Production of human alkaline phosphatase, a secreted, glycosylated protein, from a baculovirus expression system and the attachment-dependent cell line Trichoplusia ni BTI-Tn 5B1-4 using a split-flow, air-lift bioreactor

1993 ◽  
Vol 9 (6) ◽  
pp. 675-678 ◽  
Author(s):  
I. S. Chung ◽  
R. A. Taticek ◽  
M. L. Shuler
Keyword(s):  
Alkaline Phosphatase ◽  
Cell Line ◽  
Trichoplusia Ni ◽  
Expression System ◽  
Baculovirus Expression System ◽  
Baculovirus Expression ◽  
Split Flow ◽  
Dependent Cell ◽  
Air Lift

Expression and characterization of recombinant human α-3/4-fucosyltransferase III from Spodoptera frugiperda (Sf9) and Trichoplusia ni (Tn) cells using the baculovirus expression system

2001 ◽  
Vol 353 (3) ◽  
pp. 719-725 ◽  
Author(s):  
Vanessa A. MORAIS ◽  
Jacinta SERPA ◽  
Angelina S. PALMA ◽  
Teresa COSTA ◽  
Luís MARANGA ◽  
...  
Keyword(s):  
Cell Lines ◽  
Trichoplusia Ni ◽  
Secretory Pathway ◽  
Expression System ◽  
Baculovirus Expression System ◽  
Exchange Chromatography ◽  
Sf9 Cells ◽  
Type I ◽  
Baculovirus Expression ◽  
Insect Cell Lines

The human α-3/4-fucosyltransferase III (Fuc-TIII) participates in the synthesis of Lewis determinants. The enzyme from human sources is scarce and heterogeneous. In this paper we describe the expression of a secreted form of Fuc-TIII (SFT3) in two insect cell lines, Spodopterafrugiperda (Sf9) and Trichoplusiani (Tn), using the baculovirus expression system. The Sf9 cells secreted approx. 0.4unit/l (1mg/l) of the enzyme. The Tn cells secreted approx. 3-fold this amount. A large proportion of active protein was accumulated in the two cell lines (50 and 75% respectively for Sf9 and Tn cells, on the fourth day after infection) indicating a possible limitation not only of the folding machinery, but also a saturation of the secretory pathway. SFT3 was purified by cation-exchange chromatography followed by affinity chromatography. The enzyme from the Tn cell line had a lower global charge, possibly due to post-translational modifications, such as phosphorylation or sulphation. The two glycosylation sites from SFT3 were occupied. SFT3 secreted by Sf9 cells was completely deglycosylated by peptide-N-glycanase F, whereas 50% of SFT3 secreted by Tn cells was resistant to deglycosylation by this enzyme. The apparent kinetic parameters determined with the type I acceptor were kcat = 0.4s-1 and Km = 0.87mM for the SFT3 secreted by Tn cells, and kcat = 0.09s-1 and Km = 0.76mM for the SFT3 secreted by Sf9 cells, indicating that the enzymes had substrate affinities within the same order of magnitude as their mammalian counterpart. Furthermore, SFT3 secreted by either cell type showed a clear preference for type 1 carbohydrate acceptors, similarly to human Fuc-TIII.

scholarly journals The potential application of Trichoplusia ni granulovirus En3 enhancin as a synergist in baculovirus-based insecticides

2021 ◽  
Author(s):  
◽  
Adriana Ricarte Bermejo
Keyword(s):  
Mass Production ◽  
Trichoplusia Ni ◽  
Expression System ◽  
Baculovirus Expression System ◽  
Active Matter ◽  
Expression Systems ◽  
Lepidopteran Pests ◽  
Baculovirus Expression ◽  
Present Thesis

The increased costs associated with baculovirus mass-production urge the search for synergistic products that reduce the amount of active matter. In the present thesis, a synergistic factor with great potential for baculovirus-based formulations was expressed and produced using a baculovirus expression system. The main achievement of the present thesis is that the in vivo production of solubilized enhancins using baculovirus-based expression systems can be used to improve the efficacy of biological insecticides against lepidopteran pests, reducing the active matter of bioinsecticides and making them commercially competitive with chemicals.

