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Donald L Jarvis - One of the best experts on this subject based on the ideXlab platform.
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adventitious viruses in Insect Cell lines used for recombinant protein expression
Protein Expression and Purification, 2018Co-Authors: Christoph Geisler, Donald L JarvisAbstract:Abstract Insect Cells are widely used for recombinant protein expression, typically as hosts for recombinant baculovirus vectors, but also for plasmid-mediated transient transfection or stable genetic transformation. Insect Cells are used to express proteins for research, as well as to manufacture biologicals for human and veterinary medicine. Recently, several Insect Cell lines used for recombinant protein expression were found to be persistently infected with adventitious viruses. This has raised questions about how these infections might affect research performed using those Cell lines. Furthermore, these findings raised serious concerns about the safety of biologicals produced using those Cell lines. In response, new Insect Cell lines lacking adventitious viruses have been isolated for use as improved research tools and safer biological manufacturing platforms. Here, we review the scientific and patent literature on adventitious viruses found in Insect Cell lines, affected Cell lines, and new virus-free Cell lines.
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crispr cas9 vectors for genome editing and host engineering in the baculovirus Insect Cell system
Proceedings of the National Academy of Sciences of the United States of America, 2017Co-Authors: Hideaki Mabashiasazuma, Donald L JarvisAbstract:The baculovirus–Insect Cell system (BICS) has been widely used to produce many different recombinant proteins for basic research and is being used to produce several biologics approved for use in human or veterinary medicine. Early BICS were technically complex and constrained by the relatively primordial nature of Insect Cell protein glycosylation pathways. Since then, recombination has been used to modify baculovirus vectors—which has simplified the system—and transform Insect Cells, which has enhanced its protein glycosylation capabilities. Now, CRISPR-Cas9 tools for site-specific genome editing are needed to facilitate further improvements in the BICS. Thus, in this study, we used various Insect U6 promoters to construct CRISPR-Cas9 vectors and assessed their utility for site-specific genome editing in two Insect Cell lines commonly used as hosts in the BICS. We demonstrate the use of CRISPR-Cas9 to edit an endogenous Insect Cell gene and alter protein glycosylation in the BICS.
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modifying an Insect Cell n glycan processing pathway using crispr cas technology
ACS Chemical Biology, 2015Co-Authors: Hideaki Mabashiasazuma, Chuwei Kuo, Kayhooi Khoo, Donald L JarvisAbstract:Fused lobes (FDL) is an enzyme that simultaneously catalyzes a key trimming reaction and antagonizes elongation reactions in the Insect N-glycan processing pathway. Accordingly, FDL function accounts, at least in part, for major differences in the N-glycosylation patterns of glycoproteins produced by Insect and mammalian Cells. In this study, we used the CRISPR-Cas9 system to edit the fdl gene in Drosophila melanogaster S2 Cells. CRISPR-Cas9 editing produced a high frequency of site-specific nucleotide insertions and deletions, reduced the production of Insect-type, paucimannosidic products (Man3GlcNAc2), and led to the production of partially elongated, mammalian-type complex N-glycans (GlcNAc2Man3GlcNAc2) in S2 Cells. As CRISPR-Cas9 has not been widely used to analyze or modify protein glycosylation pathways or edit Insect Cell genes, these results underscore its broad utility as a tool for these purposes. Our results also confirm the key role of FDL at the major branch point distinguishing Insect and m...
