The Experts below are selected from a list of 4494 Experts worldwide ranked by ideXlab platform
Spiros N. Agathos - One of the best experts on this subject based on the ideXlab platform.
-
Insect Cell Culture
2010Co-Authors: Spiros N. AgathosAbstract:The Insect Cell-baculovirus expression vector system (IC-BEVS) is a highly versatile system because it can express gene products of practically any origin (from bacteria to human tissue), and in contrast to most industrial mammalian Cell Culture systems, it is based on engineering only the vector and not the host Cell line. As a result, the development time needed to progress from gene cloning to protein overproduction is much shorter. Compared to other biomanufacturing platforms, the IC-BEVS offers other significant advantages, including typically high product titers, a range of posttranslational modifications, and the possibility to express multimeric proteins or even several distinct proteins using the same vector. The Sf lines are adapted to suspension cultivation and are easily detached from T-flask (or other recipient) surfaces by gentle agitation without trypsinization. Small bench-scale Culture involves volumes ranging from a total of a few milliliters in wells of multiple-well plates or T-flasks for adherent Culture to shake flasks or spinners up to 250 ml for suspension Culture. A number of bioreactor systems have been investigated and proven merits of bioreactors in terms of robustness and scalability. Batch Culture remains the most common method for large scale IC-BEVS processing because of its inherent simplicity and flexibility in bioreactor equipment. The practice of Insect Cell Culture is becoming more and more widespread as an essential component of the IC-BEVS, perhaps one of the most successful and far-reaching expression systems available today for the production of recombinant proteins and viral particles.
-
Insect Cell Culture for industrial production of recombinant proteins.
2003Co-Authors: Laertis Ikonomou, Yves-jacques Schneider, Spiros N. AgathosAbstract:Insect Cells used in conjunction with the baculovirus expression vector system (BEVS) are gaining ground rapidly as a platform for recombinant protein production. Insect Cells present several comparative advantages to mammalian Cells, such as ease of Culture, higher tolerance to osmolality and by-product concentration and higher expression levels when infected with a recombinant baculovirus. Here we review some of the recent developments in protein expression by Insect Cells and their potential application in large-scale Culture. Our current knowledge of Insect Cell metabolism is summarised and emphasis is placed on elements useful in the rational design of serum-free media. The Culture of Insect Cells in the absence of serum is reaching maturity, and promising serum substitutes (hydrolysates, new growth and production-enhancing factors) are being evaluated. Proteolysis is a problem of the BEVS system due to its lytic nature, and can, therefore, be a critical issue in Insect Cell bioprocessing. Several Cell- or baculovirus proteases are involved in degradation events during protein production by Insect Cells. Methods for proteolysis control, the optimal inhibitors and Culture and storage conditions which affect proteolysis are discussed. Finally, engineering issues related to high-density Culture (new bioreactor types, gas exchange, feeding strategies) are addressed in view of their relevance to large-scale Culture.
-
Supernatant proteolytic activities of High-Five Insect Cells grown in serum-free Culture
2002Co-Authors: Laertis Ikonomou, Y. -j. Schneider, C. Peeters-joris, Spiros N. AgathosAbstract:The proteolytic activity of High-Five Insect Cell Culture supernatants was analysed using substrate gel electrophoresis (zymography). During growth in serum-free media, High-Five Cells constitutively expressed and secreted proteases that were active on casein gel but not on gelatin or bovine serum albumin gels. Two main protease bands were visible at about 41–42 kDa and 32–33 kDa. By addition of various protease inhibitors in the incubation buffer, the proteases were identified as metalloproteases as complete and specific inhibition of the proteolytic activities was only obtained by 1,10-phenanthroline.
-
Production scale Insect Cell Culture.
1991Co-Authors: Spiros N. AgathosAbstract:Abstract Insect Cells in Culture are currently commanding great interest as superior hosts for the efficient production of biologicals with applications in health care and in agriCulture. Insect Cell Culture is ripe for scale-up technologies, in order to meet future projected production requirements of (a) Insect viruses used as bioInsecticides and (b) recombinant proteins of therapeutic potential for humans and animals. The single most prominent system used in research-based and in commercial Insect Cell Culture today involves lepidopteran Cells transfected with baculovirus expression vectors for abundant formation of recombinant biologicals. However, dipteran Insect Cell lines also are beginning to emerge as useful tools in biotechnology. Current practices in bioprocess development using Insect Cell Culture, advances in media formulation and in Insect Cell bioreactor design, and emerging trends are presented and critically evaluated.
