The Experts below are selected from a list of 4095 Experts worldwide ranked by ideXlab platform
Marcus Thelen - One of the best experts on this subject based on the ideXlab platform.
-
monocyte chemotactic protein 4 mcp 4 a novel structural and functional analogue of mcp 3 and eotaxin
Journal of Experimental Medicine, 1996Co-Authors: Mariagrazia Uguccioni, S H Lima, Gianni Garotta, Beatrice Dewald, Ulf Forssmann, Pius Loetscher, H Li, Y Li, Barbara Kreider, Marcus ThelenAbstract:A novel human CC chemokine complementary DNA was identified in a library constructed from human fetal RNA, cloned into a Baculovirus Vector, and expressed in Sf9 insect cells. The mature recombinant protein that was released had the NH2-terminal sequence pyro-QPDALNVPSTC...and consisted of 75 amino acids. Minor amounts of two variants of 77 and 82 residues (NH2 termini: LAQPDA...and FNPQGLAQPDA...) were released as well. The novel chemokine was designated monocyte chemotactic protein 4 (MCP-4) and the variants were designated (LA)MCP-4 and (FNPQGLA)MCP-4. MCP-4 shares the pyroglutamic acidproline NH2-terminal motif and 56-61% sequence identity with the three known monocyte chemotactic proteins and is 60% identical to eotaxin. It has marked functional similarities to MCP-3 and eotaxin. Like MCP-3, MCP-4 is a chemoattractant of high efficacy for monocytes and T lymphocytes. On these cells, it binds to receptors that recognize MCP-1, MCP-3, and RANTES. On eosinophils, MCP-4 has similar efficacy and potency as MCP-3, RANTES, and cotaxin. It shares receptors with eotaxin and shows full cross-desensitization with this cosinophil-selective chemokine. Of the two variants, only (LA)MCP-4 could be purified in sufficient quantities for testing and was found to be at least 30-fold less potent than MCP-4 itself. This suggests that the 75-residue form with the characteristic NH2 terminus of an MCP is the biologically relevant species.
-
monocyte chemotactic protein 4 mcp 4 a novel structural and functional analogue of mcp 3 and eotaxin
Journal of Experimental Medicine, 1996Co-Authors: Mariagrazia Uguccioni, S H Lima, Gianni Garotta, Beatrice Dewald, Ulf Forssmann, Pius Loetscher, H Li, Y Li, Barbara Kreider, Marcus ThelenAbstract:A novel human CC chemokine complementary DNA was identified in a library constructed from human fetal RNA, cloned into a Baculovirus Vector, and expressed in Sf9 insect cells. The mature recombinant protein that was released had the NH2-terminal sequence pyro-QPDALNVPSTC...and consisted of 75 amino acids. Minor amounts of two variants of 77 and 82 residues (NH2 termini: LAQPDA...and FNPQGLAQPDA...) were released as well. The novel chemokine was designated monocyte chemotactic protein 4 (MCP-4) and the variants were designated (LA)MCP-4 and (FNPQGLA)MCP-4. MCP-4 shares the pyroglutamic acidproline NH2-terminal motif and 56-61% sequence identity with the three known monocyte chemotactic proteins and is 60% identical to eotaxin. It has marked functional similarities to MCP-3 and eotaxin. Like MCP-3, MCP-4 is a chemoattractant of high efficacy for monocytes and T lymphocytes. On these cells, it binds to receptors that recognize MCP-1, MCP-3, and RANTES. On eosinophils, MCP-4 has similar efficacy and potency as MCP-3, RANTES, and cotaxin. It shares receptors with eotaxin and shows full cross-desensitization with this cosinophil-selective chemokine. Of the two variants, only (LA)MCP-4 could be purified in sufficient quantities for testing and was found to be at least 30-fold less potent than MCP-4 itself. This suggests that the 75-residue form with the characteristic NH2 terminus of an MCP is the biologically relevant species.