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a new glycoengineered Insect Cell line with an inducibly mammalianized protein n glycosylation pathway
Glycobiology, 2012Co-Authors: Jared J Aumiller, Xianzong Shi, Hideaki Mabashiasazuma, Alexander Hillar, Donald L JarvisAbstract:The inability to produce recombinant glycoproteins with authentic N-glycans is a limitation of many heterologous protein expression systems. In the baculovirus-Insect Cell system, this limitation has been addressed by glycoengineering Insect Cell lines with mammalian genes encoding protein N-glycosylation functions ("glycogenes") under the transcriptional control of constitutive promoters. However, a potential problem with this approach is that the metabolic load imposed by the expression of multiple transgenes could adversely impact the growth and/or stability of glycoengineered Insect Cell lines. Thus, we created a new transgenic Insect Cell line (SfSWT-5) with an inducibly mammalianized protein N-glycosylation pathway. Expression of all six glycogenes was induced when uninfected SfSWT-5 Cells were cultured in growth medium containing doxycycline. Higher levels of expression and induction were observed when SfSWT-5 Cells were cultured with doxycycline and infected with a baculovirus. Interestingly, there were no major differences in the short-term growth properties of SfSWT-5 Cells cultured with or without doxycycline. Furthermore, there were no major differences in the phenotypic stability of these Cells after continuous culture for over 300 passages with or without doxycycline. Baculovirus-infected Sf9 and SfSWT-5 Cells produced about the same amounts of a model recombinant glycoprotein, but only the latter sialylated this product and sialylation was more pronounced when the Cells were treated with doxycycline. In summary, this is the first report of a lower eukaryotic system with an inducibly mammalianized protein N-glycosylation pathway and the first to examine how the presumed metabolic load imposed by multiple transgene expression impacts Insect Cell growth and stability.
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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.
Spiros N. Agathos - One of the best experts on this subject based on the ideXlab platform.
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Insect Cell culture for industrial production of recombinant proteins.
Applied Microbiology and Biotechnology, 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.
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Production scale Insect Cell culture.
Biotechnology Advances, 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.
David W. Murhammer - One of the best experts on this subject based on the ideXlab platform.
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evidence of oxidative stress following the viral infection of two lepidopteran Insect Cell lines
Free Radical Biology and Medicine, 2001Co-Authors: Ying Wang, Larry W Oberley, David W. MurhammerAbstract:The infection of Spodoptera frugiperda Sf-9 (Sf-9) and Trichoplusia ni BTI-Tn-5B1-4 (Tn-5B1-4) Insect Cell lines with Autographa californica multiple nucleopolyhedrovirus (AcMNPV) resulted in increased levels of lipid hydroperoxides and protein carbonyls. In addition, the viral infection resulted in a significant decrease in the reduced glutathione to oxidized glutathione (2GSH/GSSG) ratio. These results are all consistent with an increased level of oxidative stress as a result of the viral infection. It was also observed that the oxidative damage corresponded to reduced Cell viability, i.e., the results are consistent with the premise that oxidative damage contributes to Cell death. Finally, the measured intraCellular activities of most of the antioxidant enzymes, specifically manganese superoxide dismutase (MnSOD), ascorbate peroxidase (APOX), and catalase (CAT, not present in Sf-9 Cells), did not significantly decrease following viral infection. In contrast, the measured activity of copper-zinc superoxide dismutase (CuZnSOD) decreased in the Sf-9 and Tn-5B1-4 Cells following AcMNPV infection.
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Recombinant protein synthesis in Trichoplusia ni BTI-Tn-5B1-4 Insect Cell aggregates.
Biotechnology and bioengineering, 1999Co-Authors: Mark A. Saarinen, Kimberly A. Troutner, Steve G. Gladden, Christine M. Mitchell-logean, David W. MurhammerAbstract:The Trichoplusia ni BTI-Tn-5B1-4 (Tn-5B1-4) Insect Cell line has received considerable attention as a host for the baculovirus expression vector system. In the present study, suspension cultures were used to compare Tn-5B1-4 Cell aggregates and Cells selected to grow predominantly as individual Cells. No significant difference was found between Cell aggregates and Cells growing predominantly individually in regard to Cell growth rate, glucose consumption and lactate accumulation, and specific recombinant protein synthesis levels. In addition, the levels of recombinant protein synthesis were considerably higher than those produced by the commonly used Spodoptera frugiperda Sf-9 Insect Cell line.