scholarly journals A novel baculovirus-derived promoter with high activity in the Baculovirus Expression System

2016 ◽  
Author(s):  
María Martínez-Solís ◽  
Silvia Gómez-Sebastián ◽  
José M Escribano ◽  
Agata K Jakubowska ◽  
Salvador Herrero
Keyword(s):  
Recombinant Proteins ◽  
Trichoplusia Ni ◽  
Promoter Activity ◽  
Expression System ◽  
Cost Effective ◽  
Baculovirus Expression System ◽  
Start Codon ◽  
Vector System ◽  
Viral Gene ◽  
Baculovirus Expression

The baculovirus expression vector system (BEVS) has been widely used to produce a large number of recombinant proteins, and is becoming one of the most powerful, robust, and cost-effective systems for the production of eukaryotic proteins. Nevertheless, as in any other protein expression system, it is important to improve the production capabilities of this vector. The orf46 viral gene was identified among the most highly abundant sequences in the transcriptome of Spodoptera exigua larvae infected with its native baculovirus, the S. exigua multiple nucleopolyhedrovirus (SeMNPV). Different sequences upstream of the orf46 gene were cloned, and their promoter activities were tested by the expression of the GFP reporter gene using the Autographa californica nucleopolyhedrovirus (AcMNPV) vector system in different insect cell lines (Sf21, Se301, and Hi5) and in larvae from S. exigua and Trichoplusia ni. The strongest promoter activity was defined by a 120 nt sequence upstream of the ATG start codon for the orf46 gene. On average, GFP expression under this new promoter was more than two fold higher than the expression obtained with the standard polyhedrin (pph) promoter. Additionally, the orf46 promoter was also tested in combination with the pph promoter, revealing an additive effect over the pph promoter activity. In conclusion, this new characterized promoter represents an excellent alternative to the most commonly used baculovirus promoters for the efficient expression of recombinant proteins using the BEVS.

Expression of spider flagelliform silk protein in Bombyx mori cell line by a novel Bac-to-Bac/BmNPV baculovirus expression system

2006 ◽  
Vol 71 (2) ◽  
pp. 192-199 ◽  
Author(s):  
Yungen Miao ◽  
Yuansong Zhang ◽  
Koichi Nakagaki ◽  
Tianfu Zhao ◽  
Aichun Zhao ◽  
...  
Keyword(s):  
Bombyx Mori ◽  
Cell Line ◽  
Expression System ◽  
Baculovirus Expression System ◽  
Silk Protein ◽  
Baculovirus Expression

scholarly journals Expression, Purification and Sublocalization of SARS-CoV Nucleocapsid Protein in Insect Cells

2004 ◽  
Vol 36 (11) ◽  
pp. 754-758 ◽  
Author(s):  
Ai-Xia Ren ◽  
You-Hua Xie ◽  
Yu-Ying Kong ◽  
Guan-Zhen Yang ◽  
Yao-Zhou Zhang ◽  
...  
Keyword(s):  
Nucleocapsid Protein ◽  
Trichoplusia Ni ◽  
Insect Cells ◽  
Expression System ◽  
Recombinant Baculovirus ◽  
Baculovirus Expression System ◽  
Functional Study ◽  
Expression And Purification ◽  
N Protein ◽  
Baculovirus Expression

Abstract The causative agent of severe acute respiratory syndrome (SARS) is a previously unidentified coronavirus, SARS-CoV. The nucleocapsid (N) protein of SARS-CoV is a major viral protein recognized by acute and early convalescent sera from SARS patients. To facilitate the studies on the function and structure of the N protein, this report describe the expression and purification of recombinant SARS-CoV N protein using the baculovirus expression system. Recombinant hexa-histidine-tagged N protein with a molecular mass of 47 kD was produced in insect cells. Recombinant N protein was purified to near homogeneity by Ni2+-NTA affinity chromatography. In addition, we examined the subcellular localization of the N protein by confocal microscopy in Trichoplusia ni BT1 Tn 5B1–4 cells infected with recombinant baculovirus. The N protein was found localized in the cytoplasm as well as in the nucleolus. The purified recombinant N protein can be used in further functional study of SARS-CoV.

