scholarly journals Enhanced Production of Recombinant Protein by Fusion Expression with Ssp DnaB Mini-Intein in the Baculovirus Expression System

Viruses ◽  
2018 ◽  
Vol 10 (10) ◽  
pp. 523 ◽  
Author(s):  
Won Gwak ◽  
Jae Choi ◽  
Beom Han ◽  
Sung Bae ◽  
Soo Woo
Keyword(s):  
Fluorescent Protein ◽  
Expression System ◽  
Classical Swine Fever ◽  
Baculovirus Expression System ◽  
Additional Treatment ◽  
Fusion Partner ◽  
Target Proteins ◽  
Enhanced Production ◽  
Baculovirus Expression ◽  
Green Fluorescent

The baculovirus expression system (BES) is considered to be a very powerful tool for the expression of numerous difficult to express vertebrate proteins. Ssp DnaB mini-intein is a useful fusion partner for the production of recombinant proteins because it can be self-cleaved by controlling the pH and temperature, without additional treatment. To evaluate the utility of Ssp DnaB mini-intein in the BES, recombinant viruses were generated to express the enhanced green fluorescent protein, the VP2 protein of porcine parvovirus, and the E2 protein of classical swine fever virus fused to a mini-intein. As expected, intracellular self-cleavage of the mini-intein occurred during virus infection, but the cleavage initiation time varied depending on the target protein. Significantly enhanced protein production was observed for all of the target proteins that were fused to the mini-intein. This increase was enough to overcome the decrease in the fusion protein due to intracellular self-cleavage. The mini-intein in all of the recombinant fusion proteins was successfully cleaved by controlling the pH and temperature. These results suggest that the Ssp DnaB mini-intein is a useful fusion partner in the BES for easy purification and enhanced production of target proteins.

scholarly journals Rotavirus Virus-Like Particles as Surrogates in Environmental Persistence and Inactivation Studies

2004 ◽  
Vol 70 (7) ◽  
pp. 3904-3909 ◽  
Author(s):  
Santiago Caballero ◽  
F. Xavier Abad ◽  
Fabienne Loisy ◽  
Françoise S. Le Guyader ◽  
Jean Cohen ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Uv Light ◽  
Expression System ◽  
Baculovirus Expression System ◽  
Virus Removal ◽  
Virus Like Particles ◽  
Reverse Transcription Pcr ◽  
Actual Field ◽  
Uv Dose ◽  
Green Fluorescent

ABSTRACT Virus-like particles (VLPs) with the full-length VP2 and VP6 rotavirus capsid proteins, produced in the baculovirus expression system, have been evaluated as surrogates of human rotavirus in different environmental scenarios. Green fluorescent protein-labeled VLPs (GFP-VLPs) and particles enclosing a heterologous RNA (pseudoviruses), whose stability may be monitored by flow cytometry and antigen capture reverse transcription-PCR, respectively, were used. After 1 month in seawater at 20°C, no significant differences were observed between the behaviors of GFP-VLPs and of infectious rotavirus, whereas pseudovirus particles showed a higher decay rate. In the presence of 1 mg of free chlorine (FC)/liter both tracers persisted longer in freshwater at 20°C than infectious viruses, whereas in the presence of 0.2 mg of FC/liter no differences were observed between tracers and infectious rotavirus at short contact times. However, from 30 min of contact with FC onward, the decay of infectious rotavirus was higher than that of recombinant particles. The predicted Ct value for a 90% reduction of GFP-VLPs or pseudoviruses induces a 99.99% inactivation of infectious rotavirus. Both tracers were more resistant to UV light irradiation than infectious rotavirus in fresh and marine water. The effect of UV exposure was more pronounced on pseudovirus than in GFP-VLPs. In all types of water, the UV dose to induce a 90% reduction of pseudovirus ensures a 99.99% inactivation of infectious rotavirus. Recombinant virus surrogates open new possibilities for the systematic validation of virus removal practices in actual field situations where pathogenic agents cannot be introduced.

scholarly journals Expression, purification and structural analysis of functional GABA transporter 1 using the baculovirus expression system

