scholarly journals A Newly Designed EGFP-2A Peptide Monocistronic Baculoviral Vector for Concatenating the Expression of Recombinant Proteins in Insect Cells

Processes ◽  
2019 ◽  
Vol 7 (5) ◽  
pp. 291 ◽  
Author(s):  
Chih-Yu Wu ◽  
Chao-Wei Huang ◽  
Yu-Shin Nai ◽  
Pei-Yu Chu ◽  
Chung-Hsiung Wang ◽  
...  
Keyword(s):  
Recombinant Proteins ◽  
Posttranslational Modifications ◽  
Mammalian Cells ◽  
Insect Cells ◽  
Fluorescent Protein ◽  
Expression System ◽  
Recombinant Baculovirus ◽  
New Combination ◽  
Vector System ◽  
Baculovirus Expression Vector

Recombinant proteins produced by the baculovirus expression vector system (BVES) have been widely applied in the agricultural and medical fields. However, the procedure for protein expression is inefficient and needs to be improved. Herein, we propose a simple construct that incorporates a selectable marker (enhanced green fluorescent protein, EGFP) and a picorna viral-derived “self-cleaving” 2A-like peptide to separate the EGFP and target proteins in a monocistronic baculovirus vector to facilitate isolation of the recombinant baculovirus in the BVES. In this study, porcine adiponectin (ADN), a secreted, multimeric protein with insulin-sensitizing properties, was used to demonstrate its utility in our EGFP-2A-based expression system. EGFP and ADN were simultaneously expressed by a recombinant alphabaculovirus. Co-expression of EGFP facilitates the manipulation of the following processes, such as determining expression kinetics and harvesting ADN. The results showed that the 2A “self-cleaving” process does not interfere with EGFP activity or with signal peptide removal and the secretion of recombinant ADN. Posttranslational modifications, including glycosylation, of the recombinant ADN occurred in insect cells, and the formation of various multimers was further verified. Most importantly, the insect-produced ADN showed a similar bioactivity to that of mammalian cells. This concept provides a practical and economic approach that utilizes a new combination of alphabaculovirus/insect cell expression systems for future applications.

The components of shear stress affecting insect cells used with the baculovirus expression vector system

2017 ◽  
Vol 72 (9-10) ◽  
pp. 429-439 ◽  
Author(s):  
Tobias Weidner ◽  
Damir Druzinec ◽  
Martina Mühlmann ◽  
Rainer Buchholz ◽  
Peter Czermak
Keyword(s):  
Surface Area ◽  
Recombinant Proteins ◽  
Expression Vector ◽  
Insect Cells ◽  
Vector System ◽  
Baculovirus Expression Vector System ◽  
Shear Forces ◽  
Baculovirus Expression ◽  
Baculovirus Expression Vector ◽  
Infected Insect

AbstractInsect-based expression platforms such as the baculovirus expression vector system (BEVS) are widely used for the laboratory- and industrial-scale production of recombinant proteins. Thereby, major drawbacks to gain high-quality proteins are the lytic infection cycle and the shear sensitivity of infected insect cells due to turbulence and aeration. Smaller bubbles were formerly assumed to be more harmful than larger ones, but we found that cell damage is also dependent on the concentration of protective agents such as Pluronic®. At the appropriate concentration, Pluronic forms a layer around air bubbles and hinders the attachment of cells, thus limiting the damage. In this context, we used microaeration to vary bubble sizes and confirmed that size is not the most important factor, but the total gas surface area in the reactor is. If the surface area exceeds a certain threshold, the concentration of Pluronic is no longer sufficient for cell protection. To investigate the significance of shear forces, a second study was carried out in which infected insect cells were cultivated in a hollow fiber module to protect them from shear forces. Both model studies revealed important aspects of the design and scale-up of BEVS processes for the production of recombinant proteins.

scholarly journals Recombinant Baculovirus: A Flexible Drug Screening Platform for Chikungunya Virus

2021 ◽  
Vol 22 (15) ◽  
pp. 7891
Author(s):  
Muhammed Muhsin Varikkodan ◽  
Chun-Chung Chen ◽  
Tzong-Yuan Wu
Keyword(s):  
Chikungunya Virus ◽  
Fluorescent Protein ◽  
Recombinant Baculovirus ◽  
Infectious Agent ◽  
High Rate ◽  
Vector System ◽  
Structural Protein ◽  
Entry Site ◽  
Baculovirus Expression Vector