Mariagrazia Uguccioni - One of the best experts on this subject based on the ideXlab platform.
-
monocyte chemotactic protein 4 mcp 4 a novel structural and functional analogue of mcp 3 and eotaxin
Journal of Experimental Medicine, 1996Co-Authors: Mariagrazia Uguccioni, S H Lima, Gianni Garotta, Beatrice Dewald, Ulf Forssmann, Pius Loetscher, H Li, Y Li, Barbara Kreider, Marcus ThelenAbstract:A novel human CC chemokine complementary DNA was identified in a library constructed from human fetal RNA, cloned into a Baculovirus Vector, and expressed in Sf9 insect cells. The mature recombinant protein that was released had the NH2-terminal sequence pyro-QPDALNVPSTC...and consisted of 75 amino acids. Minor amounts of two variants of 77 and 82 residues (NH2 termini: LAQPDA...and FNPQGLAQPDA...) were released as well. The novel chemokine was designated monocyte chemotactic protein 4 (MCP-4) and the variants were designated (LA)MCP-4 and (FNPQGLA)MCP-4. MCP-4 shares the pyroglutamic acidproline NH2-terminal motif and 56-61% sequence identity with the three known monocyte chemotactic proteins and is 60% identical to eotaxin. It has marked functional similarities to MCP-3 and eotaxin. Like MCP-3, MCP-4 is a chemoattractant of high efficacy for monocytes and T lymphocytes. On these cells, it binds to receptors that recognize MCP-1, MCP-3, and RANTES. On eosinophils, MCP-4 has similar efficacy and potency as MCP-3, RANTES, and cotaxin. It shares receptors with eotaxin and shows full cross-desensitization with this cosinophil-selective chemokine. Of the two variants, only (LA)MCP-4 could be purified in sufficient quantities for testing and was found to be at least 30-fold less potent than MCP-4 itself. This suggests that the 75-residue form with the characteristic NH2 terminus of an MCP is the biologically relevant species.
-
monocyte chemotactic protein 4 mcp 4 a novel structural and functional analogue of mcp 3 and eotaxin
Journal of Experimental Medicine, 1996Co-Authors: Mariagrazia Uguccioni, S H Lima, Gianni Garotta, Beatrice Dewald, Ulf Forssmann, Pius Loetscher, H Li, Y Li, Barbara Kreider, Marcus ThelenAbstract:A novel human CC chemokine complementary DNA was identified in a library constructed from human fetal RNA, cloned into a Baculovirus Vector, and expressed in Sf9 insect cells. The mature recombinant protein that was released had the NH2-terminal sequence pyro-QPDALNVPSTC...and consisted of 75 amino acids. Minor amounts of two variants of 77 and 82 residues (NH2 termini: LAQPDA...and FNPQGLAQPDA...) were released as well. The novel chemokine was designated monocyte chemotactic protein 4 (MCP-4) and the variants were designated (LA)MCP-4 and (FNPQGLA)MCP-4. MCP-4 shares the pyroglutamic acidproline NH2-terminal motif and 56-61% sequence identity with the three known monocyte chemotactic proteins and is 60% identical to eotaxin. It has marked functional similarities to MCP-3 and eotaxin. Like MCP-3, MCP-4 is a chemoattractant of high efficacy for monocytes and T lymphocytes. On these cells, it binds to receptors that recognize MCP-1, MCP-3, and RANTES. On eosinophils, MCP-4 has similar efficacy and potency as MCP-3, RANTES, and cotaxin. It shares receptors with eotaxin and shows full cross-desensitization with this cosinophil-selective chemokine. Of the two variants, only (LA)MCP-4 could be purified in sufficient quantities for testing and was found to be at least 30-fold less potent than MCP-4 itself. This suggests that the 75-residue form with the characteristic NH2 terminus of an MCP is the biologically relevant species.
David N Drechsel - One of the best experts on this subject based on the ideXlab platform.
