scholarly journals Improving Baculovirus Infectivity by Efficiently Embedding Enhancing Factors into Occlusion Bodies

2017 ◽  
Vol 83 (14) ◽  
Author(s):  
Shili Yang ◽  
Lijuan Zhao ◽  
Ruipeng Ma ◽  
Wei Fang ◽  
Jia Hu ◽  
...  
Keyword(s):  
Protein Expression ◽  
Fluorescent Protein ◽  
Expression System ◽  
Agrotis Segetum ◽  
Foreign Protein ◽  
Content Type ◽  
Occlusion Bodies ◽  
Foreign Proteins ◽  
Novel Strategy

ABSTRACT The relatively low infectivity of baculoviruses to their host larvae limits their use as insecticidal agents on a larger scale. In the present study, a novel strategy was developed to efficiently embed foreign proteins into Autographa californica multiple nucleopolyhedrovirus (AcMNPV) occlusion bodies (OBs) to achieve stable expression of foreign proteins and to improve viral infectivity. A recombinant AcMNPV bacmid was constructed by expressing the 150-amino-acid (aa) N-terminal segment of polyhedrin under the control of the p10 promoter and the remaining C-terminal 95-aa segment under the control of the polyhedrin promoter. The recombinant virus formed OBs in Spodoptera frugiperda 9 cells, in which the occlusion-derived viruses were embedded in a manner similar to that for wild-type AcMNPV. Next, the 95-aa polyhedrin C terminus was fused to enhanced green fluorescent protein, and the recombinant AcMNPV formed fluorescent green OBs and was stably passaged in vitro and in vivo. The AcMNPV recombinants were further modified by fusing truncated Agrotis segetum granulovirus enhancin or truncated Cydia pomonella granulovirus ORF13 (GP37) to the C-terminal 95 aa of polyhedrin, and both recombinants were able to form normal OBs. Bioactivity assays indicated that the median lethal concentrations of these two AcMNPV recombinants were 3- to 5-fold lower than that of the control virus. These results suggest that embedding enhancing factors in baculovirus OBs by use of this novel technique may promote efficient and stable foreign protein expression and significantly improve baculovirus infectivity. IMPORTANCE Baculoviruses have been used as bioinsecticides for over 40 years, but their relatively low infectivity to their host larvae limits their use on a larger scale. It has been reported that it is possible to improve baculovirus infectivity by packaging enhancing factors within baculovirus occlusion bodies (OBs); however, so far, the packaging efficiency has been low. In this article, we describe a novel strategy for efficiently embedding foreign proteins into AcMNPV OBs by expressing N- and C-terminal (dimidiate) polyhedrin fragments (150 and 95 amino acids, respectively) as fusions to foreign proteins under the control of the p10 and polyhedrin promoters, respectively. When this strategy was used to embed an enhancing factor (enhancin or GP37) into the baculovirus OBs, 3- to 5-fold increases in baculoviral infectivity were observed. This novel strategy has the potential to create an efficient protein expression system and a highly efficient virus-based system for insecticide production in the future.

Expression of Foreign Proteins in Escherichia coli

2021 ◽  
Author(s):  
Ali Iftikhar
Keyword(s):  
Escherichia Coli ◽  
Protein Expression ◽  
Recombinant Proteins ◽  
Expression System ◽  
Maximum Yield ◽  
Protein Yield ◽  
Foreign Protein ◽  
Low Protein ◽  
Prokaryotic Expression System ◽  
Foreign Proteins

Abstract BackgroundOptimization of conditions for the recombinant production of proteins in a prokaryotic expression system is essential as the recombinant proteins impose a metabolic burden on cell's growth leading to low protein yield and low protein expression resulting from cell death.Main textThe concentration of media components is optimized to accommodate for depleted nutrients due to foreign protein expression. The temperature is optimized to reduce proteolytic degradation and accumulation of protein as inclusion bodies in Escherichia coli. The concentration of inducer and time of induction for high protein yield is also optimized. These optimization conditions depend on the promoter under which the gene of interest is present and the characteristics of the target protein.ConclusionIn the past few years, many optimization conditions for the production of recombinant proteins in Escherichia coli have been studied. These conditions depend mainly upon the promoter used to produce protein and the type of protein produced. Optimizing the expression parameters of protein produced in Escherichia coli ensures maximum yield of the desired protein.

