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1
Sasaki, Tetsuji, and Akiyoshi Taniguchi. "Development of a Non-protein and Lipid Medium Adopted Cell Line for Biopharmaceutical Recombinant Protein Expression." Open Biotechnology Journal 7, no. 1 (February 22, 2013): 1–6. http://dx.doi.org/10.2174/1874070701307010001.
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Recently, many biopharmaceuticals have been developed such as cytokines, growth factors, and antibodies. These recombinant proteins are mostly expressed by CHO cells. However, the culture medium of CHO cells requires the addition of serum, which can contain unknown biological substances such as viruses, or requires the addition of expensive growth factors. To avoid the risks of biological ingredients and to decrease the cost of biopharmaceutical production, we developed a non-protein and lipid medium adopted (NPLAd) CHO cell line using the adapted culture method. Our results indicated that autocrine EGF production and insulin addition are essential for NPLAd CHO cell growth. However, the rate of cell proliferation of NPLAd CHO cells was decreased compared with original CHO-K1 cells. The proliferation of NPLAd CHO cells was improved by GM3 addition, suggesting increased signaling efficiency of autocrine factors. No difference was found in the growth rate between original CHO-K1 and NPLAd CHO cells supplemented with insulin and GM3. The productivity of recombinant protein in NPLAd CHO cells was verified using secreting luciferase reporter system. As a result, luciferase activity in NPLAd CHO cells showed more than three times higher than in the original CHOK1 cells. The results suggested that this cell line could be useful for biopharmaceutical recombinant protein.
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Obiakor, Harold, Marion Avril, Nicholas J. MacDonald, Prakash Srinivasan, Karine Reiter, Charles Anderson, Kevin L. Holmes, et al. "Identification of VAR2CSA Domain-Specific Inhibitory Antibodies of the Plasmodium falciparum Erythrocyte Membrane Protein 1 Using a Novel Flow Cytometry Assay." Clinical and Vaccine Immunology 20, no. 3 (January 23, 2013): 433–42. http://dx.doi.org/10.1128/cvi.00638-12.
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ABSTRACTVAR2CSA, a member of thePlasmodium falciparumerythrocyte membrane protein 1 (PfEMP1) family, is a leading candidate for use in vaccines to protect first-time mothers from placental malaria (PM). VAR2CSA, which is comprised of a series of six Duffy binding-like (DBL) domains, binds chondroitin sulfate A (CSA) on placental syncytiotrophoblast. Several recombinant DBL domains have been shown to bind CSA. In order to identify and develop recombinant proteins suitable for clinical development, DBL2X and DBL3X, as well as their respective third subdomain (S3) from the FCR3 parasite clone, were expressed inEscherichia coli, refolded, and purified. All but DBL3X-S3 recombinant proteins bound to CSA expressed on Chinese hamster ovary (CHO)-K1 cells but not to CHO-pgsA745 cells, which are CSA negative as determined by flow cytometry. All but DBL3X-S3 bound to CSA on chondroitin sulfate proteoglycan (CSPG) as determined by surface plasmon resonance (SPR) analysis. Purified IgG from rats and rabbits immunized with these four recombinant proteins bound homologous and some heterologous parasite-infected erythrocytes (IE). Using a novel flow cytometry inhibition-of-binding assay (flow-IBA), antibodies against DBL3X-S3 inhibited 35% and 45% of IE binding to CSA on CHO-K1 cells compared to results for soluble CSA (sCSA) and purified multigravida (MG) IgG, respectively, from areas in Tanzania to which malaria is endemic. Antibodies generated against the other domains provided little or no inhibition of IE binding to CSA on CHO-K1 cells as determined by the flow cytometry inhibition-of-binding assay. These results demonstrate for the first time the ability to identify antibodies to VAR2CSA DBL domains and subdomains capable of inhibiting VAR2CSA parasite-IE binding to CSA by flow cytometry. The flow cytometry inhibition-of-binding assay was robust and provided an accurate, reproducible, and reliable means to identify blocking of IE binding to CSA and promises to be significant in the development of a vaccine to protect pregnant women.
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KAWASAKI, Kiyoshi, Osamu KUGE, Yoshio YAMAKAWA, and Masahiro NISHIJIMA. "Purification of phosphatidylglycerophosphate synthase from Chinese hamster ovary cells." Biochemical Journal 354, no. 1 (February 8, 2001): 9–15. http://dx.doi.org/10.1042/bj3540009.
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Phosphatidylglycerophosphate (PGP) synthase catalyses the committed step in the biosynthesis of phosphatidylglycerol and cardiolipin in mammalian cells. Recently we isolated a Chinese hamster ovary (CHO) PGS1 cDNA encoding PGP synthase. In the present study we purified this PGP synthase to near-homogeneity from the mitochondrial fraction of CHO-K1 cells; the final enzyme preparation gave a single 60kDa protein on SDS/PAGE. Polyclonal antibodies raised against a recombinant CHO PGS1 protein cross-reacted with the purified 60kDa protein and with CHO membrane proteins of 60kDa and 62kDa that increased after transfection with the PGS1 cDNA. The 60 and 62kDa protein levels in a PGP synthase-defective mutant of CHO-K1 cells were markedly lower than those in CHO-K1 cells. These results indicated that the purified 60kDa protein was PGP synthase encoded by the PGS1 gene. In addition we found that the purified PGP synthase had no PGP phosphatase activity, indicating that phosphatidylglycerol was produced from CDP-diacylglycerol through two steps catalysed by distinct enzymes, PGP synthase and PGP phosphatase.
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Gentry, L. E., N. R. Webb, G. J. Lim, A. M. Brunner, J. E. Ranchalis, D. R. Twardzik, M. N. Lioubin, H. Marquardt, and A. F. Purchio. "Type 1 transforming growth factor beta: amplified expression and secretion of mature and precursor polypeptides in Chinese hamster ovary cells." Molecular and Cellular Biology 7, no. 10 (October 1987): 3418–27. http://dx.doi.org/10.1128/mcb.7.10.3418-3427.1987.
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Recombinant type 1 transforming growth factor beta (TGF-beta) was expressed to high levels in CHO cells by using dihydrofolate reductase (dhfr) gene amplification. The expression plasmid was derived from the pSV2 vectors and contained, in tandem, the simian TGF-beta and mouse dhfr cDNAs. Transcription of both cDNAs was controlled by the simian virus 40 early promoter. Stepwise selection of transfected CHO cells in increasing concentrations of methotrexate yielded cell lines that expressed amplified TGF-beta nucleic acid sequences. The expression plasmid DNA was amplified greater than 35-fold in one of the methotrexate-selected transfectants. The major proteins secreted by these cells consisted of latent TGF-beta and TGF-beta precursor polypeptides, as judged by immunoblots by using site-specific anti-peptide antibodies derived from various regions of the TGF-beta precursor. Levels of recombinant TGF-beta protein secreted by these cells approached 30 micrograms/24 h per 10(7) cells and required prior acidification for optimal activity; nonacidified supernatants were approximately 1% as active as acidified material. Antibodies directed toward sequences present in the mature growth factor readily identified a proteolytically processed recombinant TGF-beta which, on sodium dodecyl sulfate-polyacrylamide gels, comigrated with highly purified natural TGF-beta. In addition to mature recombinant TGF-beta, site-specific antibodies demonstrated the existence of larger TGF-beta precursor polypeptides. The availability of biologically active recombinant type 1 TGF-beta and precursor forms should provide a means to examine the structure, function, and potential in vivo therapeutic use of this growth factor.
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Gentry, L. E., N. R. Webb, G. J. Lim, A. M. Brunner, J. E. Ranchalis, D. R. Twardzik, M. N. Lioubin, H. Marquardt, and A. F. Purchio. "Type 1 transforming growth factor beta: amplified expression and secretion of mature and precursor polypeptides in Chinese hamster ovary cells." Molecular and Cellular Biology 7, no. 10 (October 1987): 3418–27. http://dx.doi.org/10.1128/mcb.7.10.3418.
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Recombinant type 1 transforming growth factor beta (TGF-beta) was expressed to high levels in CHO cells by using dihydrofolate reductase (dhfr) gene amplification. The expression plasmid was derived from the pSV2 vectors and contained, in tandem, the simian TGF-beta and mouse dhfr cDNAs. Transcription of both cDNAs was controlled by the simian virus 40 early promoter. Stepwise selection of transfected CHO cells in increasing concentrations of methotrexate yielded cell lines that expressed amplified TGF-beta nucleic acid sequences. The expression plasmid DNA was amplified greater than 35-fold in one of the methotrexate-selected transfectants. The major proteins secreted by these cells consisted of latent TGF-beta and TGF-beta precursor polypeptides, as judged by immunoblots by using site-specific anti-peptide antibodies derived from various regions of the TGF-beta precursor. Levels of recombinant TGF-beta protein secreted by these cells approached 30 micrograms/24 h per 10(7) cells and required prior acidification for optimal activity; nonacidified supernatants were approximately 1% as active as acidified material. Antibodies directed toward sequences present in the mature growth factor readily identified a proteolytically processed recombinant TGF-beta which, on sodium dodecyl sulfate-polyacrylamide gels, comigrated with highly purified natural TGF-beta. In addition to mature recombinant TGF-beta, site-specific antibodies demonstrated the existence of larger TGF-beta precursor polypeptides. The availability of biologically active recombinant type 1 TGF-beta and precursor forms should provide a means to examine the structure, function, and potential in vivo therapeutic use of this growth factor.
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Naghneh, Ehsan, Es'hagh Pourmaleki, and Azam Rahimpour. "Evaluation of the Effects of Human Beta-Interferon Scaffold Attachment Region (IFN-SAR) on Expression of Vascular Endothelial Growth Factor-Fc (VEGF-Fc) Fusion Protein Expression in Chinese Hamster Ovary (CHO) Cells." Pharmaceutical Sciences 26, no. 4 (December 25, 2020): 393–98. http://dx.doi.org/10.34172/ps.2020.37.
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Background: Recombinant anti-vascular endothelial growth factor (VEGF) monoclonal antibodies and Fc-fusion proteins have been widely used for the effective treatment of retinal neovascular diseases. In this regard, VEGFR-Fc fusions, which act as strong VEGF inhibitors, have been approved for the treatment of age-related macular degeneration (AMD) and diabetic macular edema (DME). Production of monoclonal antibodies and Fc-fusion proteins relies on mammalian host systems such as Chinese hamster ovary (CHO) cells. Application of genomic regulatory elements including scaffold/matrix attachment regions (SAR/MARs) can profoundly affect recombinant protein expression in CHO cells. Methods: To construct the VEGFR-Fc expression vectors, the enhanced green fluorescent protein (EGFP) gene was replaced by the VEGFR-Fc coding sequence in pEGFP-SAR-puro and pEGFP-puro vectors. Recombinant plasmids were transfected to CHO-K1 cells using TurboFect transfection reagent. VEGFR-Fc expression was evaluated in transiently transfected cells as well as stable cell pools and clones using an enzyme-linked immunosorbent assay (ELISA). Results: IFN-SAR showed no significant effect on transient expression of VEGFR-Fc during 72 h of culture. However, a 2.2-fold enhancement in VEGFR-Fc fusion protein titer was observed in IFN-SAR containing stable cell pools. Further evaluation of the VEGFR-Fc expression level in single-cell clones also indicated that clones with the highest VEGFR-Fc expression belonged to the pools transfected with IFN-SAR construct. Conclusion: Our results indicate that the incorporation of IFN-SAR in expression vector can increase the expression of VEGFR-Fc in stable cell pools as well as single-cell clones. In contrast, transient expression of the fusion protein was not affected by IFN-SAR. More studies are needed to investigate the mechanism underlying this effect, including the analysis of mRNA expression and gene copy number in stable cell pools as well as clonal cells.