-
flexibac a versatile open source Baculovirus Vector system for protein expression secretion and proteolytic processing
BMC Biotechnology, 2019Co-Authors: Regis P Lemaitre, Aliona Bogdanova, Barbara Borgonovo, Jeffrey B Woodruff, David N DrechselAbstract:Baculovirus-mediated expression in insect cells is a powerful approach for protein production. However, many existing methods are time-consuming, offer limited options for protein tagging, and are unsuitable for secreted proteins requiring proteolytic maturation, such as TGF-β family growth factors. To overcome the limitations of traditional Baculovirus expression systems, we engineered “FlexiBAC”. This system allows recombinant Baculovirus formation inside insect cells and reduces the time between initial cloning and protein production to 13 days. FlexiBAC includes 143 shuttle Vectors that append combinations of purification tags, fluorescent markers, proteolytic cleavage sites, trafficking signals, and chemical conjugation tags to the termini of the target protein. This system also overexpresses recombinant furin convertase to allow efficient proteolytic processing of secreted proteins. We demonstrate that FlexiBAC can be used to produce high levels of mature, active forms of TGF-β family growth factors, such as Activin A, as well as other proteins that are typically difficult to reconstitute, such as proteins rich in coiled-coil, low complexity, and disordered domains. FlexiBAC is a protein expression system for production of both cytosolic proteins and secreted proteins that require proteolytic maturation. The design of FlexiBAC and its expansive complementary shuttle Vector system reduces cloning steps and simplifies Baculovirus production.
-
flexibac a versatile open source Baculovirus Vector system for protein expression secretion and proteolytic processing
BMC Biotechnology, 2019Co-Authors: Regis P Lemaitre, Aliona Bogdanova, Barbara Borgonovo, Jeffrey B Woodruff, David N DrechselAbstract:Background Baculovirus-mediated expression in insect cells is a powerful approach for protein production. However, many existing methods are time-consuming, offer limited options for protein tagging, and are unsuitable for secreted proteins requiring proteolytic maturation, such as TGF-β family growth factors.
-
FlexiBAC: a versatile, open-source Baculovirus Vector system for protein expression, secretion, and proteolytic processing
BMC Biotechnology, 2019Co-Authors: Regis P Lemaitre, Aliona Bogdanova, Barbara Borgonovo, Jeffrey B Woodruff, David N DrechselAbstract:Background Baculovirus-mediated expression in insect cells is a powerful approach for protein production. However, many existing methods are time-consuming, offer limited options for protein tagging, and are unsuitable for secreted proteins requiring proteolytic maturation, such as TGF-β family growth factors. Results To overcome the limitations of traditional Baculovirus expression systems, we engineered “FlexiBAC”. This system allows recombinant Baculovirus formation inside insect cells and reduces the time between initial cloning and protein production to 13 days. FlexiBAC includes 143 shuttle Vectors that append combinations of purification tags, fluorescent markers, proteolytic cleavage sites, trafficking signals, and chemical conjugation tags to the termini of the target protein. This system also overexpresses recombinant furin convertase to allow efficient proteolytic processing of secreted proteins. We demonstrate that FlexiBAC can be used to produce high levels of mature, active forms of TGF-β family growth factors, such as Activin A, as well as other proteins that are typically difficult to reconstitute, such as proteins rich in coiled-coil, low complexity, and disordered domains. Conclusions FlexiBAC is a protein expression system for production of both cytosolic proteins and secreted proteins that require proteolytic maturation. The design of FlexiBAC and its expansive complementary shuttle Vector system reduces cloning steps and simplifies Baculovirus production.