2017 ◽  
Vol 13 ◽  
pp. 874-882 ◽  
Author(s):  
Jing Hu ◽  
Chris Weise ◽  
Christoph Böttcher ◽  
Hua Fan ◽  
Jian Yin
Keyword(s):  
Fusion Protein ◽  
Fluorescent Protein ◽  
Expression System ◽  
Baculovirus Expression System ◽  
Size Exclusion ◽  
Coupled Transport ◽  
Gaba Transporter ◽  
Gfp Fusion Protein ◽  
Baculovirus Expression ◽  
Gaba Transporter 1

The γ-aminobutyric acid (GABA) transporter 1 (GAT1) belongs to a family of Na+ and Cl−-coupled transport proteins and possesses 12 putative transmembrane domains. To perform structural analyses of the GAT1 protein, the GAT1/green fluorescent protein (GFP) fusion protein was functionally expressed in insect Sf9 cells by the BAC-TO-BAC® baculovirus expression system. A two-step procedure to purify the GAT1/GFP fusion protein from insect Sf9 cells has been established and involves immunoaffinity chromatography using self-prepared anti-GFP antibodies and size-exclusion fast protein liquid chromatography (SE-FPLC). A yield of 200–300 μg of the GAT1/GFP protein could be purified from 400–600 mL of infected Sf9 cells. The purified protein was analyzed by transmission electron microscopy (TEM), which revealed that the GAT1/GFP fusion protein was isolated in its monomeric form.

Expression of EGFP driven by BmNPV orf4 promoter, a novel immediate early promoter

Biologia ◽  
2009 ◽  
Vol 64 (2) ◽  
Author(s):  
Wu-Jie Su ◽  
Bing Li ◽  
Wei-De Shen ◽  
Yan Wu ◽  
Shan-Ying Zhu ◽  
...  
Keyword(s):  
Bombyx Mori ◽  
Fluorescent Protein ◽  
Early Stage ◽  
Expression System ◽  
Baculovirus Expression System ◽  
Immediate Early ◽  
Early Promoter ◽  
Polyhedrin Promoter ◽  
Foreign Genes ◽  
Green Fluorescent

AbstractBombyx mori nucleopolyhedrovirus (BmNPV) orf4 has been shown to be expressed at very early stage of Bm-NPV infection cycle. In this study, using transient expression experiment, we demonstrated for the first time that orf4 promoter is an immediate early promoter, indicating that orf4 may play a role in the immediate-early stage of BmNPV infection. Moreover, with the recently developed Bac-to-Bac/BmNPV baculovirus expression system and a modified pFast-Bac1 whose polyhedrin promoter was replaced with orf4 promoter, a recombinant bacmid baculovirus expressing enhanced green fluorescent protein (EGFP) under the control of orf4 promoter in Bombyx mori (Bm) cells was successfully constructed. The result not only showed that the polyhedrin promoter can be replaced easily with other promoters to direct the expression of foreign genes by using this novel system but also laid the foundation for the rescue experiment of orf4 deletion mutant.

scholarly journals Enhanced production of Canine parvovirus VP2  protein using improved Baculovirus expression  system  and its immunogenicity

2020 ◽  
Author(s):  
Dao Chang ◽  
Yangkun Liu ◽  
Yangyang Chen ◽  
Xiaomin Hu ◽  
Andrey Burov ◽  
...  
Keyword(s):  
Expression System ◽  
Baculovirus Expression System ◽  
Canine Parvovirus ◽  
Full Length ◽  
Recombinant Viruses ◽  
Vp2 Protein ◽  
Enhanced Production ◽  
Baculovirus Expression ◽  
Yield Production ◽  
High Level

Abstract Background : Canine parvovirus (CPV) is now recognized as a serious threat to dog industry worldwide. Vaccination remains the principal tool to control CPV infection. However, due to low yield, production of VP2 protein of CPV in baculovirus expression system remains challenging. The aim of this study was to increase the VP2 protein production by using a improved baculovirus expression system (Multibac) and evaluate the immunogenicity of the purified VP2 protein in mice. Results: The results showed that CPV VP2 protein was successfully expressed in the improved baculovirus expression system efficiently. A high level of expression of the full length VP2 protein was achieved using our modified system. The recombinant virus carrying two copies of VP2 showed the highest expression level, with a productivity of 186 mg/L, which is about 1.4-1.6 fold that of the recombinant viruses carrying only one copy. The purified protein could react with Mouse anti-His tag monoclonal antibody and Rabbit anti-VP2 polyclonal antibody with good reactogenicity. The mice were then immunized with purified full length VP2 protein to evaluate its immunogenicity. After vaccination, VP2 protein could induce the mice produce high level of hemagglutination inhibition antibodies. Conclusions: Full length CPV VP2 protein was successfully expressed at high level and purified efficiently. And it stimulated mice to produce high level of antibody. The full length VP2 expressed in this study could be used as a putative economic and efficient subunit vaccine against CPV infection.