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simultaneous measurement of glucose and glutamine in Insect Cell culture media by near infrared spectroscopy
Biotechnology and Bioengineering, 1997Co-Authors: Mark R Riley, Martin Rhiel, Xiangji Zhou, Mark A Arnold, David W. MurhammerAbstract:The purpose of this study was to develop non- invasive techniques to monitor the composition of Cell culture media in Insect Cell bioreactors. Such a monitor could be used in conjunction with a fed-batch feeding scheme to ensure that Cells are maintained in an optimal environment for growth and protein production. Glucose and glutamine concentrations in an Insect Cell culture bioreactor were determined off-line with near-infrared (NIR) absorption spectroscopy. Spectra were collected from 5000 to 4000 cm ˛1 with a 1.5-mm optical path length. Partial least squares (PLS) regression was applied to correlate the collected spectra with the concentration of the desired analytes. Under the culture conditions evaluated here, glucose and glutamine concentrations ranged from 38 to 55 mM and from 3t o 13 mM, respec- tively. Accurate measurements of glucose and glutamine in Insect Cell culture samples were possible over these entire ranges. The standard error of prediction (SEP) and mean percent error (MPE) for glutamine were 0.52 mM and 5.3%, respectively. Glucose could be measured with an SEP of 1.30 mM and an MPE of 2.3%. These levels of error are quite low considering the changing complexity of the growth media due to the shifting levels of amino acids, carbohydrates, yeastolate, proteins, and Cell de- bris. This study represents an important step in the de- velopment of noninvasive on-line monitoring devices for Cell culture bioreactors. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 11-15, 1997.
Osamu Nishimura - One of the best experts on this subject based on the ideXlab platform.
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Preparation of ubiquitin-conjugated proteins using an Insect Cell-free protein synthesis system.
Journal of Biotechnology, 2010Co-Authors: Takashi Suzuki, Toshihiko Utsumi, Eiji Ando, Toru Ezure, Osamu Nishimura, Susumu TsunasawaAbstract:Ubiquitination is one of the most significant posttranslational modifications (PTMs). To evaluate the ability of an Insect Cell-free protein synthesis system to carry out ubiquitin (Ub) conjugation to in vitro translated proteins, poly-Ub chain formation was studied in an Insect Cell-free protein synthesis system. Poly-Ub was generated in the presence of Ub aldehyde (UA), a de-ubiquitinating enzyme inhibitor. In vitro ubiquitination of the p53 tumor suppressor protein was also analyzed, and p53 was poly-ubiquitinated when Ub, UA, and Mdm2, an E3 Ub ligase (E3) for p53, were added to the in vitro reaction mixture. These results suggest that the Insect Cell-free protein synthesis system contains enzymatic activities capable of carrying out ubiquitination. CBB-detectable ubiquitinated p53 was easily purified from the Insect Cell-free protein synthesis system, allowing analysis of the Ub-conjugated proteins by mass spectrometry (MS). Lys 305 of p53 was identified as one of the Ub acceptor sites using this strategy. Thus, we conclude that the Insect Cell-free protein synthesis system is a powerful tool for studying various PTMs of eukaryotic proteins including ubiqutination presented here.
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protein prenylation in an Insect Cell free protein synthesis system and identification of products by mass spectrometry
Proteomics, 2007Co-Authors: Takashi Suzuki, Toshihiko Utsumi, Eiji Ando, Susumu Tsunasawa, Toru Ezure, Osamu Nishimura, Masaaki Ito, Masamitsu ShikataAbstract:To evaluate the ability of an Insect Cell-free protein synthesis system to carry out proper protein prenylation, several CAIX (X indicates any C-terminal amino acid) sequences were introduced into the C-terminus of truncated human gelsolin (tGelsolin). Tryptic digests of these mutant proteins were analyzed by MALDI-TOF MS and MALDI-quadrupole-IT-TOF MS. The results indicated that the Insect Cell-free protein synthesis system possesses both farnesyltransferase (FTase) and geranylgeranyltransferase (GGTase) I, as is the case of the rabbit reticulocyte lysate system. The C-terminal amino acid sequence requirements for protein prenylation in this system showed high similarity to those observed in rat prenyltransferases. In the case of rhoC, which is a natural geranylgeranylated protein, it was found that it could serve as a substrate for both prenyltransferases in the presence of either farnesyl or geranylgeranyl pyrophosphate, whereas geranylgeranylation was only observed when both prenyl pyrophosphates were added to the in vitro translation reaction mixture. Thus, a combination of the Cell-free protein synthesis system with MS is an effective strategy to analyze protein prenylation.