Mattheus F A Goosen - One of the best experts on this subject based on the ideXlab platform.
-
Insect Cell Culture engineering
2020Co-Authors: Mattheus F A Goosen, Andrew J Daugulis, Peter FaulknerAbstract:Insect Cell Culture engineering - an overview, Mattheus F.A. Goosen an overview of the molecular biology and applications of baculoviruses, John Kuzio and Peter Faulkner bioreactor design for Insect Cell cultivation - a review, Janus J. Malinowski and Andrew J. Daugulis Insect Cell immobilization, Mattheus F.A. Goosen applications of Insect Cell gene expression in pharmaceutical research, Darrell R. Thomsen et al scale-up considerations and bioreactor development for animal Cell cultivation, Johannes Tramper et al serum-free media, Stefan A. Weiss et al foreign gene expression in Insect Cells, Donald L. Jarvis the future of Insect Cell Culture engineering, Spiros N. Agathos.
-
large scale Insect Cell Culture
1992Co-Authors: Mattheus F A GoosenAbstract:Significant advances have been made over the past year in our understanding of the protective mechanisms, both fluid-mechanical and biological, of media additives on suspended animal Cells. The degree of protection offered by different additives, such as pluronic polyol, appears to be Cell-type dependent, varying quite dramatically not only between Insect and mammalian Cells, but also between different Insect Cell lines themselves.
-
large scale Insect Cell Culture methods applications and products
1991Co-Authors: Mattheus F A GoosenAbstract:The primary development in large-scale Insect Cell Culture over the past year has been the continuing accumulation of documented evidence (fundamental and applied) that conventional aerated stirred-tank and air-lift bioreactors may be employed for Insect Cell cultivation and recombinant protein production, provided that air sparging, agitation, and the addition to the medium of Pluronic F-68 and methyl Cellulose polymers are carefully controlled.
William E. Bentley - One of the best experts on this subject based on the ideXlab platform.
-
investigation of sequential behavior of carboxyl protease and cysteine protease activities in virus infected sf 9 Insect Cell Culture by inhibition assay
2001Co-Authors: T Gotoh, Kenichi Kikuchi, Yoshinori Miyazaki, William E. BentleyAbstract:Proteases produced during the Culture of Spodoptera frugiperda Sf-9 Cells infected with Autographa californica nuclear polyhedrosis virus (AcNPV) were assayed with various protease inhibitors. This inhibitory analysis revealed that: (1) carboxyl and cysteine proteases were predominantly produced by the Insect Cells infected with recombinant AcNPV, the gene of which encoded a variant of green fluorescent protein in a portion of the polyhedrin gene of the baculovirus, and (2) the protease activity was almost completely blocked by pepstatin A (carboxyl protease inhibitor) and E64 (cysteine protease inhibitor) in an additive manner in the presence of EDTA. Utilizing the additive property of the inhibitors, the inhibition-based protease assay discriminated between the two protease activities and elucidated the sequential behavior of the carboxyl and cysteine proteases produced in the virus-infected Sf-9 Cell Culture. The carboxyl protease(s) existed in the virus-infected Cells all the time and their level in the medium continuously increased. Uninfected Cells also contained a carboxyl protease activity, the level of which was similar to that of the virus-infected Cells. At a certain time after virus infection, the cysteine protease activity was largely increased in the virus-infected Cells and a significant amount of the protease(s) was released into the medium, due to the Cell membranes losing their integrity. The behavior of intraCellular and extraCellular cysteine protease activities coincided with that of a recombinant protein whose expression was under the control of the viral polyhedrin promoter. Similar examinations with wt-AcNPV-infected and uninfected Insect Cells showed that the inhibition-based protease assay was useful for analyzing the carboxyl protease and cysteine protease activities emerging in the Insect Cell (Sf-9)/baculovirus expression system.
-
continuous Insect Cell sf 9 Culture with aeration through sparging
1994Co-Authors: M Y Wang, William E. BentleyAbstract:The continuous growth of Spodoptera frugiperda Sf-9 Cells in a 250-ml blown-glass jacketed spinner flask under a direct air sparging environment was investigated. Even at 220 ml working volume (about 90% of total volume), this spinner flask provided good mixing and oxygenation as demonstrated by a higher Cell density compared with fermentor Cultures. This eliminates a common limitation of the traditional spinner flask, namely much lower Cell density at high working volume. Furthermore, this spinner flask has been run with Sf-9 Cell Culture at five different dilution rates and two different air sparging rates at steady state, demonstrating its utility in research applications where Cell size, metabolic activity and environmental conditions can be constantly maintained. In addition to demonstrating the utility of the reactor, three novel points are made in this report. First, Cell density in continuous Cultures is increased significantly due to a high agitation rate and, especially, air sparging rate, which is seldom used in animal Cell or Insect Cell Culture. Second, there is no apparent difference in the specific death rate at two different sparging rates (0.0093 vvm and 0.0125 vvm). Finally, we have maintained Sf-9 Cells for more than 4 months in a continuous Culture using a serum-free medium without loss of recombinant protein expression in infected Cells.