-
flexibac a versatile open source Baculovirus Vector system for protein expression secretion and proteolytic processing
bioRxiv, 2018Co-Authors: Regis P Lemaitre, Aliona Bogdanova, Barbara Borgonovo, Jeffrey B Woodruff, David N DrechselAbstract:Baculovirus-mediated expression in insect cells is a powerful approach for protein production. However, many existing methods are time consuming, offer limited options for protein tagging, and are unsuitable for secreted proteins requiring proteolytic maturation, such as TGF-β family growth factors. To overcome these limitations, we engineered ″FlexiBAC″, a system that simplifies Baculovirus production and permits furin-driven proteolytic maturation of targets. This system allows recombinant Baculovirus formation inside insect cells and reduces the time between initial cloning and protein production to 13 days. FlexiBAC includes 146 shuttle Vectors that append combinations of purification tags, fluorescent markers, proteolytic cleavage sites, trafficking signals, and chemical conjugation tags to the termini of the target protein. We demonstrate that this system can be used to produce high levels of mature, active forms of TGF-β family growth factors, such as Activin A, as well as other proteins that are typically difficult to reconstitute, such as proteins rich in coiled-coil, low complexity, and disordered domains.
Donald L Jarvis - One of the best experts on this subject based on the ideXlab platform.
-
a novel Baculovirus Vector for the production of nonfucosylated recombinant glycoproteins in insect cells
Glycobiology, 2014Co-Authors: Hideaki Mabashiasazuma, Chuwei Kuo, Kayhooi Khoo, Donald L JarvisAbstract:Glycosylation is an important attribute of Baculovirus-insect cell expression systems, but some insect cell lines produce core α1,3-fucosylated N-glycans, which are highly immunogenic and render recombinant glycoproteins unsuitable for human use. To address this problem, we exploited a bacterial enzyme, guanosine-5'-diphospho (GDP)-4-dehydro-6-deoxy-d-mannose reductase (Rmd), which consumes the GDP-l-fucose precursor. We expected this enzyme to block glycoprotein fucosylation by blocking the production of GDP-l-fucose, the donor substrate required for this process. Initially, we engineered two different insect cell lines to constitutively express Rmd and isolated subclones with fucosylation-negative phenotypes. However, we found the fucosylation-negative phenotypes induced by Rmd expression were unstable, indicating that this host cell engineering approach is ineffective in insect systems. Thus, we constructed a Baculovirus Vector designed to express Rmd immediately after infection and facilitate the insertion of genes encoding any glycoprotein of interest for expression later after infection. We used this Vector to produce a daughter encoding rituximab and found, in contrast to an Rmd-negative control, that insect cells infected with this virus produced a nonfucosylated form of this therapeutic antibody. These results indicate that our Rmd(+) baculoviral Vector can be used to solve the immunogenic core α1,3-fucosylation problem associated with the Baculovirus-insect cell system. In conjunction with existing glycoengineered insect cell lines, this Vector extends the utility of the Baculovirus-insect cell system to include therapeutic glycoprotein production. This new Vector also extends the utility of the Baculovirus-insect cell system to include the production of recombinant antibodies with enhanced effector functions, due to its ability to block core α1,6-fucosylation.
-
Baculovirus insect cell expression systems
Methods in Enzymology, 2009Co-Authors: Donald L JarvisAbstract:In the early 1980s, the first-published reports of Baculovirus-mediated foreign gene expression stimulated great interest in the use of Baculovirus-insect cell systems for recombinant protein production. Initially, this system appeared to be the first that would be able to provide the high production levels associated with bacterial systems and the eukaryotic protein processing capabilities associated with mammalian systems. Experience and an increased understanding of basic insect cell biology have shown that these early expectations were not completely realistic. Nevertheless, Baculovirus-insect cell expression systems have the capacity to produce many recombinant proteins at high levels and they also provide significant eukaryotic protein processing capabilities. Furthermore, important technological advances over the past 20 years have improved upon the original methods developed for the isolation of Baculovirus expression Vectors, which were inefficient, required at least some specialized expertise and, therefore, induced some frustration among those who used the original Baculovirus-insect cell expression system. Today, virtually any investigator with basic molecular biology training can relatively quickly and efficiently isolate a recombinant Baculovirus Vector and use it to produce their favorite protein in an insect cell culture. This chapter will begin with background information on the basic Baculovirus-insect cell expression system and will then focus on recent developments that have greatly facilitated the ability of an average investigator to take advantage of its attributes.