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performance of expression vector ptd1 in Insect Cell free translation system
Journal of Bioscience and Bioengineering, 2006Co-Authors: Takashi Suzuki, Toru Ezure, Osamu Nishimura, Masaaki Ito, Shinichiro Kobayashi, Masamitsu Shikata, Koji TanimizuAbstract:We constructed a pTD1 vector for an Insect Cell-free translation system containing a 5' untranslated region (UTR) of a polyhedrin gene as a translational enhancer sequence. Its translational efficiency was about 50-fold higher than those of mRNAs without an enhancer sequence. Moreover, the pTD1 vector functioned as an effective expression vector not only in the Insect Cell-free translation system but also in wheat germ extract and rabbit reticulocyte lysate systems.
Takashi Suzuki - One of the best experts on this subject based on the ideXlab platform.
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strategy for comprehensive identification of human n myristoylated proteins using an Insect Cell free protein synthesis system
Proteomics, 2010Co-Authors: Takashi Suzuki, Kei Nagatoshi, Yoshinobu Ota, Koko Moriya, Eiji Ando, Susumu Tsunasawa, Toru Ezure, Toshihiko UtsumiAbstract:To establish a strategy for the comprehensive identification of human N-myristoylated proteins, the susceptibility of human cDNA clones to protein N-myristoylation was evaluated by metabolic labeling and MS analyses of proteins expressed in an Insect Cell-free protein synthesis system. One-hundred-and-forty-one cDNA clones with N-terminal Met-Gly motifs were selected as potential candidates from approximately 2000 Kazusa ORFeome project human cDNA clones, and their susceptibility to protein N-myristoylation was evaluated using fusion proteins, in which the N-terminal ten amino acid residues were fused to an epitope-tagged model protein. As a result, the products of 29 out of 141 cDNA clones were found to be effectively N-myristoylated. The metabolic labeling experiments both in an Insect Cell-free protein synthesis system and in the transfected COS-1 Cells using full-length cDNA revealed that 27 out of 29 proteins were in fact N-myristoylated. Database searches with these 27 cDNA clones revealed that 18 out of 27 proteins are novel N-myristoylated proteins that have not been reported previously to be N-myristoylated, indicating that this strategy is useful for the comprehensive identification of human N-myristoylated proteins from human cDNA resources.
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strategy for comprehensive identification of human n myristoylated proteins using an Insect Cell free protein synthesis system
Proteomics, 2010Co-Authors: Takashi Suzuki, Kei Nagatoshi, Yoshinobu Ota, Koko Moriya, Eiji Ando, Susumu Tsunasawa, Toru Ezure, Toshihiko UtsumiAbstract:To establish a strategy for the comprehensive identification of human N-myristoylated proteins, the susceptibility of human cDNA clones to protein N-myristoylation was evaluated by metabolic labeling and MS analyses of proteins expressed in an Insect Cell-free protein synthesis system. One-hundred-and-forty-one cDNA clones with N-terminal Met-Gly motifs were selected as potential candidates from ∼2000 Kazusa ORFeome project human cDNA clones, and their susceptibility to protein N-myristoylation was evaluated using fusion proteins, in which the N-terminal ten amino acid residues were fused to an epitope-tagged model protein. As a result, the products of 29 out of 141 cDNA clones were found to be effectively N-myristoylated. The metabolic labeling experiments both in an Insect Cell-free protein synthesis system and in the transfected COS-1 Cells using full-length cDNA revealed that 27 out of 29 proteins were in fact N-myristoylated. Database searches with these 27 cDNA clones revealed that 18 out of 27 proteins are novel N-myristoylated proteins that have not been reported previously to be N-myristoylated, indicating that this strategy is useful for the comprehensive identification of human N-myristoylated proteins from human cDNA resources.