-
Glutamine determination in Insect Cell Culture media
1993Co-Authors: Min Ying Wang, Kelly Wester, William E. BentleyAbstract:Glutamine is an essential nutrient in Insect Cell Culture and plays a role in maximizing protein expression in baculovirus-infected Cells. This paper presents a technique which combines two enzymatic reactions, specifically the deamination of glutamine via glutaminase and the subsequent amimation of 2-oxoglutarate to glutamate, to measure the concentration of glutamine in Insect Cell media. A specific glutamine uptake rate of 4.2×10^−2 μmol/10^6Cells/hr was determined for infected Cells by this method.
Kenichi Kikuchi - One of the best experts on this subject based on the ideXlab platform.
-
significant increase in recombinant protein production of a virus infected sf 9 Insect Cell Culture of low moi under low dissolved oxygen conditions
2002Co-Authors: Takeshi Gotoh, Yoshinori Miyazaki, Kunihiro Chiba, Kenichi KikuchiAbstract:Spodoptera frugiperda Sf-9 Insect Cells were infected with recombinant Autographa californica nuclear polyhedrosis virus at a low multiplicity of infection (MOI) (0.1), and the effect of dissolved oxygen (DO) on the production of a polyhedrin promoter-driven recombinant protein (beta-galactosidase), intrinsic proteases (carboxyl and cysteine proteases), and the virus was determined. The DO concentrations used in the present study were 45%, 25%, 5%, and 1.3% of air saturation. At 5% DO the Cell growth following viral infection was greatest and beta-galactosidase was about 5-fold increased in volumetric yield compared to that at 45% and 25% DO, whereas the growth at 1.3% DO was extremely poor. The virus titer in the medium at 4-8 d post-infection (dpi) was also highest at 5% DO, but the titer was significantly decreased by further increasing the Culture time. This was in part attributed to the fact that baculovirus is susceptible to oxidative inactivation under aerobic conditions. The DO dependency of the specific oxygen consumption rate of virus-infected and uninfected Sf-9 Cells was expressed by a Monod-type equation. A critical DO, above which the rate of oxygen utilization is not limited by DO, was estimated to be 3.5% of air saturation for virus-infected Sf-9 Cells. These results indicated that for a baculovirus-infected Sf-9 Insect Cell Culture of low MOI, the optimal DO was likely to be approximately 5% of air saturation, which is above the critical DO for the infected Sf-9 Cells but sufficiently low to reduce the possibility of the oxidative inactivation of virus. For the production of carboxyl and cysteine proteases, the accumulation behavior and concentrations did not significantly vary with DO, except that a peak of cysteine protease activity was observed intraCellularly only at 5% DO, coinciding with beta-galactosidase production.
-
investigation of sequential behavior of carboxyl protease and cysteine protease activities in virus infected sf 9 Insect Cell Culture by inhibition assay
2001Co-Authors: T Gotoh, Kenichi Kikuchi, Yoshinori Miyazaki, William E. BentleyAbstract:Proteases produced during the Culture of Spodoptera frugiperda Sf-9 Cells infected with Autographa californica nuclear polyhedrosis virus (AcNPV) were assayed with various protease inhibitors. This inhibitory analysis revealed that: (1) carboxyl and cysteine proteases were predominantly produced by the Insect Cells infected with recombinant AcNPV, the gene of which encoded a variant of green fluorescent protein in a portion of the polyhedrin gene of the baculovirus, and (2) the protease activity was almost completely blocked by pepstatin A (carboxyl protease inhibitor) and E64 (cysteine protease inhibitor) in an additive manner in the presence of EDTA. Utilizing the additive property of the inhibitors, the inhibition-based protease assay discriminated between the two protease activities and elucidated the sequential behavior of the carboxyl and cysteine proteases produced in the virus-infected Sf-9 Cell Culture. The carboxyl protease(s) existed in the virus-infected Cells all the time and their level in the medium continuously increased. Uninfected Cells also contained a carboxyl protease activity, the level of which was similar to that of the virus-infected Cells. At a certain time after virus infection, the cysteine protease activity was largely increased in the virus-infected Cells and a significant amount of the protease(s) was released into the medium, due to the Cell membranes losing their integrity. The behavior of intraCellular and extraCellular cysteine protease activities coincided with that of a recombinant protein whose expression was under the control of the viral polyhedrin promoter. Similar examinations with wt-AcNPV-infected and uninfected Insect Cells showed that the inhibition-based protease assay was useful for analyzing the carboxyl protease and cysteine protease activities emerging in the Insect Cell (Sf-9)/baculovirus expression system.
