using a disposable method. As expected, our results confirmed that MenSC-CM improves viability of both naïve targeted cells and H2O2-injured targeted cells, and suggested that 36 h is suitable for sustainable MenSC-CM preparation in which the angiogenic factors almost reach to the peak. Simultaneously, the MenSCs used to prepare the MenSC-CM for 36 h also maintained preferable cell viability and could be sustainably used for further MenSC-CM preparation. Moreover, the in vivo results further confirmed the improvement of MenSC-CM on promoting skin wound healing. Consequently, our results not only provide support for optimizing MSC-CM sustainable preparation based on various MSCs but also promote the comprehensive application of MenSCs in the clinic.
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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.
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Project description:BackgroundIn the present study, we attempted to adapt an adherent and serum-dependent Chinese hamster ovary DG44 cell line to a serum-free suspension culture and optimize the culture condition to achieve a higher yield of recombinant human follicle stimulating hormone (r-hFSH) with acceptable quality. This approach helps to mitigate the risks associated with blood-borne pathogens, reduces lot-to-lot variability, and lowers costs, making it suitable for industrial processing and scale-up.MethodsThe cell adaptation was performed using different chemically defined SFM. This process was followed by optimization through statistical experimental design, focusing on selected physicochemical parameters, including chemical supplementation of the medium and temperature shift. Both small- and large-scale cultures were conducted to test the reproducibility of the optimized condition. The expressed protein was evaluated for comparability with the standard molecule according to the Pharmacopeia guidelines.Resultsresponse surface methodology (RSM) analysis indicated that supplementation of the culture medium with galactose and sodium butyrate (NaBu), along with a temperature downshift, were the main parameters leading to increased cell viability (10%), r-hFSH level (96%), and more importantly, the glycosylation content (49%) of r-hFSH compared to the control condition.As r-hFSH isoforms generated during in vivo post-translational modifications typically exhibit different serum/plasma half-lives and bioactivity due to their incorporated sialic acid content/glycosylation, further optimizations of r-hFSH production are necessary to enhance its biological activity. In this study, following a primary screening of the studied parameters, optimization of culture conditions based on selected parameters resulted in enhanced quality and quantity of the produced r-hFSH. However, further examination is necessary before transitioning to industrial production.
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Project description:BackgroundThe conditioned medium from human dermal fibroblasts (dermal fibroblast-conditioned medium; DFCM) contains a diverse array of secretory proteins, including growth factors and wound repair-promoting proteins. Angiogenesis, a crucial process that facilitates the infiltration of inflammatory cells during wound repair, is induced by a hypoxic environment and inflammatory cytokines.MethodsIn this study, we conducted a comprehensive bioinformatic analysis of 337 proteins identified through proteomics analysis of DFCM. We specifically focused on 64 DFCM proteins with potential involvement in angiogenesis. These proteins were further classified based on their characteristics, and we conducted a detailed analysis of their protein-protein interactions.ResultsGene Ontology protein classification categorized these 64 DFCM proteins into various classes, including metabolite interconversion enzymes (N = 11), protein modifying enzymes (N = 10), protein-binding activity modulators (N = 9), cell adhesion molecules (N = 6), extracellular matrix proteins (N = 6), transfer/carrier proteins (N = 3), calcium-binding proteins (N = 2), chaperones (N = 2), cytoskeletal proteins (N = 2), RNA metabolism proteins (N = 1), intercellular signal molecules (N = 1), transporters (N = 1), scaffold/adaptor proteins (N = 1), and unclassified proteins (N = 9). Furthermore, our protein-protein interaction network analysis of DFCM proteins revealed two distinct networks: one with medium confidence level interaction scores, consisting of 60 proteins with significant connections, and another at a high confidence level, comprising 52 proteins with significant interactions.ConclusionsOur bioinformatic analysis highlights the presence of a multitude of secretory proteins in DFCM that form significant protein-protein interaction networks crucial for regulating angiogenesis. These findings underscore the critical roles played by DFCM proteins in various stages of angiogenesis during the wound repair process.
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Project description:Embryonic transfer of bovine blastocysts produced using in vitro fertilization (IVF) is widely used, although the challenge of compromised conception rates remains. Using bovine oviduct epithelial cells (BOEC) to improve embryo culture conditions has attracted attention, particularly since the recent discovery of extracellular vesicles from BOEC. The selection of embryos for transfer has also been the subject of various studies, and a set of evaluation criteria to predict pregnancy success has been suggested, in which the embryos are judged by their kinetics and morphology at the early stages. In the present study, we established a spontaneously immortalized BOEC line (SI-BOEC) and examined the effects of conditioned medium on IVF embryos, focusing on the results of the recommended criteria. A modified KSOM (mKSOM) was used to prepare conditioned media. Presumptive zygotes were cultured in mKSOM (control), SI-BOEC-conditioned medium, mKSOM supplemented with sediment (pellet) collected after the ultracentrifugation of the conditioned medium (mKSOM/sediment), and the supernatant. A significantly higher percentage of embryos satisfied the recommended criteria when grown in the conditioned medium than in the mKSOM. A higher proportion of embryos developed into blastocysts after achieving the four criteria. A similar tendency was observed when grown in mKSOM/sediment compared to mKSOM; however, this was not observed in the supernatant. Vesicles with a size similar to that of exosomes were observed in the sediment. In conclusion, the culture medium conditioned by SI-BOEC promoted the production of bovine blastocysts that satisfied the four evaluation criteria recommended for embryo selection.
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