-
protein n glycosylation in the Baculovirus insect cell expression system and engineering of insect cells to produce mammalianized recombinant glycoproteins
Advances in Virus Research, 2006Co-Authors: Robert L Harrison, Donald L JarvisAbstract:Abstract Baculovirus expression Vectors are frequently used to express glycoproteins, a subclass of proteins that includes many products with therapeutic value. The insect cells that serve as hosts for Baculovirus Vector infection are capable of transferring oligosaccharide side chains (glycans) to the same sites in recombinant proteins as those that are used for native protein N ‐glycosylation in mammalian cells. However, while mammalian cells produce compositionally more complex N ‐glycans containing terminal sialic acids, insect cells mostly produce simpler N ‐glycans with terminal mannose residues. This structural difference between insect and mammalian N ‐glycans compromises the in vivo bioactivity of glycoproteins and can potentially induce allergenic reactions in humans. These features obviously compromise the biomedical value of recombinant glycoproteins produced in the Baculovirus expression Vector system. Thus, much effort has been expended to characterize the potential and limits of N ‐glycosylation in insect cell systems. Discoveries from this research have led to the engineering of insect N ‐glycosylation pathways for assembly of mammalian‐style glycans on Baculovirus‐expressed glycoproteins. This chapter summarizes our knowledge of insect N ‐glycosylation pathways and describes efforts to engineer Baculovirus Vectors and insect cell lines to overcome the limits of insect cell glycosylation. In addition, we consider other possible strategies for improving glycosylation in insect cells.
-
mammalian glycosyltransferase expression allows sialoglycoprotein production by Baculovirus infected insect cells
Protein Expression and Purification, 2001Co-Authors: Neungseon Seo, Jason R Hollister, Donald L JarvisAbstract:Abstract The Baculovirus-insect cell expression system is widely used to produce recombinant mammalian glycoproteins, but the glycosylated end products are rarely authentic. This is because insect cells are typically unable to produce glycoprotein glycans containing terminal sialic acid residues. In this study, we examined the influence of two mammalian glycosyltransferases on N -glycoprotein sialylation by the Baculovirus-insect cell system. This was accomplished by using a novel Baculovirus Vector designed to express a mammalian α2,6-sialyltransferase early in infection and a new insect cell line stably transformed to constitutively express a mammalian β1,4-galactosyltransferase. Various biochemical assays showed that a foreign glycoprotein was sialylated by this virus-host combination, but not by a control virus-host combination, which lacked the mammalian glycosyltransferase genes. Thus, this study demonstrates that the Baculovirus-insect cell expression system can be metabolically engineered for N -glycoprotein sialylation by the addition of two mammalian glycosyltransferase genes.
-
modifying the insect cell n glycosylation pathway with immediate early Baculovirus expression Vectors
Nature Biotechnology, 1996Co-Authors: Donald L Jarvis, Eric E FinnAbstract:The Baculovirus-insect cell expression system is well-suited for recombinant glycoprotein production because Baculovirus Vectors can provide high levels of expression and insect cells can modify newly synthesized proteins in eucaryotic fashion. However, the N-glycosylation pathway of Baculovirus-infected insect cells differs from the pathway found in higher eucaryotes, as indicated by the fact that glycoproteins produced in the Baculovirus system typically lack complex biantennary N-linked oligosaccharide side chains containing penultimate galactose and terminal sialic acid residues. We recently developed a new type of Baculovirus Vector that can express foreign genes immediately after infection under the control of the viral ie 1 promoter. These immediate early Baculovirus expression Vectors can be used to modify the insect cell N-glycosylation pathway and produce a foreign glycoprotein with more extensively processed N-linked oligosaccharides. These Vectors can also be used to study the influence of the late steps in N-linked oligosaccharide processing on glycoprotein function. Further development could lead to Baculovirus-insect cell expression systems that can produce recombinant glycoproteins with complex biantennary N-linked oligosaccharides structurally identical to those produced by higher eucaryotes.