scholarly journals A novel baculovirus-derived promoter with high activity in the Baculovirus Expression System

2016 ◽  
Author(s):  
María Martínez-Solís ◽  
Silvia Gómez-Sebastián ◽  
José M Escribano ◽  
Agata K Jakubowska ◽  
Salvador Herrero
Keyword(s):  
Recombinant Proteins ◽  
Trichoplusia Ni ◽  
Promoter Activity ◽  
Expression System ◽  
Cost Effective ◽  
Baculovirus Expression System ◽  
Start Codon ◽  
Vector System ◽  
Viral Gene ◽  
Baculovirus Expression

The baculovirus expression vector system (BEVS) has been widely used to produce a large number of recombinant proteins, and is becoming one of the most powerful, robust, and cost-effective systems for the production of eukaryotic proteins. Nevertheless, as in any other protein expression system, it is important to improve the production capabilities of this vector. The orf46 viral gene was identified among the most highly abundant sequences in the transcriptome of Spodoptera exigua larvae infected with its native baculovirus, the S. exigua multiple nucleopolyhedrovirus (SeMNPV). Different sequences upstream of the orf46 gene were cloned, and their promoter activities were tested by the expression of the GFP reporter gene using the Autographa californica nucleopolyhedrovirus (AcMNPV) vector system in different insect cell lines (Sf21, Se301, and Hi5) and in larvae from S. exigua and Trichoplusia ni. The strongest promoter activity was defined by a 120 nt sequence upstream of the ATG start codon for the orf46 gene. On average, GFP expression under this new promoter was more than two fold higher than the expression obtained with the standard polyhedrin (pph) promoter. Additionally, the orf46 promoter was also tested in combination with the pph promoter, revealing an additive effect over the pph promoter activity. In conclusion, this new characterized promoter represents an excellent alternative to the most commonly used baculovirus promoters for the efficient expression of recombinant proteins using the BEVS.

scholarly journals A novel baculovirus-derived promoter with high activity in the baculovirus expression system

PeerJ ◽  
2016 ◽  
Vol 4 ◽  
pp. e2183 ◽  
Author(s):  
María Martínez-Solís ◽  
Silvia Gómez-Sebastián ◽  
José M. Escribano ◽  
Agata K. Jakubowska ◽  
Salvador Herrero
Keyword(s):  
Recombinant Proteins ◽  
Trichoplusia Ni ◽  
Promoter Activity ◽  
Expression System ◽  
Cost Effective ◽  
Baculovirus Expression System ◽  
Start Codon ◽  
Vector System ◽  
Viral Gene ◽  
Baculovirus Expression

The baculovirus expression vector system (BEVS) has been widely used to produce a large number of recombinant proteins, and is becoming one of the most powerful, robust, and cost-effective systems for the production of eukaryotic proteins. Nevertheless, as in any other protein expression system, it is important to improve the production capabilities of this vector. Theorf46viral gene was identified among the most highly abundant sequences in the transcriptome ofSpodoptera exigualarvae infected with its native baculovirus, theS. exiguamultiple nucleopolyhedrovirus (SeMNPV). Different sequences upstream of theorf46gene were cloned, and their promoter activities were tested by the expression of the GFP reporter gene using theAutographa californicanucleopolyhedrovirus (AcMNPV) vector system in different insect cell lines (Sf21, Se301, and Hi5) and in larvae fromS. exiguaandTrichoplusia ni. The strongest promoter activity was defined by a 120 nt sequence upstream of the ATG start codon for theorf46gene. On average, GFP expression under this new promoter was more than two fold higher than the expression obtained with the standard polyhedrin (polh) promoter. Additionally, theorf46promoter was also tested in combination with the polh promoter, revealing an additive effect over the polh promoter activity. In conclusion, this new characterized promoter represents an excellent alternative to the most commonly used baculovirus promoters for the efficient expression of recombinant proteins using the BEVS.

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