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Coulet, Mathilde, Oliver Kepp, Guido Kroemer, and Stéphane Basmaciogullari. "Metabolic Profiling of CHO Cells during the Production of Biotherapeutics." Cells 11, no. 12 (June 15, 2022): 1929. http://dx.doi.org/10.3390/cells11121929.
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As indicated by an ever-increasing number of FDA approvals, biotherapeutics constitute powerful tools for the treatment of various diseases, with monoclonal antibodies (mAbs) accounting for more than 50% of newly approved drugs between 2014 and 2018 (Walsh, 2018). The pharmaceutical industry has made great progress in developing reliable and efficient bioproduction processes to meet the demand for recombinant mAbs. Mammalian cell lines are preferred for the production of functional, complex recombinant proteins including mAbs, with Chinese hamster ovary (CHO) cells being used in most instances. Despite significant advances in cell growth control for biologics manufacturing, cellular responses to environmental changes need to be understood in order to further improve productivity. Metabolomics offers a promising approach for developing suitable strategies to unlock the full potential of cellular production. This review summarizes key findings on catabolism and anabolism for each phase of cell growth (exponential growth, the stationary phase and decline) with a focus on the principal metabolic pathways (glycolysis, the pentose phosphate pathway and the tricarboxylic acid cycle) and the families of biomolecules that impact these circuities (nucleotides, amino acids, lipids and energy-rich metabolites).
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Oda, Y., J. Sanders, S. Roberts, M. Maruyama, R. Kato, M. Perez, VB Petersen, N. Wedlock, J. Furmaniak, and B. Rees Smith. "Binding characteristics of antibodies to the TSH receptor." Journal of Molecular Endocrinology 20, no. 2 (April 1, 1998): 233–44. http://dx.doi.org/10.1677/jme.0.0200233.
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We have used fragments of the TSH receptor (TSHR) expressed in E. coli as glutathione S-transferase fusion proteins to produce rabbit polyclonal antibodies and a panel (n=5) of monoclonal antibodies to the extracellular fragment of the TSHR. The binding characteristics of the antibodies to linear, conformational, glycosylated and unglycosylated forms of the receptor in different assay systems have been investigated. The reactivity of these antibodies with the TSHR was assessed by Western blotting with both native and recombinant human TSHR expressed in CHO cells, immunoprecipitation of 35S-labelled full-length TSHR produced in an in vitro transcription/ translation system, immunoprecipitation of 125I-TSH/TSHR complexes, inhibition of 125I-TSH binding to the TSHR and fluorescence activated cell sorter (FACS) analysis of binding to CHO-K1 cells expressing the TSHR on their cell surface. Fab fragments of monoclonal antibodies were isolated, labelled with 125I and used to determine the affinity constants of these antibodies with receptor, bound and free Fab being separated by polyethylene glycol (PEG) precipitation. Rabbit polyclonal and mouse monoclonal antibodies reacted with the TSHR in Western blotting and one monoclonal antibody (3C7) was able to inhibit 125I-TSH binding to native human TSHR (74% inhibition), recombinant human TSHR (84% inhibition) and porcine TSHR (65% inhibition). Affinity constant values for TSHR monoclonal antibody Fab fragments calculated using Scatchard analysis were about 10(7) M(-1). Four out of five monoclonal antibodies reacted in FACS analysis with TSHR expressed on the surface of CHO-K1 cells. The FACS unreactive monoclonal (3C7) bound well to detergent solubilised TSH receptors and this emphasised the importance of using a combination of FACS analysis and radioactively-labelled probes in analysis of the TSH receptor. The monoclonal antibodies produced in this study were found to be of relatively low affinity but proved useful for detection of the receptor by Western blotting and by FACS analysis.
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Thaore, Vaishali, Dimitrios Tsourapas, Nilay Shah, and Cleo Kontoravdi. "Techno-Economic Assessment of Cell-Free Synthesis of Monoclonal Antibodies Using CHO Cell Extracts." Processes 8, no. 4 (April 12, 2020): 454. http://dx.doi.org/10.3390/pr8040454.
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Cell-free protein synthesis (CFPS) is an emerging tool for the rapid production of difficult-to-express proteins as well as for identifying protein synthesis bottlenecks. In CFPS, the biotic phase is substituted by extracts of living cells devoid of any of their own genetic material. The main advantage is that these systems delineate cell growth from recombinant protein production, enabling the expression of targets that would otherwise place too big a burden on living cells. We have conducted a techno-economic analysis of a CFPS system to produce monoclonal antibodies (mAbs) using extracts of Chinese hamster ovary (CHO) cells. We compare the performance of the CFPS system with two alternative production strategies: stable and transient gene expression in CHO cells. Our assessment shows that the viability of CFPS for mAb production requires a significant increase in the product yield and the recycling of high-cost components such as DNA. Nevertheless, CFPS shows significant promise for personalized medicine applications, providing a platform for on-demand production and simplified supply chains.
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Keysberg, Christoph, Oliver Hertel, Louise Schelletter, Tobias Busche, Chiara Sochart, Jörn Kalinowski, Raimund Hoffrogge, Kerstin Otte, and Thomas Noll. "Exploring the molecular content of CHO exosomes during bioprocessing." Applied Microbiology and Biotechnology 105, no. 9 (May 2021): 3673–89. http://dx.doi.org/10.1007/s00253-021-11309-8.
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Abstract In biopharmaceutical production, Chinese hamster ovary (CHO) cells derived from Cricetulus griseus remain the most commonly used host cell for recombinant protein production, especially antibodies. Over the last decade, in-depth multi-omics characterization of these CHO cells provided data for extensive cell line engineering and corresponding increases in productivity. However, exosomes, extracellular vesicles containing proteins and nucleic acids, are barely researched at all in CHO cells. Exosomes have been proven to be a ubiquitous mediator of intercellular communication and are proposed as new biopharmaceutical format for drug delivery, indicator reflecting host cell condition and anti-apoptotic factor in spent media. Here we provide a brief overview of different separation techniques and subsequently perform a proteome and regulatory, non-coding RNA analysis of exosomes, derived from lab-scale bioreactor cultivations of a CHO-K1 cell line, to lay out reference data for further research in the field. Applying bottom-up orbitrap shotgun proteomics and next-generation small RNA sequencing, we detected 1395 proteins, 144 micro RNA (miRNA), and 914 PIWI-interacting RNA (piRNA) species differentially across the phases of a batch cultivation process. The exosomal proteome and RNA data are compared with other extracellular fractions and cell lysate, yielding several significantly exosome-enriched species. Key points • First-time comprehensive protein and miRNA characterization of CHO exosomes. • Isolation protocol and time point of bioprocess strongly affect quality of extracellular vesicles. • CHO-derived exosomes also contain numerous piRNA species of yet unknown function.
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Stravinskiene, Dovile, Aiste Sliziene, Lina Baranauskiene, Vilma Petrikaite, and Aurelija Zvirbliene. "Inhibitory Monoclonal Antibodies and Their Recombinant Derivatives Targeting Surface-Exposed Carbonic Anhydrase XII on Cancer Cells." International Journal of Molecular Sciences 21, no. 24 (December 10, 2020): 9411. http://dx.doi.org/10.3390/ijms21249411.
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Monoclonal and recombinant antibodies are widely used for the diagnostics and therapy of cancer. They are generated to interact with cell surface proteins which are usually involved in the development and progression of cancer. Carbonic anhydrase XII (CA XII) contributes to the survival of tumors under hypoxic conditions thus is considered a candidate target for antibody-based therapy. In this study, we have generated a novel collection of monoclonal antibodies (MAbs) against the recombinant extracellular domain of CA XII produced in HEK-293 cells. Eighteen out of 24 MAbs were reactive with cellular CA XII on the surface of live kidney and lung cancer cells as determined by flow cytometry. One MAb 14D6 also inhibited the enzymatic activity of recombinant CA XII as measured by the stopped-flow assay. MAb 14D6 showed the migrastatic effect on human lung carcinoma A549 and renal carcinoma A498 cell lines in a ‘wound healing’ assay. It did not reduce the growth of multicellular lung and renal cancer spheroids but reduced the cell viability by the ATP Bioluminescence assay. Epitope mapping revealed the surface-exposed amino acid sequence (35-FGPDGENS-42) close to the catalytic center of CA XII recognized by the MAb 14D6. The variable regions of the heavy and light chains of MAb 14D6 were sequenced and their complementarity-determining regions were defined. The obtained variable sequences were used to generate recombinant antibodies in two formats: single-chain fragment variable (scFv) expressed in E. coli and scFv fused to human IgG1 Fc fragment (scFv-Fc) expressed in Chinese Hamster Ovary (CHO) cells. Both recombinant antibodies maintained the same specificity for CA XII as the parental MAb 14D6. The novel antibodies may represent promising tools for CA XII-related cancer research and immunotherapy.
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Carlos, T., N. Kovach, B. Schwartz, M. Rosa, B. Newman, E. Wayner, C. Benjamin, L. Osborn, R. Lobb, and J. Harlan. "Human monocytes bind to two cytokine-induced adhesive ligands on cultured human endothelial cells: endothelial-leukocyte adhesion molecule-1 and vascular cell adhesion molecule-1." Blood 77, no. 10 (May 15, 1991): 2266–71. http://dx.doi.org/10.1182/blood.v77.10.2266.2266.
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Abstract Vascular cell adhesion molecule-1 (VCAM-1) and endothelial-leukocyte adhesion molecule-1 (ELAM-1) are adhesive proteins induced on endothelium by cytokines. We examined the contribution of these adhesive proteins to human peripheral blood monocyte adherence to endothelium using transfected Chinese hamster ovary (CHO) cells stably expressing these proteins and monoclonal antibodies (MoAbs) to ELAM-1, VCAM-1, or CD49d/CD29 (VLA-4), the leukocyte receptor for VCAM-1. Monocytes bound to CHO cells transfected with cDNA of ELAM-1 or VCAM-1. Binding to ELAM-1 was inhibited by MoAb to ELAM-1 and binding to VCAM-1 was inhibited by MoAb to VCAM-1 or the alpha-chain of very late activation antigen-4 (VLA-4) (CD49d). Additive inhibition of adherence to unstimulated human umbilical vein endothelium (HUVE) was observed when monocytes were pretreated with both MoAb to CD49d and MoAb to CD18, the common beta-chain of the leukocyte beta 2 integrin receptors. Adherence of monocytes to HUVE stimulated by recombinant human tumor necrosis factor-alpha was not reduced by MoAbs to CD18, CD49d, or ELAM- 1 when used singly, but combinations of these MoAbs produced significant inhibition. We conclude that multiple receptor-ligand systems are involved in monocyte adherence to endothelium.
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Carlos, T., N. Kovach, B. Schwartz, M. Rosa, B. Newman, E. Wayner, C. Benjamin, L. Osborn, R. Lobb, and J. Harlan. "Human monocytes bind to two cytokine-induced adhesive ligands on cultured human endothelial cells: endothelial-leukocyte adhesion molecule-1 and vascular cell adhesion molecule-1." Blood 77, no. 10 (May 15, 1991): 2266–71. http://dx.doi.org/10.1182/blood.v77.10.2266.bloodjournal77102266.