H Li - One of the best experts on this subject based on the ideXlab platform.
-
monocyte chemotactic protein 4 mcp 4 a novel structural and functional analogue of mcp 3 and eotaxin
Journal of Experimental Medicine, 1996Co-Authors: Mariagrazia Uguccioni, S H Lima, Gianni Garotta, Beatrice Dewald, Ulf Forssmann, Pius Loetscher, H Li, Y Li, Barbara Kreider, Marcus ThelenAbstract:A novel human CC chemokine complementary DNA was identified in a library constructed from human fetal RNA, cloned into a Baculovirus Vector, and expressed in Sf9 insect cells. The mature recombinant protein that was released had the NH2-terminal sequence pyro-QPDALNVPSTC...and consisted of 75 amino acids. Minor amounts of two variants of 77 and 82 residues (NH2 termini: LAQPDA...and FNPQGLAQPDA...) were released as well. The novel chemokine was designated monocyte chemotactic protein 4 (MCP-4) and the variants were designated (LA)MCP-4 and (FNPQGLA)MCP-4. MCP-4 shares the pyroglutamic acidproline NH2-terminal motif and 56-61% sequence identity with the three known monocyte chemotactic proteins and is 60% identical to eotaxin. It has marked functional similarities to MCP-3 and eotaxin. Like MCP-3, MCP-4 is a chemoattractant of high efficacy for monocytes and T lymphocytes. On these cells, it binds to receptors that recognize MCP-1, MCP-3, and RANTES. On eosinophils, MCP-4 has similar efficacy and potency as MCP-3, RANTES, and cotaxin. It shares receptors with eotaxin and shows full cross-desensitization with this cosinophil-selective chemokine. Of the two variants, only (LA)MCP-4 could be purified in sufficient quantities for testing and was found to be at least 30-fold less potent than MCP-4 itself. This suggests that the 75-residue form with the characteristic NH2 terminus of an MCP is the biologically relevant species.
-
monocyte chemotactic protein 4 mcp 4 a novel structural and functional analogue of mcp 3 and eotaxin
Journal of Experimental Medicine, 1996Co-Authors: Mariagrazia Uguccioni, S H Lima, Gianni Garotta, Beatrice Dewald, Ulf Forssmann, Pius Loetscher, H Li, Y Li, Barbara Kreider, Marcus ThelenAbstract:A novel human CC chemokine complementary DNA was identified in a library constructed from human fetal RNA, cloned into a Baculovirus Vector, and expressed in Sf9 insect cells. The mature recombinant protein that was released had the NH2-terminal sequence pyro-QPDALNVPSTC...and consisted of 75 amino acids. Minor amounts of two variants of 77 and 82 residues (NH2 termini: LAQPDA...and FNPQGLAQPDA...) were released as well. The novel chemokine was designated monocyte chemotactic protein 4 (MCP-4) and the variants were designated (LA)MCP-4 and (FNPQGLA)MCP-4. MCP-4 shares the pyroglutamic acidproline NH2-terminal motif and 56-61% sequence identity with the three known monocyte chemotactic proteins and is 60% identical to eotaxin. It has marked functional similarities to MCP-3 and eotaxin. Like MCP-3, MCP-4 is a chemoattractant of high efficacy for monocytes and T lymphocytes. On these cells, it binds to receptors that recognize MCP-1, MCP-3, and RANTES. On eosinophils, MCP-4 has similar efficacy and potency as MCP-3, RANTES, and cotaxin. It shares receptors with eotaxin and shows full cross-desensitization with this cosinophil-selective chemokine. Of the two variants, only (LA)MCP-4 could be purified in sufficient quantities for testing and was found to be at least 30-fold less potent than MCP-4 itself. This suggests that the 75-residue form with the characteristic NH2 terminus of an MCP is the biologically relevant species.