Chikungunya virus (CHIKV) is a mosquito-transmitted infectious agent that causes an endemic or epidemic outbreak(s) of Chikungunya fever that is reported in almost all countries. This virus is an intense global threat, due to its high rate of contagion and the lack of effective remedies. In this study, we developed two baculovirus expression vector system (BEVS)-based approaches for the screening of anti-CHIKV drugs in Spodoptera frugiperda insect (Sf21) cells and U-2OS cells. First, structural protein of CHIKV was co-expressed through BEVS and thereby induced cell fusion in Sf21 cells. We used an internal ribosome entry site (IRES) to co-express the green fluorescent protein (EGFP) for identifying these fusion events. The EGFP-positive Sf21 cells fused with each other and with uninfected cells to form syncytia. We identified that ursolic acid has potential anti-CHIKV activity in vitro, by using this approach. Second, BacMam virus-based gene delivery has been successfully applied for the transient expression of non-structural proteins with a subgenomic promoter-EGFP (SP-EGFP) cassette in U-2OS cells to act as an in vitro CHIKV replicon system. Our BacMam-based screening system has identified that the potential effects of baicalin and baicalein phytocompounds can inhibit the replicon activity of CHIKV in U-2OS cells. In conclusion, our results suggested that BEVS can be a potential tool for screening drugs against CHIKV.

scholarly journals Characterization and purification of human retinoic acid receptor-γ 1 overexpressed in the baculovirus-insect cell system

1992 ◽  
Vol 287 (3) ◽  
pp. 833-840 ◽  
Author(s):  
A P Reddy ◽  
J Y Chen ◽  
T Zacharewski ◽  
H Gronemeyer ◽  
J J Voorhees ◽  
...  
Keyword(s):  
Retinoic Acid ◽  
Dna Binding ◽  
Mammalian Cells ◽  
Retinoic Acid Receptor ◽  
Expression System ◽  
Gel Filtration ◽  
Recombinant Baculovirus ◽  
Sf9 Cells ◽  
Acid Receptor ◽  
Gel Retardation

The full-length cDNA for the human retinoic acid receptor-gamma 1 (RAR-gamma 1) has been expressed to high levels in Spodoptera frugiferda (Sf9) cells using the baculovirus expression system. Western blot analysis revealed that RAR-gamma 1 expression increased between 32 and 60 h post-infection. The recombinant receptor was expressed primarily as a nuclear protein and displayed a molecular mass of 50 kDa as determined by SDS/PAGE and gel-filtration chromatography, consistent with its cDNA-deduced size. Based on ligand binding, 2 x 10(6) RAR-gamma 1 molecules were expressed per Sf9 cell, a level approx. 2000 times greater than in mammalian cells. The receptor was partially purified 300-fold by sequential anion-exchange, gel-filtration and DNA affinity chromatographies. The overexpressed receptor specifically bound all-trans-retinoic acid (RA) and the synthetic retinoid CD367 with high affinity (Kd 0.15 nM and 0.23 nM respectively). The RA metabolites 4-hydroxy-RA and 4-oxo-RA were poor competitors for [3H]CD367 binding to recombinant RAR-gamma 1 (K(i) > 1 microM), indicating that 4-oxidation of RA greatly reduces its affinity for RAR-gamma 1. Gel-retardation analysis demonstrated that RAR-gamma 1 specifically bound the RA response element of the mouse RAR-beta gene. RAR-gamma 1 species expressed from recombinant baculovirus (in Sf9 cells) and vaccinia virus (in HeLa cells) exhibited similar affinities for RA and CD367 and had comparable DNA-binding properties in gel-retardation experiments. Moreover, a similar requirement for additional DNA-binding stimulatory factor(s) was observed in both cases. These results provide a basis for the use of baculovirus-expressed RAR-gamma 1 in further functional and structural studies.

scholarly journals Construction of a Vitreoscilla Hemoglobin Promoter-Based Tunable Expression System for Corynebacterium glutamicum

Catalysts ◽  
2018 ◽  
Vol 8 (11) ◽  
pp. 561 ◽  
Author(s):  
Kei-Anne Baritugo ◽  
Hee Taek Kim ◽  
Mi Na Rhie ◽  
Seo Young Jo ◽  
Tae Uk Khang ◽  
...  
Keyword(s):  
Corynebacterium Glutamicum ◽  
Fluorescent Protein ◽  
Expression System ◽  
Vitreoscilla Hemoglobin ◽  
Aminobutyric Acid ◽  
Vector System ◽  
Efficient Production ◽  
Gamma Aminobutyric Acid ◽  
Recombinant Strains ◽  
Gaba Production