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Vascular cell adhesion molecule-1 (VCAM-1) and endothelial-leukocyte adhesion molecule-1 (ELAM-1) are adhesive proteins induced on endothelium by cytokines. We examined the contribution of these adhesive proteins to human peripheral blood monocyte adherence to endothelium using transfected Chinese hamster ovary (CHO) cells stably expressing these proteins and monoclonal antibodies (MoAbs) to ELAM-1, VCAM-1, or CD49d/CD29 (VLA-4), the leukocyte receptor for VCAM-1. Monocytes bound to CHO cells transfected with cDNA of ELAM-1 or VCAM-1. Binding to ELAM-1 was inhibited by MoAb to ELAM-1 and binding to VCAM-1 was inhibited by MoAb to VCAM-1 or the alpha-chain of very late activation antigen-4 (VLA-4) (CD49d). Additive inhibition of adherence to unstimulated human umbilical vein endothelium (HUVE) was observed when monocytes were pretreated with both MoAb to CD49d and MoAb to CD18, the common beta-chain of the leukocyte beta 2 integrin receptors. Adherence of monocytes to HUVE stimulated by recombinant human tumor necrosis factor-alpha was not reduced by MoAbs to CD18, CD49d, or ELAM- 1 when used singly, but combinations of these MoAbs produced significant inhibition. We conclude that multiple receptor-ligand systems are involved in monocyte adherence to endothelium.
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Chan, K. W., M. N. Langan, J. L. Sui, J. A. Kozak, A. Pabon, J. A. Ladias, and D. E. Logothetis. "A recombinant inwardly rectifying potassium channel coupled to GTP-binding proteins." Journal of General Physiology 107, no. 3 (March 1, 1996): 381–97. http://dx.doi.org/10.1085/jgp.107.3.381.
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GTP-binding (G) proteins have been shown to mediate activation of inwardly rectifying potassium (K+) channels in cardiac, neuronal and neuroendocrine cells. Here, we report functional expression of a recombinant inwardly rectifying channel which we call KGP (or hpKir3.4), to signify that it is K+ selective, G-protein-gated and isolated from human pancreas. KGP expression in Xenopus oocytes resulted in sizeable basal (or agonist-independent) currents while coexpression with a G-protein-linked receptor, yielded additional agonist-induced currents. Coexpression of KGP and hGIRK1 (a human brain homolog of GIRK1/Kir3.1) produced much larger basal currents than those observed with KGP or hGIRK1 alone, and upon coexpression with receptor, similarly large agonist-induced currents could be obtained. Pertussis toxin treatment significantly diminished agonist-dependent currents due to either KGP or KGP/hGIRK1 expression. Interestingly, PTX also significantly reduced basal KGP or KGP/hGIRK1 currents, suggesting that basal activity is largely the result of G-protein gating as well. When the two channels were coexpressed with receptor, the relative increase in current elicited by agonist was similar whether KGP and hGIRK1 were expressed alone or together. When in vitro translated or when expressed in Xenopus oocytes or CHO mammalian cells, KGP gave rise to a nonglycosylated 45-kD protein. Antibodies directed against either KGP or hGIRK1 coprecipitated both proteins coexpressed in oocytes, providing evidence for the heteromeric assembly of the two channels and suggesting that the current potentiation seen with coexpression of the two channel subunits is due to specific interactions between them. An endogenous oocyte protein similar in size to KGP was also coprecipitated with hGIRK1.
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Petit, François M., Catherine Serres, and Jana Auer. "Moonlighting proteins in sperm–egg interactions." Biochemical Society Transactions 42, no. 6 (November 17, 2014): 1740–43. http://dx.doi.org/10.1042/bst20140218.
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Sperm–egg interaction is a highly species-specific step during the fertilization process. The first steps consist of recognition between proteins on the sperm head and zona pellucida (ZP) glycoproteins, the acellular coat that protects the oocyte. We aimed to determine which sperm head proteins interact with ZP2, ZP3 and ZP4 in humans. Two approaches were combined to identify these proteins: immunoblotting human spermatozoa targeted by antisperm antibodies (ASAs) from infertile men and far-Western blotting of human sperm proteins overlaid by each of the human recombinant ZP (hrZP) proteins. We used a proteomic approach with 2D electrophoretic separation of sperm protein revealed using either ASAs eluted from infertile patients or recombinant human ZP glycoproteins expressed in Chinese-hamster ovary (CHO) cells. Only spots highlighted by both methods were analysed by MALDI–MS/MS for identification. We identified proteins already described in human spermatozoa, but implicated in different metabolic pathways such as glycolytic enzymes [phosphokinase type 3 (PK3), enolase 1 (ENO1), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), aldolase A (ALDOA) and triose phosphate isomerase (TPI)], detoxification enzymes [GST Mu (GSTM) and phospholipid hydroperoxide glutathione peroxidase (PHGPx) 4], ion channels [voltage-dependent anion channel 2 (VDAC2)] or structural proteins (outer dense fibre 2). Several proteins were localized on the sperm head by indirect immunofluorescence, and their interaction with ZP proteins was confirmed by co-precipitation experiments. These results confirm the complexity of the sperm–ZP recognition process in humans with the implication of different proteins interacting with the main three ZP glycoproteins. The multiple roles of these proteins suggest that they are multifaceted or moonlighting proteins.
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Omelina, Evgeniya S., Anna E. Letiagina, Lidiya V. Boldyreva, Anna A. Ogienko, Yuliya A. Galimova, Lyubov A. Yarinich, Alexey V. Pindyurin, and Evgeniya N. Andreyeva. "Slight Variations in the Sequence Downstream of the Polyadenylation Signal Significantly Increase Transgene Expression in HEK293T and CHO Cells." International Journal of Molecular Sciences 23, no. 24 (December 7, 2022): 15485. http://dx.doi.org/10.3390/ijms232415485.
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Compared to transcription initiation, much less is known about transcription termination. In particular, large-scale mutagenesis studies have, so far, primarily concentrated on promoter and enhancer, but not terminator sequences. Here, we used a massively parallel reporter assay (MPRA) to systematically analyze the influence of short (8 bp) sequence variants (mutations) located downstream of the polyadenylation signal (PAS) on the steady-state mRNA level of the upstream gene, employing an eGFP reporter and human HEK293T cells as a model system. In total, we evaluated 227,755 mutations located at different overlapping positions within +17..+56 bp downstream of the PAS for their ability to regulate the reporter gene expression. We found that the positions +17..+44 bp downstream of the PAS are more essential for gene upregulation than those located more distal to the PAS, and that the mutation sequences ensuring high levels of eGFP mRNA expression are extremely T-rich. Next, we validated the positive effect of a couple of mutations identified in the MPRA screening on the eGFP and luciferase protein expression. The most promising mutation increased the expression of the reporter proteins 13-fold and sevenfold on average in HEK293T and CHO cells, respectively. Overall, these findings might be useful for further improving the efficiency of production of therapeutic products, e.g., recombinant antibodies.
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Uchida, Nobuko, Zhi Yang, Jesse Combs, Olivier Pourquié, Megan Nguyen, Rajeev Ramanathan, Joan Fu, et al. "The Characterization, Molecular Cloning, and Expression of a Novel Hematopoietic Cell Antigen From CD34+ Human Bone Marrow Cells." Blood 89, no. 8 (April 15, 1997): 2706–16. http://dx.doi.org/10.1182/blood.v89.8.2706.
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Abstract The adhesion molecule BEN/SC1/DM-GRASP (BEN) is a marker in the developing chicken nervous system that is also expressed on the surface of embryonic and adult hematopoietic cells such as immature thymocytes, myeloid progenitors, and erythroid progenitors. F84.1 and KG-CAM, two monoclonal antibodies to rat neuronal glycoproteins with similarity to BEN, cross-react with an antigen on rat hematopoietic progenitors, but F84.1 only also recognizes human blood cell progenitors. We have defined the antigen recognized by F84.1 as the hematopoietic cell antigen (HCA). HCA expression was detected on 40% to 70% of CD34+ fetal and adult bone marrow cells and mobilized peripheral blood cells. Precursor cell activity for long-term in vitro bone marrow cell culture was confined to the subset of CD34+ cells that coexpress HCA. HCA is expressed by the most primitive subsets of CD34+ cells, including all rhodamine 123lo, Thy-1+, and CD38−/lo CD34+ adult bone marrow cells. HCA was also detected on myeloid progenitors but not on early B-cell progenitors. We also describe here the cloning and characterization of cDNAs encoding two variants of the human HCA antigen (huHCA-1 and huHCA-2) and of a cDNA clone encoding rat HCA (raHCA). The deduced amino acid sequences of huHCA and raHCA are homologous to that of chicken BEN. Recombinant proteins produced from either human or rat HCA cDNAs were recognized by F84.1, whereas rat HCA but not human HCA was recognized by antirat KG-CAM. Expression of either form of huHCA in CHO cells conferred homophilic adhesion that could be competed with soluble recombinant huHCA-Fc. The molecular cloning of HCA and the availability of recombinant HCA should permit further evaluation of its role in human and rodent hematopoiesis.
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Allander, Tobias, Katarina Drakenberg, Aster Beyene, Domenico Rosa, Sergio Abrignani, Michael Houghton, Anders Widell, Lena Grillner, and Mats A. A. Persson. "Recombinant human monoclonal antibodies against different conformational epitopes of the E2 envelope glycoprotein of hepatitis C virus that inhibit its interaction with CD81." Journal of General Virology 81, no. 10 (October 1, 2000): 2451–59. http://dx.doi.org/10.1099/0022-1317-81-10-2451.
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The antibody response to the envelope proteins of hepatitis C virus (HCV) may play an important role in controlling the infection. To allow molecular analyses of protective antibodies, we isolated human monoclonal antibodies to the E2 envelope glycoprotein of HCV from a combinatorial Fab library established from bone marrow of a chronically HCV-infected patient. Anti-E2 reactive clones were selected using recombinant E2 protein. The bone marrow donor carried HCV genotype 2b, and E2 used for selection was of genotype 1a. The antibody clones were expressed as Fab fragments in E. coli, and as Fab fragments and IgG1 in CHO cells. Seven different antibody clones were characterized, and shown to have high affinity for E2, genotype 1a. Three clones also had high affinity for E2 of genotype 1b. They all bind to conformation-dependent epitopes. Five clones compete for the same or overlapping binding sites, while two bind to one or two other epitopes of E2. Four clones corresponding to the different epitopes were tested as purified IgG1 for blocking the CD81–E2 interaction in vitro; all four were positive at 0·3–0·5 μg/ml. Thus, the present results suggest the existence of at least two conserved epitopes in E2 that mediate inhibition of the E2–CD81 interaction, of which one appeared immunodominant in this donor.
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Matthews, Alicia M., Thomas G. Biel, Uriel Ortega-Rodriguez, Vincent M. Falkowski, Xin Bush, Talia Faison, Hang Xie, Cyrus Agarabi, V. Ashutosh Rao, and Tongzhong Ju. "SARS-CoV-2 spike protein variant binding affinity to an angiotensin-converting enzyme 2 fusion glycoproteins." PLOS ONE 17, no. 12 (December 6, 2022): e0278294. http://dx.doi.org/10.1371/journal.pone.0278294.