Toni M. Kutchan - One of the best experts on this subject based on the ideXlab platform.
-
characterization and mechanism of the berberine bridge enzyme a covalently flavinylated oxidase of benzophenanthridine alkaloid biosynthesis in plants
1995Co-Authors: Toni M. Kutchan, Heinz DittrichAbstract:Abstract The berberine bridge enzyme ((S)-reticuline:oxygen oxidoreductase (methylene-bridge-forming), EC 1.5.3.9) catalyzes the oxidative cyclization of the N-methyl moiety of (S)-reticuline into the berberine bridge carbon, C-8, of (S)-scoulerine. This is a reaction that has neither an equivalent in organic chemistry nor a parallel in nature. The uniqueness of this catalytic reaction prompted an in depth study that began with the isolation of the cDNA encoding the berberine bridge enzyme followed by the overexpression of this cDNA in Insect Cell Culture. The heterologously expressed enzyme has herein been shown to contain covalently attached FAD in a molar ratio of cofactor to protein of 1:1.03. Site-directed mutagenesis and laser desorption time-of-flight mass spectrometry suggest that the site of covalent attachment is at His-104. The holoenzyme exhibited absorbance maxima at 380 and 442 nm and a fluorescence emission maximum at 628 nm (310 nm excitation). Enzymic transformation of a series of (S)-reticuline derivatives modified with respect to the stereochemistry at C-1 or in the aromatic ring substitution suggests that ring closure proceeds in two steps: formation of the methylene iminium ion and subsequent ring closure via an ionic mechanism.
-
molecular cloning and heterologous expression of a cdna encoding berbamunine synthase a c o phenol coupling cytochrome p450 from the higher plant berberis stolonifera
1995Co-Authors: Peter Kraus, Toni M. KutchanAbstract:A cDNA encoding a cytochrome P450-dependent oxidase, berbamunine synthase (EC 1.1.3.34; CYP80), from Cell suspension Cultures of the higher plant Berberis stolonifera Koehne and Wolf (barberry) has been isolated and heterologously expressed in functional form in Insect Cell Culture using a baculovirus-based expression system. This cytochrome P450-dependent enzyme is unusual in that it catalyzes the regio- and stereoselective formation of a C--O phenol couple in bisbenzylisoquinoline alkaloid biosynthesis without concomitant incorporation of activated oxygen into the product. Consistent with the function of an oxidase rather than a monooxygenase, an essential glycine residue in the distal helix, which forms the oxygen-binding pocket in the well-studied bacterial enzyme P-450cam, is replaced by proline at the equivalent position in berbamunine synthase. This oxidase was accumulated in an active form in Insect Cell microsomes and accepted electrons from the endogenous NADPH-cytochrome P450 reductase. The heterologously expressed enzyme oxidatively couples either two molecules of (R)-N-methylcoclaurine to form the (R,R) dimer guattegaumerine or one molecule each of (R)- and (S)-N-methylcoclaurine to form the (R,S) dimer berbamunine. The ratio of the two bisbenzylisoquinolines formed could be altered by reductase source or by varying the enantiomer composition of the substrates.
-
Heterologous expression of the plant proteins strictosidine synthase and berberine bridge enzyme in Insect Cell Culture
1994Co-Authors: Toni M. Kutchan, A Bock, H DittrichAbstract:The heterologous expression of cDNAs encoding the alkaloid biosynthetic enzymes, strictosidine synthase [EC 4.3.3.2] from Rauvolfia serpentina and the berberine bridge enzyme [(S)-reticuline: oxygen oxidoreductase (methylene-bridge-forming), EC 1.5.3.9] from Eschscholtzia californica, has been achieved in a Cell Culture (Sf9) of the fall army worm, Spodoptera frugiperda, using a baculovirus-based expression system. The expression resulted in the overproduction of each plant enzyme in a catalytically active form. The maximal production attained was 4 mg purified, active enzyme per litre Cell Culture for both the strictosidine synthase and berberine bridge enzymes.