scholarly journals Elizabethkingia anophelis: Molecular Manipulation and Interactions with Mosquito Hosts

2015 ◽  
Vol 81 (6) ◽  
pp. 2233-2243 ◽  
Author(s):  
Shicheng Chen ◽  
Michael Bagdasarian ◽  
Edward D. Walker
Keyword(s):  
Fluorescent Protein ◽  
Developmental Stages ◽  
Genetic Manipulation ◽  
Life Stage ◽  
Expression System ◽  
Aedes Triseriatus ◽  
Content Type ◽  
Mosquito Midgut

ABSTRACTFlavobacteria (members of the familyFlavobacteriaceae) dominate the bacterial community in theAnophelesmosquito midgut. One such commensal,Elizabethkingia anophelis, is closely associated withAnophelesmosquitoes through transstadial persistence (i.e., from one life stage to the next); these and other properties favor its development for paratransgenic applications in control of malaria parasite transmission. However, the physiological requirements ofE. anophelishave not been investigated, nor has its capacity to perpetuate despite digestion pressure in the gut been quantified. To this end, we first developed techniques for genetic manipulation ofE. anophelis, including selectable markers, reporter systems (green fluorescent protein [GFP] and NanoLuc), and transposons that function inE. anophelis. A flavobacterial expression system based on the promoter PompAwas integrated into theE. anophelischromosome and showed strong promoter activity to drive GFP and NanoLuc reporter production. Introduced, GFP-taggedE. anophelisassociated with mosquitoes at successive developmental stages and propagated inAnopheles gambiaeandAnopheles stephensibut not inAedes triseriatusmosquitoes. Feeding NanoLuc-tagged cells toA. gambiaeandA. stephensiin the larval stage led to infection rates of 71% and 82%, respectively. In contrast, a very low infection rate (3%) was detected inAedes triseriatusmosquitoes under the same conditions. Of the initialE. anopheliscells provided to larvae, 23%, 71%, and 85% were digested inA. stephensi,A. gambiae, andAedes triseriatus, respectively, demonstrating thatE. anophelisadapted to various mosquito midgut environments differently. Bacterial cell growth increased up to 3-fold when arginine was supplemented in the defined medium. Furthermore, the number of NanoLuc-tagged cells inA. stephensisignificantly increased when arginine was added to a sugar diet, showing it to be an important amino acid forE. anophelis. Animal erythrocytes promotedE. anophelisgrowthin vivoandin vitro, indicating that this bacterium could obtain nutrients by participating in erythrocyte lysis in the mosquito midgut.

scholarly journals Expression of Recombinant Fusion Protein of Newcastle Disease Virus from Escherichia coli Plasmid Clone C-2a by In-vitro Cell-free Protein Expression System

2020 ◽  
Vol 20 ◽  
pp. 04004
Author(s):  
Ahmad Pandu Satria Wiratama ◽  
Aris Haryanto
Keyword(s):  
Protein Expression ◽  
Newcastle Disease ◽  
Recombinant Plasmid ◽  
Expression System ◽  
Free Protein ◽  
F Protein ◽  
E Coli ◽  
Protein Expression System ◽  
Sds Page

Newcastle Disease Virus (NDV) is an infectious disease that infect many kinds of wild and domesticated birds. Infection of NDV become a massive problem for poultry industry around the world especially in Indonesia. Vaccination is an effort to prevent the infection of NDV in poultry. NDV vaccine that used in Indonesia is a conventional life vaccine from LaSota and B1 strains. These type of vaccine is 21%-23% genetically distinct with the virus that spread in the environment. The antibody protection provided by the vaccine is not effective. Therefore, vaccination with new local NDV strain is needed to prevent the NDV infection in Indonesia. The previously study research reported that the local isolate of NDV from Kulon Progo, Indonesia has been isolated. Fusion (F) protein encoding gene that has been inserted into pBT7-N-His expression p lasmid which isolated from clone C-2a of E. coli, then it was expressed by the Cell-free protein expression system. The aim of this study was to confirm whether clone C-2a of E.coli carrying a recombinant plasmid pBT7-N-His-Fusion NDV and to express a recombinant F protein of NDV in-vitro from expression plasmid by cell-free protein expression system. This work started by detection of recombinant plasmid pBT7-N-His-Fusion NDV by DNA plasmid extraction followed by agarose gel electrophoresis. The recombinant F protein was in-vitro expressed by cell-free protein expression kit. The expressed F protein of NDV then was visualized by SDS-PAGE and Westernblott to analyse the expression of NDV recombinant F protein. It confirmed that clone C-2a of E. coli contained plasmid pBT7-N-His (4.001 bp) inserted by recombinant F protein of NDV gene (642 bp). The visualisation of expressed recombinant F protein by SDS-PAGE and Westernblott showed the NDV recombinant F protein was a specific protein fragment with molecular weight of 25,6 kDa..