Full textAbstract:
Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), the causative agent of the Coronavirus disease 2019 (Covid-19) pandemic, continues to evolve and circulate globally. Current prophylactic and therapeutic countermeasures against Covid-19 infection include vaccines, small molecule drugs, and neutralizing monoclonal antibodies. SARS-CoV-2 infection is mainly mediated by the viral spike glycoprotein binding to angiotensin converting enzyme 2 (ACE2) on host cells for viral entry. As emerging mutations in the spike protein evade efficacy of spike-targeted countermeasures, a potential strategy to counter SARS-CoV-2 infection is to competitively block the spike protein from binding to the host ACE2 using a soluble recombinant fusion protein that contains a human ACE2 and an IgG1-Fc domain (ACE2-Fc). Here, we have established Chinese Hamster Ovary (CHO) cell lines that stably express ACE2-Fc proteins in which the ACE2 domain either has or has no catalytic activity. The fusion proteins were produced and purified to partially characterize physicochemical properties and spike protein binding. Our results demonstrate the ACE2-Fc fusion proteins are heavily N-glycosylated, sensitive to thermal stress, and actively bind to five spike protein variants (parental, alpha, beta, delta, and omicron) with different affinity. Our data demonstrates a proof-of-concept production strategy for ACE2-Fc fusion glycoproteins that can bind to different spike protein variants to support the manufacture of potential alternative countermeasures for emerging SARS-CoV-2 variants.
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Mao, Qian, Weijian Zhang, Shengming Ma, Zilong Qiu, Bingke Li, Chen Xu, Huangyu He, et al. "Fusion Expression and Immune Effect of PCV2 Cap Protein Tandem Multiantigen Epitopes with CD154/GM-CSF." Veterinary Sciences 8, no. 10 (September 29, 2021): 211. http://dx.doi.org/10.3390/vetsci8100211.
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Porcine circovirus associated diseases (PCVAD) is a contagious disease of swine caused by porcine circovirus type 2 (PCV2). The capsid protein (Cap) is the sole structural protein and the main antigen of PCV2. Cap is the principal immunogenic protein and induces humoral and cellular immunity. CD154 and GM-CSF are immune adjuvants that enhance responses to vaccines. However, whether these two cellular molecules could produce an enhanced effect in PCV2 vaccines still needs to be further studied. The results of PCR and restriction enzyme showed that the recombinant lentiviral plasmids pCDH-TB-Cap, pCDH-TB-Cap-CD154 and pCDH-TB-Cap were successfully constructed. Western blot and IFA showed that the three fusion proteins TB-Cap, TB-Cap-CD154 and TB-Cap-GM-CSF were stably expressed in CHO-K1 cells. Indirect ELISA assay showed that mice immunized with TB-Cap-CD154 and TB-Cap-GM-CSF fusion proteins produced higher PCV2-specific antibodies than mice immunized with the TB-Cap and a commercial vaccine (p < 0.0001). Moreover, lymphocyte proliferation and flow cytometry showed that the cellular immune response of each immune group was significantly enhanced (p < 0.0001). After PCV2 challenge, the results revealed that the viral loads in serum, lung and kidney of all vaccinated groups were significantly lower than the PBS group (p < 0.0001). The transcription levels of IL-2, IFN-gamma, IL-4 and IL-10 cytokines in the TB-Cap, TB-Cap-CD154 and TB-Cap-GM-CSF groups were significantly higher than those in the PBS and recombinant vaccine groups (p < 0.0001). These results indicated that CD154 and GM-CSF could enhance the ability of TB-Cap protein to induce the body to produce PCV2 specific antibodies and increase the transcription level of cytokines. Thus, CD154 and GM-CSF molecules were a powerful immunoadjuvant for PCV2 subunit vaccines. The novel TB-Cap-CD154 and TB-Cap-GM-CSF subunit vaccine has the potential to be used for the prevention and control of PCVAD.
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Baruch, Dror I., Benoit Gamain, and Louis H. Miller. "DNA Immunization with the Cysteine-Rich Interdomain Region 1 of the Plasmodium falciparum Variant Antigen Elicits Limited Cross-Reactive Antibody Responses." Infection and Immunity 71, no. 8 (August 2003): 4536–43. http://dx.doi.org/10.1128/iai.71.8.4536-4543.2003.
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ABSTRACT The variant surface antigens of Plasmodium falciparum are an important component of naturally acquired immunity and an important vaccine target. However, these proteins appear to elicit primarily variant-specific antibodies. We tested if naked DNA immunization can elicit more cross-reactive antibody responses and allow simultaneous immunization with several variant constructs. Mice immunized with plasmid DNA expressing variant cysteine-rich interdomain region 1 (CIDR1) domains of the P. falciparum erythrocyte membrane protein 1 (PfEMP1) developed antibodies that were reactive to the corresponding PfEMP1s as measured by an enzyme-linked immunosorbent assay, flow cytometry, and agglutination of parasitized erythrocytes (PEs). We observed some cross-reactive immune responses; for example, sera from mice immunized with one domain agglutinated PEs of various lines and recognized heterologous domains expressed on the surface of Chinese hamster ovary (CHO) cells. We found no significant antigenic competition when animals were immunized with a mixture of plasmids or immunized sequentially with individual constructs. Moreover, mixed or sequential immunizations resulted in greater cross-reactive agglutination responses than immunization with a single domain. Recombinant protein (Sc y179) immunization after priming with DNA (prime-boost regimen) increased antibody titers to the homologous domain substantially but seemed to diminish the cross-reactive responses somewhat. The titer of agglutinating antibodies was previously shown to correlate with protection. Surprisingly, the agglutination titers of sera from DNA immunization were high, similar to those of pooled human hyperimmune sera. These sera also appeared to give limited low-titer variant transcending agglutination. Thus, DNA immunization appears to be a very useful tool for developing variant antigen vaccines.
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Feghhi, Shirin, Adam D. Munday, Wes Tooley, Rajsekar Shreya, José A. López, and Nathan Sniadecki. "Platelet Cytoskeletal Force Transmission Through The GPIb-IX-V Complex." Blood 122, no. 21 (November 15, 2013): 197. http://dx.doi.org/10.1182/blood.v122.21.197.197.
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Abstract Platelets are the primary cellular components of the hemostatic plug that forms during primary hemostasis. The first step in this process is platelet adhesion from the flowing blood to a surface, carried out by the platelet glycoprotein (GP) Ib-IX-V binding to immobilized von Willebrand factor (VWF). Adhesion is followed by activation of integrin αIIbβ3, which mediates the attachment of platelets to each other by binding multivalent ligands such as VWF or fibrinogen. To stabilize the hemostatic plug and strengthen its attachment to the wound site, platelets must transmit contractile forces from actin and myosin proteins in their cytoskeleton to extracellular matrix proteins within the vessel wall or to the adhesive proteins between adjacent platelets. Integrin αIIbβ3 is one of the membrane proteins capable of transmitting these forces, having a direct link to the platelet cytoskeleton through talin and other focal adhesion related proteins. In the current study, we investigated whether the GPIb-IX-V complex is also capable of force transmission after binding ligand. The GPIb-IX-V complex contains 4 polypeptides, GPIbα, GPIbβ, GPIX and GPV. Only GPIbα binds VWF, which it does through VWF's A1 domain. GPIbα also attaches the complex to the actin and membrane skeletons through its cytoplasmic domain, with the large skeletal protein filamin functioning as the intermediary. There is strong evidence that the GPIbα-A1 bond is force sensitive, becoming stronger as force is applied to it, a property that defines it as a “catch bond”. For this reason, we investigated the role of GPIbα in transmitting platelet forces using a new tool that we have developed to measure contractile forces generated by platelets. This tool, composed of arrays of nanoposts separated by 2 μm (Figure 1), was fabricated using e-beam lithography. VWF was adsorbed to the tips of the nanoposts and platelets were allowed to adhere, spread, and contract. To assess the contribution of αIIbβ3 and GPIbα to force generation, we blocked these receptors with the antibodies 7E3 and AK2, respectively. Treatment with 7E3 significantly lowered the force generated, but did not eliminate it completely (57% reduction). AK2 had a smaller effect (20% reduction), and the combination of the two usually abolished force generation. We observed a similar force reduction (30%) as AK2 treatment when we blocked the VWF A1 domain with recombinant GPIbα N-terminus. Because VWF contains binding sites for more than one platelet receptor, and although purified, could have trace amounts of other plasma proteins, we also evaluated force generation on nanoposts coated with recombinant VWF A1 domain, which should only bind GPIbα. In this case, the platelets generated forces similar to those observed when αIIbβ3 was blocked by 7E3, providing further evidence that GPIbα can transmit forces by binding the A1 domain. Figure 1.Platelet bending nanoposts.Figure 1. Platelet bending nanoposts. As a final test of the ability of GPIbα to support force generation, we examined whether Chinese hamster ovary (CHO) cells expressing the GPIb-IX complex (CHOαβIX, fully functional but lacking GPV) could generate force on VWF or A1 domain (Figure 2). CHOαβIX cells adhered, spread and generated forces of similar magnitude on microposts (larger because of the larger cell size) coated with either substrate. CHOβIX cells, lacking GPIbα, did not adhere to either substrate.Figure 2.CHO cell bending microposts.Figure 2. CHO cell bending microposts. To investigate the requirement for cytoskeletal attachment of the complex in force generation, we studied a CHOαβIX line containing GPIbα truncated after residue 518 and therefore lacking almost the entire cytoplasmic domain. These cells adhered and spread on VWF-coated microposts, but generated minimal contractile force. Together, these results indicate that the GPIb-IX-V complex is able to transmit cytoskeletal contractile forces to its ligand, VWF, in a process requiring the cytoplasmic domain of GPIbα. This is the first example of a non-integrin transmitting force to an external substrate. Disclosures: Sniadecki: Stasys Medical Corporation: Equity Ownership, Founder Other.
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Manabe, Ri-ichiroh, Naoko Oh-e, Toshinaga Maeda, Tomohiko Fukuda, and Kiyotoshi Sekiguchi. "Modulation of Cell-adhesive Activity of Fibronectin by the Alternatively Spliced EDA Segment." Journal of Cell Biology 139, no. 1 (October 6, 1997): 295–307. http://dx.doi.org/10.1083/jcb.139.1.295.
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Fibronectin (FN) has a complex pattern of alternative splicing at the mRNA level. One of the alternatively spliced segments, EDA, is prominently expressed during biological processes involving substantial cell migration and proliferation, such as embryonic development, malignant transformation, and wound healing. To examine the function of the EDA segment, we overexpressed recombinant FN isoforms with or without EDA in CHO cells and compared their cell-adhesive activities using purified proteins. EDA+ FN was significantly more potent than EDA− FN in promoting cell spreading and cell migration, irrespective of the presence or absence of a second alternatively spliced segment, EDB. The cell spreading activity of EDA+ FN was not affected by antibodies recognizing the EDA segment but was abolished by antibodies against integrin α5 and β1 subunits and by Gly-Arg-Gly-Asp-Ser-Pro peptide, indicating that the EDA segment enhanced the cell-adhesive activity of FN by potentiating the interaction of FN with integrin α5β1. In support of this conclusion, purified integrin α5β1 bound more avidly to EDA+ FN than to EDA− FN. Augmentation of integrin binding by the EDA segment was, however, observed only in the context of the intact FN molecule, since the difference in integrin-binding activity between EDA+ FN and EDA− FN was abolished after limited proteolysis with thermolysin. Consistent with this observation, binding of integrin α5β1 to a recombinant FN fragment, consisting of the central cell-binding domain and the adjacent heparin-binding domain Hep2, was not affected by insertion of the EDA segment. Since the insertion of an extra type III module such as EDA into an array of repeated type III modules is expected to rotate the polypeptide up to 180° at the position of the insertion, the conformation of the FN molecule may be globally altered upon insertion of the EDA segment, resulting in an increased exposure of the RGD motif in III10 module and/or local unfolding of the module. Our results suggest that alternative splicing at the EDA exon is a novel mechanism for up-regulating integrin-binding affinity of FN operating when enhanced migration and proliferation of cells are required.