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Preparation of ubiquitin-conjugated proteins using an Insect Cell-free protein synthesis system.
Journal of Biotechnology, 2010Co-Authors: Takashi Suzuki, Toshihiko Utsumi, Eiji Ando, Toru Ezure, Osamu Nishimura, Susumu TsunasawaAbstract:Ubiquitination is one of the most significant posttranslational modifications (PTMs). To evaluate the ability of an Insect Cell-free protein synthesis system to carry out ubiquitin (Ub) conjugation to in vitro translated proteins, poly-Ub chain formation was studied in an Insect Cell-free protein synthesis system. Poly-Ub was generated in the presence of Ub aldehyde (UA), a de-ubiquitinating enzyme inhibitor. In vitro ubiquitination of the p53 tumor suppressor protein was also analyzed, and p53 was poly-ubiquitinated when Ub, UA, and Mdm2, an E3 Ub ligase (E3) for p53, were added to the in vitro reaction mixture. These results suggest that the Insect Cell-free protein synthesis system contains enzymatic activities capable of carrying out ubiquitination. CBB-detectable ubiquitinated p53 was easily purified from the Insect Cell-free protein synthesis system, allowing analysis of the Ub-conjugated proteins by mass spectrometry (MS). Lys 305 of p53 was identified as one of the Ub acceptor sites using this strategy. Thus, we conclude that the Insect Cell-free protein synthesis system is a powerful tool for studying various PTMs of eukaryotic proteins including ubiqutination presented here.
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protein prenylation in an Insect Cell free protein synthesis system and identification of products by mass spectrometry
Proteomics, 2007Co-Authors: Takashi Suzuki, Toshihiko Utsumi, Eiji Ando, Susumu Tsunasawa, Toru Ezure, Osamu Nishimura, Masaaki Ito, Masamitsu ShikataAbstract:To evaluate the ability of an Insect Cell-free protein synthesis system to carry out proper protein prenylation, several CAIX (X indicates any C-terminal amino acid) sequences were introduced into the C-terminus of truncated human gelsolin (tGelsolin). Tryptic digests of these mutant proteins were analyzed by MALDI-TOF MS and MALDI-quadrupole-IT-TOF MS. The results indicated that the Insect Cell-free protein synthesis system possesses both farnesyltransferase (FTase) and geranylgeranyltransferase (GGTase) I, as is the case of the rabbit reticulocyte lysate system. The C-terminal amino acid sequence requirements for protein prenylation in this system showed high similarity to those observed in rat prenyltransferases. In the case of rhoC, which is a natural geranylgeranylated protein, it was found that it could serve as a substrate for both prenyltransferases in the presence of either farnesyl or geranylgeranyl pyrophosphate, whereas geranylgeranylation was only observed when both prenyl pyrophosphates were added to the in vitro translation reaction mixture. Thus, a combination of the Cell-free protein synthesis system with MS is an effective strategy to analyze protein prenylation.
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performance of expression vector ptd1 in Insect Cell free translation system
Journal of Bioscience and Bioengineering, 2006Co-Authors: Takashi Suzuki, Toru Ezure, Osamu Nishimura, Masaaki Ito, Shinichiro Kobayashi, Masamitsu Shikata, Koji TanimizuAbstract:We constructed a pTD1 vector for an Insect Cell-free translation system containing a 5' untranslated region (UTR) of a polyhedrin gene as a translational enhancer sequence. Its translational efficiency was about 50-fold higher than those of mRNAs without an enhancer sequence. Moreover, the pTD1 vector functioned as an effective expression vector not only in the Insect Cell-free translation system but also in wheat germ extract and rabbit reticulocyte lysate systems.