Corynebacterium glutamicum is an industrial strain used for the production of valuable chemicals such as L-lysine and L-glutamate. Although C. glutamicum has various industrial applications, a limited number of tunable systems are available to engineer it for efficient production of platform chemicals. Therefore, in this study, we developed a novel tunable promoter system based on repeats of the Vitreoscilla hemoglobin promoter (Pvgb). Tunable expression of green fluorescent protein (GFP) was investigated under one, four, and eight repeats of Pvgb (Pvgb, Pvgb4, and Pvgb8). The intensity of fluorescence in recombinant C. glutamicum strains increased as the number of Pvgb increased from single to eight (Pvgb8) repeats. Furthermore, we demonstrated the application of the new Pvgb promoter-based vector system as a platform for metabolic engineering of C. glutamicum by investigating 5-aminovaleric acid (5-AVA) and gamma-aminobutyric acid (GABA) production in several C. glutamicum strains. The profile of 5-AVA and GABA production by the recombinant strains were evaluated to investigate the tunable expression of key enzymes such as DavBA and GadBmut. We observed that 5-AVA and GABA production by the recombinant strains increased as the number of Pvgb used for the expression of key proteins increased. The recombinant C. glutamicum strain expressing DavBA could produce higher amounts of 5-AVA under the control of Pvgb8 (3.69 ± 0.07 g/L) than the one under the control of Pvgb (3.43 ± 0.10 g/L). The average gamma-aminobutyric acid production also increased in all the tested strains as the number of Pvgb used for GadBmut expression increased from single (4.81–5.31 g/L) to eight repeats (4.94–5.58 g/L).

scholarly journals Development of a Dual-Vector System Utilizing MicroRNA Mimics of the Autographa californica miR-1 for an Inducible Knockdown in Insect Cells

2019 ◽  
Vol 20 (3) ◽  
pp. 533
Author(s):  
Krisztina Koczka ◽  
Wolfgang Ernst ◽  
Dieter Palmberger ◽  
Miriam Klausberger ◽  
Lisa Nika ◽  
...  
Keyword(s):  
Fluorescence Intensity ◽  
Insect Cell ◽  
Fluorescent Protein ◽  
Expression System ◽  
Yellow Fluorescent Protein ◽  
Mrna Level ◽  
Vector System ◽  
Sf9 Cells ◽  
Artificial Microrna ◽  
T7 Promoter

The baculovirus-insect cell expression system is a popular tool for the manufacturing of various attractive recombinant products. Over the years, several attempts have been made to engineer and further improve this production platform by targeting host or baculoviral genes by RNA interference. In this study, an inducible knockdown system was established in insect (Sf9) cells by combining an artificial microRNA precursor mimic of baculoviral origin and the bacteriophage T7 transcription machinery. Four structurally different artificial precursor constructs were created and tested in a screening assay. The most efficient artificial microRNA construct resulted in a 69% reduction in the fluorescence intensity of the target enhanced yellow fluorescent protein (eYFP). Next, recombinant baculoviruses were created carrying either the selected artificial precursor mimic under the transcriptional control of the T7 promoter or solely the T7 RNA polymerase under a baculoviral promoter. Upon co-infecting Sf9 cells with these two viruses, the fluorescence intensity of eYFP was suppressed by ~30–40% on the protein level. The reduction in the target mRNA level was demonstrated with real-time quantitative PCR. The presented inducible knockdown system may serve as an important and valuable tool for basic baculovirus-insect cell research and for the improvement of production processes using this platform.

scholarly journals Efficient and Stable Delivery of Multiple Genes to Fish Cells by a Modified Recombinant Baculovirus System

2018 ◽  
Vol 19 (12) ◽  
pp. 3767 ◽  
Author(s):  
Qian Wang ◽  
Jian Fang ◽  
Qihua Pan ◽  
Yizhou Wang ◽  
Ting Xue ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Fluorescent Protein ◽  
Recombinant Baculovirus ◽  
Transgenic Fish ◽  
Early Gene ◽  
Drug Selection ◽  
Baculovirus System ◽  
Fish Cells