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El Nemer, Wassim, Cecile Rahuel, Yves Colin, Pierre Gane, Jean Pierre Cartron, and Caroline Le Van Kim. "Organization of the Human LU Gene and Molecular Basis of the Lua/Lub Blood Group Polymorphism." Blood 89, no. 12 (June 15, 1997): 4608–16. http://dx.doi.org/10.1182/blood.v89.12.4608.
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Abstract The Lutheran (Lu) blood group antigens and the B-cell adhesion molecule (B-CAM) epithelial cancer antigen are carried by recently cloned integral glycoproteins that belong to the Ig superfamily. We have previously shown that the Lu and B-CAM antigens are encoded by the same gene, LU, and that alternative splicing of the primary transcript most likely accounts for the presence of both antigens on two isoforms that differ by the length of their cytoplasmic tails. In the present report, we isolated the human LU gene by cloning a 20-kb HindIII fragment from Lu(a − b+) genomic DNA. The LU gene is organized into 15 exons distributed over 12.5 kb. Alternative splicing of intron 13 generates the 2.5- and 4.0-kb transcript spliceoforms encoding the long tail and the short tail Lu polypeptides, respectively. Sequencing of the major mRNA species (2.5 kb) amplified from human bone marrow, kidney, placenta, and skeletal muscle did not suggest the presence of tissue-specific Lu glycoprotein isoforms. The same transcription initiation point, located 22 bp upstream from the initiation codon, was characterized in several tissues. In agreement with the wide tissue distribution of the Lu messengers, the GC-rich proximal 5′ flanking region of the LU gene does not contain TATA or CAAT boxes, but includes several potential binding sites for the ubiquitous Sp1 transcription factor. In addition, the distal 5′ region, encompassing nucleotides −673 to −764, contains clustered binding sequences for the GATA, CACCC, and Ets transcription factors. Analysis of the coding sequences amplified from genomic DNA of Lu(a + b−) or Lu(a − b+) donors showed a single nucleotide change in exon 3 (A229G) that correlates with an Aci I restriction site polymorphism and results in a His77Arg amino-acid substitution. Polymerase chain reaction/restriction fragment length polymorphism analysis indicated that the A229G mutation is associated with the Lua/Lub blood group polymorphism. When expressed in Chinese hamster ovary (CHO) cells, Lu cDNAs carrying the A229 or the G229 produced cell surface proteins that reacted with anti-Lua or anti-Lub antibodies, respectively, showing that these nucleotides specify the Lua and Lub alleles of the Lutheran blood group locus. CHO cells expressing recombinant short-tail or long-tail Lu glycoproteins reacted as well with anti-Lu as with anti–B-CAM antibodies, providing the definitive proof that the Lu blood group and B-CAM antigens are carried by the same molecules.
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Kenny, Dermot, Peter J. Newman, Patricia A. Morateck, and Robert R. Montgomery. "A Dinucleotide Deletion Results in Defective Membrane Anchoring and Circulating Soluble Glycoprotein Ibα in a Novel Form of Bernard-Soulier Syndrome." Blood 90, no. 7 (October 1, 1997): 2626–33. http://dx.doi.org/10.1182/blood.v90.7.2626.
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Abstract The platelet membrane glycoprotein (GP)Ib-V-IX complex is the receptor for von Willebrand factor and is composed of four membrane-spanning polypeptides: GPIbα, GPIbβ, GPIX, and GPV. A qualitative or quantitative deficiency in the GPIb-V-IX complex on the platelet membrane is the cause of the congenital platelet disorder Bernard-Soulier syndrome (BSS). We describe the molecular basis of a novel variant BSS in a patient in which GPIbα was absent from the platelet surface but present in a soluble form in the plasma. DNA sequence analysis showed a homozygous dinucleotide deletion in the codon for Tyr 508 (TAT) in GPIbα. This mutation (GPIbαΔAT) causes a frame shift that alters the amino acid sequence of GPIbα within its transmembrane region. The hydrophobic nature of the predicted transmembrane region and the cytoplasmic tail at the COOH terminal are altered before reaching a new premature stop codon 38 amino acids short of the wild-type peptide. Although GPIbαΔAT was not detectable on the platelet surface, immunoprecipitation of plasma with specific monoclonal antibodies (MoAbs) identified circulating GPIbα. Transient expression of recombinant GPIbαΔAT in 293T cells also generated a soluble form of the protein. Moreover, when a plasmid encoding GPIbαΔAT was transiently transfected into Chinese hamster ovary (CHO) cells stably expressing the GPβ-IX complex, it failed to be expressed on the cell surface. Thus, a dinucleotide deletion in the codon for Tyr 508 causes a frameshift that alters the amino acid sequence of GPIbα starting within its transmembrane region, changes the hydrophobicity of the normal transmembrane region, and truncates the cytoplasmic domain affecting binding to the cytoskeleton and cytoplasmic proteins. This mutation affects anchoring of the GPIbα polypeptide in platelets and causes the observed BSS phenotype with circulating soluble GPIbα.
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Kenny, Dermot, Peter J. Newman, Patricia A. Morateck, and Robert R. Montgomery. "A Dinucleotide Deletion Results in Defective Membrane Anchoring and Circulating Soluble Glycoprotein Ibα in a Novel Form of Bernard-Soulier Syndrome." Blood 90, no. 7 (October 1, 1997): 2626–33. http://dx.doi.org/10.1182/blood.v90.7.2626.2626_2626_2633.
Full textAbstract:
The platelet membrane glycoprotein (GP)Ib-V-IX complex is the receptor for von Willebrand factor and is composed of four membrane-spanning polypeptides: GPIbα, GPIbβ, GPIX, and GPV. A qualitative or quantitative deficiency in the GPIb-V-IX complex on the platelet membrane is the cause of the congenital platelet disorder Bernard-Soulier syndrome (BSS). We describe the molecular basis of a novel variant BSS in a patient in which GPIbα was absent from the platelet surface but present in a soluble form in the plasma. DNA sequence analysis showed a homozygous dinucleotide deletion in the codon for Tyr 508 (TAT) in GPIbα. This mutation (GPIbαΔAT) causes a frame shift that alters the amino acid sequence of GPIbα within its transmembrane region. The hydrophobic nature of the predicted transmembrane region and the cytoplasmic tail at the COOH terminal are altered before reaching a new premature stop codon 38 amino acids short of the wild-type peptide. Although GPIbαΔAT was not detectable on the platelet surface, immunoprecipitation of plasma with specific monoclonal antibodies (MoAbs) identified circulating GPIbα. Transient expression of recombinant GPIbαΔAT in 293T cells also generated a soluble form of the protein. Moreover, when a plasmid encoding GPIbαΔAT was transiently transfected into Chinese hamster ovary (CHO) cells stably expressing the GPβ-IX complex, it failed to be expressed on the cell surface. Thus, a dinucleotide deletion in the codon for Tyr 508 causes a frameshift that alters the amino acid sequence of GPIbα starting within its transmembrane region, changes the hydrophobicity of the normal transmembrane region, and truncates the cytoplasmic domain affecting binding to the cytoskeleton and cytoplasmic proteins. This mutation affects anchoring of the GPIbα polypeptide in platelets and causes the observed BSS phenotype with circulating soluble GPIbα.
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Fong, Karen Pei Yi, Ismail A. Ahmed, Marco Mravic, Hyunil Jo, William F. DeGrado, Feng Gai, and Joel S. Bennett. "Direct Visualization of Platelet Integrins Using ANTI-Transmembrane Domain Peptides Containing a BLUE Fluorescent Amino Acid." Blood 134, Supplement_1 (November 13, 2019): 2344. http://dx.doi.org/10.1182/blood-2019-128363.
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Antibodies containing a fluorescent label or recombinant proteins containing a fluorescent reporter are commonly used to visualize proteins in situ. However, antibodies and bulky reporters can perturb protein structure and function. To overcome this problem, we have designed a minimally perturbing blue fluorescent unnatural amino acid that enables the direct imaging of intrinsically-fluorescent proteins with their interaction partners. Specifically, we used a nitrile-derivatized tryptophan, 4-cyanotryptophan (4CNTrp), which differs by only two atoms from native tryptophan. 4CNTrp has unique photophysical properties in the visible blue region: an absorption maximum at ~325 nm, an emission maximum at ~420 nm, a large fluorescence quantum yield (>0.8 in aqueous solution), a long fluorescence lifetime (ca. 13 ns), and good photostability (Hilaire et al. PNAS 2017; 114: 6005-09). Based on these properties, 4CNTrp-labeling was recently used to visualize the binding of a model peptide pHLIP (pH-(Low) Insertion Peptide) to cell membranes via wide-field fluorescence microscopy. 4CNTrp is also a viable FRET donor for acceptor pairs in the green visible region. 4CNTrp can be prepared by a simple, high-yielding cost-effective synthetic route and because it does not inhibit bacterial growth, it may be practical for cellular applications (Zhang et al. Chem. Comm. 2019; 55: 5095-98). Here, we used 4CNTrp-labeled peptides to directly image integrins on cell surfaces. Previously, we described the synthesis of peptides, called CHAMP peptides for Computed Helical Anti-Membrane Protein, that target the transmembrane (TM) domains of integrins in a sequence-specific manner (Yin et al. Science 2007; 315: 1817-22). Thus, the CHAMP peptides anti-αIIb and anti-αv specifically bind to the TM domains of αIIb and αv in platelet membranes, causing separation of the αIIbβ3 and αvβ3 TM domains and αIIbβ3 and αvβ3 activation. Anti-αIIb also caused αIIbβ3 clustering that was visualized using the β3-specific monoclonal antibody SSA6 labeled with Alexa Fluor 488 but only after the platelets were fixed with paraformaldehyde to prevent antibody-induced clustering. To enable the direct visualization of αIIβ3, αvβ3, and α2β1 on cell surfaces, we used solid-phase peptide synthesis to generate CHAMP peptides labeled with 4CNTrp at their N-termini (CN-αIIb, CN-αv, CN-β1). We found that 4CNTrp labeling did not perturb the ability of the CHAMP peptides to bind to integrins and to cause specific integrin activation. Thus, CN-αIIb caused αIIbβ3-dependent platelet aggregation, CN-αv caused the αvβ3-dependent platelet adhesion to osteopontin, and CN-α1 caused α2β1-mediated platelet adhesion to collagen. We then used high resolution wide-field deconvolution microscopy to image 4CNTrp-containing integrins on the surface of platelets, HEL cells, and transfected CHO cells. Compared to 4CNTrp-containing random peptides that uniformly decorate cell surfaces, the CN-labeled CHAMP peptides were present as a limited number of discrete foci on the cell surface. To confirm that these foci represented CN-peptide containing integrins, we co-stained the cells with Alexa Fluor 488-labeled integrin-specific monoclonal antibodies and found that CN-peptide and antibody fluorescence coincided. Thus, these studies demonstrate the specific and direct imaging of integrins embedded in cell membranes in their activated state using 4CNTrp-labeled anti-integrin TM peptides. Because 4CNTrp can readily be incorporated into proteins and peptides with little if any structural perturbation, 4CNTrp-labeling provides a facile way to directly monitor protein behavior and protein-protein interactions in cellular environments. Disclosures No relevant conflicts of interest to declare.