scholarly journals The Maturation Process of pVP2 Requires Assembly of Infectious Bursal Disease Virus Capsids

2002 ◽  
Vol 76 (5) ◽  
pp. 2384-2392 ◽  
Author(s):  
Christophe Chevalier ◽  
Jean Lepault ◽  
Inge Erk ◽  
Bruno Da Costa ◽  
Bernard Delmas
Keyword(s):  
Disease Virus ◽  
Infectious Bursal Disease Virus ◽  
Fluorescent Protein ◽  
Expression System ◽  
Baculovirus Expression System ◽  
Infectious Bursal Disease ◽  
Maturation Process ◽  
Final Maturation ◽  
Bursal Disease ◽  
Green Fluorescent

ABSTRACT Infectious bursal disease virus (IBDV) is a nonenveloped avian virus with a two-segment double-stranded RNA genome. Its T=13 icosahedral capsid is most probably assembled with 780 subunits of VP2 and 600 copies of VP3 and has a diameter of about 60 nm. VP1, the RNA-dependent RNA polymerase, resides inside the viral particle. Using a baculovirus expression system, we first observed that expression of the pVP2-VP4-VP3 polyprotein encoded by the genomic segment IBDA results mainly in the formation of tubules with a diameter of about 50 nm and composed of pVP2, the precursor of VP2. Very few virus-like particles (VLPs) and VP4 tubules with a diameter of about 25 nm were also identified. The inefficiency of VLP assembly was further investigated by expression of additional IBDA-derived constructs. Expression of pVP2 without any other polyprotein components results in the formation of isometric particles with a diameter of about 30 nm. VLPs were observed mainly when a large exogeneous polypeptide sequence (the green fluorescent protein sequence) was fused to the VP3 C-terminal domain. Large numbers of VLPs were visualized by electron microscopy, and single particles were shown to be fluorescent by standard and confocal microscopy analysis. Moreover, the final maturation process converting pVP2 into the VP2 mature form was observed on generated VLPs. We therefore conclude that the correct scaffolding of the VP3 can be artificially induced to promote the formation of VLPs and that the final processing of pVP2 to VP2 is controlled by this particular assembly. To our knowledge, this is the first report of the engineering of a morphogenesis switch to control a particular type of capsid protein assembly.

scholarly journals Expression of functional G protein beta gamma dimers of defined subunit composition using a baculovirus expression system.

1992 ◽  
Vol 267 (19) ◽  
pp. 13123-13126 ◽  
Author(s):  
S.G. Graber ◽  
R.A. Figler ◽  
V.K. Kalman-Maltese ◽  
J.D. Robishaw ◽  
J.C. Garrison
Keyword(s):  
G Protein ◽  
Expression System ◽  
Baculovirus Expression System ◽  
Subunit Composition ◽  
Baculovirus Expression

scholarly journals Production, processing and partial purification of functional G protein βγ subunits in baculovirus-infected insect cells

1992 ◽  
Vol 286 (3) ◽  
pp. 677-680 ◽  
Author(s):  
J D Robishaw ◽  
V K Kalman ◽  
K L Proulx
Keyword(s):  
G Protein ◽  
G Proteins ◽  
Insect Cells ◽  
Expression System ◽  
Baculovirus Expression System ◽  
Partial Purification ◽  
Baculovirus Expression ◽  
Gamma Subunits ◽  
Infected Insect ◽  
Beta 2

As a result of the inability to resolve the heterogeneous mixture of G protein beta gamma subunits present in tissues, it has not been possible to compare different beta gamma subunits of the G proteins in terms of their proposed roles in receptor-effector coupling. This study was undertaken to establish the utility of the baculovirus expression system in producing homogeneous beta gamma subunits of defined composition for the comparative analysis of these subunits in reconstitution systems. In this study we report the expression, and appropriate post-translational processing, of recombinant beta 2, gamma 2 and gamma 3 subunits. In addition, we show that the recombinant beta gamma subunits can be readily purified, and can functionally interact with the alpha subunits of the G proteins.

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