scholarly journals Ingestibility, Digestibility, and Engineered Biological Control Potential of Flavobacteriumhibernum, Isolated from Larval Mosquito Habitats

2013 ◽  
Vol 80 (3) ◽  
pp. 1150-1158 ◽  
Author(s):  
Shicheng Chen ◽  
Michael G. Kaufman ◽  
Michelle L. Korir ◽  
Edward D. Walker
Keyword(s):  
Fluorescent Protein ◽  
Ingestion Rate ◽  
Expression System ◽  
Luciferase Reporter ◽  
Larval Feeding ◽  
Protein Markers ◽  
Reporter System ◽  
Content Type ◽  
Larval Habitats ◽  
Tree Hole

ABSTRACTFlavobacteriumhibernum, isolated from larval habitats of the eastern tree hole mosquito,A. triseriatus, remained suspended in the larval feeding zone much longer (8 days) than other bacteria. Autofluorescent protein markers were developed for the labeling ofF. hibernumwith a strong flavobacterial expression system. Green fluorescent protein (GFP)-taggedF. hibernumcells were quickly consumed by larval mosquitoes at an ingestion rate of 9.5 × 104/larva/h. The ingestedF. hibernumcells were observed mostly in the foregut and midgut and rarely in the hindgut, suggesting that cells were digested and did not pass the gut viably. The NanoLuc luciferase reporter system was validated for quantitative larval ingestion rate and bacterial fate analyses. Larvae digested 1.87 × 105cells/larva/h, and fewF. hibernumcells were excreted intact. Expression of the GFP::Cry11A fusion protein with the P20 chaperone protein fromBacillus thuringiensisH-14 was successfully achieved inF. hibernum. Whole-cell bioassays of recombinantF. hibernumexhibited high larvicidal activity againstA. triseriatusin microplates and in microcosms simulating tree holes.F. hibernumcells persisted in microcosms at 100, 59, 30, and 10% of the initial densities at days 1, 2, 3, and 6, respectively, when larvae were absent, while larvae consumed nearly all of theF. hibernumcells within 3 days of their addition to microcosms.

scholarly journals Development of a Tetracycline-Inducible Gene Expression System for the Study of Helicobacter pylori Pathogenesis

2013 ◽  
Vol 79 (23) ◽  
pp. 7351-7359 ◽  
Author(s):  
Aleksandra W. Debowski ◽  
Phebe Verbrugghe ◽  
Miriam Sehnal ◽  
Barry James Marshall ◽  
Mohammed Benghezal
Keyword(s):  
Helicobacter Pylori ◽  
Animal Models ◽  
Chronic Infection ◽  
Fluorescent Protein ◽  
Expression System ◽  
Content Type ◽  
Conditional Expression ◽  
H Pylori ◽  
Green Fluorescent

ABSTRACTDeletion mutants and animal models have been instrumental in the study ofHelicobacter pyloripathogenesis. Conditional mutants, however, would enable the study of the temporal gene requirement duringH. pyloricolonization and chronic infection. To achieve this goal, we adapted theEscherichia coliTn10-derived tetracycline-inducible expression system for use inH. pylori. TheureApromoter was modified by inserting one or twotetoperators to generate tetracycline-responsive promoters, nameduPtetO, and these promoters were then fused to the reportergfpmut2 and inserted into different loci. The expression of the tetracycline repressor (tetR) was placed under the control of one of three promoters and inserted into the chromosome. Conditional expression of green fluorescent protein (GFP) in strains harboringtetRanduPtetO-GFPwas characterized by measuring GFP activity and by immunoblotting. The twotet-responsiveuPtetOpromoters differ in strength, and induction of these promoters was inducer concentration and time dependent, with maximum expression achieved after induction for 8 to 16 h. Furthermore, the chromosomal location of theuPtetO-GFPconstruct and the nature of the promoter driving expression oftetRinfluenced the strength of theuPtetOpromoters upon induction. Integration ofuPtetO-GFPandtetRconstructs at different genomic loci was stablein vivoand did not affect colonization. Finally, we demonstrate tetracycline-dependent induction of GFP expressionin vivoduring chronic infection. These results open new experimental avenues for dissectingH. pyloripathogenesis using animal models and for testing the roles of specific genes in colonization of, adaptation to, and persistence in the host.