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Takamatsu, Hiroyuki, Yoshitaka Zaimoku, Tatsuhiko Ozawa, Shintaro Kawai, Hidenori Tanaka, Hiroyuki Kishi, Atsushi Muraguchi, and Shinji Nakao. "Development of Novel Human Anti-HLA-Monoclonal Antibodies for Clinical Applications Using Peripheral Blood B Cells Derived from Anti-HLA Antibody-Positive Donors." Blood 128, no. 22 (December 2, 2016): 4723. http://dx.doi.org/10.1182/blood.v128.22.4723.4723.
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Abstract Introduction: The development of monoclonal antibodies (mAbs) such as anti-CD20 mAb (rituximab) and anti-CD38 mAb (daratumumab) has revolutionized the treatment of lymphoid malignancies. Potential efficacy of HLA allele-specific mAbs in treating malignant lymphoma has been shown by several studies. However, treatment of B-cell malignancies with humanized mAbs against HLA-DR alleles is associated with infusion-related toxicities (Lin et al., Leuk Lymphoma, 2009). In addition, previous attempts to develop mAbs specific to some HLA alleles (e.g., HLA-B61) by immunizing mice with recombinant HLA proteins have failed, likely because of a wide variety of polymorphisms of HLA molecules. To overcome these problems, we recently created a novel method to develop mAbs using microarray technology and human peripheral blood (PB) B lymphocytes derived from donors who are positive for anti-HLA antibodies. The process took approximately 1 month to produce human anti-HLA mAbs. Methods: Approximately 20 ml of PB was collected from anti-HLA antibody-positive donors, and mononuclear cells (MNCs) isolated using the Ficoll-Hypaque method were cultured in RPMI-1640 + 10% FCS containing 5 μg/ml R-848, 1000 IU/ml hIL2, 2.5 μg/ml CpG2006, 2.5 μg/ml anti-CD40 antibody, 100 ng/ml hIL21, 2 ng/ml hIL17, 10 ng/ml hIL4, and 100 ng/ml hBAFF for 6 days. CD138+ cells were then isolated using anti-CD138-antibody-conjugated microbeads. A microwell array chip was manufactured using micromachining techniques, as previously described (Jin et al., Nature Medicine, 2009). Microwells (diameter, 10 μm; depth, 15 μm) were formed on a silicon surface. The chip was coated with a PBS-containing purified HLA antigen (10 μg/ml) and incubated overnight at 4°C. After removing the antigen solution, 50 μl of the CD138+ cell suspension was added to the chip, and the mixture was incubated for 4 h at 37°C. After gentle washing with PBS, 2 μg/ml of anti-human IgG Fc-Cy3 solution was added to the microwells, and the plate was left at room temperature for 15 min. Finally, the cells were stained with 1 μM Oregon Green for 2 min. The microwells were screened for the antigen-specific antibodies released from single cells under a fluorescence microscope. Antigen-specific antibody-secreting cells (ASCs) from individual wells were isolated using a micromanipulator fitted with capillaries under the fluorescence microscope and were then expelled to microtubes for reverse transcription. The antibody cDNA fragments for VH and VL fragments were amplified using the single-cell 5′-RACE method and inserted into expression vectors containing the complete constant region of cDNA for heavy or light chains. Thereafter, CHO cells were transfected with both the heavy and light chain expression vectors to obtain a supernatant containing complete antibody molecules. The antigen specificity of the recombinant antibodies was examined using ELISA and flow cytometry (FCM). The frequency of ASCs in the PB-MNCs of donors was quantified using allele-specific oligonucleotide PCR. Complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC) of the mAbs were assessed using conventional methods. Results and Discussion : Two novel human mAbs specific to HLA-A24 and HLA-B61 were successfully generated. The antigen specificity of the mAbs was confirmed using ELISA and FCM. The mAbs bound to normal and malignant blood cells that expressed corresponding HLA alleles and showed CDC/ADCC activities against five B lymphoblastoid cell lines but not against lymphocytes/monocytes/granulocytes derived from five healthy donors, probably because of low expression levels of the target HLA alleles. The frequency of ASCs in PB-MNCs of donors was less than 0.001%. Conclusion s : The present method enabled the generation of mAbs specific to HLA alleles, which can be used for detecting minimal residual diseases of hematological malignancies as well as for treating B-cell lymphoma, within 1 month. Disclosures Takamatsu: Celgene: Honoraria; Janssen Pharmaceuticals: Honoraria. Kawai:Wakunaga Pharmaceutical Co., Ltd.: Employment. Nakao:Alexion Pharmaceuticals: Honoraria, Research Funding.
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Biswas, Tapan K., and Jonathan L. Miller. "Properties of Soluble His-Tagged rGPIbα1–483 Wild-Type and Increase-of-Function Mutants." Blood 106, no. 11 (November 16, 2005): 3949. http://dx.doi.org/10.1182/blood.v106.11.3949.3949.
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Abstract The extracellular domain of human platelet GPIbα contains the binding site for von Willebrand factor (vWF) within the amino-terminal 45 kDa region comprising approximately 300 amino acids. In the present studies, we have used CHO cells to synthesize a longer fragment of His1-Phe483, representing nearly the complete extracellular domain of GPIbα, with an additional 8 histidine residues added at the carboxyl end to provide recognition by his-tag reagents. Both wild-type and the increase-of-function mutants Gly233→Val233 and/or Met239→Val239 were employed. For studies of antibody binding, modulator-induced vWF binding, or antibody inhibition of this vWF binding, capture of the recombinant GPIbα could be accomplished by nickel chelate binding of the his-tag. Both botrocetin and ristocetin induced saturable binding of vWF by ELISA, as demonstrated by HRP-conjugated anti-vWF polyclonal antibody. In the case of the different increase-of-function mutants, significant vWF binding was seen even in the absence of added modulator. Inhibition of botrocetin-induced vWF binding was greater for wild-type than for the Met239→Val239 GPIbα mutant with some mabs (e.g., AS-2), whereas the degree of inhibition was essentially equivalent with other mabs (e.g., C-34). When a rabbit polyclonal anti-his antibody was used to immobilize the recombinant proteins instead of the nickel chelate system, binding of vWF and of conformation-dependent mabs was impaired. However, when this same antibody was biotinylated and then captured on a streptavidin plate, excellent capture and subsequent binding characteristics of the recombinant GPIbα were restored. With both this latter system and with the nickel chelate system, a highly sensitive functional vWF activity assay could be established, using botrocetin-induced binding of vWF from human plasma. Linearity of binding was observed between 0.1–0.6% normal plasma, with saturation occurring around 2%. Whereas higher concentrations of plasma actually appeared to displace bound GPIbα to the nickel chelate, the streptavidin-biotin-anti-his attachment method appeared more resistant to such effects. The ELISA-based assay systems developed in this study accordingly provide an and effective approach to study functional interactions of both wild-type and mutant forms of GPIbα with its ligand vWF and also with antibodies directed against different epitopes within the entire extracellular domain of this receptor molecule. The high sensitivity, reproducibility, and rapidity of the assay system also offer the possibility of a useful test for functional assay of plasma vWF.
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Wang, Haitao, Yong Du, Rui Zhang, Jin Xu, and Longbin Liu. "A Nova EPO-Fusion Protein with Prolonged Half-Life in Circulation and Enhanced Activity In Vivo." Blood 108, no. 11 (November 16, 2006): 1301. http://dx.doi.org/10.1182/blood.v108.11.1301.1301.
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Abstract Clinical practice merits the search of long-lasting erythropoietic proteins that reduce the injection requirements. Darbepoietin, the first long-lasting EPO, was created by replacing two amino acids of EPO molecule to increase the sugar contents. Darbepoietin has a half-life of about 24 hours and enhanced functional activities. However, the structural changes of EPO molecules may increase the potential of antibody production in logn-term application. With our NovaFusion technology, we successfully created a nova EPO-fusion protein that contains two identical molecules of natural human EPO sequences and a fragment of another human protein(Nova-EPO). We produced recombinant Nova-EPO protein by CHO cells and then conducted extensive laboratory and anemial studies. Pharmacokinetic studies in primates showed that the half-life of Nova-EPO was about 35 hours comparing to about 8 hours of that of recombinant EPO. In pharmacodynamic studies, we compared, on the same molar basis, the erythropoietic effects of Nova-EPO, recombinant EPO and Darbepoietin in normal mice or rats with experimental renal anemia. The experimental renal anemia was created by the surgical removal of 5/6 renal tissues in rats. Five doses (0.1, 0.5, 2.5, 12.5 or 62.5 ug/kg), two administration methods (subcutaneouly or intravenously) and three administration intervials(three injections per week, one injection per week or one injection per two weeks) were selected in different combinations. With three injections per week, both Nova-EPO and EPO induced dose-dependent elevation of hemoglobulin (HB) levels in normal mice. Lower doses of Nova-EPO increased the HB levels to the extent only achived by the higher doses of EPO, and the highest elevation of HB levels was induced by Nova-EPO. With one injection per week in normal mice, the dose-dependent elevation of HB levels was more significant in the Nova-EPO groups than in the EPO groups, and 12.5ug/kg of Nova-EPO induced the increase of HB levels to the same extent by 62.5ug/kg of EPO. The elevation of HB levels in Nova-EPO groups also remained longer than that in EPO groups after the termination of treatment. These data demonstrated that Nova-EPO has functional activities in vivo and stimulates stronger erythropoiesis. The erythropoietic properties of Nova-EPO were further eveluated in rats with experimental anemia, and compared with EPO and Darbepoietin. When administrated once per week, Nova-EPO, EPO and Darbepoietin all induced the increase of HB levels and corrected the anemia. The levels of HB in Nova-EPO and Darbepoietin groups, however, were higher and decreased more slowly than those in EPO groups. The highest HB levels were observed in Nova-EPO groups. After reducing the injection to once per two weeks, Nova-EPO and Darbepoietin still induced satisfactory elevation of HB levels. Similarly, Nova-EPO induced stronger erythropoietic responses than Darbepoietin. In immunogenisis studies in primates, injection of 5ug/kg of Nova-EPO three times per week for four weeks failed to produce antibodies against Nova-EPO molecules. Acute toxic studies in mice showed that injection of up to 13mg/kg of Nova-EPO intravenously did not result in pathologic changes. Collectively these data indicated that Nova-EPO has significantly prolonged half-life in vivo and enhanced erythropoietic activities. With no immunogenisis in primates, Nova-EPO has the potential to become a better alternative than the long-lasting erythropoietic therapeutics on the market and is justified to be further evaluated in clinical studies.
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Shestopal, Svetlana A., James H. Kurasawa, Jian-Jiang Hao, Elena Karnaukhova, Yideng Liang, Min Lin, Mikhail V. Ovanesov, Timothy K. Lee, John H. McVey, and Andrey G. Sarafanov. "Structural and Functional Characterization of a Codon Optimized Coagulation Factor VIII." Blood 128, no. 22 (December 2, 2016): 3765. http://dx.doi.org/10.1182/blood.v128.22.3765.3765.