The recombinant baculovirus has been widely used as an efficient tool to mediate gene delivery into mammalian cells but has barely been used in fish cells. In the present study, we constructed a recombinant baculovirus containing the dual-promoter cytomegalovirus (CMV) and white spot syndrome virus (WSSV) immediate-early gene 1 (ie1) (WSSV ie1), followed by a puromycin–green fluorescent protein (Puro-GFP, pf) or puromycin–red fluorescent protein (Puro-RFP, pr) cassette, which simultaneously allowed for easy observation, rapid titer determination, drug selection, and exogenous gene expression. This recombinant baculovirus was successfully transduced into fish cells, including Mylopharyngodon piceus bladder (MPB), fin (MPF), and kidney (MPK); Oryzias latipes spermatogonia (SG3); and Danio rerio embryonic fibroblast (ZF4) cells. Stable transgenic cell lines were generated after drug selection, which was further verified by Western blot. A cell monoclonal formation assay proved the stable heredity of transgenic MPB cells. In addition, a recombinant baculovirus containing a pr cassette and four transcription factors for induced pluripotent stem cells (iPSC) was constructed and transduced into ZF4 cells, and these exogenous genes were simultaneously delivered and transcribed efficiently in drug-selected ZF4 cells, proving the practicability of this modified recombinant baculovirus system. We also proved that the WSSV ie1 promoter had robust activity in fish cells in vitro and in vivo. Taken together, this modified recombinant baculovirus can be a favorable transgenic tool to obtain transient or stable transgenic fish cells.

scholarly journals Biosynthesis of recombinant human pro-α1(III) chains in a baculovirus expression system: production of disulphide-bonded and non-disulphide-bonded species containing full-length triple helices

1995 ◽  
Vol 312 (3) ◽  
pp. 847-853 ◽  
Author(s):  
M Tomita ◽  
N Ohkura ◽  
M Ito ◽  
T Kato ◽  
P M Royce ◽  
...  
Keyword(s):  
Insect Cells ◽  
Incubation Temperature ◽  
Expression System ◽  
Significant Proportion ◽  
Recombinant Baculovirus ◽  
Baculovirus Expression System ◽  
Dermal Fibroblasts ◽  
Full Length ◽  
Type Iii Collagen ◽  
Procollagen Iii

We have investigated the expression of human procollagen III by insect cells infected with a recombinant baculovirus carrying cDNA for the pro-alpha1(III) chain of type-III collagen. A high level of expression was obtained, and a small proportion of the heterologously expressed pro-alpha1(III) chains formed normally disulphide-bonded procollagen III, which was secreted into the culture medium. This species displayed a melting temperature (Tm) of approx. 38 degrees C as assessed by its resistance to digestion by a mixture of trypsin and chymotrypsin, slightly lower than that of 39.5 degrees C for procollagen III synthesized by cultured human dermal fibroblasts, and reflected a slight degree of under-hydroxylation of prolyl residues. This is possibly a consequence of the lower incubation temperature of insect cells, or of an insufficiency of prolyl hydroxylase activity within them. A significant proportion of the expressed chains formed trimeric molecules of similar thermal stability containing an apparently full-length triple-helical region, but were not disulphide-bonded and not secreted. In addition to providing a source of recombinant human procollagen III, the system promises to be useful in the study of procollagen chain association and subsequent folding.

scholarly journals Unique features of a recombinant haemagglutinin influenza vaccine that influence vaccine performance

npj Vaccines ◽  
2021 ◽  
Vol 6 (1) ◽  
Author(s):  
Arun B. Arunachalam ◽  
Penny Post ◽  
Deborah Rudin
Keyword(s):  
Influenza Vaccine ◽  
Adverse Reactions ◽  
Insect Cells ◽  
Age Groups ◽  
Structural Features ◽  
Vector System ◽  
Baculovirus Expression Vector System ◽  
Baculovirus Expression ◽  
Baculovirus Expression Vector ◽  
Protective Antibodies

AbstractThe influenza vaccine field has been constantly evolving to improve the speed, scalability, and flexibility of manufacturing, and to improve the breadth and longevity of the protective immune response across age groups, giving rise to an array of next generation vaccines in development. Among these, the recombinant influenza vaccine tetravalent (RIV4), using a baculovirus expression vector system to express recombinant haemagglutinin (rHA) in insect cells, is the only one to have reached the market and has been studied extensively. We describe how the unique structural features of rHA in RIV4 improve protective immune responses compared to conventional influenza vaccines made from propagated influenza virus. In addition to the sequence integrity, characteristic of recombinant proteins, unique post-translational processing of the rHA in insect cells instills favourable tertiary and quaternary structural features. The absence of protease-driven cleavage and addition of simple N-linked glycans help to preserve and expose certain conserved epitopes on HA molecules, which are likely responsible for the high levels of broadly cross-reactive and protective antibodies with rare specificities observed with RIV4. Furthermore, the presence of uniform compact HA oligomers and absence of egg proteins, viral RNA or process impurities, typically found in conventional vaccines, are expected to eliminate potential adverse reactions to these components in susceptible individuals with the use of RIV4. These distinct structural features and purity of the recombinant HA vaccine thus provide a number of benefits in vaccine performance which can be extended to other viral targets, such as for COVID-19.

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