scholarly journals Holotoxin Activity of Botulinum Neurotoxin Subtype A4 Originating from a Nontoxigenic Clostridium botulinum Expression System

2014 ◽  
Vol 80 (23) ◽  
pp. 7415-7422 ◽  
Author(s):  
Marite Bradshaw ◽  
William H. Tepp ◽  
Regina C. M. Whitemarsh ◽  
Sabine Pellett ◽  
Eric A. Johnson
Keyword(s):  
Clostridium Botulinum ◽  
Botulinum Neurotoxin ◽  
Expression System ◽  
Single Amino Acid ◽  
Content Type ◽  
Expression Host ◽  
Toxin Complex ◽  
Human Neuronal Cells

ABSTRACTClostridium botulinumsubtype A4 neurotoxin (BoNT/A4) is naturally expressed in the dual-toxin-producingC. botulinumstrain 657Ba at 100× lower titers than BoNT/B. In this study, we describe purification of recombinant BoNT/A4 (rBoNT/A4) expressed in a nonsporulating and nontoxigenicC. botulinumexpression host strain. The rBoNT/A4 copurified with nontoxic toxin complex components provided intransby the expression host and was proteolytically cleaved to the active dichain form. Activity of the recombinant BoNT/A4 in mice and in human neuronal cells was about 1,000-fold lower than that of BoNT/A1, and the recombinant BoNT/A4 was effectively neutralized by botulism heptavalent antitoxin. A previous report using recombinant truncated BoNT/A4 light chain (LC) expressed inEscherichia colihas indicated reduced stability and activity of BoNT/A4 LC compared to BoNT/A1 LC, which was surmounted by introduction of a single-amino-acid substitution, I264R. In order to determine whether this mutation would also affect the holotoxin activity of BoNT/A4, a recombinant full-length BoNT/A4 carrying this mutation as well as a second mutation predicted to increase solubility (L260F) was produced in the clostridial expression system. Comparative analyses of thein vitro, cellular, andin vivoactivities of rBoNT/A4 and rBoNT/A4-L260F I264R showed 1,000-fold-lower activity than BoNT/A1 in both the mutated and nonmutated BoNT/A4. This indicates that these mutations do not alter the activity of BoNT/A4 holotoxin. In summary, a recombinant BoNT from a dual-toxin-producing strain was expressed and purified in an endogenous clostridial expression system, allowing analysis of this toxin.

scholarly journals Promotion and Rescue of Intracellular Brucella neotomae Replication during Coinfection with Legionella pneumophila

2017 ◽  
Vol 85 (5) ◽  
Author(s):  
Yoon-Suk Kang ◽  
James E. Kirby
Keyword(s):  
Legionella Pneumophila ◽  
Fluorescent Protein ◽  
Life Cycles ◽  
Wild Type ◽  
Intracellular Replication ◽  
Content Type ◽  
Type Iv ◽  
Reporter Systems ◽  
Brucella Neotomae

ABSTRACT We established a new Brucella neotomae in vitro model system for study of type IV secretion system-dependent (T4SS) pathogenesis in the Brucella genus. Importantly, B. neotomae is a rodent pathogen, and unlike B. abortus, B. melitensis, and B. suis, B. neotomae has not been observed to infect humans. It therefore can be handled more facilely using biosafety level 2 practices. More particularly, using a series of novel fluorescent protein and lux operon reporter systems to differentially label pathogens and track intracellular replication, we confirmed T4SS-dependent intracellular growth of B. neotomae in macrophage cell lines. Furthermore, B. neotomae exhibited early endosomal (LAMP-1) and late endoplasmic reticulum (calreticulin)-associated phagosome maturation. These findings recapitulate prior observations for human-pathogenic Brucella spp. In addition, during coinfection experiments with Legionella pneumophila, we found that defective intracellular replication of a B. neotomae T4SS virB4 mutant was rescued and baseline levels of intracellular replication of wild-type B. neotomae were significantly stimulated by coinfection with wild-type but not T4SS mutant L. pneumophila. Using confocal microscopy, it was determined that intracellular colocalization of B. neotomae and L. pneumophila was required for rescue and that colocalization came at a cost to L. pneumophila fitness. These findings were not completely expected based on known temporal and qualitative differences in the intracellular life cycles of these two pathogens. Taken together, we have developed a new system for studying in vitro Brucella pathogenesis and found a remarkable T4SS-dependent interplay between Brucella and Legionella during macrophage coinfection.