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Abstract Background. Production of recombinant factor VIII (FVIII) is challenging due to its low expression. Previously, it was shown that codon optimization of a B domain-deleted (BDD) FVIII resulted in its increased expression (Ward et al, Blood 2011; 117: 798-807). However, in some cases, synonymous mutations are known to affect the protein's post-translation modifications, conformation, fidelity of amino acid sequence, and functions. Thus, for each particular codon optimization of a given protein, confirmation of its biochemical characteristics is necessary. Recently, we established conditions for expression and purification of a codon optimized BDD-FVIII (CO), in parallel, testing the BDD-FVIII (WT) expressed from the wild-type cDNA sequence (Shestopal et al, ISTH-2015 meeting, Abstract PO196-WED). In present work, we verified if the characteristics of the CO remain unchanged upon modification of its coding sequence. Objective. To characterize structural and functional properties of the BDD-FVIII encoded by either a codon-optimized or wild-type cDNA sequence (CO and WT, respectively). Experimental Approach. Several preparations of each WT and CO, purified from independent CHO cells clonal lines, were analyzed by: polyacrylamide gel electrophoresis (PAGE), before and after thrombin treatment; Western-blot analysis; ELISA; mass-spectrometry upon specific protein fragmentation; circular dichroism; chromogenic, clotting and thrombin generation assays to test the FVIII activity; and by surface plasmon resonance to test the binding to von Willebrand factor and a fragment of the low-density lipoprotein receptor-related protein 1 (LRP). Results. The average purification yield of CO was approximately 7-fold higher than that of WT. The proteins were identical in the amino acid sequences, covered by 99% by mass-spectrometry, and were very similar in: i) patterns of the molecular fragments, including those produced upon thrombin cleavage by PAGE, ii) recognition by anti-FVIII antibodies by both Western-blot and ELISA, iii) glycosylation and tyrosine sulfation by mass spectrometry, iv) secondary structures by circular dichroism and v) binding to von Willebrand factor, and vi) to a fragment of the low-density lipoprotein receptor-related protein 1 by surface plasmon resonance. By chromogenic, clotting and thrombin generation assays, the CO had about 1.5-fold higher FVIII specific activity (activity normalized to protein mass) than WT. Conclusions. The higher specific activity of CO was attributed to better preservation of its structure due to consistently higher concentrations than WT at all steps of the production. Thus, we concluded that the codon optimization of the BDD-FVIII resulted in a significant increase of its expression, while did not affect the protein's properties. Disclosures No relevant conflicts of interest to declare.
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Skipper, Kristian Alsbjerg, Anne Kruse Hollensen, Michael N. Antoniou, and Jacob Giehm Mikkelsen. "Sustained transgene expression from sleeping beauty DNA transposons containing a core fragment of the HNRPA2B1-CBX3 ubiquitous chromatin opening element (UCOE)." BMC Biotechnology 19, no. 1 (November 9, 2019). http://dx.doi.org/10.1186/s12896-019-0570-2.
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Abstract Background DNA transposon-based vectors are effective nonviral tools for gene therapy and genetic engineering of cells. However, promoter DNA methylation and a near-random integration profile, which can result in transgene integration into heterochromatin, renders such vectors vulnerable to transcriptional repression. Therefore, to secure persistent transgene expression it may be necessary to protect transposon-embedded transgenes with anti-transcriptional silencing elements. Results We compare four different protective strategies in CHO-K1 cells. Our findings show robust protection from silencing of transgene cassettes mediated by the ubiquitous chromatin-opening element (UCOE) derived from the HNRPA2B1-CBX3 locus. Using a bioinformatic approach, we define a shorter HNRPA2B1-CBX3 UCOE core fragment and demonstrate that this can robustly maintain transgene expression after extended passaging of CHO-K1 cells carrying DNA transposon vectors equipped with this protective feature. Conclusions Our findings contribute to the understanding of the mechanism of HNRPA2B1-CBX3 UCOE-based transgene protection and support the use of a correctly oriented core fragment of this UCOE for DNA transposon vector-based production of recombinant proteins in CHO-K1 cells.
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Donaldson, James S., Matthew P. Dale, and Susan J. Rosser. "Decoupling Growth and Protein Production in CHO Cells: A Targeted Approach." Frontiers in Bioengineering and Biotechnology 9 (June 2, 2021). http://dx.doi.org/10.3389/fbioe.2021.658325.
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Fed-batch cultures of Chinese Hamster Ovary cells have been used to produce high quantities of biotherapeutics, particularly monoclonal antibodies. However, a growing number of next-generation biotherapeutics, such as bi-specific antibodies and fusion proteins, are difficult to express using standard fed-batch processes. Decoupling cell growth and biotherapeutic production is becoming an increasingly desired strategy for the biomanufacturing industry, especially for difficult-to-express products. Cells are grown to a high cell density in the absence of recombinant protein production (the growth phase), then expression of the recombinant protein is induced and cell proliferation halted (the production phase), usually by combining an inducible gene expression system with a proliferation control strategy. Separating the growth and production phases allows cell resources to be more efficiently directed toward either growth or production, improving growth characteristics and enhancing the production of difficult to express proteins. However, current mammalian cell proliferation control methods rely on temperature shifts and chemical agents, which interact with many non-proliferation pathways, leading to variable impacts on product quality and culture viability. Synthetic biology offers an alternative approach by strategically targeting proliferation pathways to arrest cell growth but have largely remained unused in industrial bioproduction. Due to recent developments in microbial decoupling systems and advances in available mammalian cell engineering tools, we propose that the synthetic biology approach to decoupling growth and production needs revisiting.
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Sharker, Shazid Md, and Md Atiqur Rahman. "Review of the current methods of Chinese Hamster Ovary (CHO) cells cultivation for production of therapeutic protein." Current Drug Discovery Technologies 17 (March 12, 2020). http://dx.doi.org/10.2174/1570163817666200312102137.
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Most of clinical approved protein-based drugs or under in clinical trial have a profound impact in the treatment of critical diseases. The mammalian eukaryotic cells culture approaches, particularly the CHO (Chinese Hamster Ovary) cells are mainly used in the biopharmaceutical industry for the mass-production of therapeutic protein. Recent advances in CHO cell bioprocessing to yield recombinant proteins and monoclonal antibodies have enabled the expression of quality protein. The developments of cell lines are possible to upgrade specific productivity. As a result, it holds an interesting area for academic as well as industrial researchers around the world. This review will concentrate on the recent progress of the mammalian CHO cells culture technology and the future scope of further development for the mass-production of protein therapeutics.
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ÖZDEMİR BAHADIR, Aylin. "Developing selection strategy for CHO-K1 cell line that secretes scfv-Fc fusion antibodies using ClonePix2." International Journal of Life Sciences and Biotechnology, September 2, 2022. http://dx.doi.org/10.38001/ijlsb.1112823.
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In the pharmaceutical industry, biopharmaceuticals (biologics) are gaining market share. There has been a dramatic increase in the sale and market penetration of monoclonal antibodies in particular. Typically, therapeutic antibodies are produced using high-expression, clonal, or recombinant CHO cell lines. CHO cells dominate the market as a commercial production host due to their ease of use, built-in regulatory records, and security profiles. While traditional limiting-dilution and cloning-ring regulations are frequently used to select mammalian cell lines that produce high levels of proteins, they have a number of drawbacks. ClonePix2 is a fully automated, single cell-based clone selector that significantly increases the likelihood of rapidly selecting high-production clones with high monoclonality. Scfv-Fc recombinant antibody structures with a variety of therapeutic advantages have gained prominence in recent years. Single cell cloning of CHO cells expressing the scfv-Fc fusion protein, which differs from the classical immunoglobulin structure, was performed in situ using the ClonePix2 device using FITC-tagged anti-Fc and anti-H+L antibodies. The fluorescent intensity parameters of the resulting cell clones were analyzed. Additionally, ELISA was used to determine the production capacities of the best clones. As a result, it was established that anti-Fc antibody recognizes the scfv-Fc fusion protein in a semi-solid environment, enabling the identification of higher production clones.
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Karimi, Sahar, Shahram Nazarian, Fattah Sotoodehnejadnematalahi, Roohollah Dorostkar, and Jafar Amani. "Antigenicity and immunogenicity of SARS-CoV-2 surface glycoprotein fragment in CHO cells." Iranian Journal of Microbiology, February 13, 2023. http://dx.doi.org/10.18502/ijm.v15i1.11929.
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Background and Objectives: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S) glycoprotein that projects from the virus surface is highly immunogenic. It is considered to be the target of many neutralizing antibodies as well as a target in vaccine design efforts. Evaluation the immunogenicity of a recombinant fragment of the spike protein (rfsp) that is comprised of Receptor Binding Domain (RBD), S1/S2 cleavage site, and fusion peptide (FP) as immunogenic proteins of SARS-COV-2, in BALB/c mice and evaluation of the efficacy of epitopes rfsp as a multi-subunit chimeric vaccine.
Materials and Methods: The present study made use of CHO-K1 (Chinese hamster ovary K1) cells to create a cell line for constant expression rfsp. The rfsp was purified with Ni-NTA chromatography and confirmed by Western blotting. The immunogenicity and neutralizing antibody efficacy of rfsp were evaluated in BALB/c mice. ELISA was employed to test rfsp via sera of COVID-19 convalescent patients infected with SARS-CoV-2 alpha and delta variants.
Results: Our results showed significant differences in antibody titers in immunized mice compared to the control groups and neutralizing antibodies were positive, sera from mice immunized are capable of bound SARS-CoV-2 virus, chimer peptide is capable bound antibodies patients infected with SARS-CoV-2 and patients infected with delta variant SARS-CoV-2.
Conclusion: Overall, these results indicate that rfsp protein would be a novel potential antigen candidate for the development of a subunit SARS CoV-2 vaccine and rfsp has the potential to be a useful option for the development of the assays for serodiagnosis of SARS-CoV-2 infection.
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Plotnik, David, Wenjin Guo, Brad Cleveland, Priska von Haller, Jimmy K. Eng, Miklos Guttman, Kelly K. Lee, James Arthos, and Shiu-Lok Hu. "Extracellular Matrix Proteins Mediate HIV-1 gp120 Interactions with α4β7." Journal of Virology 91, no. 21 (August 16, 2017). http://dx.doi.org/10.1128/jvi.01005-17.
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ABSTRACT Gut-homing α4β7 high CD4+ T lymphocytes have been shown to be preferentially targeted by human immunodeficiency virus type 1 (HIV-1) and are implicated in HIV-1 pathogenesis. Previous studies demonstrated that HIV-1 envelope protein gp120 binds and signals through α4β7 and that this likely contributes to the infection of α4β7 high T cells and promotes cell-to-cell virus transmission. Structures within the second variable loop (V2) of gp120, including the tripeptide motif LDV/I, are thought to mediate gp120-α4β7 binding. However, lack of α4β7 binding has been reported in gp120 proteins containing LDV/I, and the precise determinants of gp120-α4β7 binding are not fully defined. In this work, we report the novel finding that fibronectins mediate indirect gp120-α4β7 interactions. We show that Chinese hamster ovary (CHO) cells used to express recombinant gp120 produced fibronectins and other extracellular matrix proteins that copurified with gp120. CHO cell fibronectins were able to mediate the binding of a diverse panel of gp120 proteins to α4β7 in an in vitro cell binding assay. The V2 loop was not required for fibronectin-mediated binding of gp120 to α4β7, nor did V2-specific antibodies block this interaction. Removal of fibronectin through anion-exchange chromatography abrogated V2-independent gp120-α4β7 binding. Additionally, we showed a recombinant human fibronectin fragment mediated gp120-α4β7 interactions similarly to CHO cell fibronectin. These findings provide an explanation for the apparently contradictory observations regarding the gp120-α4β7 interaction and offer new insights into the potential role of fibronectin and other extracellular matrix proteins in HIV-1 biology. IMPORTANCE Immune tissues within the gut are severely damaged by HIV-1, and this plays an important role in the development of AIDS. Integrin α4β7 plays a major role in the trafficking of lymphocytes, including CD4+ T cells, into gut lymphoid tissues. Previous reports indicate that some HIV-1 gp120 envelope proteins bind to and signal through α4β7, which may help explain the preferential infection of gut CD4+ T cells. In this study, we demonstrate that extracellular matrix proteins can mediate interactions between gp120 and α4β7. This suggests that the extracellular matrix may be an important mediator of HIV-1 interaction with α4β7-expressing cells. These findings provide new insight into the nature of HIV-1–α4β7 interactions and how these interactions may represent targets for therapeutic intervention.