scholarly journals A Cytochrome b 5 -Containing Plastid-Located Fatty Acid Desaturase from Chlamydomonas reinhardtii

2012 ◽  
Vol 11 (7) ◽  
pp. 856-863 ◽  
Author(s):  
Simone Zäuner ◽  
Wibke Jochum ◽  
Tara Bigorowski ◽  
Christoph Benning
Keyword(s):  
Fatty Acid ◽  
Chlamydomonas Reinhardtii ◽  
Fluorescent Protein ◽  
Acyl Chain ◽  
Transit Peptide ◽  
Primary Electron ◽  
Chloroplast Targeting ◽  
Content Type ◽  
Cell Extracts

ABSTRACT Monogalactosyldiacylglycerol (MGDG) in Chlamydomonas reinhardtii and other green algae contains hexadeca-4,7,10,13-tetraenoic acid (16:4) in the glycerol sn- 2 position. While many genes necessary for the introduction of acyl chain double bonds have been functionally characterized, the Δ4-desaturase remained unknown. Using a phylogenetic comparison, a candidate gene encoding the MGDG-specific Δ4-desaturase from Chlamydomonas (CrΔ4FAD) was identified. CrΔ4FAD shows all characteristic features of a membrane-bound desaturase, including three histidine boxes and a transit peptide for chloroplast targeting. But it also has an N-terminal cytochrome b 5 domain, distinguishing it from other known plastid desaturases. Cytochrome b 5 is the primary electron donor for endoplasmic reticulum (ER) desaturases and is often fused to the desaturase domain in desaturases modifying the carboxyl end of the acyl group. Difference absorbance spectra of the recombinant cytochrome b 5 domain of CrΔ4FAD showed that it is functional in vitro . Green fluorescent protein fusions of CrΔ4FAD localized to the plastid envelope in Chlamydomonas . Interestingly, overproduction of CrΔ4FAD in Chlamydomonas not only increased levels of 16:4 acyl groups in cell extracts but specifically increased the total amount of MGDG. Vice versa, the amount of MGDG was lowered in lines with reduced levels of CrΔ4FAD. These data suggest a link between MGDG molecular species composition and galactolipid abundance in the alga, as well as a specific function for this fatty acid in MGDG.

scholarly journals Visualization of Periplasmic and Cytoplasmic Proteins with a Self-Labeling Protein Tag

2016 ◽  
Vol 198 (7) ◽  
pp. 1035-1043 ◽  
Author(s):  
Na Ke ◽  
Dirk Landgraf ◽  
Johan Paulsson ◽  
Mehmet Berkmen
Keyword(s):  
Escherichia Coli ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Living Cells ◽  
Functional Protein ◽  
Content Type ◽  
Additional Tool

ABSTRACTThe use of fluorescent and luminescent proteins in visualizing proteins has become a powerful tool in understanding molecular and cellular processes within living organisms. This success has resulted in an ever-increasing demand for new and more versatile protein-labeling tools that permit light-based detection of proteins within living cells. In this report, we present data supporting the use of the self-labeling HaloTag protein as a light-emitting reporter for protein fusions within the model prokaryoteEscherichia coli. We show that functional protein fusions of the HaloTag can be detected bothin vivoandin vitrowhen expressed within the cytoplasmic or periplasmic compartments ofE. coli. The capacity to visually detect proteins localized in various prokaryotic compartments expands today's molecular biologist toolbox and paves the path to new applications.IMPORTANCEVisualizing proteins microscopically within living cells is important for understanding both the biology of cells and the role of proteins within living cells. Currently, the most common tool is green fluorescent protein (GFP). However, fluorescent proteins such as GFP have many limitations; therefore, the field of molecular biology is always in need of new tools to visualize proteins. In this paper, we demonstrate, for the first time, the use of HaloTag to visualize proteins in two different compartments within the model prokaryoteEscherichia coli. The use of HaloTag as an additional tool to visualize proteins within prokaryotes increases our capacity to ask about and understand the role of proteins within living cells.

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