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Lopez-Perez, Mary, Mads Delbo Larsen, Rafael Bayarri-Olmos, Paulina Ampomah, Liz Stevenson, Myriam Arévalo-Herrera, Sócrates Herrera, and Lars Hviid. "IgG Responses to the Plasmodium falciparum Antigen VAR2CSA in Colombia Are Restricted to Pregnancy and Are Not Induced by Exposure to Plasmodium vivax." Infection and Immunity 86, no. 8 (May 21, 2018). http://dx.doi.org/10.1128/iai.00136-18.
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ABSTRACT Clinical immunity to malaria is associated with the acquisition of IgG specific for members of the Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) family of clonally variant antigens on the surface of infected erythrocytes (IEs). The VAR2CSA subtype of PfEMP1 mediates IE binding in the placenta. VAR2CSA-specific IgG is normally acquired only after exposure to placental parasites. However, it was recently reported that men and children from Colombia often have high levels of functional VAR2CSA-specific IgG. This potentially undermines the current understanding of malaria immunity in pregnant women, and we thus conducted a study to assess further the levels of VAR2CSA-specific IgG in pregnant and nonpregnant Colombians. Plasma IgG against two full-length recombinant PfEMP1 proteins (one of the VAR2CSA type and one not) produced in baculovirus-transfected insect cells was detected frequently among Colombian men, children, and pregnant women with acute or previous malaria exposure. In contrast, IgG reactivity to a homologous full-length VAR2CSA-type protein expressed in Chinese hamster ovary (CHO) cells was low and infrequent among the Colombian plasma samples, as was reactivity to both corresponding native PfEMP1 proteins. Moreover, human and rabbit antibodies specific for Plasmodium vivax Duffy-binding protein (PvDBP), a protein with some homology to PfEMP1, did not react with VAR2CSA-type recombinant or native proteins, although the mouse monoclonal and PvDBP-specific antibody 3D10 was weakly reactive with recombinant proteins expressed in baculovirus-transfected insect cells. Our data indicate that the previously reported Colombian IgG reactivity to recombinant VAR2CSA is not malaria specific and that the acquisition of VAR2CSA-specific IgG is restricted to pregnancy, in Colombia and elsewhere.
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Wang, Shixia, Yegor Voronin, Peng Zhao, Mayumi Ishihara, Nickita Mehta, Mindy Porterfield, Yuxin Chen, et al. "Glycan Profiles of gp120 Protein Vaccines from Four Major HIV-1 Subtypes Produced from Different Host Cell Lines under Non-GMP or GMP Conditions." Journal of Virology 94, no. 7 (January 15, 2020). http://dx.doi.org/10.1128/jvi.01968-19.
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ABSTRACT Envelope (Env) glycoprotein of human immunodeficiency virus type 1 (HIV-1) is an important target for the development of an HIV vaccine. Extensive glycosylation of Env is an important feature that both protects the virus from antibody responses and serves as a target for some highly potent broadly neutralizing antibodies. Therefore, analysis of glycans on recombinant Env proteins is highly significant. Here, we present glycosylation profiles of recombinant gp120 proteins from four major clades of HIV-1 (A, B, C, and AE), produced either as research-grade material in 293 and CHO cells or as two independent lots of clinical material under good manufacturing practice (GMP) conditions. Almost all potential N-linked glycosylation sites were at least partially occupied in all proteins. The occupancy rates were largely consistent among proteins produced under different conditions, although a few sites showed substantial variability even between the two GMP lots. Our data confirmed previous studies in the field, showing an abundance of oligomannose on Env protein, with 40 to 50% of glycans being Man5 to Man9 on all four proteins under all production conditions. Overall, the differences in occupancy and glycan forms among different Env subtypes produced under different conditions were less dramatic than anticipated, and antigenicity analysis with a panel of six monoclonal antibodies, including antibodies that recognize glycan forms, showed that all four gp120s maintained their antibody-binding profiles. Such findings have major implications for the final production of a clinical HIV vaccine with Env glycoprotein components. IMPORTANCE HIV-1 Env protein is a major target for the development of an HIV-1 vaccine. Env is covered with a large number of sugar-based glycan forms; about 50% of the Env molecular weight is composed of glycans. Glycan analysis of recombinant Env is important for understanding its roles in viral pathogenesis and immune responses. The current report presents the first extensive comparison of glycosylation patterns of recombinant gp120 proteins from four major clades of HIV-1 produced in two different cell lines, grown either under laboratory conditions or at 50-liter GMP scale in different lots. Information learned in this study is valuable for the further design and production of HIV-1 Env proteins as the critical components of HIV-1 vaccine formulations.
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Ringe, Rajesh P., Gabriel Ozorowski, Anila Yasmeen, Albert Cupo, Victor M. Cruz Portillo, Pavel Pugach, Michael Golabek, et al. "Improving the Expression and Purification of Soluble, Recombinant Native-Like HIV-1 Envelope Glycoprotein Trimers by Targeted Sequence Changes." Journal of Virology 91, no. 12 (April 5, 2017). http://dx.doi.org/10.1128/jvi.00264-17.
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ABSTRACT Soluble, recombinant native-like envelope glycoprotein (Env) trimers of various human immunodeficiency virus type 1 (HIV-1) genotypes are being developed for structural studies and as vaccine candidates aimed at the induction of broadly neutralizing antibodies (bNAbs). The prototypic design is designated SOSIP.664, but many HIV-1 env genes do not yield fully native-like trimers efficiently. One such env gene is CZA97.012 from a neutralization-resistant (tier 2) clade C virus. As appropriately purified, native-like CZA97.012 SOSIP.664 trimers induce autologous neutralizing antibodies (NAbs) efficiently in immunized rabbits, we sought to improve the efficiency with which they can be produced and to better understand the limitations to the original design. By using structure- and antigenicity-guided mutagenesis strategies focused on the V2 and V3 regions and the gp120-gp41 interface, we developed the CZA97 SOSIP.v4.2-M6.IT construct. Fully native-like, stable trimers that display multiple bNAb epitopes could be expressed from this construct in a stable CHO cell line and purified at an acceptable yield using either a PGT145 or a 2G12 bNAb affinity column. We also show that similar mutagenesis strategies can be used to improve the yields and properties of SOSIP.664 trimers of the DU422, 426c, and 92UG037 genotypes. IMPORTANCE Recombinant trimeric proteins based on HIV-1 env genes are being developed for future vaccine trials in humans. A feature of these proteins is their mimicry of the envelope glycoprotein (Env) structure on virus particles that is targeted by neutralizing antibodies, i.e., antibodies that prevent cells from becoming infected. The vaccine concept under exploration is that recombinant trimers may be able to elicit virus-neutralizing antibodies when delivered as immunogens. Because HIV-1 is extremely variable, a practical vaccine may need to incorporate Env trimers derived from multiple different virus sequences. Accordingly, we need to understand how to make recombinant trimers from many different env genes. Here, we show how to produce trimers from a clade C virus, CZA97.012, by using an array of protein engineering techniques to improve a prototypic construct. We also show that the methods may have wider utility for other env genes, thereby further guiding immunogen design.
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Arévalo-Herrera, Myriam, Kazutoyo Miura, Nora Cespedes, Carlos Echeverry, Eduardo Solano, Angélica Castellanos, Juan Sebastián Ramirez, et al. "Immunoreactivity of Sera From Low to Moderate Malaria-Endemic Areas Against Plasmodium vivax rPvs48/45 Proteins Produced in Escherichia coli and Chinese Hamster Ovary Systems." Frontiers in Immunology 12 (June 24, 2021). http://dx.doi.org/10.3389/fimmu.2021.634738.
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P48/45 is a conserved gametocyte antigen involved in Plasmodium parasite fertilization. A recombinant Plasmodium vivax P48/45 (Pvs48/45) protein expressed in Escherichia coli (E. coli) was highly antigenic and immunogenic in experimental animals and elicited specific transmission-blocking (TB) antibodies in a previous pilot study. Here, a similar Pvs48/45 gene was expressed in Chinese Hamster Ovary (CHO) cells and we compared its immunoreactivity with the E. coli product. Specific antibody titers were determined using plasma from Colombian individuals (n=227) living in endemic areas where both P. vivax and P. falciparum are prevalent and from Guatemala (n=54) where P. vivax is highly prevalent. In Colombia, plasma seroprevalence to CHO-rPvs48/45 protein was 46.3%, while for E. coli-rPvs48/45 protein was 36.1% (p<0.001). In Guatemala, the sero prevalence was 24.1% and 14.8% (p<0.001), respectively. Reactivity index (RI) against both proteins showed an age-dependent increase. IgG2 was the predominant subclass and the antibody avidity index evaluated by ELISA ranged between 4-6 mol/L. Ex vivo P. vivax mosquito direct membrane feeding assays (DMFA) performed in presence of study plasmas, displayed significant parasite transmission-blocking (TB), however, there was no direct correlation between antibody titers and oocysts transmission reduction activity (%TRA). Nevertheless, DMFA with CHO rPvs48/45 affinity purified IgG showed a dose response; 90.2% TRA at 100 μg/mL and 71.8% inhibition at 10 μg/mL. In conclusion, the CHO-rPvs48/45 protein was more immunoreactive in most of the malaria endemic places studied, and CHO-rPvs48/45 specific IgG showed functional activity, supporting further testing of the protein vaccine potential.
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Wang, Hanlu, Tiantian Yang, Wenhong Jiang, Meng Qin, Ziyong Sun, Wei Dai, and Yongping Jiang. "Identification and characterization of a novel cell binding and cross-reactive region on spike protein of SARS-CoV-2." Scientific Reports 12, no. 1 (September 19, 2022). http://dx.doi.org/10.1038/s41598-022-19886-y.
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AbstractGiven that COVID-19 continues to wreak havoc around the world, it is imperative to search for a conserved region involved in viral infection so that effective vaccines can be developed to prevent the virus from rapid mutations. We have established a twelve-fragment library of recombinant proteins covering the entire region of spike protein of both SARS-CoV-2 and SARS-CoV from Escherichia coli. IgGs from murine antisera specifically against 6 spike protein fragments of SARS-CoV-2 were produced, purified, and characterized. We found that one specific IgG against the fusion process region, named COVID19-SF5, serologically cross-reacted with all twelve S-protein fragments. COVID19-SF5, with amino acid sequences from 880 to 1084, specifically bound to VERO-E6 and BEAS-2B cells, with Kd values of 449.1 ± 21.41 and 381.9 ± 31.53 nM, and IC50 values of 761.2 ± 28.2 nM and 862.4 ± 32.1 nM, respectively. In addition, COVID19-SF5 greatly enhanced binding of the full-length CHO cell-derived spike protein to the host cells in a concentration-dependent manner. Furthermore, COVID19-SF5 and its IgGs inhibited the infection of the host cells by pseudovirus. The combined data from our studies reveal that COVID19-SF5, a novel cell-binding fragment, may contain a common region(s) for mediating viral binding during infection. Our studies also provide valuable insights into how virus variants may evade host immune recognition. Significantly, the observation that the IgGs against COVID19-SF5 possesses cross reactivity to all other fragments of S protein, suggesting that it is possible to develop universal neutralizing monoclonal antibodies to curb rapid mutations of COVID-19.